Association of autoantibodies with the IFN signature and NETosis in patients with systemic lupus erythematosus.

Objective: Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by a variety of disease symptoms and an unpredictable clinical course. To improve treatment outcome, stratification based on immunological manifestations commonly seen in patients with SLE such as autoantibodies, type I interferon (IFN) signature and neutrophil extracellular trap (NET) release may help. It is assumed that there is an association between these immunological phenomena, since NET release induces IFN production and IFN induces autoantibody formation via B-cell activation. Here we studied the association between autoantibodies, the IFN signature, NET release, and clinical manifestations in patients with SLE. Methods: We performed principal component analysis (PCA) and hierarchical clustering of 57 SLE-related autoantibodies in 25 patients with SLE. We correlated each autoantibody to the IFN signature and NET inducing capacity. Results: We observed two distinct clusters: one cluster contained mostly patients with a high IFN signature. Patients in this cluster often present with cutaneous lupus, and have higher anti-dsDNA concentrations. Another cluster contained a mix of patients with a high and low IFN signature. Patients with high and low NET inducing capacity were equally distributed between the clusters. Variance between the clusters is mainly driven by antibodies against histones, RibP2, RibP0, EphB2, RibP1, PCNA, dsDNA, and nucleosome. In addition, we found a trend towards increased concentrations of autoantibodies against EphB2, RibP1, and RNP70 in patients with an IFN signature. We found a negative correlation of NET inducing capacity with anti-FcER (r = -0.530; p = 0.007) and anti-PmScl100 (r = -0.445; p = 0.03). Conclusion: We identified a subgroup of patients with an IFN signature that express increased concentrations of antibodies against DNA and RNA-binding proteins, which can be useful for further patient stratification and a more targeted therapy. We did not find positive associations between autoantibodies and NET inducing capacity. Our study further strengthens the evidence of a correlation between RNA-binding autoantibodies and the IFN signature.

Memory B-cell derived donor-specific antibodies do not predict outcome in sensitized kidney transplant recipients: a retrospective single-center study.

Background: Repeated exposure to sensitizing events can activate HLA-specific memory B cells, leading to the production of donor-specific memory B cell antibodies (DSAm) that pose a risk for antibody-mediated rejection (ABMR) in kidney transplant recipients (KTRs). This single-center retrospective study aimed to identify DSAm and assess their association with outcomes in a cohort of KTRs with pretransplant serum donor-specific antibodies (DSA). Methods: We polyclonally activated pretransplant peripheral blood mononuclear cells (PBMCs) from 60 KTRs in vitro, isolated and quantified IgG from the culture supernatant using ELISA, and analyzed the HLA antibodies of eluates with single antigen bead (SAB) assays, comparing them to the donor HLA typing for potential DSAm. Biopsies from 41 KTRs were evaluated for rejection based on BANFF 2019 criteria. Results: At transplantation, a total of 37 DSAm were detected in 26 of 60 patients (43%), of which 13 (35%) were found to be undetectable in serum. No significant association was found between pretransplant DSAm and ABMR (P=0.53). Similar results were observed in a Kaplan-Meier analysis for ABMR within the first year posttransplant (P=0.29). Additionally, MFI levels of DSAm showed no significant association with ABMR (P=0.28). Conclusion: This study suggests no significant association between DSAm and biopsy-proven clinical ABMR. Further prospective research is needed to determine whether assessing DSAm could enhance existing immunological risk assessment methods for monitoring KTRs, particularly in non-sensitized KTRs.

A 21-bp deletion in the complement regulator CD55 promotor region is associated with multifocal motor neuropathy and its disease course.

BACKGROUND AND AIMS: To further substantiate the role of antibody-mediated complement activation in multifocal motor neuropathy (MMN) immunopathology, we investigated the distribution of promotor polymorphisms of genes encoding the membrane-bound complement regulators CD46, CD55, and CD59 in patients with MMN and controls, and evaluated their association with disease course. METHODS: We used Sanger sequencing to genotype five common polymorphisms in the promotor regions of CD46, CD55, and CD59 in 133 patients with MMN and 380 controls. We correlated each polymorphism to clinical parameters. RESULTS: The genotype frequencies of rs28371582, a 21-bp deletion in the CD55 promotor region, were altered in patients with MMN as compared to controls (p .009; Del/Del genotype 16.8% vs. 7.7%, p .005, odds ratio: 2.43 [1.27-4.58]), and patients carrying this deletion had a more favorable disease course (mean difference 0.26 Medical Research Council [MRC] points/year; 95% confidence interval [CI]: 0.040-0.490, p .019). The presence of CD59 rs141385724 was associated with less severe pre-diagnostic disease course (mean difference 0.940 MRC point/year; 95% CI: 0.083-1.80, p .032). INTERPRETATION: MMN susceptibility is associated with a 21-bp deletion in the CD55 promotor region (rs2871582), which is associated with lower CD55 expression. Patients carrying this deletion may have a more favorable long-term disease outcome. Taken together, these results point out the relevance of the pre-C5 level of the complement cascade in the inflammatory processes underlying MMN.

Ellipro scores of donor epitope specific HLA antibodies are not associated with kidney graft survival.

In kidney transplantation, donor HLA antibodies are a risk factor for graft loss. Accessibility of donor eplets for HLA antibodies is predicted by the ElliPro score. The clinical usefulness of those scores in relation to transplant outcome is unknown. In a large Dutch kidney transplant cohort, Ellipro scores of pretransplant donor antibodies that can be assigned to known eplets (donor epitope specific HLA antibodies [DESAs]) were compared between early graft failure and long surviving deceased donor transplants. We did not observe a significant Ellipro score difference between the two cohorts, nor significant differences in graft survival between transplants with DESAs having high versus low total Ellipro scores. We conclude that Ellipro scores cannot be used to identify DESAs associated with early versus late kidney graft loss in deceased donor transplants.

Determination of the clinical relevance of donor epitope-specific HLA-antibodies in kidney transplantation.

In kidney transplantation, survival rates are still partly impaired due to the deleterious effects of donor specific HLA antibodies (DSA). However, not all luminex-defined DSA appear to be clinically relevant. Further analysis of DSA recognizing polymorphic amino acid configurations, called eplets or functional epitopes, might improve the discrimination between clinically relevant vs. irrelevant HLA antibodies. To evaluate which donor epitope-specific HLA antibodies (DESAs) are clinically important in kidney graft survival, relevant and irrelevant DESAs were discerned in a Dutch cohort of 4690 patients using Kaplan-Meier analysis and tested in a cox proportional hazard (CPH) model including nonimmunological variables. Pre-transplant DESAs were detected in 439 patients (9.4%). The presence of certain clinically relevant DESAs was significantly associated with increased risk on graft loss in deceased donor transplantations (p < 0.0001). The antibodies recognized six epitopes of HLA Class I, 3 of HLA-DR, and 1 of HLA-DQ, and most antibodies were directed to HLA-B (47%). Fifty-three patients (69.7%) had DESA against one donor epitope (range 1-5). Long-term graft survival rate in patients with clinically relevant DESA was 32%, rendering DESA a superior parameter to classical DSA (60%). In the CPH model, the hazard ratio (95% CI) of clinically relevant DESAs was 2.45 (1.84-3.25) in deceased donation, and 2.22 (1.25-3.95) in living donation. In conclusion, the developed model shows the deleterious effect of clinically relevant DESAs on graft outcome which outperformed traditional DSA-based risk analysis on antigen level.

The predictive value of antinuclear antibodies in patients with suspected connective tissue disease for the development of connective tissue diseases.

OBJECTIVES: Early confirmation of the diagnosis of connective tissue diseases (CTD) is important, as prolonged disease activity can result in irreversible organ damage. Although antinuclear antibodies (ANAs) have been shown to precede the diagnosis of SLE, this has not been investigated in large cohorts for other CTDs. In this study, we investigated whether the presence of antinuclear autoantibodies in undiagnosed patients suspected of having CTDs is predictive of development of a future CTD. METHODS: We screened the Electronic Health Records of a cohort of 1030 patients, who were tested for ANAs and their specificity in 2013/2014, to evaluate whether new CTD diagnoses had been recorded by a clinician between the original blood draw date and 2020. We compared the prevalence of ANAs in patients who developed a new CTD diagnosis during follow-up with patients with similar symptoms at baseline who did not receive a subsequent CTD diagnosis and with patients with an established CTD at baseline. RESULTS: Sixteen out of 1030 patients had developed a new CTD in the studied time period. The mean time period between baseline blood draw and subsequent CTD diagnosis of these patients was approximately 2.3 years. Eleven out of 16 (69%) newly diagnosed patients had positive ANA screening tests, compared to 54% of patients with a CTD diagnosis at baseline (p=ns) and 30% of symptomatic undiagnosed patients (p<0.001). This resulted in a positive predictive value (PPV) of 7% and a negative predictive value (NPV) of 98% for the development of a new CTD in undiagnosed symptomatic patients. For specific ANAs associated with the suspected CTD, the PPV was 12%, with a NPV of 98%. CONCLUSIONS: Progression to a CTD diagnosis is rare in undiagnosed patients. Undiagnosed patients with symptoms associated with a CTD who progress to a CTD more often have ANAs than patients with similar symptoms who do not progress to a CTD. ANA testing can be used to more stringently select patients who should remain in follow-up.

In Vitro Effectiveness of Soft Contact Lens Solutions Available on the Dutch Market against Acanthamoeba Species.

Acanthamoeba keratitis is almost universally associated with contact lens (CL) use. Until today, however, CL solution manufacturing protocols lack testing of anti-amoebic activity. This study investigates the effectiveness of CL solutions available on the Dutch market against trophozoites and cysts of Acanthamoeba castellanii and Acanthamoeba polyphaga. Sixteen CL solutions were tested: 13 multiple purpose solutions (MPS), 2 hydrogen peroxidase solutions (HPS) and 1 povidone-iodine-based solution (PIS). The Spearman-Karber (SK) log reduction method and an XTT colorimetric assay were used to evaluate the effectiveness at the manufacturer's minimum recommended disinfection time (MMRDT) and after eight hours. At the MMRDT, one MPS showed an SK mean log reduction (MLR) of >3.0 against A. castellanii trophozoites. Two additional MPS and both HPS reached this threshold after eight hours. The SK MLR values for A. polyphaga trophozoites were between 1 and 3 at all time points. Using the XTT colorimetric assay, only HPS 1 showed >99.9% reduction (equivalent to 3 log reduction) in metabolic activity of A. castellanii trophozoites after eight hours. For A. polyphaga, both HPS and PIS showed a metabolic reduction of >99.9% after eight hours. Cysts were resistant against all solutions. We conclude that following the manufacturer's guidelines, few solutions provide sufficient effectiveness against Acanthamoeba trophozoites and none against cysts. The results underline the importance of adequate hygiene when handling CLs.

Complement component C3 and C5b-9 deposition on hypoxia reperfused endothelial cells by non-HLA antibodies against RhoGDI2: A player involved in graft failure?

Antibodies against Rho GDP-dissociation inhibitor 2 (RhoGDI2) are associated with inferior graft survival in transplant patients receiving a kidney from deceased donors. Although this suggests that these antibodies contribute to graft injury because of ischemia, it remains unknown whether they are also pathogenically involved in the process of graft loss. To study this, we firstly analyzed the IgG subclass profile of anti-RhoGDI2 antibodies in kidney transplant recipients, and whether antibody titers change over time or because of acute rejection. Next, we investigated the expression of RhoGDI2 on primary kidney and lung endothelial cells (ECs) upon hypoxia reperfusion. In addition, the complement-fixing properties of anti-RhoGDI2 antibodies were studied using imaging flow cytometry. Anti-RhoGDI2 antibodies in patients are mainly IgG1, and titers remained stable and seemed not be changed because of rejection. Antibodies against RhoGDI2, which surface expression seemed to increase upon hypoxia reperfusion, co-localized with C3 on ECs. Binding of human IgG1 monoclonal anti-RhoGDI2 antibodies as well as patient derived antibodies, resulted in complement activation, suggesting that these antibodies are complement fixing. This study suggested a potential pathogenic role of anti-RhoGDI2 antibodies in kidney graft loss. During ischemia reperfusion, the ability of these antibodies to fix complement could be one of the mechanisms resulting in tissue injury.

The number of donor HLA-derived T cell epitopes available for indirect antigen presentation determines the risk for vascular rejection after kidney transplantation.

The role of the indirect T-cell recognition pathway of allorecognition in acute T cell-mediated rejection (aTCMR) is not well defined. The amount of theoretical T-cell epitopes available for indirect allorecognition can be quantified for donor-recipient combinations by the Predicted Indirectly ReCognizable HLA Epitopes algorithm (PIRCHE-II). The PIRCHE-II score was calculated for 688 donor kidney-recipient combinations and associated with the incidence of first-time diagnosed cases of TCMR. A diagnosis of TCMR was made in 182 cases; 121 cases of tubulo-interstitial rejection cases (79 cases of borderline TCMR, 42 cases of TCMR IA-B) and 61 cases of vascular TCMR (TCMR II-III). The PIRCHE-II score for donor HLA-DR/DQ (PIRCHE-II DR/DQ) was highly associated with vascular rejection. At one year after transplantation, the cumulative percentage of recipients with a vascular rejection was 12.7%, 8.6% and 2.1% within respectively the high, medium and low tertile of the PIRCHE-II DR/DQ score (p<0.001). In a multivariate regression analysis this association remained significant (p<0.001 for PIRCHE-II DR/DQ tertiles). The impact of a high PIRCHE-II DR/DQ score was mitigated by older recipient age and a living donor kidney. In conclusion, indirect antigen presentation of donor HLA-peptides may significantly contribute to the risk for acute vascular rejection.

Microarray analysis of autoantibodies can identify future Systemic Lupus Erythematosus patients.

OBJECTIVE: Reliable early ascertainment in patients with SLE is important to prevent the accumulation of irreversible organ damage. Autoantibodies are often present in the serum of patients before the first symptoms arise, therefore they are of potential use as early diagnostic tools. METHODS: We used a custom-made antibody microarray containing 57 autoantigens to analyze serum samples of 1519 patients previously tested for anti-dsDNA and 361 samples of self-reported healthy blood bank donors (BBD). The 1519 patients included 483 patients with SLE, 346 patients with other immune mediated inflammatory diseases (IMID), 218 patient controls without relevant clinical symptoms (Non-IMID), and 472 patients that did not fit in any of the previous groups (Rest). The Non-IMID and BBD groups were used individually to create multivariable prediction models to distinguish samples of patients with SLE from these control groups. We subsequently used these models to predict the outcome for samples of patients who developed SLE while in follow-up (pre-SLE). RESULTS: Out of 1036 patients with no diagnosis of SLE at the moment of sample collection, 17 patients developed SLE while in follow-up (mean time to diagnosis 7.2 months). The best performing model (AUC 0.83) identified 9 out of 17 (53%) pre-SLE samples as SLE, with a specificity of 94%. CONCLUSION: Serum samples of patients who will develop SLE in the future already show a shift of the autoantibody profile prior to diagnosis. In this study, we show that these autoantibody profiles can be used to identify these future SLE patients.

Measurement of IgE to hazelnut allergen components cannot replace hazelnut challenge in Dutch adults.

BACKGROUND: Component-resolved diagnostics (CRD) help predict hazelnut allergy (HA) in children, but are of unknown diagnostic value in adults. This study aimed to evaluate the diagnostic accuracy of IgE to hazelnut extract and components in adults. METHODS: A Dutch population of consecutively presenting adults suspected of HA, who underwent a double-blind placebo-controlled food challenge, were included. Serum IgE to hazelnut extract and Cor a 1, 8, 9, and 14 was measured on ImmunoCAP. Diagnostic accuracy was assessed by area under the curve (AUC) analysis. RESULTS: Of 89 patients undergoing challenge, 46 had challenge-confirmed HA: 17 based on objective and 29 based on subjective symptoms. At commonly applied cutoffs 0.1 and 0.35 kUA /L, high sensitivity was observed for IgE to hazelnut extract and Cor a 1 (range 85-91%), and high specificity for IgE to Cor a 8, 9 and 14 (range 77-95%). However, the AUCs for hazelnut extract and components were too low for accurate prediction of HA (range 0.50-0.56). Combining hazelnut extract and component IgE measurements did not significantly improve accuracy. Higher IgE levels to Cor a 9 and 14 were tentatively associated with HA with objective symptoms, but the corresponding AUCs still only reached 0.68 and 0.63, respectively. CONCLUSIONS: Although hazelnut allergic adults are generally sensitized to hazelnut extract and Cor a 1, and hazelnut tolerant adults are usually not sensitized to Cor a 8, 9, or 14, challenge testing is still needed to accurately discriminate between presence and absence of HA in adults from a birch-endemic country.

T-Cell Epitopes Shared Between Immunizing HLA and Donor HLA Associate With Graft Failure After Kidney Transplantation.

CD4+ T-helper cells play an important role in alloimmune reactions following transplantation by stimulating humoral as well as cellular responses, which might lead to failure of the allograft. CD4+ memory T-helper cells from a previous immunizing event can potentially be reactivated by exposure to HLA mismatches that share T-cell epitopes with the initial immunizing HLA. Consequently, reactivity of CD4+ memory T-helper cells toward T-cell epitopes that are shared between immunizing HLA and donor HLA could increase the risk of alloimmunity following transplantation, thus affecting transplant outcome. In this study, the amount of T-cell epitopes shared between immunizing and donor HLA was used as a surrogate marker to evaluate the effect of donor-reactive CD4+ memory T-helper cells on the 10-year risk of death-censored kidney graft failure in 190 donor/recipient combinations using the PIRCHE-II algorithm. The T-cell epitopes of the initial theoretical immunizing HLA and the donor HLA were estimated and the number of shared PIRCHE-II epitopes was calculated. We show that the natural logarithm-transformed PIRCHE-II overlap score, or Shared T-cell EPitopes (STEP) score, significantly associates with the 10-year risk of death-censored kidney graft failure, suggesting that the presence of pre-transplant donor-reactive CD4+ memory T-helper cells might be a strong indicator for the risk of graft failure following kidney transplantation.

Adult mouse and human organoids derived from thyroid follicular cells and modeling of Graves' hyperthyroidism.

The thyroid maintains systemic homeostasis by regulating serum thyroid hormone concentrations. Here we report the establishment of three-dimensional (3D) organoids from adult thyroid tissue representing murine and human thyroid follicular cells (TFCs). The TFC organoids (TFCOs) harbor the complete machinery of hormone production as visualized by the presence of colloid in the lumen and by the presence of essential transporters and enzymes in the polarized epithelial cells that surround a central lumen. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at comparable levels to tissue. Single-cell RNA sequencing and transmission electron microscopy confirm that TFCOs phenocopy primary thyroid tissue. Thyroid hormones are readily detectable in conditioned medium of human TFCOs. We show clinically relevant responses (increased proliferation and hormone secretion) of human TFCOs toward a panel of Graves' disease patient sera, demonstrating that organoids can model human autoimmune disease.

Microarray testing in patients with systemic lupus erythematosus identifies a high prevalence of CpG DNA-binding antibodies.

OBJECTIVE: Many autoantibodies are known to be associated with SLE, although their role in clinical practice is limited because of low sensitivity and weak associations with clinical manifestations. There has been great interest in the discovery of new autoantibodies to use in clinical practice. In this study, we investigated 57 new and known antibodies and their potential for diagnostics or risk stratification. METHODS: Between 2014 and 2017, residual sera of all anti-dsDNA tests in the UMC Utrecht were stored in a biobank. This included sera of patients with SLE, patients with a diagnosis of another immune-mediated inflammatory disease (IMID), patients with low (non-IMID) or medium levels of clinical suspicion of SLE but no IMID diagnosis (Rest), and self-reported healthy blood bank donors. Diagnosis and (presence of) symptoms at each blood draw were retrospectively assessed in the patient records with the Utrecht Patient-Oriented Database using a newly developed text mining algorithm. Sera of patients were analysed for the presence of 57 autoantibodies with a custom-made immunofluorescent microarray. Signal intensity cut-offs for all antigens on the microarray were set to the 95th percentile of the non-IMID control group. Differences in prevalence of autoantibodies between patients with SLE and control groups were assessed. RESULTS: Autoantibody profiles of 483 patients with SLE were compared with autoantibody profiles of 1397 patients from 4 different control groups. Anti-dsDNA was the most distinguishing feature between patients with SLE and other patients, followed by antibodies against Cytosine-phosphate-Guanine (anti-CpG) DNA motifs (p<0.0001). Antibodies against CMV (cytomegalovirus) and ASCA (anti-Saccharomyces cerevisiae antibodies) were more prevalent in patients with SLE with (a history of) lupus nephritis than patients with SLE without nephritis. CONCLUSION: Antibodies against CpG DNA motifs are prevalent in patients with SLE. Anti-CMV antibodies are associated with lupus nephritis.

Systemic and local evidence for complement involvement in chronic spontaneous urticaria.

BACKGROUND: The pathogenesis of chronic spontaneous urticaria (CSU), including the mechanism of action of omalizumab, remain unclear. We hypothesized complement system involvement given the often fast clinical response induced by treatment, including omalizumab. Therefore, we assessed the role of various complement factors surrounding omalizumab treatment. METHODS: Thirty CSU patients (median age 42 [range 21-70]; 73 % female) with a median once daily Urticaria Activity Score over 7 days (UAS7) score at baseline of 31.5 points were enrolled. Treatment consisted of six administrations of 300 mg omalizumab every 4 weeks succeeded by a follow-up period of 12 weeks. Four punch skin biopsies were taken per patient; at baseline from lesional skin, at baseline from nonlesional skin, and after 1 and 7 days from formerly lesional skin. Complement activity, including C1q, C3, C3bc/C3, C4, C4bc/C4, C5a, and Membrane Attack Complex in peripheral blood were analyzed and complement activation in the skin was determined by the analysis of C4d deposition. Results were related to the clinical response to omalizumab. RESULTS: Fifteen patients showed a UAS7 score of 6 or lower (median 0) at Week 24, 15 patients did not (median 16). Lesional skin biopsies at baseline revealed complement deposition (C4d) in blood vessels in the papillary dermis of 53% (16/30) of the patients, which suggests involvement of immune complexes in the pathogenesis of urticaria. Moreover, indication of increased complement activation in CSU was substantiated by increased C5a levels in peripheral blood compared to healthy controls (p = 0.010). The clinical effect of omalizumab could not be linked to the variation of complement components. CONCLUSIONS: Both C4d deposition in lesional skin and elevated C5a levels in peripheral blood indicate the involvement of complement activation in the pathogenesis of CSU. No correlation was found between omalizumab and activation of complement indicative of independent processes in the immunopathogenesis of CSU.

Distinction between peanut allergy and tolerance by characterization of B cell receptor repertoires.

BACKGROUND: Specific IgE against a peanut 2S albumin (Ara h 2 or 6) is the best predictor of clinically relevant peanut sensitization. However, sIgE levels of peanut allergic and those of peanut sensitized but tolerant patients partly overlap, highlighting the need for improved diagnostics to prevent incorrect diagnosis and consequently unnecessary food restrictions. Thus, we sought to explore differences in V(D)J gene transcripts coding for peanut 2S albumin-specific monoclonal antibodies (mAbs) from allergic and sensitized but tolerant donors. METHODS: 2S albumin-binding B-cells were single-cell sorted from peripheral blood of peanut allergic (n=6) and tolerant (n=6) donors sensitized to Ara h2 and/or 6 (≥ 0.1 kU/l) and non-atopic controls (n=5). h 2 and/or 6 (≥ 0.1 kU/l). Corresponding h heavy and light chain gene transcripts were heterologously expressed as mAbs and tested for specificity to native Ara h2 and 6. HCDR3 sequence motifs were identified by Levenshtein distances and hierarchically clustering. RESULTS: The frequency of 2S albumin-binding B cells was increased in allergic (median: 0.01%) compared to tolerant (median: 0.006%) and non-atopic donors (median: 0.0015%, p = 0.008). The majority of mAbs (74%, 29/39) bound specifically to Ara h 2 and/or 6. Non-specific mAbs (9/10) were mainly derived from non-atopic controls. In allergic donors, 89% of heavy chain gene transcripts consisted of VH3 family genes, compared with only 54% in sensitized but tolerant and 63% of non-atopic donors. Additionally, certain HCDR3 sequence motifs were associated with allergy (n = 4) or tolerance (n = 3) upon hierarchical clustering of their Levenshtein distances. CONCLUSIONS: Peanut allergy is associated with dominant VH3 family gene usage and certain public antibody sequences (HCDR3 motifs).

Comparison of Two Strategies to Generate Antigen-Specific Human Monoclonal Antibodies: Which Method to Choose for Which Purpose?

Human monoclonal antibodies (mAbs) are valuable tools to link genetic information with functional features and to provide a platform for conformational epitope mapping. Additionally, combined data on genetic and functional features provide a valuable mosaic for systems immunology approaches. Strategies to generate human mAbs from peripheral blood have been described and used in several studies including single cell sequencing of antigen-binding B cells and the establishment of antigen-specific monoclonal Epstein-Barr Virus (EBV) immortalized lymphoblastoid cell lines (LCLs). However, direct comparisons of these two strategies are scarce. Hence, we sought to set up these two strategies in our laboratory using peanut 2S albumins (allergens) and the autoantigen anti-Rho guanosine diphosphate dissociation inhibitor 2 (RhoGDI2, alternatively 'ARHGDIB') as antigen targets to directly compare these strategies regarding costs, time expenditure, recovery, throughput and complexity. Regarding single cell sequencing, up to 50% of corresponding V(D)J gene transcripts were successfully amplified of which 54% were successfully cloned into expression vectors used for heterologous expression. Seventy-five percent of heterologously expressed mAbs showed specific binding to peanut 2S albumins resulting in an overall recovery of 20.3%, which may be increased to around 29% by ordering gene sequences commercially for antibody cloning. In comparison, the establishment of monoclonal EBV-LCLs showed a lower overall recovery of around 17.6%. Heterologous expression of a mAb carrying the same variable region as its native counterpart showed comparable concentration-dependent binding abilities. By directly comparing those two strategies, single cell sequencing allows a broad examination of antigen-binding mAbs in a moderate-throughput manner, while the establishment of monoclonal EBV-LCLs is a powerful tool to select a small number of highly reactive mAbs restricted to certain B cell subpopulations. Overall, both strategies, initially set-up for peanut 2S albumins, are suitable to obtain human mAbs and they are easily transferrable to other target antigens as shown for ARHGDIB.

High-resolution mapping identifies HLA class II associations with multifocal motor neuropathy.

OBJECTIVE: To gain further insight in the immunopathology underlying multifocal motor neuropathy (MMN) by exploring the association between MMN and the human leukocyte antigen (HLA) class II DRB1, DQB1, and DQA loci in depth and by correlating associated haplotypes to detailed clinical and anti-ganglioside antibody data. METHODS: We performed high-resolution HLA-class II typing for the DRB1, DQB1, and DQA1 loci in 126 well-characterized MMN patients and assessed disease associations with haplotypes. We used a cohort of 1305 random individuals as a reference for haplotype distribution in the Dutch population. RESULTS: The DRB1*15:01-DQB1*06:02 haplotype (OR 1.6 [95% CI 1.1-2.2], p < 0.05) and the DRB1*12:01-DQB1*03:01 haplotype (OR 2.7 [95% CI 1.2-5.5], p < 0.05) were more frequent in patients with MMN than in controls. These haplotypes were not associated with disease course, response to treatment or anti-ganglioside antibodies. CONCLUSIONS: MMN is associated with the DRB1*15:01-DQB1*06:02 and DRB1*12:01-DQB1*03:01 haplotypes. These HLA molecules or gene variants in their immediate vicinity may promote the specific inflammatory processes underlying MMN.

Text Mining of Electronic Health Records Can Accurately Identify and Characterize Patients With Systemic Lupus Erythematosus.

OBJECTIVE: Electronic health records (EHR) are increasingly being recognized as a major source of data reusable for medical research and quality monitoring, although patient identification and assessment of symptoms (characterization) remain challenging, especially in complex diseases such as systemic lupus erythematosus (SLE). Current coding systems are unable to assess information recorded in the physician's free-text notes. This study shows that text mining can be used as a reliable alternative. METHODS: In a multidisciplinary research team of data scientists and medical experts, a text mining algorithm on 4607 patient records was developed to assess the diagnosis of 14 different immune-mediated inflammatory diseases and the presence of 18 different symptoms in the EHR. The text mining algorithm included key words in the EHR, while mining the context for exclusion phrases. The accuracy of the text mining algorithm was assessed by manually checking the EHR of 100 random patients suspected of having SLE for diagnoses and symptoms and comparing the outcome with the outcome of the text mining algorithm. RESULTS: After evaluation of 100 patient records, the text mining algorithm had a sensitivity of 96.4% and a specificity of 93.3% in assessing the presence of SLE. The algorithm detected potentially life-threatening symptoms (nephritis, pleuritis) with good sensitivity (80%-82%) and high specificity (97%-97%). CONCLUSION: We present a text mining algorithm that can accurately identify and characterize patients with SLE using routinely collected data from the EHR. Our study shows that using text mining, data from the EHR can be reused in research and quality control.