A novel functional mast cell assay for the detection of allergies.

BACKGROUND: Clinical management of allergic diseases has been hampered by the lack of safe and convenient tests to reliably identify culprit allergens and to closely follow changes in disease activity over time. Because allergy diagnosis is a complex and laborious multistep procedure, there is an urgent need for simpler but still functionally accurate ex vivo assays allowing objective diagnosis, substantiating treatment choices, and quantifying therapeutic responses. OBJECTIVE: In this study, we sought to develop a novel functional cell-based assay that relies on passive sensitization of allergic effector cells with patient serum, circumventing current limitations in allergy diagnosis. METHODS: We genetically engineered a conditional homeobox B8 (Hoxb8)-immortalized progenitor line from the bone marrow of mice that are transgenic for the human high-affinity IgE receptor (FcεRIα). These cells can be reproducibly differentiated into mature Hoxb8 mast cells within 5 days of culture in virtually unlimited numbers. RESULTS: We demonstrate that the established Hoxb8 mast cell assay can be used to accurately measure total IgE levels, identify culprit allergens, longitudinally monitor allergen-specific immunotherapy, and potentially determine the time point of tolerance induction upon allergen-specific immunotherapy in patients with allergy. To facilitate the analysis of large testing volumes, we demonstrate a proof-of-concept for a high-throughput screening application based on fluorescent cell barcoding using the engineered Hoxb8 mast cells. CONCLUSIONS: Our results indicate that this novel mast cell assay could represent a valuable tool to support clinicians in the identification of IgE-mediated allergies and in the quantification of treatment efficacy as well as duration of therapeutic response.

Serological testing for SARS-CoV-2 antibodies in clinical practice: A comparative diagnostic accuracy study.

BACKGROUND: Serological tests are a powerful tool in the monitoring of infectious diseases and the detection of host immunity. However, manufacturers often provide diagnostic accuracy data generated through biased studies, and the performance in clinical practice is essentially unclear. OBJECTIVES: We aimed to determine the diagnostic accuracy of various serological testing strategies for (a) identification of patients with previous coronavirus disease-2019 (COVID-19) and (b) prediction of neutralizing antibodies against SARS-CoV-2 in real-life clinical settings. METHODS: We prospectively included 2573 consecutive health-care workers and 1085 inpatients with suspected or possible previous COVID-19 at a Swiss University Hospital. Various serological immunoassays based on different analytical techniques (enzyme-linked immunosorbent assays, ELISA; chemiluminescence immunoassay, CLIA; electrochemiluminescence immunoassay, ECLIA; and lateral flow immunoassay, LFI), epitopes of SARS-CoV-2 (nucleocapsid, N; receptor-binding domain, RBD; extended RBD, RBD+; S1 or S2 domain of the spike [S] protein, S1/S2), and antibody subtypes (IgG, pan-Ig) were conducted. A positive real-time PCR test from a nasopharyngeal swab was defined as previous COVID-19. Neutralization assays with live SARS-CoV-2 were performed in a subgroup of patients to assess neutralization activity (n = 201). RESULTS: The sensitivity to detect patients with previous COVID-19 was ≥85% in anti-N ECLIA (86.8%) and anti-S1 ELISA (86.2%). Sensitivity was 84.7% in anti-S1/S2 CLIA, 84.0% in anti-RBD+LFI, 81.0% in anti-N CLIA, 79.2% in anti-RBD ELISA, and 65.6% in anti-N ELISA. The specificity was 98.4% in anti-N ECLIA, 98.3% in anti-N CLIA, 98.2% in anti-S1 ELISA, 97.7% in anti-N ELISA, 97.6% in anti-S1/S2 CLIA, 97.2% in anti-RBD ELISA, and 96.1% in anti-RBD+LFI. The sensitivity to detect neutralizing antibodies was ≥85% in anti-S1 ELISA (92.7%), anti-N ECLIA (91.7%), anti-S1/S2 CLIA (90.3%), anti-RBD+LFI (87.9%), and anti-RBD ELISA (85.8%). Sensitivity was 84.1% in anti-N CLIA and 66.2% in anti-N ELISA. The specificity was ≥97% in anti-N CLIA (100%), anti-S1/S2 CLIA (97.7%), and anti-RBD+LFI (97.9%). Specificity was 95.9% in anti-RBD ELISA, 93.0% in anti-N ECLIA, 92% in anti-S1 ELISA, and 65.3% in anti-N ELISA. Diagnostic accuracy measures were consistent among subgroups. CONCLUSIONS: The diagnostic accuracy of serological tests for SARS-CoV-2 antibodies varied remarkably in clinical practice, and the sensitivity to identify patients with previous COVID-19 deviated substantially from the manufacturer's specifications. The data presented here should be considered when using such tests to estimate the infection burden within a specific population and determine the likelihood of protection against re-infection.

Cutaneous involvement in anti-HMGCR positive necrotizing myopathy.

OBJECTIVE: Anti-3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) positive immune-mediated necrotizing myopathy (IMNM) is a rare disease. It is induced by exogenous substances, most often by statins. Little is known about cutaneous manifestations of HMGCR positive IMNM and about HMGCR antibody positivity in other diseases. METHODS: The characteristics of patients with anti-HMGCR autoantibodies measured at our laboratory between January 2012 and September 2020 were studied. Characteristics of patients with IMNM were compared to those patients with positive antibodies but without muscle involvement. Associations of IMNM with other organ involvements were searched for. RESULTS: Of the 32 patients studied, 23 showed characteristics of IMNM, 9 did not fulfill current classification criteria but most showed signs of connective tissue diseases. Patients with IMNM were older (66 and 35 years, respectively; 0.92 (0.73-0.98); p < 0.001), had more frequent statin exposure (87% and 33%, respectively; 0.84 (0.61-0.94); p = 0.005) and higher mean peak CK (8717U/l and 329U/l, respectively; 1.0 (0.85-1.0); p < 0.001). 13/23 (56%) of IMNM patients showed cutaneous lesions; none of the patients suffered from cancer; only three IMNM patients showed drug-free complete remission. Incidence of IMNM in the catchment area of our center is at least 2.7/Mio/year. CONCLUSION: Cutaneous lesions were found to be more frequent in anti-HMRCR positive IMNM than previously reported. Titer of anti-HMGCR antibodies and CK levels were significantly higher in IMNM than in other autoimmune connective tissue diseases. The data support the hypothesis of an antigen-driven response in IMNM, and suggests an activation of autoreactive B-lymphocytes in non-IMNM patients.

Accuracy of serological testing for SARS-CoV-2 antibodies: First results of a large mixed-method evaluation study.

BACKGROUND: Serological immunoassays that can identify protective immunity against SARS-CoV-2 are needed to adapt quarantine measures, assess vaccination responses, and evaluate donor plasma. To date, however, the utility of such immunoassays remains unclear. In a mixed-design evaluation study, we compared the diagnostic accuracy of serological immunoassays that are based on various SARS-CoV-2 proteins and assessed the neutralizing activity of antibodies in patient sera. METHODS: Consecutive patients admitted with confirmed SARS-CoV-2 infection were prospectively followed alongside medical staff and biobank samples from winter 2018/2019. An in-house enzyme-linked immunosorbent assay utilizing recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike protein was developed and compared to three commercially available enzyme-linked immunosorbent assays (ELISAs) targeting the nucleoprotein (N), the S1 domain of the spike protein (S1), and a lateral flow immunoassay (LFI) based on full-length spike protein. Neutralization assays with live SARS-CoV-2 were performed. RESULTS: One thousand four hundred and seventy-seven individuals were included comprising 112 SARS-CoV-2 positives (defined as a positive real-time PCR result; prevalence 7.6%). IgG seroconversion occurred between day 0 and day 21. While the ELISAs showed sensitivities of 88.4% for RBD, 89.3% for S1, and 72.9% for N protein, the specificity was above 94% for all tests. Out of 54 SARS-CoV-2 positive individuals, 96.3% showed full neutralization of live SARS-CoV-2 at serum dilutions ≥ 1:16, while none of the 6 SARS-CoV-2-negative sera revealed neutralizing activity. CONCLUSIONS: ELISAs targeting RBD and S1 protein of SARS-CoV-2 are promising immunoassays which shall be further evaluated in studies verifying diagnostic accuracy and protective immunity against SARS-CoV-2.

Machine Learning in Antibody Diagnostics for Inflammatory Bowel Disease Subtype Classification.

Antibody testing in inflammatory bowel disease (IBD) can add to diagnostic accuracy of the main subtypes Crohn's disease (CD) and ulcerative colitis (UC). Whether modern modeling techniques such as supervised and unsupervised machine learning are of value for finer distinction of subtypes such as IBD-unclassified (IBD-U) is not known. We determined the antibody profile of 100 adult IBD patients from the Swiss IBD cohort study with known subtype (50 CD, 50 UC) as well as of 76 IBD-U patients. We included ASCA IgG and IgA, p-ANCA, MPO- and PR3-ANCA, and xANCA measurements for computing different antibody panels as well as machine learning models. The AUC of an optimized antibody panel was 85% (95%CI, 78-92%) to distinguish CD from UC patients. The antibody profile of IBD-U patients was closely related to UC. No specific antibody profile was predictive for IBD-U nor for re-classification. The panel diagnostic was in favor of UC reclassification prediction with a correct assignment rate of 69.2-73.1% depending on the cut-off applied. Supervised machine learning could not distinguish between CD, UC, and IBD-U. More so, unsupervised machine learning suggested only two distinct clusters as a likely number of IBD subtypes. Antibodies in IBD are supportive in confirming clinical determined subtypes CD and UC but have limited capacity to predict IBD-U and reclassification during follow-up. In terms of antibody profiles, IBD-U is not a distinct subtype of IBD.

Clinical Validity of Anti-Proteinase 3 Antibodies in Patients with Inflammatory Bowel Disease: A Short Meta-Analysis.

Anti-neutrophil cytoplasmic antibodies (ANCA) directed to proteinase 3 (PR3) represent highly established markers for patients with ANCA-associated vasculitis (AAV). PR3-ANCA have also demonstrated utility in the management of inflammatory bowel disease (IBD). More specifically, PR3-ANCA discriminate individuals with ulcerative colitis (UC) from Crohn's disease (CD) patients and are associated with disease severity, activity, and treatment non-response. Here, we aim to summarize the current data on the diagnostic utility of PR3-ANCA in IBD. A structured, systematic literature review, including three electronic databases, was conducted on June 6th, 2023, to identify studies assessing the diagnostic accuracy of the QUANTA Flash® PR3 assay in UC vs. CD patients. Electronic searches were supplemented by hand searching. A hierarchical, bivariate, mixed-effect meta-analysis was conducted using the metandi function, as per the Cochrane collaboration recommendations. Study quality was assessed using the QUADAS-2 tool, which considers the risk of bias and applicability. Six out of a hundred and eleven citations met the inclusion criteria and reported QUANTA Flash® PR3 diagnostic accuracy in UC vs. CD (UC, n = 667, CD, n = 682 patients). The sensitivity/specificity point estimate for UC was 34.9%/95.9%. This resulted in a Diagnostic Odds Ratio (DOR) of 12.6. The risk of bias was low in the index test and reference standard domains. Four of the six studies (67%) showed an unclear risk of bias in patient selection and in flow and timing domains. All studies had low concerns about applicability in all the domains. PR3-ANCA measured with the QUANTA Flash® PR3 assay represent novel diagnostic markers in IBD and enables discrimination between UC and CD.

[CME: Rational Laboratory Diagnostics in the Context of COVID-19]. / CME: Rationale Labordiagnostik im Kontext von COVID-19.

CME: Rational Laboratory Diagnostics in the Context of COVID-19 Abstract. In the management of COVID-19, a variety of laboratory tests are available to the general practitioner. The choice is difficult, and some of these laboratory tests are discussed controversially in the lay press. In this article, we discuss the important clinical questions and the contribution of laboratory tests to answering these questions. We describe the most common test principles and discuss their diagnostic value. In doing so, we provide the practicing physician with a simple overview for the clinical practice.

Rational development and application of biomarkers in the field of autoimmunity: A conceptual framework guiding clinicians and researchers.

Clear guidance is needed in the development and implementation of laboratory biomarkers in medicine. So far, no standardized phased approach is established that would pilot researchers and clinicians in this process. This leads to often incompletely validated biomarkers, which can bear the consequence of wrong applications, misinterpretation and inadequate management in the clinical context. In this conceptual article, we describe a stepwise approach to develop and comprehensively validate laboratory biomarkers. We will delineate basic steps including technical performance, pre-analytical issues, and biological variation, as well as advanced aspects of biomarker utility comprising interpretability, diagnostic and prognostic accuracy, and health-care outcomes. These aspects will be illustrated by using well-known examples from the field of immunology. The application of this conceptual framework will guide researchers in conducting meaningful projects to develop and evaluate biomarkers for the use in clinical practice. Furthermore, clinicians will be able to adequately interpret pre-clinical and clinical diagnostic literature and rationally apply biomarkers in clinical practice. Improvement in the implementation and application of biomarkers might relevantly change the management and outcomes of our patients for the better.

Humoral and cellular immune responses on SARS-CoV-2 vaccines in patients with anti-CD20 therapies: a systematic review and meta-analysis of 1342 patients.

BACKGROUND: Immune responses on SARS-CoV-2 vaccination in patients receiving anti-CD20 therapies are impaired but vary considerably. We conducted a systematic review and meta-analysis of the literature on SARS-CoV-2 vaccine induced humoral and cell-mediated immune response in patients previously treated with anti-CD20 antibodies. METHODS: We searched PubMed, Embase, Medrxiv and SSRN using variations of search terms 'anti-CD20', 'vaccine' and 'COVID' and included original studies up to 21 August 2021. We excluded studies with missing data on humoral or cell-mediated immune response, unspecified methodology of response testing, unspecified timeframes between vaccination and blood sampling or low number of participants (≤3). We excluded individual patients with prior COVID-19 or incomplete vaccine courses. Primary endpoints were humoral and cell-mediated immune response rates. Subgroup analyses included time since anti-CD20 therapy, B cell depletion and indication for anti-CD20 therapy. We used random-effects models of proportions. FINDINGS: Ninety studies were assessed. Inclusion criteria were met by 23 studies comprising 1342 patients. Overall rate of humoral response was 0.40 (95% CI 0.35 to 0.47). Overall rate of cell-mediated immune responses was 0.71 (95% CI 0.57 to 0.87). A time interval >6 months since last anti-CD20 therapy was associated with higher humoral response rates with 0.63 (95% CI 0.53 to 0.72) versus <6 months 0.2 (95% CI 0.03 to 0.43); p=0<01. Similarly, patients with circulating B cells more frequently showed humoral responses. Anti-CD20-treated kidney transplant recipients showed lower humoral response rates than patients with haematological malignancies or autoimmune disease. INTERPRETATION: Patients on anti-CD20 therapies can develop humoral and cell-mediated immune responses after SARS-CoV-2 vaccination, but subgroups such as kidney transplant recipients or those with very recent therapy and depleted B cell are at high risk for non-seroconversion and should be individually assessed for personalised SARS-CoV-2 vaccination strategies. Potential limitations are small patient numbers and heterogeneity of studies included. FUNDING: This study was funded by Bern University Hospital.

Humoral response to mRNA vaccines against SARS-CoV-2 in patients with humoral immunodeficiency disease.

OBJECTIVES: Although mRNA-based vaccines against SARS-CoV-2 induce a robust immune response and prevent infections and hospitalizations, there are limited data on the antibody response in individuals with humoral immunodeficiency. The aim of this study was to evaluate the humoral immune response after two vaccine doses with BNT162b2 or mRNA-1273 in patients with humoral immunodeficiency disease. METHODS: This cross-sectional study assessed 39 individuals with hypogammaglobulinemia under immunoglobulin replacement therapy. IgG anti-SARS-CoV-2 spike protein antibodies (anti-S) were measured 4 weeks to 4 months after two doses of an mRNA vaccine against SARS-CoV-2. The proportion of patients, who developed a humoral immune response to the spike protein were evaluated and compared to 19 healthy controls. RESULTS: After vaccination with two vaccine doses, 26/39 patients (66.7%) with humoral immunodeficiency disease and all healthy controls developed anti-S. In subjects with baseline IgG <3 g/l, only 1/5 (20%) showed a humoral immune response. 10 out of 26 with CVID (38.5%) and 7/9 under immunosuppressive drugs (77.8%) developed no immune response (13 subjects with no response) compared to 0/19 in healthy controls. Subgroup analysis in patients without immunosuppressive drugs revealed lower anti-S in patients with moderate to severe humoral immunodeficiency disease: baseline IgG <3 g/l: 12.0 AU/ml (95%CI 12.0-125.0), baseline IgG 3-5 g/l: 99.9 AU/ml (95%CI 14.4-400.0), baseline IgG >5 g/l: 151.5 AU/ml (95%CI 109.0-400.0), healthy controls 250.0 AU/ml (95%CI 209.0-358.0), p = 0.007. CONCLUSION: In most patients with mild to moderate humoral immunodeficiency we found only slightly lower anti-S antibodies compared with healthy controls after two vaccine doses with BNT162b2 and mRNA-1273. However, in patients with a decreased baseline IgG below 3 g/l and/or under immunosuppressive drugs, we found severely impaired humoral immune responses.

Trajectories of humoral and cellular immunity and responses to a third dose of mRNA vaccines against SARS-CoV-2 in patients with a history of anti-CD20 therapy.

BACKGROUND: The majority of patients with B-cell-depleting therapies show compromised vaccination-induced immune responses. Herein, we report on the trajectories of anti-SARS-CoV-2 immune responses in patients of the RituxiVac study compared with healthy volunteers and investigate the immunogenicity of a third vaccination in previously humoral non-responding patients. METHODS: We investigated the humoral and cell-mediated immune response after SARS-CoV-2 messanger RNA vaccination in patients with a history with anti-CD20 therapies. Coprimary outcomes were antispike and SARS-CoV-2-stimulated interferon-γ concentrations in vaccine responders 4.3 months (median; IQR: 3.6-4.8 months) after first evaluation, and humoral and cell-mediated immunity (CMI) after a third vaccine dose in previous humoral non-responders. Immunity decay rates were compared using analysis of covariance in linear regression. RESULTS: 5.6 months (IQR: 5.1-6.7) after the second vaccination, we detected antispike IgG in 88% (29/33) and CMI in 44% (14/32) of patients with a humoral response after two-dose vaccination compared with 92% (24/26) healthy volunteers with antispike IgG and 69% (11/16) with CMI 6.8 months after the second vaccination (IQR: 6.0-7.1). Decay rates of antibody concentrations were comparable between patients and controls (p=0.70). In two-dose non-responders, a third SARS-CoV-2 vaccine elicited humoral responses in 19% (6/32) and CMI in 32% (10/31) participants. CONCLUSION: This study reveals comparable immunity decay rates between patients with anti-CD20 treatments and healthy volunteers, but inefficient humoral or CMI after a third SARS-CoV-2 vaccine in most two-dose humoral non-responders calling for individually tailored vaccination strategies in this population.Trial registration numberNCT04877496; ClinicalTrials.gov number.

The Release Kinetics of Eosinophil Peroxidase and Mitochondrial DNA Is Different in Association with Eosinophil Extracellular Trap Formation.

Eosinophils are a subset of granulocytes characterized by a high abundance of specific granules in their cytoplasm. To act as effector cells, eosinophils degranulate and form eosinophil extracellular traps (EETs), which contain double-stranded DNA (dsDNA) co-localized with granule proteins. The exact molecular mechanism of EET formation remains unknown. Although the term "EET release" has been used in scientific reports, it is unclear whether EETs are pre-formed in eosinophils and subsequently released. Moreover, although eosinophil degranulation has been extensively studied, a precise time-course of granule protein release has not been reported until now. In this study, we investigated the time-dependent release of eosinophil peroxidase (EPX) and mitochondrial DNA (mtDNA) following activation of both human and mouse eosinophils. Unexpectedly, maximal degranulation was already observed within 1 min with no further change upon complement factor 5 (C5a) stimulation of interleukin-5 (IL-5) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-primed eosinophils. In contrast, bulk mtDNA release in the same eosinophil populations occurred much slower and reached maximal levels between 30 and 60 min. Although no single-cell analyses have been performed, these data suggest that the molecular pathways leading to degranulation and mtDNA release are at least partially different. Moreover, based on these data, it is likely that the association between the mtDNA scaffold and granule proteins in the process of EET formation occurs in the extracellular space.

Serum calprotectin as new biomarker for disease severity in idiopathic pulmonary fibrosis: a cross-sectional study in two independent cohorts.

BACKGROUND: Non-invasive biomarkers for the assessment of disease severity in idiopathic pulmonary fibrosis (IPF) are urgently needed. Calprotectin belongs to the S-100 proteins produced by neutrophils, which likely contribute to IPF pathogenesis. Calprotectin is a well-established biomarker in inflammatory bowel diseases. In this cross-sectional study, we aimed to establish the potential role of calprotectin as a biomarker in IPF. Specifically, we hypothesised that patients with IPF have higher serum calprotectin levels compared with healthy controls, and that calprotectin levels are associated with disease severity. METHODS: Blood samples were obtained from healthy volunteers (n=26) and from two independent IPF cohorts (derivation cohort n=26, validation cohort n=66). Serum calprotectin levels were measured with a commercial kit adapted for that purpose and compared between healthy controls and patients with IPF. Clinical parameters, including forced vital capacity, diffusing capacity for carbon monoxide (DLCO) and the Composite Physiologic Index (CPI), were correlated with calprotectin serum levels. RESULTS: The IPF derivation cohort showed increased serum calprotectin levels compared with healthy controls (2.47±1.67 vs 0.97±0.53 µg/mL, p<0.001). In addition, serum calprotectin levels correlated with DLCO% predicted (r=-0.53, p=0.007) and with CPI (r=0.66, p=0.007). These findings were confirmed in an independent IPF validation cohort. CONCLUSION: Serum calprotectin levels are significantly increased in patients with IPF compared with healthy controls and correlate with DLCO and CPI. Calprotectin might be a potential new biomarker for disease severity in IPF.

Evaluation of diagnostic criteria and red flags of myelin oligodendrocyte glycoprotein encephalomyelitis in a clinical routine cohort.

AIMS: Myelin oligodendrocyte glycoprotein antibodies (MOG-IgG) have been proposed to define "MOG encephalomyelitis" (MOG-EM), with published diagnostic and "red flag" criteria. We aimed to evaluate these criteria in a routine clinical setting. METHODS: We retrospectively analyzed patients with borderline/positive MOG-IgG and applied the diagnostic and red flag criteria to determine likelihood of MOG-EM diagnosis. Para-/clinical parameters were described and analyzed with chi-square test. RESULTS: In total, 37 patients fulfilled MOG-EM diagnostic criteria (female-to-male ratio: 1.6:1, median onset age: 28.0 years [IQR 18.5-40.5], n = 8 with pediatric onset). In 24/37, red flags were present, predominantly MOG-IgG at assay cutoff and/or MRI lesions suggestive of multiple sclerosis (MS). As proposed in the consensus criteria, these patients should rather be described as "possible" MOG-EM. Of these, we classified 13 patients as "unlikely" MOG-EM in the presence of the red flag "borderline MOG-IgG" with negative MOG-IgG retest or coincidence of ≥1 additional red flag. This group mainly consisted of patients diagnosed with MS (n = 11). Frequency of cerebrospinal fluid (CSF-)-specific oligoclonal bands (OCB) is significantly lower in definite vs possible and unlikely MOG-EM (P = .0005). CONCLUSION: Evaluation of diagnostic and red flag criteria, MOG-IgG retesting (incl. change of assay), and CSF-specific OCB are relevant in clinical routine cohorts to differentiate MOG-EM from MS.

Humoral and cellular responses to mRNA vaccines against SARS-CoV-2 in patients with a history of CD20 B-cell-depleting therapy (RituxiVac): an investigator-initiated, single-centre, open-label study.

BACKGROUND: B-cell-depleting therapies increase the risk of morbidity and mortality due to COVID-19. Evidence-based SARS-CoV-2 vaccination strategies for patients on B-cell-depleting therapies are scarce. We aimed to investigate humoral and cell-mediated immune responses to SARS-CoV-2 mRNA-based vaccines in patients receiving CD20-targeted B-cell-depleting agents for autoimmune disease, malignancy, or transplantation. METHODS: The RituxiVac study was an investigator-initiated, single-centre, open-label study done at the Bern University Hospital (Bern, Switzerland). Patients with a treatment history of anti-CD20-depleting agents (rituximab or ocrelizumab) and with no previous history of SARS-CoV-2 infection were enrolled between April 26 and June 30, 2021, for analysis of humoral and cell-mediated immune responses (by interferon-γ [IFNγ] release assay) at least 4 weeks after completing vaccination against SARS-CoV-2. Healthy controls without a history of SARS-CoV-2 infection were also enrolled at least 4 weeks after completing vaccination against SARS-CoV-2. All study participants received two doses of either the Pfizer-BioNTech BNT162b2 vaccine or the Moderna mRNA-1273 vaccine. The primary outcome was the proportion of patients with a history of anti-CD20 treatment who showed a humoral immune response against the SARS-CoV-2 spike protein in comparison with immunocompetent controls. Prespecified secondary endpoints were the effect of anti-CD20 therapy (including time since last treatment and cumulative dose) on humoral or cell-mediated immune responses to SARS-CoV-2 vaccination, and biomarkers of immunocompetence. This study is registered with ClinicalTrials.gov, NCT04877496. FINDINGS: The final study population comprised 96 patients and 29 immunocompetent controls. The median age of patients was 67 years (IQR 57-72) and of controls was 54 years (45-62), and 51 (53%) of 96 patients and 19 (66%) of 29 controls were female. The median time since last anti-CD20 treatment was 1·07 years (IQR 0·48-2·55) and the median cumulative dose of an anti-CD20 depleting agent was 2·80 g (1·50-5·00). Anti-spike IgG antibodies were detected in 47 (49%) of 96 patients 1·79 months (IQR 1·16-2·48) after the second vaccine dose compared to 29 (100%) of 29 controls 1·81 months (1·17-2·48) after the second vaccine dose (p<0·001). SARS-CoV-2-specific IFNγ release was detected in 13 (20%) of 66 patients and 21 (75%) of 28 of healthy controls (p<0·001). Only nine (14%) of 66 patients were double positive for anti-SARS-CoV-2 spike IgG and cell-mediated responses, compared with 21 (75%) of 28 healthy controls (p<0·001). Time since last anti-CD20 therapy (>7·6 months; positive predictive value 0·78), peripheral CD19+ cell count (>27 cells per µL; positive predictive value 0·70), and CD4+ lymphocyte count (>653 cells per µL; positive predictive value 0·71) were predictive of humoral vaccine response (area under the curve [AUC] 67% [95% CI 56-78] for time since last anti-CD20 therapy, 67% [55-80] for peripheral CD19+ count, and 66% [54-79] for CD4+ count). INTERPRETATION: This study provides further evidence of blunted humoral and cell-mediated immune responses elicited by SARS-CoV-2 mRNA vaccines in patients with a history of CD20 B-cell-depleting treatment. Lymphocyte subpopulation counts were associated with vaccine response in this highly vulnerable population. On validation, these results could help guide both the administration of SARS-CoV-2 vaccines and B-cell-depleting agents in this population. FUNDING: Bern University Hospital.

Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11.

Pseudomonas aeruginosa infection in ventilator-associated pneumonia is a serious and often life-threatening complication in intensive care unit patients, and new treatment options are needed. We used B-cell-enriched peripheral blood lymphocytes from a volunteer immunized with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine to generate human hybridoma cell lines producing monoclonal antibodies specific for individual P. aeruginosa lipopolysaccharide serotypes. The fully human monoclonal antibody secreted by one of these lines, KBPA101, is an IgM/kappa antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 x 10(7) M(-1) +/- 2.8 x 10(7) M(-1)) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges with P. aeruginosa at doses as low as 5 microg/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any toxicity of KBPA101. Based on these preclinical findings, the first human clinical trials have been initiated.

Conventional autoantibodies against brain antigens are not routinely detectable in serum and CSF of narcolepsy type 1 and 2 patients.

Narcolepsy with cataplexy (NT1) is a chronic hypothalamic disorder with a presumed immune-mediated etiology leading to a loss of hypocretin neurons. Previous studies reported conflicting results in terms of presence of auto-antibodies involved in narcolepsy pathophysiology. A total of 86 patients with primary/idiopathic narcolepsy (74 NT1, 12 NT2) and 23 control patients with excessive daytime sleepiness due to other causes were tested for the presence of a wide range of anti-neuronal antibodies in both serum and cerebrospinal fluid (CSF). Anti-neuronal antibodies were rarely found in patients with narcolepsy (n = 2) and in controls (n = 1). Our results are in line with previous reports. We can therefore support the current evidence, that conventional anti-neuronal antibodies are not routinely detected during the workup of NT1 and other CDH patients.

Pharmacokinetics and safety profile of the human anti-Pseudomonas aeruginosa serotype O11 immunoglobulin M monoclonal antibody KBPA-101 in healthy volunteers.

KBPA-101 is a human monoclonal antibody of the immunoglobulin M isotype, which is directed against the O-polysaccharide moiety of Pseudomonas aeruginosa serotype O11. This double-blind, dose escalation study evaluated the safety and pharmacokinetics of KBPA-101 in 32 healthy volunteers aged 19 to 46 years. Each subject received a single intravenous infusion of KBPA-101 at a dose of 0.1, 0.4, 1.2, or 4 mg/kg of body weight or placebo infused over 2 h. Plasma samples for pharmacokinetic assessments were taken before infusion as well as 0.25, 0.5, 1, 2, 2.5, 4, 6, 8, 12, 24, 36, and 48 h and 4, 7, 10, and 14 days after start of dosing. Plasma concentrations of KBPA-101 were detected with mean maximum concentrations of drug in plasma of 1,877, 7,571, 24,923, and 83,197 ng/ml following doses of 0.1, 0.4, 1.2, and 4.0 mg/kg body weight, respectively. The mean elimination half-life was between 70 and 95 h. The mean volume of distribution was between 4.76 and 5.47 liters. Clearance ranged between 0.039 and 0.120 liters/h. At the highest dose of 4.0 mg/kg, plasma KBPA-101 levels were greater than 5,000 ng/ml for 14 days. KBPA-101 exhibited linear kinetics across all doses. No anti-KBPA-101 antibodies were detected after dosing in any subject. Overall, the human monoclonal antibody KBPA-101 was well tolerated over the entire dose range in healthy volunteers, and no serious adverse events have been reported.

PR3-ANCA and panel diagnostics in pediatric inflammatory bowel disease to distinguish ulcerative colitis from Crohn's disease.

BACKGROUND: Accurate classification of patients with inflammatory bowel disease into the subtypes ulcerative colitis (UC) and Crohn's disease (CD) is still a challenge, but important for therapy and prognosis. OBJECTIVES: To evaluate the diagnostic utility of anti-neutrophil cytoplasmic antibodies specific for proteinase-3 (PR3-ANCA) for ulcerative colitis (UC) and the value of an antibody panel incorporating PR3-ANCA to differentiate between Crohn's disease (CD) and UC. STUDY DESIGN: In this cohort study, 122 pediatric and adolescent individuals were retrospectively included (61 IBD patients of two clinical centers, 61 non-IBD controls). All subjects had a comprehensive antibody profile done from stored sera taken close to time of diagnosis. By employing quasi-exhaustive logistic regression the best discriminative model for UC and CD,subjects was determined in a training cohort and confirmed in a validation cohort. RESULTS: PR3-ANCA was specifically associated with UC (odds ratio (OR), 17.6; 95% confidence interval (CI); 3.6, 87); P < .001). A four antibody-panel including PR3-ANCA had an AUC of 90.81% (95%CI; 81.93, 99.69) to distinguish between UC and CD in the training cohort. In a smaller external validation cohort, the AUC was 84.13% (95%CI; 64.21, 100) for accurate diagnosis of CD and UC. CONCLUSION: PR3-ANCA is highly specific for UC. The differentiating capability of a panel, which contains PR3-ANCA and weighs broadly available antibodies, is superior and utilization of the panel can support accurate classification in the work-up of pediatric and adolescent patients with IBD patients.

Rebound After Fingolimod and a Single Daclizumab Injection in a Patient Retrospectively Diagnosed With NMO Spectrum Disorder-MRI Apparent Diffusion Coefficient Maps in Differential Diagnosis of Demyelinating CNS Disorders.

Objective: Differential diagnosis of neuromyelitis optica spectrum disorders (NMOSD) and multiple sclerosis (MS) or mimics can be challenging, especially in patients with atypical presentations and negative serostatus for aquaporin-4 antibodies (AQP4-Ab). This brief research report describes magnetic resonance imaging (MRI) findings focusing on quantitative apparent diffusion coefficient (ADC) histogram analysis as a potential tool to differentiate NMOSD from MS. Methods: Longitudinal MRI data obtained during routine clinical examinations were retrospectively analyzed in a patient with histologically determined cerebral NMOSD, a patient with an acute tumefactive MS lesion, and a patient with ischemic stroke. Histogram analyses of ADC maps were evaluated. Results: A patient diagnosed with MS experienced a severe rebound after fingolimod withdrawal and a single daclizumab injection. Cerebral NMOSD manifestation was confirmed by brain biopsy. However, the patient did not fulfill consensus criteria for NMOSD and was AQP4-Ab negative. Comparison of ADC histogram analyses of this patient with those from a patient with MS and one with ischemic stroke revealed differential ADC characteristics: namely a more pronounced and prolonged ADC leftward shift in inflammatory than in ischemic pathology, even more accentuated in NMOSD versus MS. Conclusion: ADC map histograms and ADC threshold values for different conditions may be useful for differentiation of large inflammatory brain lesions and further studies are merited.