SDHA gain-of-function engages inflammatory mitochondrial retrograde signaling via KEAP1-Nrf2.

Whether screening the metabolic activity of immune cells facilitates discovery of molecular pathology remains unknown. Here we prospectively screened the extracellular acidification rate as a measure of glycolysis and the oxygen consumption rate as a measure of mitochondrial respiration in B cells from patients with primary antibody deficiency. The highest oxygen consumption rate values were detected in three study participants with persistent polyclonal B cell lymphocytosis (PPBL). Exome sequencing identified germline mutations in SDHA, which encodes succinate dehydrogenase subunit A, in all three patients with PPBL. SDHA gain-of-function led to an accumulation of fumarate in PPBL B cells, which engaged the KEAP1-Nrf2 system to drive the transcription of genes encoding inflammatory cytokines. In a single patient trial, blocking the activity of the cytokine interleukin-6 in vivo prevented systemic inflammation and ameliorated clinical disease. Overall, our study has identified pathological mitochondrial retrograde signaling as a disease modifier in primary antibody deficiency.

Circovirus Hepatitis in Immunocompromised Patient, Switzerland.

We identified a novel human circovirus in an immunocompromised 66-year-old woman with sudden onset of self-limiting hepatitis. We detected human circovirus 1 (HCirV-1) transcripts in hepatocytes and the HCirV-1 genome long-term in the patient's blood, stool, and urine. HCirV-1 is an emerging human pathogen that persists in susceptible patients.

Post-transplant Inflammatory Bowel Disease Associated with Donor-Derived TIM-3 Deficiency.

Inflammatory bowel disease (IBD) occurring following allogeneic stem cell transplantation (aSCT) is a very rare condition. The underlying pathogenesis needs to be better defined. There is currently no systematic effort to exclude loss- or gain-of-function mutations in immune-related genes in stem cell donors. This is despite the fact that more than 100 inborn errors of immunity may cause or contribute to IBD. We have molecularly characterized a patient who developed fulminant inflammatory bowel disease following aSCT with stable 100% donor-derived hematopoiesis. A pathogenic c.A291G; p.I97M HAVCR2 mutation encoding the immune checkpoint protein TIM-3 was identified in the patient's blood-derived DNA, while being absent in DNA derived from the skin. TIM-3 expression was much decreased in the patient's serum, and in vitro-activated patient-derived T cells expressed reduced TIM-3 levels. In contrast, T cell-intrinsic CD25 expression and production of inflammatory cytokines were preserved. TIM-3 expression was barely detectable in the immune cells of the patient's intestinal mucosa, while being detected unambiguously in the inflamed and non-inflamed colon from unrelated individuals. In conclusion, we report the first case of acquired, "transplanted" insufficiency of the regulatory TIM-3 checkpoint linked to post-aSCT IBD.

Major low-energy trauma results in non-specific immunoglobulin generation without evidence for specific autoantibody production: A prospective cohort study.

Cellular debris resulting from large trauma might overwhelm the scavenger mechanisms and lead to autoimmune reactions. We analysed whether a major well-defined trauma in humans induces laboratory signs of transient autoimmunity in the months after the insult. We included 50 patients with pertrochanteric femur fracture undergoing intramedullary nail osteosynthesis in a prospective cohort study and followed them at 3-4 days, 6 weeks, 12 weeks and 12 months postoperatively. By standard techniques, we assessed levels of total immunoglobulins, anti-nuclear antibodies (ANA), anti-cardiolipin antibodies, anti-dsDNA antibodies and anti-C1q antibodies, as well as antibodies against cytomegalovirus (CMV) as a control. Blood leukocyte differential and lymphocyte subpopulations were determined at baseline and in the first two postoperative samples. The mean age of the patients reached 80.1 years, and 23 (46%) completed all visits. Serum concentrations of total IgG, IgM and IgA increased at all follow-up time points. The ANA fluorescence light intensity units increased at 12 weeks and 12 months postoperatively (p < 0.0001), but the proportion of ANA-positive patients did not change (35%). The values of anti-C1q mildly increased at all follow-up visits, but not the ratio to total IgG. Anti-dsDNA remained negative in all patients, and anti-cardiolipin IgG/IgM antibodies did not change. Anti-CMV IgG antibodies increased significantly at all follow-up visits, without change in the ratio to total IgG. Flow cytometry showed an increased proportion of B-cells 3-4 days postoperatively. In conclusion, major musculoskeletal trauma in elderly patients induces a generalized non-specific increase in immunoglobulin production without laboratory signs for enhanced systemic autoimmunity.

Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency.

BACKGROUND: Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVES: This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS: Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. RESULTS: A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSIONS: This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.

Immunologic and Genetic Contributors to CD46-Dependent Immune Dysregulation.

Mutations in CD46 predispose to atypical hemolytic uremic syndrome (aHUS) with low penetrance. Factors driving immune-dysregulatory disease in individual mutation carriers have remained ill-understood. In addition to its role as a negative regulator of the complement system, CD46 modifies T cell-intrinsic metabolic adaptation and cytokine production. Comparative immunologic analysis of diseased vs. healthy CD46 mutation carriers has not been performed in detail yet. In this study, we comprehensively analyzed clinical, molecular, immune-phenotypic, cytokine secretion, immune-metabolic, and genetic profiles in healthy vs. diseased individuals carrying a rare, heterozygous CD46 mutation identified within a large single family. Five out of six studied individuals carried a CD46 gene splice-site mutation causing an in-frame deletion of 21 base pairs. One child suffered from aHUS and his paternal uncle manifested with adult-onset systemic lupus erythematosus (SLE). Three mutation carriers had no clinical evidence of CD46-related disease to date. CD4+ T cell-intrinsic CD46 expression was uniformly 50%-reduced but was comparable in diseased vs. healthy mutation carriers. Reconstitution experiments defined the 21-base pair-deleted CD46 variant as intracellularly-but not surface-expressed and haploinsufficient. Both healthy and diseased mutation carriers displayed reduced CD46-dependent T cell mitochondrial adaptation. Diseased mutation carriers had lower peripheral regulatory T cell (Treg) frequencies and carried potentially epistatic, private rare variants in other inborn errors of immunity (IEI)-associated proinflammatory genes, not found in healthy mutation carriers. In conclusion, low Treg and rare non-CD46 immune-gene variants may contribute to clinically manifest CD46 haploinsufficiency-associated immune-dysregulation.

Intrathecal IgM Synthesis Is Associated with Spinal Cord Manifestation and Neuronal Injury in Early MS.

OBJECTIVE: Intrathecal Immunoglobulin M synthesis (IgMIntrathecal Fraction (IF) + ) and spinal MRI lesions are both strong independent predictors of higher disease activity and severity in multiple sclerosis (MS). We investigated whether IgMIF + is associated with spinal cord manifestation and higher neuroaxonal damage in early MS. METHODS: In 122 patients with a first demyelinating event associations between (1) spinal versus (vs) non-spinal clinical syndrome (2) spinal vs cerebral T2-weighted (T2w) and (3) contrast-enhancing (CE) lesion counts with IgGIF + (vs IgGIF - ) or IgMIF + (vs IgMIF - ) were investigated by logistic regression adjusted for age and sex, respectively. For serum neurofilament light chain (sNfL) analysis patients were categorized for presence or absence of oligoclonal IgG bands (OCGB), IgGIF and IgMIF (>0% vs 0%, respectively): (1) OCGB- /IgGIF - /IgMIF - ; (2) OCGB+ /IgGIF - /IgMIF - ; (3) OCGB+ /IgGIF + /IgMIF - ; and (4) OCGB+ /IgGIF + /IgMIF + . Associations between categories 2 to 4 vs category 1 with sNfL concentrations were analyzed by robust linear regression, adjusted for sex and MRI parameters. RESULTS: Patients with a spinal syndrome had a 8.36-fold higher odds of IgMIF + (95%CI 3.03-23.03; p < 0.01). Each spinal T2w lesion (odds Ratio 1.39; 1.02-1.90; p = 0.037) and CE lesion (OR 2.73; 1.22-6.09; p = 0.014) was associated with an increased risk of IgMIF + (but not of IgGIF + ); this was not the case for cerebral lesions. OCGB+ /IgGIF + /IgMIF + category patients showed highest sNfL levels (estimate:1.80; 0.55-3.06; p < 0.01). INTERPRETATION: Intrathecal IgM synthesis is strongly associated with spinal manifestation and independently more pronounced neuroaxonal injury in early MS, suggesting a distinct clinical phenotype and pathophysiology. ANN NEUROL 2022;91:814-820.

Staphylococcus aureus enterotoxin A- and B-specific IgE in chronic obstructive pulmonary disease.

Sensitization to Staphylococcus aureus enterotoxins A (SEA) and B (SEB) has been associated with asthma severity, exacerbations, and disease control. Our study aimed to investigate if there are differences in serum SEA-IgE and SEB-IgE levels between patients with chronic obstructive pulmonary disease (COPD), asthma, and controls, and to assess the association between SE sensitization and COPD clinical parameters and Th2 inflammation biomarkers in two well-defined COPD cohorts. Our findings suggest that COPD patients do not exhibit higher SEA and SEB sensitization compared to asthma patients and controls. However, in COPD patients, the presence of atopy and allergy is associated with positivity for SEA-IgE and SEB-IgE. Consequently, these allergens may aid in identifying atopic or allergic subgroups within the COPD population, but they are not directly associated with the diagnosis of COPD, elevated circulating blood eosinophils, or fractional exhaled nitric oxide (FENO) levels.

Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Standardisation of ACPA tests: evaluation of a new candidate reference preparation.

INTRODUCTION: Commercial assays measuring antibodies to citrullinated protein/peptide (ACPA) show poor quantitative agreement. The diagnostic industry has never adopted the International Union of Immunological Societies-Centers for Disease Control and Prevention (IUIS-CDC) ACPA reference standard. Recently, the National Institute for Biological Standards and Control (NIBSC) prepared a new candidate ACPA standard (18/204). We evaluated both reference materials using different commercially available ACPA assays. MATERIALS AND METHODS: This is an international study in which the NIBSC candidate ACPA standard and the IUIS-CDC ACPA reference material were analysed together with 398 diagnostic samples from individuals with rheumatoid arthritis (RA) and in 1073 individuals who did not have RA using nine commercial ACPA assays. RESULTS: For both reference materials and samples from individuals with RA and individuals who did not have RA, there were large differences in quantitative ACPA results between assays. For most assays, values for the IUIS-CDC standard were lower than values for NIBSC 18/204 and the IUIS-CDC/NIBSC ratio was comparable for several, but not all assays. When NIBSC 18/204 was used as a calibrator, an improvement in alignment of ACPA results across several of the evaluated assays was obtained. Moreover, NIBSC 18/204 could align clinical interpretation for some but not all assays. CONCLUSION: Adoption of an international standard for ACPA determination is highly desirable. The candidate NIBSC 18/204 standard improved the standardisation and alignment of most ACPA assays and might therefore be recommended to be used as reference in commercial assays.

Intrathecal Immunoglobulin M Synthesis is an Independent Biomarker for Higher Disease Activity and Severity in Multiple Sclerosis.

OBJECTIVE: We aimed to determine in relapsing multiple sclerosis (MS) whether intrathecal synthesis of immunoglobulin (Ig) M and IgG is associated with outcomes reflecting inflammatory activity and chronic worsening. METHODS: We compared cerebrospinal fluid analysis, clinical and magnetic resonance imaging data, and serum neurofilament light chain (sNfL) levels at baseline and follow-up in 530 patients with relapsing MS. Patients were categorized by the presence of oligoclonal IgG bands (OCGB) and intrathecal synthesis of IgG and IgM (intrathecal fraction [IF]: IgGIF and IgMIF ). Relationships with the time to first relapse, sNfL concentrations, T2-weighted (T2w) lesions, MS Severity Score (MSSS), and time to initiation of high-efficacy therapy were analyzed in covariate-adjusted statistical models. RESULTS: By categorical analysis, in patients with IgMIF the median time to first relapse was 28 months shorter and MSSS on average higher by 1.11 steps compared with patients without intrathecal immunoglobulin synthesis. Moreover, patients with IgMIF had higher sNfL concentrations, more new/enlarging T2w lesions, and higher total T2w lesion counts (all p ≤ 0.01). These associations were absent or equally smaller in patients who were positive for only OCGB or OCGB/IgGIF . Furthermore, quantitative analyses revealed that in patients with IgMIF ≥ median, the time to first relapse and to initiation of high-efficacy therapy was shorter by 32 and by 203 months, respectively (both p < 0.01), in comparison to patients with IgMIF < median. Dose-dependent associations were also found for IgMIF but not for IgGIF with magnetic resonance imaging-defined disease activity and sNfL. INTERPRETATION: This large study supports the value of intrathecal IgM synthesis as an independent biomarker of disease activity and severity in relapsing MS. ANN NEUROL 2021;90:477-489.

Repository of intra- and inter-run variations of quantitative autoantibody assays: a European multicenter study.

OBJECTIVES: No reference data are available on repositories to measure precision of autoantibody assays. The scope of this study was to document inter- and intra-run variations of quantitative autoantibody assays based on a real-world large international data set. METHODS: Members of the European Autoimmunity Standardisation Initiative (EASI) group collected the data of intra- and inter-run variability obtained with assays quantifying 15 different autoantibodies in voluntary participating laboratories from their country. We analyzed the impact on the assay performances of the type of immunoassay, the number of measurements used to calculate the coefficient of variation (CVs), the nature and the autoantibody level of the internal quality control (IQC). RESULTS: Data were obtained from 64 laboratories from 15 European countries between February and October 2021. We analyzed 686 and 1,331 values of intra- and inter-run CVs, respectively. Both CVs were significantly dependent on: the method of immunoassay, the level of IQC with higher imprecision observed when the antibody levels were lower than 2-fold the threshold for positivity, and the nature of the IQC with commercial IQCs having lower CVs than patients-derived IQCs. Our analyses also show that the type of autoantibody has low impact on the assay' performances and that 15 measurements are sufficient to establish reliable intra- and inter-run variations. CONCLUSIONS: This study provides for the first time an international repository yielding values of intra- and inter-run variation for quantitative autoantibody assays. These data could be useful for ISO 15189 accreditation requirements and will allow clinical diagnostic laboratories to assure quality of patient results.

IgA rheumatoid factor in rheumatoid arthritis.

OBJECTIVES: Rheumatoid factor (RF) is a well-established marker for the diagnosis and classification of rheumatoid arthritis (RA). Most studies evaluated IgM RF or isotype-nonspecific total RF assays. We evaluated the added value of IgA RF in this context. METHODS: An international sample cohort consisting of samples from 398 RA patients and 1073 controls was tested for IgA RF with 3 commercial assays. For all RA patients and 100 controls essential clinical and serological data for ACR/EULAR classification were available. RESULTS: The sensitivity of IgA RF for diagnosing RA was lower than the sensitivity of IgM RF. Differences in numerical values between IgA RF assays were observed. With all assays, the highest IgA RF values were found in patients with primary Sjögren's syndrome. Double positivity for IgM RF and IgA RF had a higher specificity for RA than either IgM RF or IgA RF. The sensitivity of double positivity was lower than the sensitivity of either IgA RF or IgM RF. Single positivity for IgA RF was at least as prevalent in controls than in RA patients. Adding IgA RF to IgM RF and anti-citrullinated protein antibodies (ACPA) did not affect RA classification. However, combined positivity for IgA RF, IgM RF and IgG ACPA had a higher specificity and lower sensitivity for RA classification than positivity for either of the antibodies. CONCLUSIONS: IgA RF showed a lower sensitivity than IgM RF. Combining IgA RF with IgM RF and ACPA did not improve sensitivity of RA classification. Combined positivity (IgA-RF/IgM-RF/ACPA) increased specificity.

Prediction of Acute COPD Exacerbation in the Swiss Multicenter COPD Cohort Study (TOPDOCS) by Clinical Parameters, Medication Use, and Immunological Biomarkers.

BACKGROUND AND OBJECTIVE: Whether immunological biomarkers combined with clinical characteristics measured during an exacerbation-free period are predictive of acute exacerbation of chronic obstructive pulmonary disease (AECOPD) frequency and severity is unknown. METHOD: We measured immunological biomarkers and clinical characteristics in 271 stable chronic obstructive pulmonary disease (COPD) patients (67% male, mean age 63 years) from "The Obstructive Pulmonary Disease Outcomes Cohort of Switzerland" cohort on a single occasion. One-year follow-up data were available for 178 patients. Variables independently associated with AECOPD frequency and severity were identified by multivariable regression analyses. Receiver operating characteristic analysis was used to obtain optimal cutoff levels and measure the area under the curve (AUC) in order to assess if baseline data can be used to predict future AECOPD. RESULTS: Higher number of COPD medications (adjusted incident rate ratio [aIRR] 1.17) and platelet count (aIRR 1.03), and lower FEV1% predicted (aIRR 0.84) and IgG2 (aIRR 0.84) were independently associated with AECOPD frequency in the year before baseline. Optimal cutoff levels for experiencing frequent (>1) AECOPD were ≥3 COPD medications (AUC = 0.72), FEV1 ≤40% predicted (AUC = 0.72), and IgG2 ≤2.6 g/L (AUC = 0.64). The performance of a model using clinical and biomarker parameters to predict future, frequent AECOPD events in the same patients was fair (AUC = 0.78) but not superior to a model using only clinical parameters (AUC = 0.79). The IFN-lambda rs8099917GG-genotype was more prevalent in patients who had severe AECOPD. CONCLUSIONS: Clinical and biomarker parameters assessed at a single point in time correlated with the frequency of AECOPD events during the year before and the year after assessment. However, only clinical parameters had fair discriminatory power in identifying patients likely to experience frequent AECOPD.

Variable and inaccurate serum IgG4 levels resulting from lack of standardization in IgG subclass assay calibration.

Background The quantification of serum IgG4 is commonly performed during the diagnostic workup of IgG4-related diseases (IgG4-RD). According to recent literature, IgG4 values above 1.35 g/L are characteristic of IgG4-RD and support its diagnosis at initial presentation. The purpose of this study was to evaluate comparability and accuracy of the two main commercially available IgG4 assays (Siemens Healthineers and The Binding Site). Methods Method comparison was performed for IgG and IgG subclasses using a collective of selected samples with elevated serum IgG4. In addition, we assessed the accuracy of both assays using purified polyclonal and monoclonal IgG4 preparations. Results Our data show significant discrepancies between the two IgG subclass assays for the measurement of IgG4 and, to a lesser extent, IgG3. Conclusions The lack of standardization between the two main providers of commercially available IgG4 assays leads to significant inter-assay result discrepancies, which might potentially cause unnecessary clinical workup. We conclude that serum IgG4 assay-specific decision limits, and not an assay-independent single cut-off level for IgG4 (e.g. 1.35 g/L), should be used when assessing patients for IgG4-RD. An internationally recognized, certified reference material for IgG subclasses is urgently needed, and assay manufactures are encouraged to undertake steps toward standardization of measurements of IgG4 and other IgG subclasses.

Delayed-type allergy to cobalt-comparison of a flow cytometric lymphocyte proliferation test with patch testing.

BACKGROUND: The patch test is the standard procedure for diagnosing delayed-type sensitization. If a patch test is not possible, the flow cytometric lymphocyte proliferation test (LPT), which determines the number and type of cells responding to a specific antigen in vitro, might be considered as an alternative. OBJECTIVES: Our aim was to establish a flow cytometric LPT for the detection of delayed-type allergic responses to cobalt, and to determine the correlation between stimulation indices (SIs) in LPT and the grade of patch test reactions. With the patch test as a diagnostic reference, we also assessed the sensitivity and specificity of the LPT. METHODS: Fifty-four patients patch tested with the baseline series including cobalt (CoCl2 ) were additionally tested with the flow cytometric LPT with CoCl2 . RESULTS: There was a significant correlation between the results of both tests: rs = 0.43; P = .001. The LPT with CoCl2 showed a sensitivity of 52.6% and a specificity of 85.7%. Corresponding to the low sensitivity of the LPT, high likelihood ratios for a positive patch test reaction were reached only in cases of strong lymphocyte proliferation (SI ≥ 10). CONCLUSIONS: In cases of clearly increased SIs, the flow cytometric LPT with CoCl2 gives relevant diagnostic information, and represents a valuable alternative to patch testing.

Thaumatin and gum arabic allergy in chewing gum factory workers.

Thaumatin is a sweetener and flavor modifier commonly used in the food industry. Likewise, gum arabic is widely used as a food stabilizer and thickening agent. We report here that a powder mixture composed of 10% thaumatin and 90% gum arabic led to allergic symptoms in the upper airways in occupationally exposed individuals: four of eight workers of a chewing gum factory exposed to this powder mixture had pronounced rhinitis. A positive skin prick test result for pure thaumatin was obtained in all four individuals with rhinitis of whom two also had a positive skin prick test result for pure gum arabic and gum arabic-specific IgE. Subsitution of a powdered thaumatin with a liquid form reduced symptoms among the rhinitic workers. Although gum arabic is a well-known potential allergen, we were unable to find prior documentation of allergic symptoms to thaumatin when it is used in the food industry.