DFS70 Autoantibodies: Clinical Utility in Antinuclear Antibody Testing.

BACKGROUND: Screening for antinuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cells is helpful for the diagnosis and classification of ANA-associated rheumatic diseases, including systemic lupus erythematosus, Sjögren syndrome, mixed connective tissue disease, systemic sclerosis, and inflammatory myopathies. The dense fine speckled (DFS) pattern is a special HEp-2 IIF pattern (produced by anti-DFS70 antibodies) because it is not associated with a specific medical condition and therefore can obfuscate interpretation. CONTENT: In this paper, detection methods for and clinical associations of anti-DFS70 antibodies are reviewed. SUMMARY: The target antigen of the antibodies that cause the DFS pattern is a 70 kDa protein (DFS70). Commercial methods that detect antibodies to full-length or truncated DFS70 are available for use in clinical laboratories (ELISA, chemiluminescence, dot/line blot). Anti-DFS70 can be found in (apparently) healthy individuals (with a higher frequency in young individuals and in females), in several (inflammatory) conditions and in malignancy. There is no clinical association that is well-established. Special attention (and critical reflection) is given to the observation that monospecific anti-DFS70 (i.e., in the absence of antibodies that are linked to ANA-associated rheumatic diseases) is rarely found in ANA-associated rheumatic diseases.

Automated Urinary Chemokine Assays for Noninvasive Detection of Kidney Transplant Rejection: A Prospective Cohort Study.

RATIONALE & OBJECTIVE: Prior studies have demonstrated the diagnostic potential of urinary chemokines C-X-C motif ligand 9 (CXCL9) and CXCL10 for kidney transplant rejection. However, their benefit in addition to clinical information has not been demonstrated. We evaluated the diagnostic performance for detecting acute rejection of urinary CXCL9 and CXCL10 when integrated with clinical information. STUDY DESIGN: Single-center prospective cohort study. SETTING & PARTICIPANTS: We analyzed 1,559 biopsy-paired urinary samples from 622 kidney transplants performed between April 2013 and July 2019 at a single transplant center in Belgium. External validation was performed in 986 biopsy-paired urinary samples. TESTS COMPARED: We quantified urinary CXCL9 (uCXCL9) and CXCL10 (uCXCL10) using an automated immunoassay platform and normalized the values to urinary creatinine. Urinary chemokines were incorporated into a multivariable model with routine clinical markers (estimated glomerular filtration rate, donor-specific antibodies, and polyoma viremia) (integrated model). This model was then compared with the tissue diagnosis according to the Banff classification for acute rejection. OUTCOME: Acute rejection detected on kidney biopsy using the Banff classification. RESULTS: Chemokines integrated with routine clinical markers had high diagnostic value for detection of acute rejection (n=150) (receiver operating characteristic area under the curve 81.3% [95% CI, 77.6-85.0]). The integrated model would help avoid 59 protocol biopsies per 100 patients when the risk for rejection is predicted to be below 10%. The performance of the integrated model was similar in the external validation cohort. LIMITATIONS: The cross-sectional nature obviates investigating the evolution over time and prediction of future rejection. CONCLUSIONS: The use of an integrated model of urinary chemokines and clinical markers for noninvasive monitoring of rejection could enable a reduction in the number of biopsies. Urinary chemokines may be useful noninvasive biomarkers whose use should be further studied in prospective randomized trials to clarify their role in guiding clinical care and the use of biopsies to detect rejection after kidney transplantation. PLAIN-LANGUAGE SUMMARY: Urinary chemokines CXCL9 and CXCL10 have been suggested to be good noninvasive biomarkers of kidney transplant rejection. However, defining a context of use and integration with clinical information is necessary before clinical implementation can begin. In this study, we demonstrated that urinary chemokines CXCL9 and CXCL10, together with clinical information, have substantial diagnostic accuracy for the detection of acute kidney transplant rejection. Application of urinary chemokines together with clinical information may guide biopsy practices following kidney transplantation and potentially reduce the need for kidney transplant biopsies.

Human Autosomal Recessive DNA Polymerase Delta 3 Deficiency Presenting as Omenn Syndrome.

The DNA polymerase δ complex (PolD), comprising catalytic subunit POLD1 and accessory subunits POLD2, POLD3, and POLD4, is essential for DNA synthesis and is central to genome integrity. We identified, by whole exome sequencing, a homozygous missense mutation (c.1118A > C; p.K373T) in POLD3 in a patient with Omenn syndrome. The patient exhibited severely decreased numbers of naïve T cells associated with a restricted T-cell receptor repertoire and a defect in the early stages of TCR recombination. The patient received hematopoietic stem cell transplantation at age 6 months. He manifested progressive neurological regression and ultimately died at age 4 years. We performed molecular and functional analysis of the mutant POLD3 and assessed cell cycle progression as well as replication-associated DNA damage. Patient fibroblasts showed a marked defect in S-phase entry and an enhanced number of double-stranded DNA break-associated foci despite normal expression levels of PolD components. The cell cycle defect was rescued by transduction with WT POLD3. This study validates autosomal recessive POLD3 deficiency as a novel cause of profound T-cell deficiency and Omenn syndrome.

Peptides From the Variable Domain of Immunoglobulin G as Biomarkers in Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

BACKGROUND AND OBJECTIVES: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a clinically heterogeneous immune-mediated disease. Diagnostic biomarkers for CIDP are currently lacking. Peptides derived from the variable domain of circulating immunoglobulin G (IgG) have earlier been shown to be shared among patients with the same immunologic disease. Because humoral immune factors are hypothesized to be involved in the pathogenesis of CIDP, we evaluated IgG variable domain-derived peptides as diagnostic biomarkers in CIDP (primary objective) and whether IgG-derived peptides could cluster objective clinical entities in CIDP (secondary objective). METHODS: IgG-derived peptides were determined in prospectively collected sera of patients with CIDP and neurologic controls by means of mass spectrometry. Peptides of interest were selected through statistical analysis in a discovery cohort followed by sequence determination and confirmation. Diagnostic performance was evaluated for individual selected peptides and for a multipeptide model incorporating selected peptides, followed by performance reassessment in a validation cohort. Clustering of patients with CIDP based on IgG-derived peptides was evaluated through unsupervised sparse principal component analysis followed by k-means clustering. RESULTS: Sixteen peptides originating from the IgG variable domain were selected as candidate biomarkers in a discovery cohort of 44 patients with CIDP and 29 neurologic controls. For all 16 peptides, univariate logistic regressions and ROC curve analysis demonstrated increasing peptide abundances to associate with increased odds for CIDP (area under the curves [AUCs] ranging from 64.6% to 79.6%). When including age and sex in the logistic regression models, this remained the case for 13/16 peptides. A model composed of 5/16 selected peptides showed strong discriminating performance between patients with CIDP and controls (AUC 91.5%; 95% CI 84.6%-98.4%; p < 0.001). In the validation cohort containing 45 patients and 43 controls, 2/16 peptides demonstrated increasing abundances to associate with increased odds for CIDP, while the five-peptide model demonstrated an AUC of 61.2% (95% CI 49.3%-73.2%; p = 0.064). Peptide-based patient clusters did not associate with clinical features. DISCUSSION: IgG variable domain-derived peptides showed a valid source for diagnostic biomarkers in CIDP, albeit with challenges toward replication. Our proof-of-concept findings warrant further study of IgG-derived peptides as biomarkers in more homogeneous cohorts of patients with CIDP and controls. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that the pattern of serum IgG-derived peptide clusters may help differentiate between patients with CIDP and those with other peripheral neuropathies.

Soluble Urokinase Plasminogen Activator Receptor (suPAR) in Autoimmune Rheumatic and Non Rheumatic Diseases.

The soluble urokinase plasminogen activator receptor (suPAR) is the bioactive form of uPAR, a membrane-bound glycoprotein, and it is primarily expressed on the surface of immunologically active cells. Mirroring local inflammation and immune activation, suPAR has gained interest as a potential prognostic biomarker in several inflammatory diseases. Indeed, in many diseases, including cancer, diabetes, cardiovascular diseases, kidney diseases, and inflammatory disorders, higher suPAR concentrations have been associated with disease severity, disease relapse, and mortality. Our review describes and discusses the supporting literature concerning the promising role of suPAR as a biomarker in different autoimmune rheumatic and non-rheumatic diseases.

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.

Antinuclear antibodies in individuals with COVID-19 reflect underlying disease: Identification of new autoantibodies in systemic sclerosis (CDK9) and malignancy (RNF20, RCC1, TRIP13).

A high prevalence of antinuclear antibodies (ANA) in COVID-19 has been insinuated, but the nature of the target antigens is poorly understood. We studied ANA by indirect immunofluorescence in 229 individuals with COVID-19. The target antigens of high titer ANA (≥1:320) were determined by immunoprecipitation (IP) combined with liquid-chromatography-mass spectrometry (MS). High titer ANA (≥1:320) were found in 14 (6%) of the individuals with COVID-19. Of the 14 COVID-19 cases with high titer ANA, 6 had an underlying autoimmune disease and 5 a malignancy. IP-MS revealed known target antigens associated with autoimmune disease as well as novel autoantigens, including CDK9 (in systemic sclerosis) and RNF20, RCC1 and TRIP13 (in malignancy). The novel autoantigens were confirmed by IP-Western blotting. In conclusion, in depth analysis of the targets of high titer ANA revealed novel autoantigens in systemic sclerosis and in malignant disease.

Circulating Calprotectin (cCLP) in autoimmune diseases.

BACKGROUND AND AIM: Calprotectin (CLP) is a heterodimeric complex formed by two S100 proteins (S100A8/A9), which plays a pivotal role in innate immunity. Due to its intrinsic cytotoxic and proinflammatory properties, CLP controls cell differentiation, proliferation and NETosis and has been associated with a wide range of rheumatic diseases. Our review summarizes the widespread interest in circulating CLP (cCLP) as a biomarker of neutrophil-related inflammation, in autoimmune rheumatic disease (ARD) and non-ARD. METHODS: A thorough literature review was performed using PubMed and EMBASE databases searching for circulating calprotectin and synonyms S100A8/A9, myeloid-related protein 8/14 (MRP8/MRP14), calgranulin A/B and L1 protein in addition to specific ARDs and autoimmune non-rheumatic diseases. We selected only English-language articles and excluded abstracts without the main text. RESULTS: High cCLP serum levels are associated with worse structural outcomes in rheumatoid arthritis and to a lesser extent, in spondyloarthritis. In addition, cCLP can predict disease relapse in some autoimmune diseases including systemic lupus erythematosus (SLE), anti-neutrophil cytoplasmic antibodies-associated vasculitis (AAV) and some severe manifestations of connective tissue diseases, such as glomerulonephritis in SLE, AAV, juvenile idiopathic arthritis, adult-onset Still's disease and lung fibrosis in systemic sclerosis. Therefore, cCLP levels enable the identification of patients who need an accurate and tight follow-up. The clinical usefulness of cCLP as an inflammatory marker has been suggested for inflammatory/autoimmune non-rheumatic diseases, and especially for the monitoring of the inflammatory bowel diseases patients. Currently, there are only a few studies that evaluated the cCLP efficacy as a clinical biomarker in inflammatory/autoimmune non-rheumatic diseases with controversial results. Future studies are warranted to better clarify the role of cCLP in relation to the disease severity in myasthenia gravis, multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, Graves' orbitopathy, autoimmune bullous diseases and uveitis. CONCLUSION: Our literature review supports a relevant role of cCLP as potential prognostic biomarker mirroring local or systemic inflammation, especially in chronic inflammatory rheumatic diseases.

Mass spectrometry-based identification of new anti-Ly and known antisynthetase autoantibodies.

OBJECTIVES: To discover new and detect known antisynthetase autoantibodies (ASAs) through protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS). METHODS: IP-MS was performed using sera of individuals showing features of antisynthetase syndrome (ASyS) without (n=5) and with (n=12) previously detected ASAs, and healthy controls (n=4). New candidate aminoacyl-tRNA-synthetase (ARS) autoantigens identified through unbiased IP-MS were confirmed by IP-western blot. A targeted IP-MS assay for various ASA specificities was developed and validated with sera of patients with known ASAs (n=16), disease controls (n=20) and healthy controls (n=25). The targeted IP-MS assay was applied in an additional cohort of patients with multiple ASyS features or isolated myositis without previously detected ASAs (n=26). RESULTS: Autoantibodies to cytoplasmic cysteinyl-tRNA-synthetase (CARS1) were identified by IP-MS and confirmed by western blot as a new ASA specificity, named anti-Ly, in the serum of a patient with ASyS features. Rare ASAs, such as anti-OJ, anti-Zo and anti-KS, and common ASAs could also be identified by IP-MS. A targeted IP-MS approach for ASA detection was developed and validated. Application of this method in an additional cohort identified an additional patient with anti-OJ autoantibodies that were missed by line and dot immunoassays. DISCUSSION: CARS1 is the dominant cognate ARS autoantigen of the newly discovered anti-Ly ASA specificity. Rare and common ASA specificities could be detected by both unbiased and targeted IP-MS. Unbiased and targeted IP-MS are promising methods for discovery and detection of autoantibodies, especially autoantibodies that target complex autoantigens.

Laboratory biomarkers in the diagnosis and follow-up of treatment of allergic bronchopulmonary aspergillosis in cystic fibrosis.

Allergic bronchopulmonary aspergillosis (ABPA), a severe inflammatory respiratory disease, is caused by a hypersensitivity reaction to the colonization of the airways with Aspergillus fumigatus. It is most often described in patients with asthma or cystic fibrosis. The diagnosis of ABPA is based on a combination of clinical, radiological, and immunological findings that have been included in different diagnostic criteria over the years. In this paper, we review the biomarkers included in these diagnostic criteria and novel research biomarkers that may be used in the diagnosis and treatment follow-up of ABPA in cystic fibrosis.

Common variable immunodeficiency in two kindreds with heterogeneous phenotypes caused by novel heterozygous NFKB1 mutations.

NFKB1 haploinsufficiengcy was first described in 2015 in three families with common variable immunodeficiency (CVID), presenting heterogeneously with symptoms of increased infectious susceptibility, skin lesions, malignant lymphoproliferation and autoimmunity. The described mutations all led to a rapid degradation of the mutant protein, resulting in a p50 haploinsufficient state. Since then, more than 50 other mutations have been reported, located throughout different domains of NFKB1 with the majority situated in the N-terminal Rel homology domain (RHD). The clinical spectrum has also expanded with possible disease manifestations in almost any organ system. In silico prediction tools are often used to estimate the pathogenicity of NFKB1 variants but to prove causality between disease and genetic findings, further downstream functional validation is required. In this report, we studied 2 families with CVID and two novel variants in NFKB1 (c.1638-2A>G and c.787G>C). Both mutations affected mRNA and/or protein expression of NFKB1 and resulted in excessive NLRP3 inflammasome activation in patient macrophages and upregulated interferon stimulated gene expression. Protein-protein interaction analysis demonstrated a loss of interaction with NFKB1 interaction partners for the p.V263L mutation. In conclusion, we proved pathogenicity of two novel variants in NFKB1 in two families with CVID characterized by variable and incomplete penetrance.

Child Neurology: Familial Hemophagocytic Lymphohistiocytosis Underlying Isolated CNS Inflammation.

Encephalitis and encephalopathy in children represent a diagnostic challenge. We describe a patient with relapsing encephalitis in whom the differential diagnosis included acute disseminated encephalomyelitis, human herpesvirus 6 encephalitis, and hemophagocytic lymphohistiocytosis (HLH). Because of its rarity, HLH is often overlooked as a differential diagnosis in encephalitis, especially in the isolated CNS forms. As this case illustrates, inborn errors of immunity can underlie isolated encephalitis and should be included in the differential diagnosis of these presentations.

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal α-toxin.

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.

Multicentre study to improve clinical interpretation of rheumatoid factor and anti-citrullinated protein/peptide antibodies test results.

BACKGROUND: Rheumatoid factor (RF) and anti-citrullinated protein/peptide antibodies (ACPA) are important biomarkers for diagnosis of rheumatoid arthritis (RA). However, there is poor harmonisation of RF and ACPA assays. The aim of this study was to refine RF and ACPA interpretation across commercial assays. MATERIALS AND METHODS: Six total RF isotype-non-specific assays, 3 RF IgM isotype-specific assays and 9 ACPA immunoglobulin G assays of 13 different companies were evaluated using 398 diagnostic samples from patients with RA and 1073 disease controls. RESULTS: Using cut-offs proposed by the manufacturer, there was a large variability in diagnostic sensitivity and specificity between assays. Thresholds of antibody levels were determined based on predefined specificities and used to define test result intervals. Test result interval-specific likelihood ratios (LRs) were concordant across the different RF and ACPA assays. For all assays, the LR for RA increased with increasing antibody level. Higher LRs were found for ACPA than for RF. ACPA levels associated with LRs >80 were found in a substantial fraction (>22%) of patients with RA. CONCLUSION: Defining thresholds for antibody levels and assigning test result interval-specific LRs allows alignment of clinical interpretation for all RF and ACPA assays.

Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases.

Antinuclear antibodies (ANAs) are valuable laboratory markers to screen for and support the diagnosis of various rheumatic diseases (known as ANA-associated rheumatic diseases). The importance of ANA testing has been reinforced by the inclusion of ANA positivity as an entry criterion in the 2019 systemic lupus erythematosus classification criteria. In addition, specific ANAs (such as antibodies to Sm, double-stranded DNA (dsDNA), SSA/Ro60, U1RNP, topoisomerase I, centromere protein B (CENPB), RNA polymerase III and Jo1) are included in classification criteria for other rheumatic diseases. A number of techniques are available for detecting antibodies to a selection of clinically relevant antigens (such as indirect immunofluorescence and solid phase assays). In this Review, we discuss the advantages and limitations of these techniques, as well as the clinical relevance of the differences between the techniques, to provide guidance in understanding and interpreting ANA test results. Such understanding not only necessitates insight into the sensitivity and specificity of each assay, but also into the importance of the disease context and antibody level. We also highlight the value of titre-specific information (such as likelihood ratios).

2020 international consensus on ANCA testing beyond systemic vasculitis.

This document follows up on a 2017 revised international consensus on anti-neutrophil cytoplasm antibodies (ANCA) testing in granulomatosis with polyangiitis and microscopic polyangiitis and focuses on the clinical and diagnostic value of ANCA detection in patients with connective tissue diseases, idiopathic interstitial pneumonia, autoimmune liver diseases, inflammatory bowel diseases, anti-glomerular basement membrane (GBM) disease, infections, malignancy, and during drug treatment. Current evidence suggests that in certain settings beyond systemic vasculitis, ANCA may have clinical, pathogenic and/or diagnostic relevance. Antigen-specific ANCA targeting proteinase-3 and myeloperoxidase should be tested by solid phase immunoassays in any patient with clinical features suggesting ANCA-associated vasculitis and in all patients with anti-GBM disease, idiopathic interstitial pneumonia, and infective endocarditis associated with nephritis, whereas in patients with other aforementioned disorders routine ANCA testing is not recommended. Among patients with autoimmune liver diseases or inflammatory bowel diseases, ANCA testing may be justified in patients with suspected autoimmune hepatitis type 1 who do not have conventional autoantibodies or in case of diagnostic uncertainty to discriminate ulcerative colitis from Crohn's disease. In these cases, ANCA should be tested by indirect immunofluorescence as the target antigens are not yet well characterized. Many questions concerning the optimal use of ANCA testing in patients without ANCA-associated vasculitis remain to be answered.

International Consensus on ANCA Testing in Eosinophilic Granulomatosis with Polyangiitis.

An international consensus on anti-neutrophil cytoplasm antibodies (ANCA) testing in eosinophilic granulomatosis with polyangiitis (EGPA) is presented. ANCA, specific for myeloperoxidase (MPO), can be detected in 30-35% of EGPA patients. MPO-ANCA should be tested with antigen-specific immunoassays in any patient with eosinophilic asthma and clinical features suggesting EGPA, including constitutional symptoms, purpura, polyneuropathy, unexplained heart, gastrointestinal or kidney disease, and/or pulmonary infiltrates or hemorrhage. A positive MPO-ANCA result contributes to the diagnostic work­up for EGPA. Patients with MPO-ANCA associated EGPA have more frequently vasculitis features, such as glomerulonephritis, neuropathy, and skin manifestations than patients with ANCA negative EGPA. However, the presence of MPO-ANCA is neither sensitive nor specific enough to identify whether a patient should be subclassified as having "vasculitic" or "eosinophilic" EGPA. At present, ANCA status cannot guide treatment decisions, that is, whether cyclophosphamide, rituximab or mepolizumab should be added to conventional glucocorticoid treatment. In EGPA, monitoring of ANCA is only useful when MPO-ANCA was tested positive at disease onset.