Quantification of polyreactive immunoglobulin G facilitates the diagnosis of autoimmune hepatitis.

BACKGROUND AND AIMS: Detection of autoantibodies is a mainstay of diagnosing autoimmune hepatitis (AIH). However, conventional autoantibodies for the workup of AIH lack either sensitivity or specificity, leading to substantial diagnostic uncertainty. We aimed to identify more accurate serological markers of AIH with a protein macroarray. APPROACH AND RESULTS: During the search for more-precise autoantibodies to distinguish AIH from non-AIH liver diseases (non-AIH-LD), IgG antibodies with binding capacities to many human and foreign proteins were identified with a protein macroarray and confirmed with solid-phase ELISAs in AIH patients. Subsequently, polyreactive IgG (pIgG) was exemplarily quantified by reactivity against human huntingtin-interacting protein 1-related protein in bovine serum albumin blocked ELISA (HIP1R/BSA). The diagnostic fidelity of HIP1R/BSA binding pIgG to diagnose AIH was assessed in a retrospective training, a retrospective multicenter validation, and a prospective validation cohort in cryoconserved samples from 1,568 adults from 10 centers from eight countries. Reactivity against HIP1R/BSA had a 25% and 14% higher specificity to diagnose AIH than conventional antinuclear and antismooth muscle antibodies, a significantly higher sensitivity than liver kidney microsomal antibodies and antisoluble liver antigen/liver pancreas antigen, and a 12%-20% higher accuracy than conventional autoantibodies. Importantly, HIP1R/BSA reactivity was present in up to 88% of patients with seronegative AIH and in up to 71% of AIH patients with normal IgG levels. Under therapy, pIgG returns to background levels of non-AIH-LD. CONCLUSIONS: pIgG could be used as a promising marker to improve the diagnostic workup of liver diseases with a higher specificity for AIH compared to conventional autoantibodies and a utility in autoantibody-negative AIH. Likewise, pIgG could be a major source of assay interference in untreated AIH.

IgA antibodies against CD74 are associated with structural damage in the axial skeleton in patients with axial spondyloarthritis.

OBJECTIVES: To study the association between the presence of antibodies against CD74 and structural damage in the sacroiliac joints and spine in patients with axial spondyloarthritis (axSpA). METHODS: Antibodies against CD74 were measured in the sera of patients with axSpA from 2 cohorts: 1. An observational cohort from Damp in Northern Germany and 2. from a clinical trial (ENRADAS), in which the modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) had been evaluated by two readers blinded to the time point at baseline and two years later. The presence of antibodies against CD74 was correlated with the presence and grade of radiographic sacroiliitis in the observational cohort, and with baseline mSASSS in the ENRADAS cohort. RESULTS: The sensitivity of IgA anti-CD74 antibodies for axSpA was 50% in the Damp cohort and 42% in ENRADAS. The presence of IgA antibodies against CD74 was associated with a higher grade of sacroiliitis (observational cohort) and a higher baseline mSASSS in the ENRADAS cohort. CONCLUSIONS: IgA antibodies against CD74 are not only markers of AS, but are associated with structural damage development in the sacroiliac joints and in the spine.

Presence of Antibodies Binding to Negative Elongation Factor E in Sarcoidosis.

Sarcoidosis is characterized by multiorgan involvement and granulomatous inflammation. Its origin is unknown and the potential role of autoimmunity has not been sufficiently determined. We investigated the presence of autoantibodies in sarcoidosis using protein array technology. The derivation cohort consisted of patients with sarcoidosis (n = 25) and controls including autoimmune disease and blood donors (n = 246). In addition, we tested a validation cohort including pulmonary sarcoidosis patients (n = 58) and healthy controls (n = 13). Initially, sera of three patients with sarcoidosis were screened using a protein array with 28.000 proteins against controls. Thereby we identified the Negative Elongation Factor E (NELF-E) as an autoantigen. With confirmatory Enzyme-linked Immunosorbent Assay (ELISA)testing, 29/82 patients (35%) with sarcoidosis had antibodies against NELF-E of the Immunoglobulin (Ig) G type, whereas 18/253 (7%) sera of the controls were positive for NELF-E. Clinically, there was an association of the frequency of NELF-E antibody detection with lung parenchymal involvement and corresponding x-ray types. NELF-E autoantibodies are associated with sarcoidosis and should be further investigated.

Impaired proteolysis by SPPL2a causes CD74 fragment accumulation that can be recognized by anti-CD74 autoantibodies in human ankylosing spondylitis.

Ankylosing spondylitis (AS) is associated with autoantibody production to class II MHC-associated invariant chain peptide, CD74/CLIP. In this study, we considered that anti-CD74/CLIP autoantibodies present in sera from AS might recognize CD74 degradation products that accumulate upon deficiency of the enzyme signal peptide peptidase-like 2A (SPPL2a). We analyzed monocytes from healthy controls (n = 42), psoriatic arthritis (n = 25), rheumatoid arthritis (n = 16), and AS patients (n = 15) for SPPL2a enzyme activity and complemented the experiments using SPPL2a-sufficient and -deficient THP-1 cells. We found defects in SPPL2a function and CD74 processing in a subset of AS patients, which culminated in CD74 and HLA class II display at the cell surface. These findings were verified in SPPL2a-deficient THP-1 cells, which showed expedited expression of MHC class II, total CD74 and CD74 N-terminal degradation products at the plasma membrane upon receipt of an inflammatory trigger. Furthermore, we observed that IgG anti-CD74/CLIP autoantibodies recognize CD74 N-terminal degradation products that accumulate upon SPPL2a defect. In conclusion, reduced activity of SPPL2a protease in monocytes from AS predisposes to endosomal accumulation of CD74 and CD74 N-terminal fragments, which, upon IFN-γ-exposure, is deposited at the plasma membrane and can be recognized by anti-CD74/CLIP autoantibodies.

CD74 is a T cell antigen in spondyloarthritis.

OBJECTIVES: Spondyloarthritis (SpA) is a chronic inflammatory disease of unknown aetiology. Previously, we identified autoantibodies against CD74 in sera of SpA patients. The aim of this study was to evaluate CD74 as a T cell antigen in SpA. METHODS: Recombinant CD74 protein and a panel of selected peptides representing its amino acid residues were examined for their capability to stimulate peripheral blood mononuclear cells from patients with SpA. In particular, cytokine production by CD4+ T cells was evaluated with flow cytometric detection of intracellular TNF-α, IFNγ, TGFß and IL-17A. Patients' sera were tested for antibodies against CD74 using ELISA. Samples from patients with rheumatoid arthritis and healthy blood donors were similarly tested as controls. RESULTS: Significantly more CD4+ T cells from SpA patients produced TNF-α, IFNγ and IL-17A in response to recombinant CD74 than patients with rheumatoid arthritis or healthy blood donors. Among evaluated epitopes, the most promiscuous one lies within the peptide of the amino acid residues 142-185, which appeared more immunogenic. Further, the proportion of cytokine producing CD4+ T cells was significantly higher among SpA patients with autoantibodies against CD74. CONCLUSIONS: CD74 is a T cell antigen in SpA, eliciting Th1 and Th17 responses, which may be relevant in disease pathogenesis. Recognition of the highly immunogenic amino acid residues of CD74 may contribute to our understanding of autoimmune responses of T helper cells in SpA.

Added Value of Anti-CD74 Autoantibodies in Axial SpondyloArthritis in a Population With Low HLA-B27 Prevalence.

Axial spondyloarthritis (axSpA) is often diagnosed late due to the non-specific nature of its main symptom [chronic back pain (CBP)] and to the paucity of diagnostic markers, particularly in regions with low HLA-B27 prevalence, such as the Middle-East. We tested the performance of IgG4 and IgA anti-CD74 antibodies as an early diagnostic marker for axSpA, compared with the performance of HLA-B27, in Lebanon. Sera of axSpA patients diagnosed by the rheumatologist and also fulfilling the imaging arm of the ASAS criteria (patients) and of blood donors (BD) (controls) were analyzed for HLA-B27, IgG4 and IgA anti-CD74, blinded to clinical characteristics. Receiver Operating Characteristic curves were constructed to identify an optimal cut-off point for anti-CD74 antibodies. Diagnostic properties were calculated (sensitivity, specificity, positive, and positive predictive values (PPV, NPV), Likelihood ratios) for each marker. Forty-nine axSpA patients and 102 BD were included in the final analysis. IgA anti-CD74 correlated poorly with axSpA (Area Under the Curve (AUC) 0.657), whereas IgG4 anti-CD74 had a good discriminative value (AUC 0.837). Respectively, for HLA-B27, IgG4 anti-CD74, and the combination of both, we found a sensitivity of 33-92-33%, specificity of 96-79-98%, PPV 80-68-89%, NPV 75-95-75%, and LR+ 8.2-4.4-16.5. IgG4 anti-CD 74 were positive in 88% of HLA-B27 negative axSpA patients, and correlated with BASDAI. In this first study in a population with low HLA-B27 prevalence, IgG4 anti-CD74 antibodies combined with HLA-B27 showed higher diagnostic value than HLA-B27 alone for early axSpA. IgG4 anti-CD74 should be considered for further evaluation as an early axSpA diagnostic marker in future dedicated research, particularly in patients with CBP.

Sensitivity and Specificity of Autoantibodies Against CD74 in Nonradiographic Axial Spondyloarthritis.

OBJECTIVE: Autoantibodies against CD74 (anti-CD74) are associated with ankylosing spondylitis (AS). The present multicenter study, the International Spondyloarthritis Autoantibody (InterSpA) trial, was undertaken to compare the sensitivity and specificity of anti-CD74 and HLA-B27 in identifying patients with nonradiographic axial spondyloarthritis (axSpA). METHODS: Patients ages 18-45 years with inflammatory back pain of ≤2 years' duration and a clinical suspicion of axSpA were recruited. HLA-B27 genotyping and magnetic resonance imaging of sacroiliac joints were performed in all patients. One hundred forty-nine patients with chronic inflammatory back pain (IBP) not caused by axSpA served as controls, and additional controls included 50 AS patients and 100 blood donors whose specimens were analyzed. RESULTS: One hundred patients with inflammatory back pain received a diagnosis of nonradiographic axSpA from the investigators and fulfilled the Assessment of SpondyloArthritis international Society (ASAS) criteria. The mean age was 29 years, and the mean symptom duration was 12.5 months. The sensitivity of IgA anti-CD74 and IgG anti-CD74 for identifying the 100 axSpA patients was 47% and 17%, respectively. The specificity of both IgA anti-CD74 and IgG anti-CD74 was 95.3%. The sensitivity of HLA-B27 was 81%. The positive likelihood ratios were 10.0 (IgA anti-CD74), 3.6 (IgG anti-CD74), and 8.1 (HLA-B27). Assuming a 5% pretest probability of axSpA in chronic back pain patients, the posttest probability, after consideration of the respective positive test results, was 33.3% for IgA anti-CD74, 15.3% for IgG anti-CD74, and 28.8% for HLA-B27. A combination of IgA anti-CD74 and HLA-B27 results in a posttest probability of 80.2%. CONCLUSION: IgA anti-CD74 may be a useful tool for identifying axSpA. The diagnostic value of the test in daily practice requires further confirmation.

Anti-CD74 antibodies have no diagnostic value in early axial spondyloarthritis: data from the spondyloarthritis caught early (SPACE) cohort.

BACKGROUND: Anti-CD74 IgG antibodies are reported to be elevated in patients with axial spondyloarthritis (axSpA). This study assessed the diagnostic value of anti-CD74 antibodies in patients with early axSpA. METHODS: Anti-CD74 IgG and IgA antibodies were first measured in an exploratory cohort of patients with radiographic axSpA (138 patients with ankylosing spondyloarthritis (AS)) and 57 healthy controls and then were measured in patients with early axSpA (n = 274) and with non-SpA chronic back pain (CBP) (n = 319), participating in the spondyloarthritis caught early (SPACE) prospective cohort study of patients under 45 years old with early back pain (for ≥ 3 months, but ≤ 2 years). RESULTS: In the exploratory cohort, anti-CD74 IgG antibodies were present in 79.7% of patients with AS vs. 43.9% of healthy controls (p < 0.001). Anti-CD74 IgA antibodies were present in 28.5% of patients with AS vs. 5.3% of healthy controls (p < 0.001). In the SPACE cohort, anti-CD74 IgG antibody levels were present in 46.4% of the patients with axSpA vs. 47.9% of the patients with CBP (p = 0.71). Anti-CD74 IgA antibodies were present in 54.7% of the patients with axSpA and 37.0% of the patients with CBP (p < 0.001). This resulted in a positive predictive value of 58.8% (compared to a prior probability of 46.2%) and a negative predictive value of 59.1% (compared to a prior probability of 53.8%). In a regression model, total serum IgA was associated with axSpA odds ratio (OR) 1.19, p < 0.001) whereas anti-CD74 IgA was not (OR) 1.01, p = 0.33). Furthermore, anti-CD74 IgA was associated with sacroiliitis on magnetic resonance imaging (MRI) (OR) = 2.50, p = 0.005) and heel enthesitis (OR) = 2.56, p = 0.002). CONCLUSIONS: Albeit anti-CD74 IgA is elevated in patients with early axSpA, this elevation is not sufficiently specific to yield significant diagnostic value in patients under 45 years old presenting with early back pain.

Antibodies against MYC-Associated Zinc Finger Protein: An Independent Marker in Acute Coronary Syndrome?

INTRODUCTION: Atherosclerosis is considered the pathophysiology underlying cardiovascular (CVD), cerebrovascular, and peripheral vascular diseases. Evidence supporting an autoimmune component is emerging, with imaging studies correlating MYC-associated zinc finger protein antibody (MAZ-Ab) optical density (OD) with plaque activity. This study compares MAZ-Ab OD on ELISA testing among patients presenting with acute coronary syndromes (ACSs) to healthy controls and investigates the association of MAZ-Ab to traditional CVD risk factors. METHODS: Patients admitted with ACSs between August 2007 and July 2011 were included. Serum samples taken at presentation were retrospectively tested for MAZ-Ab and compared with serum from healthy volunteers with no CVD risk factors. Large-scale assessment of post-ACS prognostic relevance was performed using the established PLATO cohort. RESULTS: In total 174 ACS patients and 96 controls were included. Among ACS patients, median MAZ-Ab OD was higher compared with controls (0.46 vs. 0.27; p = 0.001). Although the majority of ACS patients (116/174; 67%) had suffered from a ST-elevation myocardial infarction, no significant differences in MAZ-Ab titers were evident between ACS subtypes (p = 0.682). No associations between MAZ-Ab OD and conventional CVD risk factors were identified. Large-scale testing revealed no prognostic stratification regarding reinfarction (OR 1.04 [95% CI: 0.94-1.16]; p = 0.436). CONCLUSION: MAZ-Ab OD was higher or all ACS phenotypes compared with controls. Given current understanding of MAZ-Ab function, these findings support an autoimmune component to CVD independent of conventional risk factors and indeed the extent of end-organ damage.

Anti-MYC-associated zinc finger protein antibodies are associated with inflammatory atherosclerotic lesions on 18F-fluorodeoxyglucose positron emission tomography.

BACKGROUND AND AIMS: Atherosclerosis is a chronic inflammatory process of vessel walls responsible for coronary, cerebrovascular and peripheral vascular disease, which together account for the majority of non-infective global deaths. Whilst great emphasis has been placed on lifestyle factors, a growing body of evidence supports an autoimmune component to atherosclerosis. This study evaluates a novel autoantibody against MYC-associated zinc finger protein (MAZ-Ab) as a potential marker of atherosclerosis. It compares MAZ-Ab to activity on whole-body positron-emission tomography/computed tomography (PET/CT) attributable to atherosclerosis. METHODS: Antibody screening using protein arrays was performed in patients with angiographically-proven ischaemic heart disease. Following MAZ-Ab detection, an ELISA for large-scale testing was developed. An a priori group of unselected patients attending for unrelated 18F-fluorodeoxyglucose (FDG) PET/CT was prospectively enrolled. Each completed a structured questionnaire under supervision and provided serum for analysis. PET/CT scans were evaluated for inflammatory arterial lesions. Whole-body arterial inflammatory burden was then correlated with ELISA optical density for MAZ-Ab. RESULTS: Protein array testing identified IgG anti-MAZ antibodies in 4/6 (67%) patients with ischemic heart disease, versus 0/10 controls. Significant positive correlations between MAZ-Ab and both increasing number of PET positive inflammatory atherosclerostic lesions (p = 0.023) and whole-body arterial inflammatory burden (p = 0.002) were shown. No traditional atherosclerotic risk factor correlated with MAZ-Ab. CONCLUSIONS: A quantitative association between MAZ-Ab optical density on ELISA and the cumulative inflammatory burden of atherosclerosis on 18F-FDG PET/CT could be shown. These findings provide further evidence for an autoimmune component in atherosclerosis and suggest MAZ-Abs as a potential biomarker for atherosclerotic disease.

Evaluation of RAG1 mutations in an adult with combined immunodeficiency and progressive multifocal leukoencephalopathy.

Here we describe novel mutations in recombination activation gene 1 (RAG1) in a compound heterozygous male patient with combined T and B cell immunodeficiency (CID). Clinical manifestations besides antibody deficiency included airway infections, granulomatosis and autoimmune features. He died at the age of 37 due to PML caused by JC virus infection. By targeted next-generation sequencing we detected post mortem in this patient three mutations in RAG1. One allele harbored two novel mutations (c.1123C>G, p.H375D and c.1430delC, p.F478Sfs*14), namely a missense variant and a frameshift deletion, of which the latter leads to a truncated RAG1 protein. The other allele revealed a previously described missense mutation (c.1420C>T, p.R474C, rs199474678). Functional analysis of the p.R474C variant in an in vitro V(D)J recombination assay exhibited reduced recombination activity compared to a wild-type control. Our findings suggest that mutations in RAG1, specifically the p.R474C variant, can be associated with relatively mild clinical symptoms or delayed occurrence of T cell and B cell deficiencies but may predispose to PML.

Gain-of-function STAT1 mutations are associated with intracranial aneurysms.

Chronic mucocutaneous candidiasis, characterized by persistent or recurrent fungal infections, represents the clinical hallmark in gain-of-function (GOF) signal transducer and activator of transcription 1 (STAT1) mutation carriers. Several cases of intracranial aneurysms have been reported in patients with GOF STAT1 mutation but the paucity of reported cases likely suggested this association still as serendipity. In order to endorse this association, we link the development of intracranial aneurysms with STAT1 GOF mutation by presenting the two different cases of a patient and her mother, and demonstrate upregulated phosphorylated STAT4 and IL-12 receptor ß1 upon stimulation in patient's blood cells. We also detected increased transforming growth factor (TGF)-ß type 2 receptor expression, particularly in CD14+ cells, and a slightly higher phosphorylation rate of SMAD3. In addition, the mother of the patient developed disseminated bacille Calmette-Guérin disease after vaccination, speculating that GOF STAT1 mutations may confer a predisposition to weakly virulent mycobacteria.

Essential mixed cryoglobulinemia type III with leukocytoclastic vasculitis: remission by rituximab.

We report about a 39-year-old female patient with severe essential mixed cryoglobulinemia of type III with leukocytoclastic vasculitis. The patient was admitted into our hospital with mesenteric lymphangiitis, which caused enteral perforation, sepsis, and pneumonia. Cryoglobulins, cryocrit, Ig-titers, and biopsy were positive for mixed cryoglobulinemia type III. We detected no signs of hepatitis C, B, or any other infectious disease. At first, disease activity could be kept under control with high doses of glucocorticoids and multiple cyclophosphamide pulses. However, after therapy with three pulses of rituximab, steroids were stopped, and the patient has not presented any symptoms for 2 years. Therefore, we suggest that rituximab affected her disease rapidly and effectively. In conclusion, rituximab is an alternative therapy for mixed cryoglobulinemia of type III with leukocytoclastic vasculitis.

Engineering of adipose tissue by injection of human preadipocytes in fibrin.

BACKGROUND: Despite efforts of plastic surgeons in recent years to discover new alternatives, the techniques currently used for restoration of soft tissue defects still have disadvantages. The gold standard for soft tissue reconstruction remains autologous pedicled/free tissue transfer. This technique often results in high rates of operative morbidity and donor site deformity. Results obtained by autologous fat tissue transfer usually are disappointing because of a high graft resorption rate with unpredictable outcomes. Different tissue engineering approaches have been used in the past to generate adipose tissue. However, long-term results in terms of volume persistence have been disappointing. METHODS: In this study, different concentrations of undifferentiated human preadipocytes in fibrin were injected into athymic nude mice (n = 8). Mice that had fibrin injection without cells served as control subjects (n = 8). The specimens were explanted after 1, 2, 6, and 9 months, with subsequent qualitative and quantitative analysis of adipose tissue formation by histologic and image analysis. RESULTS: Within the first 4 weeks after initial volume reduction of the implants, the volume and shape of the implants with preadipocytes remained stable. The implants without cells were completely resorbed within 3 weeks. Histologic analysis demonstrated generation of stable adipose tissue with no signs of an inflammatory response or evidence of tissue necrosis in the implants containing preadipocytes. The best results were obtained after implantation of 30 million preadipocytes. Adipose tissue formation was not observed in the control group. CONCLUSIONS: The findings demonstrate that long-term stable adipose tissue can be engineered in vivo by simple injection of human preadipocytes using fibrin as a carrier material. After further investigation, this approach may represent an alternative to the techniques currently used for soft tissue restoration.