Identification of DAGLA as an autoantibody target in cerebellar ataxia.

BACKGROUND: We aimed to investigate the clinical, imaging and fluid biomarker characteristics in patients with antidiacylglycerol lipase alpha (DAGLA)-autoantibody-associated cerebellitis. METHODS: Serum and cerebrospinal fliud (CSF) samples from four index patients were subjected to comprehensive autoantibody screening by indirect immunofluorescence assay (IIFA). Immunoprecipitation, mass spectrometry and recombinant protein assays were used to identify the autoantigen. Sera from 101 patients with various neurological symptoms and a similar tissue staining pattern as the index patient samples, and 102 healthy donors were analysed in recombinant cell-based IIFA (RC-IIFA) with the identified protein. Epitope characterisation of all positive samples was performed via ELISA, immunoblot, immunoprecipitation and RC-IIFA using different DAGLA fragments. RESULTS: All index patients were relatively young (age: 18-34) and suffered from pronounced gait ataxia, dysarthria and visual impairments. Paraclinical hallmarks in early-stage disease were inflammatory CSF changes and cerebellar cortex hyperintensity in MRI. Severe cerebellar atrophy developed in three of four patients within 6 months. All patient samples showed the same unclassified IgG reactivity with the cerebellar molecular layer. DAGLA was identified as the target antigen and confirmed by competitive inhibition experiments and DAGLA-specific RC-IIFA. In RC-IIFA, serum reactivity against DAGLA was also found in 17/101 disease controls, including patients with different clinical phenotypes than the one of the index patients, and in 1/102 healthy donors. Epitope characterisation revealed that 17/18 anti-DAGLA-positive control sera reacted with a C-terminal intracellular DAGLA 583-1042 fragment, while the CSF samples of the index patients targeted a conformational epitope between amino acid 1 and 157. CONCLUSIONS: We propose that anti-DAGLA autoantibodies detected in CSF, with a characteristic tissue IIFA pattern, represent novel biomarkers for rapidly progressive cerebellitis.

Laminin ß4 is a constituent of the cutaneous basement membrane zone and additional autoantigen of anti-p200 pemphigoid.

BACKGROUND: Anti-p200 pemphigoid is a subepidermal autoimmune blistering disease (AIBD) characterized by autoantibodies against a 200 kDa protein. Laminin γ1 has been described as target antigen in 70% to 90% of patients. No diagnostic assay is widely available for anti-p200 pemphigoid, which might be due to the unclear pathogenic relevance of anti-laminin γ1 autoantibodies. OBJECTIVE: To identify a target antigen with higher clinical and diagnostic relevance. METHODS: Immunoprecipitation, mass spectrometry, and immunoblotting were employed for analysis of skin extracts and sera of patients with anti-p200 pemphigoid (n = 60), other AIBD (n = 33), and healthy blood donors (n = 29). To localize the new antigen in skin, cultured keratinocytes and fibroblasts, quantitative real-time polymerase chain reaction and immunofluorescence microscopy were performed. RESULTS: Laminin ß4 was identified as target antigen of anti-p200 pemphigoid in all analyzed patients. It was located at the level of the basement membrane zone of the skin with predominant expression in keratinocytes. LIMITATIONS: A higher number of sera needs to be tested to verify that laminin ß4 is the diagnostically relevant antigen of anti-p200 pemphigoid. CONCLUSION: The identification of laminin ß4 as an additional target antigen in anti-p200 pemphigoid will allow its differentiation from other AIBD and as such, improve the management of these rare disorders.

A Spectrum of Neural Autoantigens, Newly Identified by Histo-Immunoprecipitation, Mass Spectrometry, and Recombinant Cell-Based Indirect Immunofluorescence.

BACKGROUND: A plurality of neurological syndromes is associated with autoantibodies against neural antigens relevant for diagnosis and therapy. Identification of these antigens is crucial to understand the pathogenesis and to develop specific immunoassays. Using an indirect immunofluorescence assay (IFA)-based approach and applying different immunoprecipitation (IP), chromatographic and mass spectrometric protocols was possible to isolate and identify a spectrum of autoantigens from brain tissue. METHODS: Sera and CSF of 320 patients suspected of suffering from an autoimmune neurological syndrome were comprehensively investigated for the presence of anti-neural IgG autoantibodies by IFA using mosaics of biochips with brain tissue cryosections and established cell-based recombinant antigen substrates as well as immunoblots. Samples containing unknown brain tissue-specific autoantibodies were subjected to IP with cryosections of cerebellum and hippocampus (rat, pig, and monkey) immobilized to glass slides or with lysates produced from homogenized tissue, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tryptic digestion, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Identifications were confirmed by IFA with recombinant HEK293 cells and by neutralizing the patients' autoantibodies with the respective recombinantly expressed antigens in the tissue-based immunofluorescence test. RESULTS: Most samples used in this study produced speckled, granular, or homogenous stainings of the hippocampal and cerebellar molecular and/or granular layers. Others exclusively stained the Purkinje cells. Up to now, more than 20 different autoantigens could be identified by this approach, among them ATP1A3, CPT1C, Flotillin1/2, ITPR1, NBCe1, NCDN, RGS8, ROCK2, and Syntaxin-1B as novel autoantigens. DISCUSSION: The presented antigen identification strategy offers an opportunity for identifying up to now unknown neural autoantigens. Recombinant cell substrates containing the newly identified antigens can be used in serology and the clinical relevance of the autoantibodies can be rapidly evaluated in cohort studies.

Reliable Serological Testing for the Diagnosis of Emerging Infectious Diseases.

Climate change, increased urbanization and international travel have facilitated the spread of mosquito vectors and the viral species they carry. Zika virus (ZIKV) is currently spreading in the Americas, while dengue virus (DENV) and chikungunya virus (CHIKV) have already become firmly established in most tropical and also many non-tropical regions. ZIKV, DENV and CHIKV overlap in their endemic areas and cause similar clinical symptoms, especially in the initial stages of infection. Infections with each of these viruses can lead to severe complications, and co-infections have been reported. Therefore, laboratory analyses play an important role in differential diagnostics. A timely and accurate diagnosis is crucial for patient management, prevention of unnecessary therapies, rapid adoption of vector control measures, and collection of epidemiological data.There are two pillars to diagnosis: direct pathogen detection and the determination of specific antibodies. Serological tests provide a longer diagnostic window than direct methods, and are suitable for diagnosing acute and past infections, for disease surveillance and for vaccination monitoring. ELISA and indirect immunofluorescence test (IIFT) systems based on optimized antigens enable sensitive and specific detection of antibodies against ZIKV, DENV and CHIKV in patient serum or plasma. In recent years, Euroimmun (Lübeck, Germany) has developed numerous test systems for the serological diagnosis of (re-)emerging diseases, including a very sensitive and specific anti-ZIKV ELISA.

Rho-associated protein kinase 2 (ROCK2): a new target of autoimmunity in paraneoplastic encephalitis.

Onconeural antibodies are associated with cancer and paraneoplastic encephalitis. While their pathogenic role is still largely unknown, their high diagnostic value is undisputed. In this study we describe the discovery of a novel target of autoimmunity in an index case of paraneoplastic encephalitis associated with urogenital cancer.A 75-year-old man with a history of invasive bladder carcinoma 6 years ago with multiple recurrences and a newly discovered renal cell carcinoma presented with seizures and progressive cognitive decline followed by super-refractory status epilepticus. Clinical and ancillary findings including brain biopsy suggested paraneoplastic encephalitis. Immunohistochemistry of the brain biopsy was used to characterize the inflammatory response. Indirect immunofluorescence assay (IFA) was used for autoantibody screening. The autoantigen was identified by histo-immunoprecipitation and mass spectrometry and was validated by expressing the recombinant antigen in HEK293 cells and neutralization tests. Sera from 125 control patients were screened using IFA to test for the novel autoantibodies.IFA analysis of serum revealed a novel autoantibody against brain tissue. An intracellular enzyme, Rho-associated protein kinase 2 (ROCK2), was identified as target-antigen. ROCK2 was expressed in affected brain tissue and archival bladder tumor samples of this patient. Brain histopathology revealed appositions of cytotoxic CD8+ T cells on ROCK2-positive neurons. ROCK2 antibodies were not found in the sera of 20 patients with bladder cancer and 17 with renal cancer, both without neurological symptoms, 49 healthy controls, and 39 patients with other antineuronal autoantibodies. In conclusion, novel onconeural antibodies targeting ROCK2 are associated with paraneoplastic encephalitis and should be screened for when paraneoplastic neurological syndromes, especially in patients with urogenital cancers, occur.

Enhanced Protective Immunogenicity of Homodimeric Borrelia burgdorferi Outer Surface Protein C.

Lyme borreliosis is caused by tick-transmitted spirochetes of the Borrelia burgdorferi sensu lato group and is the most common vector-borne disease in the United States and Europe. Outer surface protein C (OspC) is a 23-kDa outer surface lipoprotein expressed during spirochete transmission from the tick to the vertebrate host. In a previous study, we found that immunization with a recombinant disulfide-bridged dimeric form of OspC (D-OspC) stimulates increased antibody responses relative to immunization with commonly employed monomeric OspC. Here, we report that mice immunized with dimeric OspC proteins also exhibited enhanced protection against infection with the cognate B. burgdorferi strain. Mice were protected by four immunizations containing as little as 100 ng of dimeric OspC, suggesting that this form of the protein can induce protective immunity within a dose range reasonable for a human or veterinary vaccine. In contrast, monomeric OspC was only partially protective at much higher doses. IgG subclass analysis revealed that D-OspC-immunized animals mainly possessed anti-OspC-IgG1. In contrast, infected animals develop anti-OspC restricted to the IgG3 isotype. A subset of antibodies generated by dimeric OspC immunization did not recognize the monomeric variant, indicating that unique epitopes exist on the dimeric form. Moreover, monoclonal antibodies that recognized only dimeric OspC protected mice from B. burgdorferi challenge, whereas another monoclonal that recognized both immunogens was not protective. These studies suggest that this dimeric OspC presents distinctive epitopes that generate antibodies protective against B. burgdorferi infection and could be a useful vaccine component.

Serodiagnosis of Zika virus (ZIKV) infections by a novel NS1-based ELISA devoid of cross-reactivity with dengue virus antibodies: a multicohort study of assay performance, 2015 to 2016.

Serological diagnosis of Zika virus (ZIKV) infections is challenging due to high cross-reactivity between flaviviruses. We evaluated the diagnostic performance of a novel anti-ZIKV ELISA based on recombinant ZIKV non-structural protein 1 (NS1). Assay sensitivity was examined using sera from 27 patients with reverse transcription (RT)-PCR-confirmed and 85 with suspected ZIKV infection. Specificity was analysed using sera from 1,015 healthy individuals. Samples from 252 patients with dengue virus (n = 93), West Nile virus (n = 34), Japanese encephalitis virus (n = 25), chikungunya virus (n = 19) or Plasmodium spp. (n = 69) infections and from 12 yellow fever-vaccinated individuals were also examined. In confirmed ZIKV specimens collected ≥ 6 days after symptom onset, ELISA sensitivity was 58.8% (95% confidence interval (CI): 36.0-78.4) for IgM, 88.2% (95% CI: 64.4-98.0) for IgG, and 100% (95% CI: 78.4-100) for IgM/IgG, at 99.8% (95% CI: 99.2-100) specificity. Cross-reactivity with high-level dengue virus antibodies was not detected. Among patients with potentially cross-reactive antibodies anti-ZIKV positive rates were 0.8% (95% CI: 0-3.0) and 0.4% (95% CI: 0-2.4) for IgM and IgG, respectively. Providing high specificity and low cross-reactivity, the NS1-based ELISA has the potential to aid in counselling patients, pregnant women and travellers after returning from ZIKV-endemic areas.

Development of a recombinant cell-based indirect immunofluorescence assay for the determination of autoantibodies against soluble liver antigen in autoimmune hepatitis.

Autoantibodies against soluble liver antigen (SLA) are specific markers for autoimmune hepatitis (AIH) type 1. In contrast to the determination of other AIH-associated autoantibodies by indirect immunofluorescence assay (IFA), detection of anti-SLA relied up to now on ELISA or immunoblot based on bacterially expressed recombinant protein. In order to develop a complementary IFA substrate, SLA isoform 1 was recombinantly produced in the human cell line HEK293 and controlled by a rabbit hyperimmune serum against SLA. The recombinant cells were used in IFA (RC-IFA) to analyze sera from 20 AIH patients with anti-SLA positivity predetermined by ELISA together with 80 controls (20 anti-SLA negative AIH, 15 primary biliary cirrhosis, 15 HCV, and 30 healthy blood donors). Using RC-IFA, anti-SLA was detected in all ELISA positive AIH sera but in none of the controls. Furthermore, a cytosolic fraction of HEK293 containing SLA was able to neutralize the autoantibodies in all positive sera in a dose-dependent manner. HEK293 cells expressing SLA are a valid substrate for the serodiagnosis of AIH relevant autoantibodies by IFA. In concert with cryosections of primate liver, rat kidney, rat liver, rat stomach, and HEp-2 cells, they enable the parallel determination of all autoantibodies associated with autoimmune liver diseases.

Development of a recombinant cell-based indirect immunofluorescence assay (RC-IFA) for the determination of autoantibodies against "rings and rods"-associated inosine-5'-monophosphate dehydrogenase 2 in viral hepatitis C.

BACKGROUND AND AIMS: Autoantibodies against so-called "rings and rods" structures, as determined by indirect immunofluorescence assay (IFA) using the human cell line HEp-2, have been described in chronic hepatitis C virus (HCV) infected patients treated with interferon/ribavirin. Recently, cytidine triphosphate synthase (CTPS) and inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), the enzyme inhibited by ribavirin, were proposed as the target antigens. We wanted to confirm the identification and setup a robust system for autoantibody testing in routine laboratories. METHODS: CTPS and IMPDH2 were individually expressed in HEK293 cells and the recombinant cells were used in IFA (RC-IFA) to analyze sera from 33 anti-"rings and rods" antibody positive individuals with unknown diagnosis, 50 patients with chronic HCV infection, 100 with autoimmune hepatitis, 50 with primary biliary cirrhosis and 50 healthy blood donors. RESULTS: We found that all sera with anti-"rings and rods" reacted with recombinant IMPDH2 but none with CTPS. In western blot or ELISA, anti-IMPDH2 positive sera reacted only weakly, if at all, with Escherichia coli derived recombinant IMPDH2 indicating that the autoantibody reaction probably depends on the 3-dimensional conformation of the antigen. A rabbit hyperimmune serum raised against bacterially expressed IMPDH2 produced the ring/rods pattern in IFA using HEp-2. CONCLUSIONS: We conclude, that IMPDH2 is indeed the main target of anti-"rings and rods" while CTPS is an unlikely target. Moreover, the novel RC-IFA test system allows a standardized semi-quantitative determination of anti-IMPDH2 basing on a defined recombinant antigen.