Fine specificity of antibodies against AQP4: Epitope mapping reveals intracellular epitopes

The autoantibody to aquaporin-4 (AQP4) is a marker and a pathogenetic factor in Neuromyelitis Optica (NMO) (Devic’s syndrome). Our aim was to identify B-cell antigenic linear epitopes of the AQP4 protein and investigate similarities with other molecules. To this end, we screened sera from 21 patients positive for anti-AQP4 antibodies (study group), from 23 SLE and 23 pSS patients without neurologic involvement (disease controls) and from 28 healthy individuals (normal controls). Eleven peptides, spanning the entire intracellular and extracellular domains of the AQP4 molecule, were synthesized, and all sera were screened for anti-peptide antibodies by ELISA. Specificity was evaluated by homologous inhibition assays. NMO positive sera exhibited reactivity against 3 different peptides spanning the sequences aa1e22 (AQPpep1) (42.9% of patients), aa88e113 (AQPpep4) (33%) and aa252e275 (AQPpep8) (23.8%). All epitopes were localized in the intracellular domains of AQP4. Homologous inhibition rates were ranging from 71.1% to 84.3%. A 73% sequence homology was observed between AQPpep80 aa257e271, a 15-mer peptide part of the AQPpep8 aa252e275, and the aa219e233 domain of the Tax1-HTLV-1 binding protein (TAX1BP1), a host protein associated with replication of the Human T-Lymphotropic Virus 1 (HTLV-1). Antibodies against the AQP4 and the TAX1BP1 15-mer peptides were detected in 26.3% (N ¼ 5) and 31.6% (N ¼ 6) of NMO positive sera (rs ¼ 0.81, P < 0.0001). Healthy controls did not react with these peptides, while homologous and cross-inhibition assays confirmed binding specificity. This first epitope mapping for AQP4 reveals that a significant proportion of anti-AQP4 antibodies target linear epitopes localized in the intracellular domains of the channel. One of the epitopes displays high similarity with a portion of TAX1BP1 protein

Reactivity to AQP4 epitopes in relapsing–remitting multiple sclerosis

Autoantibodies against the water channel AQP4, expressed predominately in central nervous system astrocytes, are markers and pathogenic factors in Devic's disease. In this study we examined whether Multiple Sclerosis (MS) patients recognize antigenic epitopes on AQP4 that may define distinct disease subsets. We screened sera from 45 patients with relapsing–remitting MS (RRMS) and 13 patients with primary progressive MS (PMS). 23 Neuromyelitis Optica (NMO) patients previously characterized were used as assay positive/negative controls. Sera from 23 patients with Systemic Lupus Erythematosus, 23 with primary Sjogren syndrome without neurological involvement and from 28 healthy individuals were also used as controls. NMO-positive sera exhibited reactivity against the intracellular epitope AQPaa252-275, confirming previous observations. All RRMS sera tested negative for anti-AQP4 antibodies using a cell-based assay, but surprisingly, 13% of them reacted with the epitope AQPaa252-275. PMS, healthy and disease controls showed no specific reactivity. Whether these antibodies define distinct MS subsets and have a pathogenic potential pointing to convergent pathogenetic mechanism with NMO, or are simply markers of astrocytic damage, remains to be determined

Incidence and prevalence of major central nervous system involvement in Systemic Lupus Erythematosus: A 3-year prospective study of 370 patients

Background: The incidence and prevalence of CNS involvement in SLE remains unclear owing to conflicting results in the published studies. The aim of the study was to evaluate the incidence and prevalence of major definite CNS events in SLE patients. Methods: 370 SLE patients with no previous history of CNS involvement were prospectively evaluated in a tertiary hospital referral center for 3 years. Major CNS manifestations were codified according to ACR definitions, including chorea, aseptic meningitis, psychosis, seizures, myelopathy, demyelinating syndrome, acute confusional state and strokes. Minor CNS events were excluded. ECLAM and SLEDAI-SELENA Modification scores were used to evaluate disease activity and SLICC/ACR Damage Index was used to assess accumulated damage. Results: 16/370 (4.3%) patients presented with a total of 23 major CNS events. These included seizures (35%), strokes (26%), myelopathy (22%), optic neuritis (8.7%), aseptic meningitis (4.3%) and acute psychosis (4.3%). Incidence was 7.8/100 person years. Among hospitalizations for SLE, 13% were due to CNS manifestations. Epileptic seizures were associated with high disease activity, while myelopathy correlated with lower disease activity and NMO-IgG antibodies (P#0.05). Stroke incidence correlated with APS coexistence (P = 0.06). Overall, CNS involvement correlated with high ECLAM and SLEDAI scores (P,0.001). Conclusions: Clinically severe CNS involvement is rare in SLE patients, accounting for 7.8/100 person years. CNS involvement correlates with high disease activity and coexistence of specific features that define the respective CNS syndromes

Long-term stability of transparent n/p ZnO homojunctions grown by rf-sputtering at room-temperature

ZnO-based n/p homojunctions were fabricated by sputtering from a single zinc nitride target at room temperature on metal or ITO-coated glass and Si substrates. A multi-target rf-sputtering system was used for the growth of all oxide films as multilayers in a single growth run without breaking the vacuum in the growth chamber. The nitrogen-containing films (less than 1.5 at.% of nitrogen) were n-type ZnO when deposited in oxygen-deficient Ar plasma (10% O2) and p-type ZnO when deposited in oxygen-rich Ar plasma (50% O2). The all-oxide homojunction ITO/n-ZnO/p-ZnO/ITO/glass was fabricated in a single deposition run and exhibited visible transparency in the range of 75–85%. The n/p ZnO homojunctions, having metallic contacts, formed on conventionally processed substrates showed a fairly unstable behavior concerning the current-voltage characteristics. However, the same homojunctions formed on Si3N4-patterned substrates and stored in atmosphere for a period of five months were stable exhibiting a turn-on voltage of around 1.5 V. The realization of a room temperature sputtered transparent and stable ZnO homojunction paves the way to the realization of all-oxide transparent optoelectronic devices

Disease interception with interleukin-17 inhibition in high-risk psoriasis patients with subclinical joint inflammation—data from the prospective IVEPSA study

Background A specific subset of psoriasis patients is characterized by subclinical inflammatory changes. These patients frequently present with arthralgia and have a higher risk to develop psoriatic arthritis (PsA). We hypothesized that IL-17A inhibition in this subset of patients can intercept the link between skin and joint disease and resolves pain and inflammatory changes. Methods Psoriasis, but no PsA, patients were included in the open prospective exploratory Interception in very early PsA (IVEPSA) study. Patients had to have nail or scalp involvement or a high psoriasis area severity index (PASI) (> 6) as well as inflammatory or erosive changes in MRI or CT. Patients received treatment with the anti-interleukin (IL)-17A antibody secukinumab over 24 weeks. Clinical assessments of skin and joint disease were done at baseline and after 12 and 24 weeks, MRI and CT at baseline and after 24 weeks. Results Twenty patients were included, 85% of them reporting arthralgia and 40% had tender joints at the examination. Eighty-three percent had at least one inflammatory lesion in the MRI, most of them synovitis/enthesitis. Skin disease (PASI: p < 0.002; BSA: p < 0.003) and arthralgia (VAS pain: p < 0.003) significantly improved after 24 weeks. Total PsAMRIS (p = 0.005) and synovitis subscore (p = 0.008) also significantly improved. Erosions and enthesiophytes did not progress, while bone mass in the distal radius significantly (p = 0.020) increased after 24 weeks. Conclusions These data suggest that very early disease interception in PsA is possible leading to a comprehensive decline in skin symptoms, pain, and subclinical inflammation. IVEPSA therefore provides rationale for future early interventions with the concept to prevent the onset of PsA in high-risk individuals. Trial registration Trial registry name PSARTROS; trial registry number: NCT02483234; June 26, 2015

Effect of disease-modifying anti-rheumatic drugs on bone structure and strength in psoriatic arthritis patients

Objectives To address whether the use of methotrexate (MTX) and biological disease-modifying anti-rheumatic drugs (bDMARDs) impacts bone structure and biomechanical properties in patients with psoriatic arthritis (PsA). Methods This is a cross-sectional study in PsA patients receiving no DMARDs, MTX, or bDMARDs. Volumetric bone mineral densities (vBMDs), microstructural parameters, and biomechanical properties (stiffness/failure load) were determined by high-resolution peripheral quantitative CT and micro-finite element analysis in the respective groups. Bone parameters were compared between PsA patients with no DMARDs and those receiving any DMARDs, MTX, or bDMARDs, respectively. Results One hundred sixty-five PsA patients were analyzed, 79 received no DMARDs, 86 received DMARDs, of them 52 bDMARDs (TNF, IL-17- or IL-12/23 inhibitors) and 34 MTX. Groups were balanced for age, sex, comorbidities, functional index, and bone-active therapy, while disease duration was longest in the bDMARD group (7.8 ± 7.4 years), followed by the MTX group (4.6 ± 7.4) and the no-DMARD group (2.9 ± 5.2). No difference in bone parameters was found between the no-DMARD group and the MTX group. In contrast, the bDMARD group revealed significantly higher total (p = 0.001) and trabecular vBMD (p = 0.005) as well as failure load (p = 0.012) and stiffness (p = 0.012). In regression models, age and bDMARDs influenced total vBMD, while age, sex, and bDMARDs influenced failure load and stiffness. Conclusion Despite longer disease duration, bDMARD-treated PsA patients benefit from higher bone mass and better bone strength than PsA patients receiving MTX or no DMARDs. These data support the concept of better control of PsA-related bone disease by bDMARDs

Increased T-cell immunity against aquaporin-4 and proteolipid protein in neuromyelitis optica.

In neuromyelitis optica (NMO), B-cell autoimmunity to aquaporin-4 (AQP4) has been shown to be essential. However, the role of T cells remains ambiguous. Here, we first showed an increase in CD69+ activated T cells in PBMCs during NMO relapses. Next, T-cell responses to AQP4 and myelin peptides were studied in 12 NM0 patients, 10 multiple sclerosis (MS) patients and 10 healthy subjects (HS). Four hours after adding 1 of 28 overlapping AQP4 peptides, a mixture of AQP4 peptides (AQP4-M) or one of six distinct myelin peptides to 2-day cultured PBMC, CD69 expression on CD4+ T cells was examined. Data were analyzed by paired t-test, frequency of samples with 3-fold increase of CD69 on CD4+ cells (fSI3) and mean stimulation index (mSI). The T-cell response to AQP4-M was significantly increased in NMO (fSI3 = 10/12, mSI = 5.50), with AQP4 (11-30) and AQP4 (91-110) representing the two major epitopes (AQP4 (11-30), fSI3 = 11/12, mSI = 16.0 and AQP4 (91-110), fSI3 = 11/12, mSI = 13.0). Significant but less extensive responses to these two epitopes were also observed in MS and HS. Significant reactivities against AQP4 (21-40), AQP4 (61-80), AQP4 (101-120), AQP4 (171-190) and AQP4 (211-230) were exclusively found in NMO. In addition, responses to AQP4 (81-100) were higher and more frequently detected in NMO, without reaching statistical significance. Interestingly, among the six myelin peptides studied, proteolipid protein (95-116) induced a significant T-cell response in NMO (fSI3 = 7/12, mSI = 4.60). Our study suggests that cellular as well as humoral responses to AQP4 are necessary for NMO development and that the immune response to myelin protein may contribute to disease pathogenesis

Critical role of interleukin (IL)-17 in inflammatory and immune disorders: An updated review of the evidence focusing in controversies

Interleukin 17 (IL-17) is a proinflammatory cytokine that has been the focus of intensive research because of its crucial role in the pathogenesis of different diseases across many medical specialties. In this context, the present review in which a panel of 13 experts in immunology, dermatology, rheumatology, neurology, hematology, infectious diseases, hepatology, cardiology, ophthalmology and oncology have been involved, puts in common the mechanisms through which IL-17 is considered a molecular target for the development of novel biological therapies in these different fields. A comprehensive review of the literature and analysis of the most outstanding evidence have provided the basis for discussing the most relevant data related to IL-17A blocking agents for the treatment of different disorders, such as psoriasis, psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, cardiovascular disorders, non alcoholic fatty liver disease, multiple sclerosis, inflammatory bowel disease, uveitis, hematological and solid cancer. Current controversies are presented giving an opening line for future research.This work was supported by Novartis Pharmaceuticals Spain

Characterization of the binding pattern of human aquaporin-4 autoantibodies in patients with neuromyelitis optica spectrum disorders

BACKGROUND: The discovery of a highly specific antibody against the aquaporin-4 (AQP4) water channel (AQP4-IgG) unified the spectrum of neuromyelitis optica spectrum disorders (NMOSD), which are considered to be antibody-mediated autoimmune diseases. The AQP4 water channel is located on astrocytic end-feet processes and consists of six transmembrane helical domains forming three extracellular loops A, C, and E in which defined amino acids were already proven to be critical for AQP4-IgG binding. However, the clinical relevance of these findings is unclear. Therefore, we have characterized the epitope specificity of AQP4-IgG-positive NMOSD patients. METHODS: We established a cell-based flow cytometry assay for the quantitative detection of AQP4-IgG-positive serum samples. Human embryonic kidney (HEK) cells were transiently transfected with an EmGFP-tagged AQP4-M23, AQP4-M1, or six AQP4-M23 extracellular loop mutants including two mutations in loop A (serial AA substitution, insertion of a myc-tag), two in loop C (N153Q, insertion of a myc-tag), and two in loop E (H230G, insertion of a myc-tag). Fourty-seven baseline and 49 follow-up serum samples and six paired cerebrospinal fluid (CSF) baseline samples of 47 AQP4-IgG-positive Austrian NMOSD patients were then tested for their binding capability to AQP4-M1 and AQP4-M23 isoforms and these six extracellular loop mutants. RESULTS: Overall, we could identify two broad patterns of antibody recognition based on differential sensitivity to mutations in extracellular loop A. Pattern A was characterized by reduced binding to the two mutations in loop A, whereas pattern B had only partial or no reduced binding to these mutations. These two patterns were not associated with significant differences in demographic and clinical parameters or serum titers in this retrospective study. Interestingly, we found a change of AQP4-IgG epitope recognition pattern in seven of 20 NMOSD patients with available follow-up samples. Moreover, we found different binding patterns in five of six paired CSF versus serum samples, with a predominance of pattern A in CSF. CONCLUSIONS: Our study demonstrates that AQP4-IgG in sera of NMOSD patients show distinct patterns of antibody recognition. The clinical and diagnostic relevance of these findings have to be addressed in prospective studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-016-0642-3) contains supplementary material, which is available to authorized users