Identification of a unique anti-Ro60 subset with restricted serological and molecular profiles.

Anti-Ro60 is one of the most common and clinically important serum autoantibodies that has a number of diagnostic and predictive capabilities. Most diagnostic laboratories report this simply as a qualitative positive/negative result. The objective of this study was to examine the clinical and serological relevance of a novel subset of anti-Ro60 in patients who display low levels of anti-Ro60 (anti-Ro60low ). We retrospectively identified anti-Ro60 sera during a 12-month period at a major immunopathology diagnostic laboratory in Australia. These all were anti-Ro60-precipitin-positive on the diagnostic gold standard counter-immuno-electrophoresis (CIEP). Lineblot immunoassay was used to stratify patients into either anti-Ro60low or anti-Ro60high subsets. We compared the medical and laboratory parameters associated with each group. Enzyme-linked immunosorbent assay (ELISA) and mass spectrometry techniques were used to analyse the serological and molecular basis behind the two subsets. Anti-Ro60low patients displayed less serological activity than anti-Ro60high patients with less intermolecular spreading, hypergammaglobulinaemia and less tendency to undergo anti-Ro60 isotype-switching than anti-Ro60high patients. Mass spectrometric typing of the anti-Ro60low subset showed restricted variable heavy chain subfamily usage and amino acid point mutations. This subset also displayed clinical relevance, being present in a number of patients with systemic autoimmune rheumatic diseases (SARD). We identify a novel anti-Ro60low patient subset that is distinct from anti-Ro60high patients serologically and molecularly. It is not clear whether they arise from common or separate origins; however, they probably have different developmental pathways to account for the stark difference in immunological maturity. We hence demonstrate significance to anti-Ro60low and justify accurate detection in the diagnostic laboratory.

Diagnostic Utility of Separate Anti-Ro60 and Anti-Ro52/TRIM21 Antibody Detection in Autoimmune Diseases.

Anti-SS-A antibodies are often sought for in autoimmune diseases diagnosis. Two different target proteins have actually been identified: Ro52 and Ro60. Clinical and immunological associations seem different depending on anti-Ro52 or anti-Ro60 antibodies presence. However, due to a heterogeneous presentation in the literature, some immunology laboratories in France have stopped providing anti-Ro52 antibody findings. We report here a new hospital study designed to determine the diagnostic utility of the separate detection of anti-Ro52 and anti-Ro60 antibodies. We conducted a retrospective, observational study, including every adult patient with positive antinuclear antibodies (ANA) tested in our immunology laboratory, and associated with anti-Ro52 and/or anti-Ro60 antibodies, between 2011 and 2014. Out of 13032 sera tested for ANA, 399 adults had antibodies to Ro52 and/or Ro60; 81.7% were female, with a mean age of 54.5 ± 17.0 years. Anti-Ro52 antibodies were found in 75.7% of the patients and anti-Ro60 antibodies in 56.9%. Among them, 43.1% were classified in the Ro52 + Ro60- group, 32.6% in the Ro52 + Ro60 + group and 24.3% in the Ro52-Ro60+ group. In the Ro52-Ro60+ group, systemic lupus was the most frequent diagnosis (48.5%), with a possible association with antiphospholipid antibodies (anti-cardiolipin antibodies: OR 2.5 (CI95 [1.0-5.0], p = 0.05) and lupus anticoagulant {OR 3.6 (CI95 [1.10-10.0] p = 0.02)}. In the Ro52+Ro60+, primary Sjögren Syndrome was the most likely (OR 4.2 95% CI [2.1-8.3] p < 10-4), especially in patients Ro52+Ro60+La+. Patients with isolated anti-Ro52 had a wider variety of diseases associated, but among auto-immune diseases they were more prone to inflammatory myositis (OR 10.5 [1.4-81.7], p = 0.02) and inflammatory rheumatism (OR 4.6 [1.6-13.8], p = 0.006) in contrast to systemic lupus (OR 0.2 [0.1-0.3], p < 10-4) or primary Sjögren's syndrome (OR 0.1 [0.06-0.2], p < 10-4). We therefore suggest that, when anti-ENA antibodies are prescribed, it should include separate anti-Ro52 and anti-Ro60 antibodies determination. To go even further, we would like to suggest a change in ENA nomenclature to avoid confusion, abandoning the anti-SS-A label in favor of the anti-Ro52/TRIM21 or anti-Ro60 antibody for a clearer designation.

Parasite specific 7SL-derived small RNA is an effective target for diagnosis of active trypanosomiasis infection.

Human and animal African trypanosomiasis (HAT & AAT, respectively) remain a significant health and economic issue across much of sub-Saharan Africa. Effective control of AAT and potential eradication of HAT requires affordable, sensitive and specific diagnostic tests that can be used in the field. Small RNAs in the blood or serum are attractive disease biomarkers due to their stability, accessibility and available technologies for detection. Using RNAseq, we have identified a trypanosome specific small RNA to be present at high levels in the serum of infected cattle. The small RNA is derived from the non-coding 7SL RNA of the peptide signal recognition particle and is detected in the serum of infected cattle at significantly higher levels than in the parasite, suggesting active processing and secretion. We show effective detection of the small RNA in the serum of infected cattle using a custom RT-qPCR assay. Strikingly, the RNA can be detected before microscopy detection of parasitaemia in the blood, and it can also be detected during remission periods of infection when no parasitaemia is detectable by microscopy. However, RNA levels drop following treatment with trypanocides, demonstrating accurate prediction of active infection. While the small RNA sequence is conserved between different species of trypanosome, nucleotide differences within the sequence allow generation of highly specific assays that can distinguish between infections with Trypanosoma brucei, Trypanosoma congolense and Trypanosoma vivax. Finally, we demonstrate effective detection of the small RNA directly from serum, without the need for pre-processing, with a single step RT-qPCR assay. Our findings identify a species-specific trypanosome small RNA that can be detected at high levels in the serum of cattle with active parasite infections. This provides the basis for the development of a cheap, non-invasive and highly effective diagnostic test for trypanosomiasis.

HULC and 7SL RNA expression levels in patients with Crimean-Congo hemorrhagic fever.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by the Crimean-Congo hemorrhagic fever virus. Long non-coding RNAs (lncRNAs) are generally classified as transcripts longer than 200 nucleotides (nt). The various lncRNAs expressed in infected cells are responsible for regulating the expression of viral and host genes. This is the first study to investigate hepatocellular carcinoma upregulated long non-coding RNA (HULC) and 7SL RNA expression levels in patients with CCHF. Blood samples were taken from 100 individuals (60 patients and 40 controls), and total RNA isolation was performed. Quantitative polymerase chain reaction (qPCR) was performed using the SYBR Green method to determine HULC and 7SL RNA expression levels in the study population. Compared the patient and control groups, HULC was upregulated statistically significantly (P = 0.04) and 7SL RNA was downregulated (P = 0.93) in patients. Also, there was a statistically significant difference between fatal cases and surviving patients for HULC and 7SL RNA (P < 0.01 and P = 0.03, respectively). In addition, HULC expression was increased statistically significantly in fatal cases compared with surviving patients in terms of clinical parameters such as aspartate aminotransferase (P < 0.01), alanine aminotransferase (P < 0.01), international normalized ratio (P = 0.05), prothrombin time (P = 0.01), active partial thromboplastin time (P < 0.01), and lactate dehydrogenase (P < 0.01). These findings highlighted that HULC and 7SL RNA could be important mediators for studying the pathogenesis of CCHF and significant therapeutic targets of the disease.

7SL RNA in vertebrate red blood cells.

We report that 7SL, the RNA component of the signal recognition particle (SRP), is an abundant noncoding RNA (ncRNA) in mature red blood cells (RBCs) of human, mouse, and the frog Xenopus. 7SL RNA in RBCs is not associated with the canonical proteins of the SRP. Instead, it coimmunoprecipitates from a lysate of RBCs with a number of membrane-binding proteins. Human and mouse RBCs also contain a previously undescribed 68 nt RNA, sRN7SL, derived from the "S domain" of 7SL RNA. We discuss the possibility that 7SL RNA is selectively protected from nucleases by association with the RBC membrane. Because 7SL is not associated with the canonical proteins of the SRP, it could represent a nonfunctional remnant of the protein synthetic machinery. Alternatively, it could play a new, as yet undefined role in RBC metabolism.

Blood-Borne RNA Correlates with Disease Activity and IFN-Stimulated Gene Expression in Systemic Lupus Erythematosus.

The loss of tolerance and the presence of circulating autoantibodies directed against nuclear Ags is the hallmark of systemic lupus erythematosus (SLE). Many of these Ags are complexed with short, noncoding RNAs, such as U1 and Y1. The amount of U1 and Y1 RNA complexed with SLE patient Abs and immune complexes was measured in a cross-section of 228 SLE patients to evaluate the role of these RNA molecules within the known biochemical framework of SLE. The study revealed that SLE patients had significantly elevated levels of circulating U1 and/or Y1 RNA compared with healthy volunteers. In addition, the blood-borne RNA molecules were correlated with SLE disease activity and increased expression of IFN-inducible genes. To our knowledge, this study provides the first systematic examination of the role of circulating RNA in a large group of SLE patients and provides an important link with IFN dysregulation.

miRNAs Regulation and Its Role as Biomarkers in Endometriosis.

MicroRNAs (miRNAs) are small non-coding RNAs (18-22 nt) that function as modulators of gene expression. Since their discovery in 1993 in C. elegans, our knowledge about their biogenesis, function, and mechanism of action has increased enormously, especially in recent years, with the development of deep-sequencing technologies. New biogenesis pathways and sources of miRNAs are changing our concept about these molecules. The study of the miRNA contribution to pathological states is a field of great interest in research. Different groups have reported the implication of miRNAs in pathologies such as cancer, diabetes, cardiovascular, and gynecological diseases. It is also well-known that miRNAs are present in biofluids (plasma, serum, urine, semen, and menstrual blood) and have been proposed as ideal candidates as disease biomarkers. The goal of this review is to highlight the current knowledge in the field of miRNAs with a special emphasis to their role in endometriosis and the newest investigations addressing the use of miRNAs as biomarkers for this gynecological disease.

Long-term Ro60 humoral autoimmunity in primary Sjögren's syndrome is maintained by rapid clonal turnover.

Long-term humoral autoimmunity to RNA-protein autoantigens is considered a hallmark of systemic autoimmune diseases. We use high resolution Orbitrap mass spectrometric autoantibody sequencing to track the evolution of a Ro60-specific public clonotypic autoantibody in 4 patients with primary Sjögren's syndrome. This clonotype is specified by a VH3-23/VK3-20 heavy and light chain pairing. Despite apparent stability by conventional immunoassay, analysis of V-region molecular signatures of clonotypes purified from serum samples collected retrospectively over 7years revealed sequential clonal replacement. Prospective longitudinal studies confirmed clonotype loss and replacement at approximately three-monthly intervals. Levels of secreted anti-Ro60 clonotypes fluctuated markedly over time, despite minimal changes in clonal affinity. Our novel findings indicate a relentless turnover of short-lived clonotypic variants, masquerading as long-lived Ro60 humoral autoimmunity.

Salivary anti-Ro60 and anti-Ro52 antibody profiles to diagnose Sjogren's Syndrome.

Simple and non-invasive saliva-based diagnostics may be useful for the identification, understanding, and monitoring of autoimmune and infectious diseases. Previously, Luciferase Immunoprecipitation Systems (LIPS) were used for sensitive detection of patient serum autoantibodies in Sjögren's Syndrome (SjS), a chronic autoimmune disease affecting the salivary and lacrimal glands. Here we explored the ability of LIPS to diagnose SjS based on IgG autoantibodies in patient saliva. From LIPS testing, anti-Ro60 autoantibodies were detected in the saliva of 70% (19/27) of SjS patients with 96% specificity. Positive anti-Ro60 autoantibodies were also found in 70% of the matched serum samples (96% specificity). LIPS detected Ro52 autoantibodies in the saliva and serum of 67% of SjS patients with 100% specificity. Overall, the autoantibody titers in saliva were approximately 4000-fold lower by volume than serum, but still distinguished seropositive patients from controls. These results suggest that LIPS salivary-based testing for SjS autoantibodies is a practical alternative to serum and compatible with point-of-care testing.

A new biodosimetric method: branched DNA-based quantitative detection of B1 DNA in mouse plasma.

A simple and accurate method for measuring the biological effects of radiation is of increasing importance, especially in mass casualty scenarios. We have therefore developed a new biodosimetric technique targeting circulating B1 DNA in mouse plasma by branched DNA signal amplification for rapid quantification of plasma DNA. This technology targets repetitive elements of the B1 retrotransposon in the mouse genome, followed by signal amplification using Panomics Quantigene 2.0 reagents. Evaluation was conducted concerning precision, accuracy and linearity. Plasma samples were collected from mice 0-24 h after 0-10 Gy total body irradiation (TBI). The average inter- and intra-assay coefficients of variance were 8.7% and 12.3%, respectively. The average recovery rate of spiked DNA into plasma was 89.5%. This assay revealed that when BALB/c and NIH Swiss mice were exposed to 6 Gy TBI, plasma B1 DNA levels increased significantly at 3 h post-TBI, peaked at 9 h and gradually returned toward baseline levels in 24 h. A dose-dependent change in plasma DNA was observed at 9 h post-TBI; the dose-response relation was monotonic, exhibiting linearity for BALB/c mice from 3 to 6 Gy (r = 0.993) and NIH Swiss mice from 3 to 7 Gy (r = 0.98). This branched DNA-based assay is reliable, accurate and sensitive in detecting plasma B1 DNA quantitatively. A radiation dose-correlated increase in plasma B1 DNA was demonstrated in BALB/c and NIH Swiss mice in the dose range from 3 to 6 Gy, suggesting that plasma B1 DNA has potential as a biomarker for radiation biological effect.

Rapid serological detection of autoantibodies associated with Sjögren's syndrome.

BACKGROUND: Sjögren's syndrome (SjS) is a relatively common autoimmune disease characterized by oral and ocular dryness. There is an increasing need for simple, sensitive and rapid technologies for the diagnosis of SjS and other autoimmune diseases. Here we investigated whether a quick version of luciferase immunoprecipitation systems (QLIPS) could be used to produce a rapid, specific and quantitative test to detect autoantibodies associated with SjS. METHODS: Using QLIPS, which requires only ten minutes of incubation, a cohort of control and SjS sera were tested for antibodies to three SjS autoantigens (La, Ro60 and Ro52). Sensitivity and specificity of the QLIPS tests were compared with LIPS and existing ELISA data. The QLIPS test for Ro52 was then evaluated with a new validation cohort and its diagnostic performance determined. RESULTS: Using QLIPS, autoantibodies to three SjS antigens, La, Ro60, and Ro52 were detected in 49%, 56% and 70%, respectively, of the SjS patients and none of the controls (100% specificity). With antibody titers in the Ro52-seropositive SjS samples approximately 1,000 times higher than the healthy controls, not only was Ro52 the most informative, but detection of anti-Ro52 antibodies under these non-equilibrium conditions was improved compared to the standard 2 hour LIPS format. Validation of the anti-Ro52 QLIPS test in a new, independent cohort of SjS and control serum samples showed 66% sensitivity and 100% specificity. CONCLUSION: Together these results suggest that the QLIPS format for Ro52 yields both a more rapid and more discriminating test for detecting Ro52 autoantibodies than existing immunoassays and has the potential to be adapted for point-of-care evaluation of patients with SjS and other rheumatologic diseases.

Transient heart block in a neonate associated with previously undiagnosed maternal anti-Ro/SSA and anti-La/SSB antibodies.

This report presents the case of an infant who was born with transient complete heart block. The cardiac rhythm converted to normal sinus rhythm within 12 hours of life. Following the diagnosis in this infant of congenital heart block, both the mother and the infant were tested for autoantibodies. Both were found to be strongly positive for anti-Ro/SSA antibodies. The infant was also weakly positive for the anti-La/SSB antibodies and her mother moderately positive for the same. Congenital heart block associated with these maternal antibodies is well documented in the literature; however, this is the only reported case that documents a transient nature of the complete heart block.

Scleroderma and repeated spontaneous abortions treated with vitamin E--a case report--.

A 33-year-old woman was referred to our hospital due to repeated spontaneous abortions and positive autoantibodies. She had noticed Raynaud's phenomenon 13 years earlier. We diagnosed scleroderma based on the presence of Raynaud's phenomenon, proximal scleroderma, presence of anti-centromere antibodies, and histological findings on skin biopsy. Neither lupus anticoagulant nor anti-cardiolipin-beta2-glycoprotein 1 antibody was detected. We administered tocopherol nicotinate. Five months after the initiation of the treatment, she became pregnant and later delivered a healthy baby.

Zinc ion dependent B-cell epitope, associated with primary Sjogren's syndrome, resides within the putative zinc finger domain of Ro60kD autoantigen: physical and immunologic properties.

The Ro/La ribonucleoprotein (RNP) complex is composed of the proteins Ro60kD, Ro52kD, and La48kD that are in association with one small cytoplasmic RNA (YRNA). Specific protein-RNA and protein-protein interactions are thought to occur through the RNP and zinc-finger secondary structure elements of the Ro60kD protein. The aim of our study was to investigate the antigenic properties of the zinc finger domain of the Ro60KD autoantigen and its contribution to the formation of Ro/La RNP complex. It was found that the peptide VSLVCEKLCNEKLLKKARIHPFHILIA (Zif-1), which corresponds to the natural sequence of the zinc finger domain (301-327), and the peptide C(Acm)NEKLLKKARIC(Acm), analogous to the intermediate loop 310-319 (Zif-3) of the same domain of Ro60KD, are recognized by the majority of anti-Ro/SSA and anti-La/SSB positive sera (82.6% and 77.1%, respectively) in the absence of zinc ions. The same sera failed to react with Zif-1 peptide in the presence of Zn2+. In contrast, the addition of zinc ions was necessary for the binding of Zif-1 to recombinant Ro52KD as shown by direct binding experiments of the recombinant protein with synthetic peptides. Our data suggest the zinc finger domain of Ro60kD contains a B-cell epitope with high specificity for primary Sjogren's syndrome. Furthermore, depending on the presence of zinc ions, the zinc finger domain of the Ro60KD protein can exist in two different conformational states favoring either an interaction with the Ro52KD protein or binding with autoantibodies.