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Contains fulltext : 207009.pdf (Publisher’s version ) (Closed access) The diagnosis and classification of idiopathic inflammatory myopathies are based mainly on clinical and histological features. The discovery of myositis-specific and myositis-associated antibodies has simplified the (sub)classification of inflammatory myopathies. Patients suspected of having an idiopathic inflammatory myopathy should undergo routine antibody testing to gain more insight into distinct phenotypes, comorbidities, treatment response and prognosis. Furthermore, autoantibody testing can help in patients with atypical patterns of weakness or with an unresolved limb-girdle myopathic phenotype, or interstitial lung disease. However, some important technical and methodological issues can hamper the interpretation of antibody testing; for example, some antibodies are not included in the widely available line blots. We aim to provide a practical review of the use of autoantibody testing in idiopathic inflammatory myopathies in clinical practice.

Contains fulltext : 238504.pdf (Publisher’s version ) (Open Access) OBJECTIVE: To assess anti-cytosolic 5'-nucleotidase 1A (anti-cN-1A) autoantibodies in children with juvenile dermatomyositis (DM) and healthy controls, using 3 different methods of antibody detection, as well as verification of the results in an independent cohort. METHODS: Anti-cN-1A reactivity was assessed in 34 Dutch juvenile DM patients and 20 healthy juvenile controls using the following methods: a commercially available full-length cN-1A enzyme-linked immunosorbent assay (ELISA), a synthetic peptide ELISA, and immunoblotting with a lysate from cN-1A-expressing HEK 293 cells. Sera from juvenile DM patients with active disease and those with disease in remission were analyzed. An independent British cohort of 110 juvenile DM patients and 43 healthy juvenile controls was assessed using an in-house full-length cN-1A ELISA. RESULTS: Anti-cN-1A reactivity was not present in sera from juvenile DM patients or healthy controls when tested with the commercially available full-length cN-1A ELISA or by immunoblotting, in either active disease or disease in remission. Additionally, in the British juvenile DM cohort, anti-cN-1A reactivity was not detected. Three Dutch juvenile DM patients had weakly positive results for 1 of 3 synthetic cN-1A peptides measured by ELISA. CONCLUSION: Juvenile DM patients and young healthy individuals did not show anti-cN-1A reactivity as assessed by different antibody detection techniques.

Background: FSHD is caused by specific genetic mutations resulting in activation of the Double Homeobox 4 gene (DUX4). DUX4 targets hundreds of downstream genes eventually leading to muscle atrophy, oxidative stress, abnormal myogenesis, and muscle inflammation. We hypothesized that DUX4-induced aberrant expression of genes triggers a sustained autoimmune response against skeletal muscle cells. Objective: This study aimed at the identification of autoantibodies directed against muscle antigens in FSHD. Moreover, a possible relationship between serum antibody reactivity and DUX4 expression was also investigated. Methods: FSHD sera (N = 138, 48±16 years, 48% male) and healthy control sera (N = 20, 47±14 years, 50% male) were analyzed by immunoblotting for antibodies against several skeletal muscle protein extracts: healthy muscle, FSHD muscle, healthy and FSHD myotubes, and inducible DUX4 expressing myoblasts. In addition, DUX4 expressing myoblasts were analyzed by immunofluorescence with FSHD and healthy control sera. Results: The results showed that the reactivity of FSHD sera did not significantly differ from that of healthy controls, with all the tested muscle antigen extracts. Besides, the immunofluorescent staining of DUX4-expressing myoblasts was not different when incubated with either FSHD or healthy control sera. Conclusion: Since the methodology used did not lead to the identification of disease-specific autoantibodies in the FSHD cohort, we suggest that autoantibody-mediated pathology may not be an important disease mechanism in FSHD. Nevertheless, it is crucial to further unravel if and which role the immune system plays in FSHD pathogenesis. Other innate as well as adaptive immune players could be involved in the complex DUX4 cascade of events and could become appealing druggable targets.

Two types of clinically important nucleic acid biomarkers, microRNA (miRNA) and circulating tumor DNA (ctDNA) were detected and quantified from human serum using an amplification-free fluorescence hybridization assay. Specifically, miRNAs hsa-miR-223-3p and hsa-miR-486-5p with relevance for rheumatoid arthritis and cancer related mutations BRAF and KRAS of ctDNA were directly measured. The required oligonucleotide probes for the assay were rationally designed and synthesized through a novel "clickable" approach which is time and cost-effective. With no need for isolating nucleic acid components from serum, the fluoresence-based assay took only 1 hour. Detection and absolute quantification of targets was successfully achieved despite their notoriously low abundance, with a precision down to individual nucleotides. Obtained miRNA and ctDNA amounts showed overall a good correlation with current techniques. With appropriate probes, our novel assay and signal boosting approach could become a useful tool for point-of-care measuring other low abundance nucleic acid biomarkers.

Contains fulltext : 226658.pdf (Publisher’s version ) (Open Access)

Women with silicone gel-filled breast implants are exposed to organosilicon compounds, in particular methylsiloxanes, as a result of ‘gel bleed’ and implant rupture. Although these silicones were originally considered to be inert, increasing evidence indicates that they can cause serious health problems. Here, we have analyzed the effects of microdroplets of the methylcyclosiloxanes, in particular D4, on the viability of cultured human cells. The exposure of Jurkat suspension and HeLa monolayer cells to D4 resulted in morphological changes of the cells. The analysis of molecular markers for apoptotic and necrotic processes not only demonstrated that caspases were activated and DNA was fragmented in Jurkat cells exposed to D4, but that also the permeability of the plasma membrane was altered. The induction of apoptotic pathways by D4 was substantiated by the inhibition of caspase activation in cells overexpressing Bcl-2. Cleavage of the caspase-3 substrate U1-70K appeared to be dependent on the D4 content and the efficiency of cleavage decreased with increasing size of the methylcyclosiloxanes (D4, D5 and D6). In addition to Jurkat cells, D4-induced U1-70K cleavage was also observed in HeLa cells, but not in HEp-2 cells. Taken together, these results indicate that D4 and, to a lesser extent, D5 can activate cell-death-related pathways in a cell type-specific fashion and suggest that this phenomenon may contribute to the development of Breast Implant Illness.

Neutrophil extracellular traps (NETs) are networks of extracellular chromatin decorated with antimicrobial proteins, formed by neutrophils to entrap pathogens. NETs have been implicated in the generation of autoimmune reactions. Here, we investigate the reactivity of rheumatoid arthritis (RA) serum antibodies with NETs and explore whether anti-NET antibodies (ANETA) have a potential as biomarker in RA. To quantify ANETA, we developed an ELISA with NETs isolated from stimulated human neutrophils and verified the results by immunofluorescence staining of NETs. ANETA were detected in 22%-69% of RA sera. No significant differences were observed in the reactivity of RA sera with NETs originating from RA patients and healthy control neutrophils, nor with NETs induced by phorbol 12-myristate 13-acetate or the calcium ionophore A23187. ANETA were detected already at baseline in newly diagnosed RA patients and both increased and decreased levels were observed in samples with a median follow-up of 7 years. By ANETA ELISA, we showed that ANETA are also present in sera of patients with systemic lupus erythematosus (36%), Sjögren's syndrome (76%) and scleroderma (61%). In addition to antibodies to NETs, also the presence of NETs or NET fragments in RA sera was determined using a sandwich ELISA. Elevated levels of NETs or NET fragments were detected in 32% of the sera. To assess the potency of ANETA as a biomarker in RA, we compared ANETA positivity with other clinical features. The presence of ANETA was significantly higher in rheumatoid factor (RF)-positive patients, but did not correlate with anti-citrullinated protein antibodies (ACPA), nor with the presence of NET fragments in serum. In addition, no correlation was observed with age, gender, onset of the disease, disease activity and inflammatory markers. These findings suggest that ANETA may be an independent biomarker in RA and possibly also in other autoimmune diseases.

Autoreactive B cells are thought to play a pivotal role in many autoimmune diseases. Rheumatoid arthritis (RA) is an autoimmune disease affecting ∼1% of the Western population and is hallmarked by the presence of anticitrullinated proteins antibodies (ACPA) produced by autoreactive B cells. We intend to develop a method to target and selectively eliminate these autoreactive B cells using a sequential antigen prodrug targeting strategy. As ACPA-expressing B cells are thought to play essential roles in RA-disease pathogenesis, we used this B cell response as a prototype to analyze the feasibility to generate a construct consisting of a biologically silenced, that is, blocked, antigen connected to a cytotoxic prodrug. Blocking of the antigen is considered relevant as it is anticipated that circulating autoantibodies will otherwise clear the antigen-prodrug before it can reach the target cell. The antigen-prodrug can only bind to the autoantigen-specific B cell receptor (BCR) upon enzymatic removal of the blocking group in close proximity of the B cell surface. BCR binding ultimately induces antigen-specific cytotoxicity after internalization of the antigen. We have synthesized a cyclic citrullinated peptide (CCP) antigen suitable for BCR binding and demonstrated that binding by ACPA was impaired upon introduction of a carboxy- p-nitrobenzyl (CNBz) blocking group at the side chain of the citrulline residue. Enzymatic removal of the CNBz moiety by nitroreductase fully restored citrulline-specific recognition by both ACPA and ACPA-expressing B cells and showed targeted cell death of CCP-recognizing B cells only. These results mark an important step toward antigen-specific B cell targeting in general and more specifically in RA, as successful blocking and activation of citrullinated antigens forms the basis for subsequent use of such construct as a prodrug in the context of autoimmune diseases.

Mitochondrial protein synthesis involves an intricate interplay between mitochondrial DNA encoded RNAs and nuclear DNA encoded proteins, such as ribosomal proteins and aminoacyl-tRNA synthases. Eukaryotic cells contain 17 mitochondria-specific aminoacyl-tRNA synthases. WARS2 encodes mitochondrial tryptophanyl-tRNA synthase (mtTrpRS), a homodimeric class Ic enzyme (mitochondrial tryptophan-tRNA ligase; EC 6.1.1.2). Here, we report six individuals from five families presenting with either severe neonatal onset lactic acidosis, encephalomyopathy and early death or a later onset, more attenuated course of disease with predominating intellectual disability. Respiratory chain enzymes were usually normal in muscle and fibroblasts, while a severe combined respiratory chain deficiency was found in the liver of a severely affected individual. Exome sequencing revealed rare biallelic variants in WARS2 in all affected individuals. An increase of uncharged mitochondrial tRNA(Trp) and a decrease of mtTrpRS protein content were found in fibroblasts of affected individuals. We hereby define the clinical, neuroradiological, and metabolic phenotype of WARS2 defects. This confidently implicates that mutations in WARS2 cause mitochondrial disease with a broad spectrum of clinical presentation.

Citrullination, the post-translational conversion of arginines to citrullines, may contribute to rheumatoid arthritis development given the generation of anti-citrullinated protein antibodies (ACPAs). However, it is not known which peptidylarginine deiminase (PAD) catalyzes the citrullination seen in inflammation. PAD4 exacerbates inflammatory arthritis and is critical for neutrophil extracellular traps (NETs). NETs display citrullinated antigens targeted by ACPAs and thus may be a source of citrullinated protein. However, PAD4 is not required for citrullination in inflamed lungs. PAD2 is important for citrullination in healthy tissues and is present in NETs, but its role in citrullination in the inflamed joint, NETosis and inflammatory arthritis is unknown. Here we use mice with TNFα-induced inflammatory arthritis, a model of rheumatoid arthritis, to identify the roles of PAD2 and PAD4 in citrullination, NETosis, and arthritis. In mice with TNFα-induced arthritis, citrullination in the inflamed ankle was increased as determined by western blot. This increase was unchanged in the ankles of mice that lack PAD4. In contrast, citrullination was nearly absent in the ankles of PAD2-deficient mice. Interestingly, PAD2 was not required for NET formation as assessed by immunofluorescence or for killing of Candida albicans as determined by viability assay. Finally, plasma cell numbers as assessed by flow cytometry, IgG levels quantified by ELISA, and inflammatory arthritis as determined by clinical and pathological scoring were all reduced in the absence of PAD2. Thus, PAD2 contributes to TNFα-induced citrullination and arthritis, but is not required for NETosis. In contrast, PAD4, which is critical for NETosis, is dispensable for generalized citrullination supporting the possibility that NETs may not be a major source of citrullinated protein in arthritis.

Summary Small non-coding RNAs have emerged as possible biomarkers for various diseases including autoimmune diseases. A number of studies have demonstrated that the expression of specific microRNAs (miRNAs) is dysregulated in rheumatoid arthritis (RA). So far, all studies on miRNAs in RA patients have been performed using either microarray or reverse transcription–quantitative polymerase chain reaction (RT–qPCR) analyses. Compared to RT–qPCR and microarray analyses, next-generation sequencing (NGS) allows the genome-wide analysis of small RNAs and the differentiation between miRNAs that differ by a single nucleotide. The application of NGS to the analysis of small RNAs circulating in sera of RA patients has not been reported. This study provides a global overview of the circulating small RNAs in the sera of RA patients and healthy subjects and identifies differences between these groups using NGS. Several classes of small RNAs, including hY RNA-derived fragments, tRNA-derived fragments and miRNAs, were determined. Differentially expressed individual small RNAs were verified by RT-qPCR. The levels of two miRNAs, miR-223-3p and miR-16-5p, were significantly lower in the sera from early RA patients than in those from established RA patients and healthy controls. In contrast, the serum level of miR-16-5p was higher in patients with established RA than in healthy control samples. These miRNAs may not only serve as biomarkers, but may also shed more light on the pathophysiology of RA.

Introduction: Autoantibodies to cytosolic 5'-nucleotidase 1A (cN-1A; NT5C1A) have a high specificity when differentiating sporadic inclusion body myositis from polymyositis and dermatomyositis. In primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE) anti-cN-1A autoantibodies can be detected as well. However, various frequencies of anti-cN-1A reactivity have been reported in SLE and pSS, which may at least in part be explained by the different assays used. Here, we determined the occurrence of anti-cN-1A reactivity in a large number of patients with pSS and SLE using one standardized ELISA. Methods: Sera from pSS (n = 193) and SLE patients (n = 252) were collected in five European centers. Anti-cN-1A, anti-Ro52, anti-nucleosome, and anti-dsDNA reactivities were tested by ELISA (Euroimmun AG) in a single laboratory. Correlations of anti-cN-1A reactivity with demographic data and clinical data (duration of disease at the moment of serum sampling, autoimmune comorbidity and presence of muscular symptoms) were analyzed using SPSS software. Results: Anti-cN-1A autoantibodies were found on average in 12% of pSS patients, with varying frequencies among the different cohorts (range: 7-19%). In SLE patients, the anti-cN-1A positivity on average was 10% (range: 6-21%). No relationship was found between anti-cN-1A reactivity and the presence or absence of anti-Ro52, anti-nucleosome, and anti-dsDNA reactivity in both pSS and SLE. No relationship between anti-cN-1A reactivity and duration of disease at the moment of serum sampling and the duration of serum storage was observed. The frequency of muscular symptoms or viral infections did not differ between anti-cN-1A-positive and -negative patients. In both disease groups anti-cN-1A-positive patients suffered more often from other autoimmune diseases than the anti-cN-1A-negative patients (15 versus 5% (p = 0.05) in pSS and 50 versus 30% (p = 0.02) in SLE). Conclusion: Our results confirm the relatively frequent occurrence of anti-cN-1A in pSS and SLE patients and the variation in anti-cN-1A reactivity between independent groups of these patients. The explanation for this variation remains elusive. The correlation between anti-cN-1A reactivity and polyautoimmunity should be evaluated in future studies. We conclude that anti-cN-1A should be classified as a myositis-associated-, not as a myositis-specific-autoantibody based on its frequent presence in SLE and pSS.

The aim of this study was to evaluate the presence of autoantibodies to cyclic citrullinated synthetic peptides (ACPAs) in the sputum of patients with long-standing rheumatoid arthritis (RA).Nineteen consecutive RA patients and 16 age- and sex-matched control subjects participated in this cross-sectional study. All underwent complete lung function tests and provided induced sputum. Antibodies to citrullinated (CitP) and the corresponding norleucine-containing (NorP) peptides in the sputum of the RA patients and control subjects, as well as in the serum of the RA patients, were determined by enzyme-linked immunosorbent assay.Patients with RA had the following characteristics: mean disease duration of 12 years, Disease Activity Score for 28 joints of 3.44, and Sharp-van der Heijde score of 57.5. Ten of the 19 RA patients showed high titers of ACPAs in their sera. Four of the seropositive (40%), none of the seronegative RA patients, and only 1 of the control subjects showed detectable levels of ACPAs in their sputum. The ratio between the reactivity with CitP and NorP peptides in the sputum was significantly higher in RA sputum than in control sputum (1.33 ± 1.2 vs. 0.64 ± 0.14, P = 0.02). A positive correlation was found between sputum ACPAs and age, serum ACPAs, sputum anti-NorP, serum anti-CitP/NorP reactivity ratio, and the proportion of neutrophils and lymphocytes in the sputum. No significant correlation was found between sputum ACPAs and disease severity, history of smoking, lung function tests, or treatment for RA.Anticitrullinated protein/peptide antibodies can be detected in the sputum of RA patients and are correlated with the presence in the serum.

Purpose of review The purpose of this article is to discuss recent advances in serological testing for sporadic inclusion body myositis (sIBM) and to provide a review of their diagnostic utility and disease-specificity of auto-antibodies in sIBM. Recent findings The identification, prevalence and diagnostic utility of a new auto-antibody targeting cytosolic 5'-nucleotidase 1A (cN-1A) in the serum of sIBM patients have recently been published. These studies have shown that anti-cN-1A auto-antibodies have diagnostic utility for differentiating sIBM from other forms of myositis and from other neuromuscular diseases. Anti-cN-1A-positive patient sera are directed to multiple epitopes of cN-1A and contain, in addition to IgG, IgA and IgM, anti-cN-1A auto-antibodies. Recent studies have also shown a relatively high prevalence of these auto-antibodies in sera form Sjogren's syndrome and systemic lupus erythematosus patients. Summary The recent discovery of auto-antibodies to cN-1A provides a serological tool to aid the differentiation between inflammatory myopathies and supports the idea that apart from degeneration, an adaptive immune response may also play a role in sIBM pathophysiology. Future research will need to focus on standardization of methods to detect these auto-antibodies in order to further explore their specificity and diagnostic utility for sIBM.

ObjectivesAutoantibodies directed against cytosolic 5′-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5′-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis.Materials and methodsData from various European inclusion body myositis registries were pooled. Anticytosolic 5′-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression.ResultsData from 311 patients were available for analysis; 102 (33%) had anticytosolic 5′-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients.InterpretationDifferences were observed in clinical and histopathological features between anticytosolic 5′-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5′-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.

Mutations in the RMRP-gene, encoding the lncRNA component of the RNase MRP complex, are the origin of cartilage-hair hypoplasia. Cartilage-hair hypoplasia is associated with severe dwarfism caused by impaired skeletal development. However, it is not clear why mutations in RMRP RNA lead to skeletal dysplasia. Since chondrogenic differentiation of the growth plate is required for development of long bones, we hypothesized that RMRP RNA plays a pivotal role in chondrogenic differentiation. Expression of Rmrp RNA and RNase MRP protein subunits was detected in the murine growth plate and during the course of chondrogenic differentiation of ATDC5 cultures, where Rmrp RNA expression was found to be correlated with chondrocyte hypertrophy. Genetic interference with Rmrp RNA expression in ATDC5 cultures caused a deregulation of chondrogenic differentiation, with a prominent impact on hypertrophy and changes in pre-rRNA processing and rRNA levels. Promoter reporter studies showed that Rmrp RNA expression responds to chondrogenic morphogens. Chondrogenic trans-differentiation of cartilage-hair hypoplasia fibroblasts was impaired with a pronounced impact on hypertrophic differentiation. Together, our data show that RMRP RNA expression is regulated during different stages of chondrogenic differentiation and indicate that RMRP RNA may play a pivotal role in chondrocyte hypertrophy, with potential consequences for CHH pathobiology.

The findings that citrullinated proteins are targeted by the most specific immune response in rheumatoid arthritis, and that citrullination also plays an important role in neurodegenerative diseases and certain cancers, have triggered many researchers to study various aspects of this form of posttranslational modification. This chapter is focused on the conditions that are needed for peptidylarginine deiminases to become active citrullinating enzymes, in particular in relation to joint inflammation as observed in rheumatoid arthritis. After briefly discussing the available methods to study the activity of peptidylarginine deiminases and their substrate specificity, the isoforms that are most relevant for citrullination during inflammation and the factors that are mediating their activation are addressed. Citrullination is crucial for one of the processes that are tightly associated with inflammation, NETosis, which is more extensively discussed in Chap. 8. Finally, peptidylarginine deiminase activation and the resulting citrullination in the context of the inflamed joints in rheumatoid arthritis are described.

Breast implants are applied to correct the size, form, and feel of a woman’s breasts in post-mastectomy breast reconstruction, for correcting congenital defects and deformities of the chest wall, for aesthetic breast augmentation, and for creating breasts in the male-to-female transsexual patient. The most widely applied silicone implants have an elastomer silicone shell filled with viscous silicone gel. It has become increasingly clear that it is important for plastic surgeons to know and to provide patients with the latest information on silicone breast implants. This article was therefore written to provide more insight into the composition of silicone breast implants and their characteristics. After describing the chemical properties of silicones and silicone implants, gel bleeding and the consequences of aging of silicone implants will be discussed. The information provided can also give a hint as to why silicone implants have been consistently implicated in health issues ever since the late 1960s. Level of Evidence: Not ratable.

OBJECTIVE: Sporadic inclusion body myositis (sIBM) is an inflammatory myopathy characterized by both degenerative and autoimmune features. In contrast to other inflammatory myopathies, myositis-specific autoantibodies had not been found in sIBM patients until recently. We used human skeletal muscle extracts as a source of antigens to detect autoantibodies in sIBM and to characterize the corresponding antigen. METHODS: Autoantibodies to skeletal muscle antigens were detected by immunoblotting. The target antigen was immunoaffinity-purified from skeletal muscle extracts and characterized by mass spectrometry. A cDNA encoding this protein was cloned and expressed in vitro, and its recognition by patient sera was analyzed in an immunoprecipitation assay. Epitopes were mapped using microarrays of overlapping peptides. RESULTS: An Mr 44,000 polypeptide (Mup44) was frequently targeted by sIBM autoantibodies. The target protein was purified, and subsequent mass spectrometry analysis revealed that Mup44 is the cytosolic 5'-nucleotidase 1A (cN1A). By immunoprecipitation of recombinant cN1A, high concentrations of anti-Mup44 autoantibodies were detected in 33% of sIBM patient sera, whereas their prevalence in dermatomyositis, polymyositis, and other neuromuscular disorders appeared to be rare (4.2%, 4.5%, and 3.2%, respectively). Low concentrations of anti-Mup44 antibodies were found in myositis as well as other neuromuscular disorders, but not in healthy controls. Three major autoepitope regions of cN1A were mapped by using microarrays containing a set of overlapping peptides covering the complete cN1A amino acid sequence. INTERPRETATION: Anti-Mup44 autoantibodies, which are targeted to cN1A, represent the first serological biomarker for sIBM and may facilitate the diagnosis of this type of myositis.

Contains fulltext : 125273pub.pdf (Publisher’s version ) (Closed access) alphaB-crystallin is regarded as a biomarker for triple-negative and/or basal-like breast cancer. In normal breast cells, overexpression of alphaB-crystallin leads to neoplastic-like changes, which likely relate to enhanced expression of phosphorylated ERK1/2 (pERK1/2). In this study, we investigated whether alphaB-crystallin expression is correlated to pERK1/2 expression in breast cancer. In a balanced tissue microarray the expression of alphaB-crystallin and pERK1/2 kinase were determined immunohistochemically, together with the triple-negativity and basal-like markers CK5/6 and SMA and the signaling molecules pAKT, pmTOR, EGFR, and IGF-1R. alphaB-crystallin expression significantly correlated with triple negativity and basal-like markers CK5/6 and SMA (Pearson Chi-square test p=0.004, p=0.001, and p

Citrullination, the conversion of peptidylarginine to peptidylcitrulline is catalyzed by peptidylarginine deiminases (PAD). The expression of PAD isoforms displays great variation among different tissues as demonstrated by PAD mRNA analyses. Here we have analyzed the differential expression of PAD2, PAD4 and PAD6 in mouse tissues at the protein level and by enzymatic activity assays using PAD2 and PAD4 knock-out strains. As expected, no PAD2 expression was detected in the PAD2−/− mice. In contrast, the PAD4 protein was observed in several tissues of the PAD4 knock-out mice, albeit at reduced levels in most tissues, and are therefore referred to as PAD4-low mice. In material from PAD2−/− mice, except for leukocyte lysates, hardly any PAD activity was found and no citrullinated proteins were detected after incubation in the presence of calcium. PAD activity in the PAD4-low mice was similar to that in wild-type mice. In both PAD knock-out strains the expression of PAD6 appeared to be up-regulated in all tissues analyzed, with the exception of spleen and testis. Our data demonstrate that the PAD2 protein is expressed in brain, spinal cord, spleen, skeletal muscle and leukocytes, but not detectably in liver, lung, kidney and testis. PAD4 was detected in each of these tissues, although the expression levels varied. In all tissues where PAD2 was detected, except for blood cells, this PAD isoform appeared to be responsible for virtually all peptidylarginine deiminase activity.

Background: Antibodies against citrullinated proteins (ACPA) are common in patients with rheumatoid arthritis (RA). ACPA can appear before disease onset and target many self-antigens. Citrullinated fibrin/fibrinogen represents a classical ACPA target antigen, and mass spectrometry of RA synovial fluid reveals elevated citrullinated (cit) fibrinogen (Fib) peptides compared to non-RA controls. We investigated the extent to which these less-studied peptides represent autoantibody targets and sought to visualize the corresponding cit-Fib-reactive B cells in RA patients. Methods: An in-house ELISA was established against four cit-Fib alpha-subunit peptides (cit-Fib alpha-35; cit-Fib alpha-216,218; cit-Fib alpha-263,271 and cit-Fib alpha-425,426) and serum from patients with established RA (n = 347) and disease controls with psoriatic arthritis (PsA) or ankylosing spondylitis (AS) (n = 236) were analyzed. RA patients were genotyped for HLA-DR alleles, PTPN22 R620W and screened for anti-CCP2 and cit-Fib protein antibodies. The cit-Fib peptides were also used to assemble antigen tetramers to identify cit-Fib-reactive B cells in peripheral blood by flow cytometry. Results: The frequencies of autoantibodies against different cit-Fib epitopes in RA patients compared to PsA/AS patients were: cit-Fib alpha-35 (RA 20%, vs PsA/AS 1%); cit-Fib alpha-216,218 (13% vs 0.5%); cit-Fib alpha-263,271 (21% vs 0.5%) and cit-Fib alpha-425,426 (17% vs 1%). The presence of autoantibodies against these peptides was associated with presence of anti-CCP2 and anti-cit-Fib protein antibodies. No association was found between HLA-DR shared epitope and antibodies to the different cit-Fib peptides. However, association was observed between the PTPN22 risk allele and positivity to cit-Fib alpha-35 and cit-Fib alpha-263,271. B cells carrying surface Ig reactive to these cit-Fib peptides were found in RA peripheral blood and these tend to be more common in PTPN22 risk allele carriers. Conclusions: Our data show that several cit-Fib peptides are targeted by autoantibodies in RA, but not in PsA/AS, implicating that these are not due to arthritis but more specific for RA etiology. The RA-associated anti-cit protein response is broad with many parallel immune responses. The association between cit-Fib autoantibodies and the PTPN22 R620W risk allele supports the hypothesis of altered B cell regulation, such as autoreactive B cells evading tolerance checkpoints.

ObjectivesThe diagnosis of inclusion body myositis (IBM) can be challenging as it can be difficult to clinically distinguish from other forms of myositis, particularly polymyositis (PM). Recent studies have shown frequent presence of autoantibodies directed against cytosolic 5′-nucleotidase 1A (cN-1A) in patients with IBM. We therefore, examined the autoantigenicity and disease specificity of major epitopes of cN-1A in patients with sporadic IBM compared with healthy and disease controls.MethodsSerum samples obtained from patients with IBM (n=238), PM and dermatomyositis (DM) (n=185), other autoimmune diseases (n=246), other neuromuscular diseases (n=93) and healthy controls (n=35) were analysed for the presence of autoantibodies using immunodominant cN-1A peptide ELISAs.ResultsAutoantibodies directed against major epitopes of cN-1A were frequent in patients with IBM (37%) but not in PM, DM or non-autoimmune neuromuscular diseases (ConclusionsIn summary, we found frequent anti-cN-1A autoantibodies in sera from patients with IBM. Heterogeneity in reactivity with the three immunodominant epitopes indicates that serological assays should not be limited to a distinct epitope region. The similar reactivities observed for SjS and SLE demonstrate the need to further investigate whether distinct IBM-specific epitopes exist.

ObjectivesTo understand the molecular features distinguishing anti-citrullinated protein antibodies (ACPA) from ‘conventional’ antibodies in rheumatoid arthritis (RA).MethodsSerum of ACPA-positive RA patients was fractionated by size exclusion chromatography and analysed for the presence of ACPA-IgG by ELISA. ACPA-IgG and non-citrulline-specific IgG were affinity purified from serum, plasma and/or synovial fluid and analysed by gel electrophoresis. Electrophoresis bands were excised, enzymatically digested and analysed by mass spectrometry. Binding affinity to citrullinated antigens was measured by ELISA and imaging surface plasmon resonance using recombinant monoclonal ACPA with molecular modifications.ResultsIn all donor samples studied (n=24), ACPA-IgG exhibited a 10–20 kDa higher molecular weight compared with non-autoreactive IgG. This feature also distinguished ACPA-IgG from antibodies against recall antigens or other disease-specific autoantibodies. Structural analysis revealed that a high frequency of N-glycans in the (hyper)variable domains of ACPA is responsible for this observation. In line with their localisation, these N-glycans were found to modulate binding avidity of ACPA to citrullinated antigens.ConclusionsThe vast majority of ACPA-IgG harbour N-glycans in their variable domains. As N-linked glycosylation requires glycosylation consensus sites in the protein sequence and as these are lacking in the ‘germline-counterparts’ of identified variable domains, our data indicate that the N-glycosylation sites in ACPA variable domains have been introduced by somatic hypermutation. This finding also suggests that ACPA-hyperglycosylation confers a selective advantage to ACPA-producing B cells. This unique and completely novel feature of the citrulline-specific immune response in RA elucidates our understanding of the underlying B cell response.

Testing for autoantibodies (AABs) is becoming more and more relevant, not only for diagnosing autoimmune diseases (AIDs) but also for the differentiation of defined AID subtypes with different clinical manifestations, course and prognosis as well as the very early diagnosis for adequate management in the context of personalized medicine. A major challenge to improve diagnostic accuracy is to harmonize or even standardize AAB analyses. This review presents the results of the 12th Dresden Symposium on Autoantibodies that focused on several aspects of improving autoimmune diagnostics. Topics that are addressed include the International Consensus on ANA Patterns (ICAP) and the International Autoantibody Standardization (IAS) initiatives, the optimization of diagnostic algorithms, the description and evaluation of novel disease-specific AABs as well as the development and introduction of novel assays into routine diagnostics. This review also highlights important developments of recent years, most notably the improvement in diagnosing and predicting the course of rheumatoid arthritis, systemic sclerosis, idiopathic inflammatory myopathies, and of autoimmune neurological, gastrointestinal and liver diseases; the potential diagnostic role of anti-DFS70 antibodies and tumor-associated AABs. Furthermore, some hot topics in autoimmunity regarding disease pathogenesis and management are described.

Purpose Sporadic inclusion body myositis (sIBM) is an autoimmune degenerative disease of the muscle, with inflammatory infiltrates and inclusion vacuoles. Its pathogenesis is not fully understood and the diagnosis is hampered by its imprecise characteristics, at times indistinguishable from other idiopathic inflammatory myopathies such as polymyositis and dermatomyositis. The diagnosis may be assisted by the detection of autoantibodies targeting Mup44, a skeletal muscle antigen identified as cytosolic 5′-nucleotidase 1A (cN-1A, NT5C1A). A novel standardized anti-cN-1A IgG ELISA was developed and its diagnostic performance was evaluated by two reference laboratories. Methods Recombinant human full-length cN-1A was expressed and purified, and subsequently utilized to set up a standardized ELISA. To evaluate the novel assay, laboratory A examined sera from North American patients with clinically and pathologically diagnosed definite sIBM (n = 17), suspected sIBM (n = 14), myositis controls (n = 110), non-myositis autoimmune controls (n = 93) and healthy subjects (n = 52). Laboratory B analyzed a Dutch cohort of definite sIBM patients (n = 51) and healthy controls (n = 202). Results Anti-cN-1A reactivity was most frequent in definite sIBM (39.2–47.1%), but absent in biopsy-proven classic polymyositis or dermatomyositis. Overall diagnostic sensitivity and specificity amounted to 35.5 and 96.1% (laboratory A) and 39.2 and 96.5% (laboratory B). Conclusions Anti-cN-1A autoantibodies were detected by ELISA with moderate sensitivity, but high specificity for sIBM and may therefore help diagnose this infrequent and difficult-to-diagnose myopathy. The novel anti-cN-1A IgG ELISA can improve and accelerate the diagnosis of sIBM using sera where muscle biopsy is delayed or unfeasible.

Objective To generate a catalog of citrullinated proteins that are present in the synovia of patients with rheumatoid arthritis (RA) and to elucidate their relevance for the anti–citrullinated protein antibody response in RA. Methods Polypeptides isolated from the synovial fluid of patients with RA were identified by mass spectrometry. Three proteins (apolipoprotein E [Apo E], myeloid nuclear differentiation antigen [MNDA], and β-actin) were studied in more detail, using immunoprecipitation and Western blotting. The presence of autoantibodies to synthetic peptides derived from these proteins in sera from patients with RA, sera from patients with other diseases, and sera from healthy control subjects was studied by enzyme-linked immunosorbent assay (ELISA). Results RA synovial fluid samples displayed several distinct patterns of citrullinated proteins. Using mass spectrometry, (fragments of) 192 proteins were identified, including 53 citrullinated proteins, some of which contained multiple citrullinated residues. In addition to previously reported citrullinated proteins in RA synovia (e.g., vimentin and fibrinogen), a series of novel citrullinated proteins, including Apo E, MNDA, β-actin, and cyclophilin A, was identified. Immunoprecipitation experiments confirmed the citrullination of Apo E and MNDA. ELISAs demonstrated the presence of autoreactive citrullinated epitopes in Apo E, MNDA, and β-actin. Conclusion Synovial fluid samples from the inflamed joints of patients with RA contain many citrullinated proteins. Citrullinated Apo E, MNDA, and β-actin are novel antigens identified in RA synovial fluid, and only a limited number of their citrullinated epitopes are targeted by the immune system in RA.

Perlman syndrome is a congenital overgrowth syndrome inherited in an autosomal recessive manner that is associated with Wilms tumor susceptibility. We mapped a previously unknown susceptibility locus to 2q37.1 and identified germline mutations in DIS3L2, a homolog of the Schizosaccharomyces pombe dis3 gene, in individuals with Perlman syndrome. Yeast dis3 mutant strains have mitotic abnormalities. Yeast Dis3 and its human homologs, DIS3 and DIS3L1, have exoribonuclease activity and bind to the core RNA exosome complex. DIS3L2 has a different intracellular localization and lacks the PIN domain found in DIS3 and DIS3L1; nevertheless, we show that DIS3L2 has exonuclease activity. DIS3L2 inactivation was associated with mitotic abnormalities and altered expression of mitotic checkpoint proteins. DIS3L2 overexpression suppressed the growth of human cancer cell lines, and knockdown enhanced the growth of these cells. We also detected evidence of DIS3L2 mutations in sporadic Wilms tumor. These observations suggest that DIS3L2 has a critical role in RNA metabolism and is essential for the regulation of cell growth and division.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by autoantibodies against citrullinated antigens. The importance of citrulline for the epitopes bound by these autoantibodies, referred to as ACPA (anti-citrullinated peptide/protein antibodies), was first described in 1998. In addition to citrullinated proteins, cyclic citrullinated peptides (CCP) can also be used as test substrates for detecting ACPA. The standard test for these antibodies is the second-generation CCP (CCP2) test, which is one of the best in terms of sensitivity and specificity. The generation of ACPA is an early event in the disease course, and is dependent on the presence of certain MHC class II alleles. ACPA in the inflamed synovium have been shown to associate with citrullinated antigens to form immune complexes, resulting in progression of the inflammatory process. The involvement of ACPA in the chronicity of RA is probably the reason why ACPA-positive patients have a more erosive disease course than ACPA-negative patients. The presence of ACPA has been included in the 2010 RA classification criteria. Thus, it is important to further standardize ACPA testing, for example by including an internal serum standard, which may lead to a better distinction between low and high ACPA levels.

OBJECTIVES: /st> To determine the fine specificity of anti-citrullinated protein antibodies (ACPA) in the early phase of arthritis development, the ACPA repertoire in arthralgia patients and the association with arthritis development were studied. METHODS: /st> A total of 244 patients with arthralgia positive for anti-cyclic citrullinated peptide antibodies (aCCPs) and/or IgM rheumatoid factor (IgM-RF), without arthritis were included. Development of arthritis was defined as presence of one or more swollen joints at clinical examination during follow-up. Sera were tested at baseline for reactivity to five citrullinated peptides derived from fibrinogen (three), vimentin (one) and α-enolase (one) and five corresponding arginine peptides in an ELISA. RESULTS: /st> In all, 69 patients (28%) developed arthritis in a median of 3 joints after a median follow-up of 11 (IQR 5-20) months. Reactivity to each peptide was significantly associated with arthritis development (p Arthritis development is not associated with recognition of a specific citrullinated peptide once joint complaints are present. The ACPA repertoire in some patients with arthralgia is expanded. High aCCP levels are associated with a qualitatively broad ACPA repertoire. Patients with an extended ACPA repertoire have a higher risk of developing arthritis

Background Anti-citrullinated protein antibodies (ACPA) are the most predictive factor for the development of rheumatoid arthritis (RA). Objective To investigate whether the recognition of citrullinated epitopes changes during disease onset or progression, by studying the fine specificity of ACPA in serum samples collected throughout the disease course, from before the onset of arthritis to longstanding RA. Methods Antibodies recognising five distinct citrullinated antigens were determined by enzyme-linked immunosorbent assay. Serum samples from 36 individuals who had donated blood before and after disease manifestation were used to investigate the development of citrullinated antigen recognition before disease onset. The association of ACPA reactivities with disease outcome was studied using sera from anti-cyclic citrullinated peptide-2 (CCP2)-positive patients with undifferentiated arthritis (UA) who did or did not progress to RA (UA-RA n = 81, or UA-UA n = 35). To investigate the ACPA recognition profile in patients with RA over a prolonged period of time, baseline serum samples from 68 patients were compared with samples obtained 7 years later. Results The number of recognised citrullinated peptides increased in the period preceding disease onset. At the time of disease manifestation, patients with UA who later developed RA recognised significantly more peptides than UA-UA patients. At later stages of the disease course, the ACPA fine specificity did not change. Conclusion Epitope spreading with an increase in the recognition of citrullinated antigens occurs before the onset of RA. Immunological differences in ACPA fine specificity between UA-UA patients and UA-RA patients are present at baseline and are associated with the future disease course.

Contains fulltext : 83310.pdf (Publisher’s version ) (Closed access) Combined oxidative phosphorylation (OXPHOS) system deficiencies are a group of mitochondrial disorders that are associated with a range of clinical phenotypes and genetic defects. They occur in approximately 30% of all OXPHOS disorders and around 4% are combined complex I, III and IV deficiencies. In this study we present two mutations in the mitochondrial tRNA(Trp) (MT-TW) and tRNA(Arg) (MT-TR) genes, m.5556G>A and m.10450A>G, respectively, which were detected in two unrelated patients showing combined OXPHOS complex I, III and IV deficiencies and progressive multisystemic diseases. Both mitochondrial tRNA mutations were almost homoplasmic in fibroblasts and muscle tissue of the two patients and not present in controls. Patient fibroblasts showed a general mitochondrial translation defect. The mutations resulted in lowered steady-state levels and altered conformations of the tRNAs. Cybrid cell lines showed similar tRNA defects and impairment of OXPHOS complex assembly as patient fibroblasts. Our results show that these tRNA(Trp) and tRNA(Arg) mutations cause the combined OXPHOS deficiencies in the patients, adding to the still expanding group of pathogenic mitochondrial tRNA mutations. 01 maart 2010 6 p.

The most specific biomarker associated with the diagnosis of rheumatoid arthritis (RA) is autoantibodies to citrullinated peptides/proteins (ACPA). Though recognized as an important marker of progressive erosive disease its use has been hampered by doubt about what is a positive versus a negative reaction in the several assays that have become available commercially. This review intends to indicate that the CCP2 assay has the highest specificity and sensitivity in stratified studies that encompass sera from RA patients and non-RA inflammatory controls compared to other ACPA tests. Still, larger and strictly stratified studies are highly warranted to substantiate this conclusion.

Cartilage hair hypoplasia (CHH; MIM 250250) is an autosomal recessive disease with diverse clinical manifestations. It is caused by mutations in RMRP gene, the RNA component of the ribonucleoprotein complex RNase MRP. Mutations in RMRP have been found in patients in the core promoter region or in the transcribed region, but the pathogenetic effect of the mutations is unclear. Real-time PCR assays confirmed that both promoter (c.-16_-1 dup and c.-15_+2 dup) and transcribed mutations (c.168G > A and c.218A > G) lower the expression level of RMRP. Experiments with 5'RACE, showed that the reduced transcription in the promoter mutants was accompanied by shifting of the transcription initiation sites to nucleotides 5'-upstream of the authentic site. Low levels of RMRP expression levels with transcript mutations were also seen when constructs encoding the wild-type and mutant genes were transfected into cultured cells. The reduced transcription was correlated with greater instability of mutant RMRP transcripts compared to controls. A comparable reduction was seen when a mouse gene containing the c.70A > G mutation (the major mutation in humans with CHH) was introduced into ES cells in place of one of the wild-type alleles. The low expression level of the c.70A > G Rmrp RNA was confirmed by expression assays into cultured cells, and was again correlated with RNA instability. Our results indicate that a loss of mutant RNA transcripts is a critical feature of pathogenesis.

Purpose Recent studies have demonstrated that serum/plasma DNA and RNA molecules in addition to proteins can serve as biomarkers. Elevated levels of these nucleic acids have been found not only in acute, but also in chronic conditions, including autoimmune diseases. The aim of this study was to assess cell-free DNA (cfDNA) levels in sera of rheumatoid arthritis (RA) patients compared to controls. Methods cfDNA was extracted from sera of patients with early and established RA, relapsing-remitting multiple sclerosis patients (RRMS) and healthy subjects, and its concentration was determined by quantitative PCR using two amplicons, Alu115 and β-actin205, corresponding to Alu repetitive elements and the β-actin single-copy gene, respectively. Serum DNase activity was measured by a single radial enzyme diffusion method. Results Reduced levels of cfDNA were observed in patients with established RA in comparison with healthy controls, early RA patients and RRMS patients. There were no significant differences in cfDNA concentration between healthy controls, early RA and RRMS patients. Total DNase activity appeared to be similar in the sera of all tested groups. Conclusions Our results demonstrate that cfDNA levels are strongly reduced in the sera of established RA patients, which is not caused by changes in DNase activity. Measurement of cfDNA can distinguish established RA patients from early RA patients. Thus, cfDNA may serve as a biomarker in RA. Electronic supplementary material The online version of this article (doi:10.1007/s13317-015-0066-6) contains supplementary material, which is available to authorized users.

The discovery that citrullination was crucial for the recognition of antigens by the most disease-specific class of autoantibodies in rheumatoid arthritis had a huge impact on studies aimed at understanding autoimmunity in this disease. In addition to the detailed characterization of anti-citrullinated protein antibodies, various studies have addressed the identity of citrullinated antigens. These investigations were facilitated by new methods to characterize these proteins, the analysis of protein citrullination by peptidylarginine deiminases, the generation of a catalogue of citrullinated proteins present in the inflamed joints of patients and the finding that the formation of extracellular traps is dependent on the activity of peptidylarginine deiminase activity. Recently, it was found that in addition to citrullination also carbamylation, which results in chemically highly related modified proteins, yields antigens that are targeted by rheumatoid arthritis patient sera. Here, all of these aspects will be discussed, culminating in current ideas about the involvement of citrullination and carbamylation in pathophysiological processes in autoimmunity, especially rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common, systemic autoimmune disease characterized by chronic inflammation of the synovium, that can lead to progressive joint destruction and in many cases results in severe disability and poor quality of life. With the availability of more sophisticated and effective therapies and with increasing evidence that the first few months of disease represent an unique therapeutic opportunity and that such early therapeutic intervention is crucial in preventing irreversible joint damage, it is widely accepted that early and accurate diagnosis of RA is critical in disease management. Within the last three years a growing number of publications have reported that the second generation anti-CCP (cyclic citrullinated peptide) test may become the marker of choice for diagnosing early RA as it appears to be highly specific for the disease with a sensitivity comparable to the widely used but less specific rheumatoid factor test. Additionally, anti-CCP2 positivity can predict future development of RA in both asymptomatic individuals and in patients with undifferentiated arthritis. Furthermore, antibody levels at presentation can correlate with progression to erosive disease.

Rheumatoid arthritis (RA) is a common systemic autoimmune disease with a prevalence of about 1% worldwide [1]. The American College of Rheumatology (ACR) criteria for the classification of RA [2] are not very well suited to diagnose RA at an early stage of the disease [3, 4], because these criteria rely heavily on the expression of clinical symptoms of RA. In early RA these clinical parameters are often not (yet) manifest. Therefore, a specific and sensitive (serological) marker, which is present very early in the disease, is needed. A good marker should ideally not only indicate the development of the disease, but also be able to predict its erosive or non-erosive progression. The serological parameter that meets these requirements for a good and useful marker for early RA is the anti-citrullinated protein antibody. The sensitivity of this antibody is comparable to that of the rheumatoid factor (RF) (approximately 80%), but its specificity is much higher, about 98%. Several assays have been developed to detect this class of autoantibodies, which are termed anti-CCP because the most sensitive test is based upon cyclic citrullinated peptides. This review will discuss the potential of this autoantibody system for the diagnosis and prognosis of RA.

Contains fulltext : 58385.pdf (Publisher’s version ) (Closed access) Rheumatoid arthritis (RA) is a common, systemic autoimmune disease of which the exact etiology is not known. In the past 10 years, substantial progress has been made in the identification of the antigens specifically recognized by the autoantibodies of RA patients. A central factor in this respect is citrullination, a form of post-translational modification that is strongly associated with autoimmunity in RA. Here, we summarize and discuss our current knowledge on (i) autoantibody systems in RA, (ii) the occurrence of peptidylarginine deiminases and (iii) citrullinated proteins in natural and diseased environments, and (iv) genetic factors involved in RA that may influence the generation and presentation of citrullinated proteins and the resulting antibody production against these modified proteins. Citrullination of proteins may play a key role in the initiation and/or the progression of RA. The onset of citrulline-specific autoimmunity in RA is probably mediated by both environmental and genetic factors, and future studies will learn whether therapeutic intervention at the level of citrullination may provide new possibilities to treat RA.

Contains fulltext : 224588.pdf (Publisher’s version ) (Closed access) Autoantibodies targeting nucleolar autoantigens (ANoA) are most frequently found in sera from patients with systemic sclerosis (SSc, also designated scleroderma) or with SSc overlap syndromes. During the last decade an extensive number of nucleolar components have been identified and this allowed a more detailed analysis of the identity of nucleolar autoantigens. This review intends to give an overview of the molecular composition of the major (families of) autoantigenic nucleolar complexes, to provide some insight into their functions and to summarise the data concerning their autoantigenicity.

Summary Sera from patients suffering from systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) have been shown to contain reactivities to nuclear components. Autoantibodies specifically targeting nucleolar antigens are found most frequently in patients suffering from SSc or SSc overlap syndromes. We determined the prevalence and clinical significance of autoantibodies directed to nucleolar RNA-protein complexes, the so-called small nucleolar ribonucleoprotein complexes (snoRNPs). A total of 172 patient sera with antinucleolar antibodies were analysed by immunoprecipitation. From 100 of these patients clinical information was obtained by chart review. Autoantibodies directed to snoRNPs were detected not only in patients suffering from SSc and primary Raynaud's phenomenon (RP), but also in patients suffering from SLE, rheumatoid arthritis (RA) and myositis (PM/DM). Antibodies against box C/D small snoRNPs can be subdivided in antifibrillarin positive and antifibrillarin negative reactivity. Antifibrillarin-positive patient sera were associated with a poor prognosis in comparison with antifibrillarin negative (reactivity with U3 or U8 snoRNP only) patient sera. Anti-Th/To autoantibodies were associated with SSc, primary RP and SLE and were found predominantly in patients suffering from decreased co-diffusion and oesophagus motility and xerophthalmia. For the first time autoantibodies that recognize box H/ACA snoRNPs are described, identifying this class of snoRNPs as a novel autoantigenic activity. Taken together, our data show that antinucleolar patient sera directed to small nucleolar ribonucleoprotein complexes are found frequently in other diseases than SSc and that categorization of diagnoses and clinical manifestations based on autoantibody profiles seems particularly informative in patient sera recognizing box C/D snoRNPs.

SUMMARY Coimmunoprecipitation studies with SW5, a frequently used and specific mouse monoclonal antibody (mAb) directed against the human La autoantigen, led to the identification of a functionally unrelated 80 000 MW protein, designated early endosome antigen 2 (EEA2). EEA2 appeared to be directly targeted by mAb SW5. Because an RNA-binding domain, a structural element of La containing the SW5-epitope, was not discernable in the primary structure of EEA2, the SW5-epitope on EEA2 was determined. Coiled-coil region 3 of EEA2 appeared to contain the epitope recognized by SW5. The SW5 epitope regions of La and EEA2 share a limited sequence homology and probably share a higher degree of structural similarity at the tertiary level. Most likely, the most critical determinants for recognition by SW5 reside in elements adopting alpha-helical conformations. These data indicate that the application of specific mAbs to purify and characterize (functionally) interacting proteins can be severely obscured by the cross-reactivity of mAbs with structurally, but not functionally, similar proteins.

A supramolecular approach was undertaken to create functionally activatable cell-penetrating peptides. Two tetra-arginines were assembled into an active cell-penetrating peptide by heterodimerizing leucine zippers. Three different leucine-zipper pairs were evaluated: activation was found to depend on the association constant of the coiled-coil peptides. The weaker-binding peptides required an additional disulfide linkage to induce cell-penetrating capability, whereas for the most-stable coiled-coil no additional stabilization was needed. The latter zipper pair was used to show that the induced formation of the coiled coils allows control over the uptake of an oligoarginine CPP-conjugated cargo protein.

Contains fulltext : 136500.pdf (Publisher’s version ) (Closed access) Increasing epidemiologic evidence supports a link between periodontitis and rheumatoid arthritis. The actual involvement of periodontitis in the pathogenesis of rheumatoid arthritis and the underlying mechanisms remain, however, poorly understood. We investigated the influence of concomitant periodontitis on clinical and histopathologic characteristics of T cell-mediated experimental arthritis and evaluated modulation of type II collagen (CII)-reactive Th cell phenotype as a potential mechanism. Repeated oral inoculations of periodontal pathogens Porphyromonas gingivalis and Prevotella nigrescens induced periodontitis in mice, as evidenced by alveolar bone resorption. Interestingly, concurrent periodontitis induced by both bacteria significantly aggravated the severity of collagen-induced arthritis. Exacerbation of arthritis was characterized by increased arthritic bone erosion, whereas cartilage damage remained unaffected. Both P. gingivalis and P. nigrescens skewed the CII-specific T cell response in lymph nodes draining arthritic joints toward the Th17 phenotype without affecting Th1. Importantly, the levels of IL-17 induced by periodontal pathogens in CII-specific T cells directly correlated with the intensity of arthritic bone erosion, suggesting relevance in pathology. Furthermore, IL-17 production was significantly correlated with periodontal disease-induced IL-6 in lymph node cell cultures. The effects of the two bacteria diverged in that P. nigrescens, in contrast to P. gingivalis, suppressed the joint-protective type 2 cytokines, including IL-4. Further in vitro studies showed that the Th17 induction strongly depended on TLR2 expression on APCs and was highly promoted by IL-1. Our data provide evidence of the involvement of periodontitis in the pathogenesis of T cell-driven arthritis through induction of Ag-specific Th17 response.

The enzyme peptidylarginine deiminase 2 (PAD2) has been associated with inflammatory diseases, such as rheumatoid arthritis and neurodegenerative diseases including multiple sclerosis. To investigate the association of various diseases with extracellular PAD2, we raised monoclonal antibodies (mAbs) against rabbit PAD2 and evaluated their cross-reactivity with human PAD2 by indirect enzyme-linked immunosorbent assay (ELISA), western blotting and immunohistological staining of inflamed synovial tissue. Moreover, we established a sandwich ELISA detecting human PAD2, based on two different monoclonal antibodies, mAbs DN2 and DN6. The assay had a lower detection limit of 200pg/mL in serum and plasma samples, and showed dilution linearity and recovery ranging from 95 to 106%. The mAbs and the ELISA showed isotype specificity for PAD2. Circulating PAD2 was found in 8/28 (29%) serum samples from healthy donors. In conclusion, several of our mAbs proved useful in western blotting and immunohistochemistry, and the ELISA described here reliably measures PAD2 levels in blood. This allows investigation of PAD2 as a possible biomarker and further investigation of PAD2's involvement in various inflammatory diseases.

Contains fulltext : 137883.pdf (Publisher’s version ) (Open Access) BACKGROUND: The presence of hypoxia in head and neck squamous cell carcinoma (HNSCC) is associated with therapeutic resistance and increased risk of metastasis formation. alphaB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether alphaB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces alphaB-crystallin expression in vitro and in this way may confer hypoxic cell survival. METHODS: In 38 HNSCC biopsies, the overlap between immunohistochemically stained alphaB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of alphaB-crystallin were analyzed in HNSCC cell lines under hypoxia and reoxygenation conditions and after exposure to reactive oxygen species (ROS) and the ROS scavenger N-acetylcysteine (NAC). siRNA-mediated knockdown was used to determine the influence of alphaB-crystallin on cell survival under hypoxic conditions. RESULTS: In all biopsies alphaB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased alphaB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, alphaB-crystallin expression was increased. alphaB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced alphaB-crystallin upregulation. Moreover, it was found that decreased alphaB-crystallin levels reduced cell survival under hypoxic conditions. CONCLUSIONS: We provide the first evidence that hypoxia stimulates upregulation of alphaB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of alphaB-crystallin may lead to prolonged survival of these cells under hypoxic conditions.

INTRODUCTION: Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients.METHODS: An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined.RESULTS: Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2) than of rhPAD4 were required for citrullination of fibrinogen. PAD activity was detected in four of five synovial fluid samples from RA patients and correlated with PAD2 concentrations in the samples (r = 0.98, P = 0.003). The calcium requirement for half-maximal activities of PAD2 and PAD4 were found in a range from 0.35 to 1.85 mM, and synovial fluid was found to contain sufficient calcium levels for the citrullination process to occur.CONCLUSIONS: We present an assay with high specificity for PAD2 activity and show that citrullination of fibrinogen can occur in cell-free synovial fluid from RA patients.

SUMMARY Immunization of BALB/c mice with purified recombinant human Ro52 protein resulted in three anti-Ro52 MoAbs termed 2E7, 4C6 and 4F11. All anti-Ro52 MoAbs specifically reacted with recombinant human Ro52 protein, and also with Ro52 protein in total extracts of all human cell lines analysed, including the epithelial cell line HeLa, the B cell line Raji, the bladder carcinoma cell line RT112, and a fibroblast cell line derived from patients with xeroderma pigmentosum. The anti-Ro52 MoAbs were able to immunoprecipitate the recombinant human Ro52 protein expressed in wheat germ extract, but failed to precipitate hY RNAs from cell extracts. The staining pattern of the MoAbs strongly differed between the RT112 cells and the fibroblast cell line. RT112 cells displayed an intense cytoplasmic staining and in addition distinct fine nuclear speckles. In contrast, in the fibroblast cell line no cytoplasmic staining but only staining of distinct nuclear speckles was observed. Using deletion mutants of Ro52 the epitopes recognized by the anti-Ro52 MoAbs 2E7, 4C6 and 4F11 were partially mapped. All three MoAbs appeared to recognize distinct epitopes, that are located in the regions of Ro52 bordered by amino acids 136–164, 208–363 and 136–190, respectively. These MoAbs can be of great use in studying the cellular processes in which the Ro52 protein is involved.

Citrullinated histone epitopes are involved in the very early stages of inflammatory responses. An important early event is the activation of neutrophils. It has been shown that Peptidyl Arginine Deiminase (PAD) expression levels increase upon pro-inflammatory signalling followed by activation of neutrophils. Subsequently, PAD enzymes cause histone citrullination in the activated neutrophils. Histone citrullination is involved in various processes. One of the most important is NETosis, which results in the release of citrullinated histones to the extracellular space. There, they are involved in Neutrophil Extracellular Trap (NET) formation, which intensifies the inflammatory response. The central role of citrullinated histones in early inflammation makes NETs an attractive target for inflammatory disease intervention. Moreover, the safety profile is expected to be superior to immune-suppressing biologicals, like anti- Tumour Necrosis Factor (TNF) drugs. It is anticipated that shielding citrullinated histone epitopes from the immune system, as well as interfering with their putative roles in the inflammatory response, will have a broad applicability in preventing and treating various inflammatory diseases, including multiple sclerosis.