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1.  Clinical and Serological Features of Patients Referred through a Rheumatology Triage System because of Positive Antinuclear Antibodies 
PLoS ONE  2014;9(4):e93812.
Background
The referral of patients with positive anti-nuclear antibody (ANA) tests has been criticized as an inappropriate use of medical resources. The utility of a positive ANA test in a central triage (CT) system was studied by determining the autoantibody profiles and clinical diagnoses of patients referred to rheumatologists through a CT system because of a positive ANA test.
Methods
Patients that met three criteria were included: (1) referred to Rheumatology CT over a three year interval; (2) reason for referral was a “positive ANA”; (3) were evaluated by a certified rheumatologist. The CT clinical database was used to obtain demographic and clinical information and a serological database was used to retrieve specific ANA and/or extractable nuclear antigen (ENA) test results. Clinical information was extracted from the consulting rheumatologist's report.
Results
15,357 patients were referred through the CT system; 643 (4.1%) of these because of a positive ANA and of these 263 (40.9%) were evaluated by a certified rheumatologist. In 63/263 (24%) of ANA positive patients, the specialist provided a diagnosis of an ANA associated rheumatic disease (AARD) while 69 (26.2%) had no evidence of any disease; 102 (38.8%) had other rheumatologic diagnoses and 29 (11%) had conditions that did not meet AARD classification criteria. Of ANA positive archived sera, 15.1% were anti-DFS70 positive and 91.2% of these did not have an AARD.
Conclusions
This is the first study to evaluate the serological and clinical features of patients referred through a CT system because of a positive ANA. The spectrum of autoantibody specificities was wide with anti-Ro52/TRIM21 being the most common autoantibody detected. Approximately 15% of referrals had only antibodies to DFS70, the vast majority of which did not have clinical evidence for an AARD. These findings provide insight into the utility of autoantibody testing in a CT system.
doi:10.1371/journal.pone.0093812
PMCID: PMC3976309  PMID: 24705829
2.  B cell depletion with rituximab in patients with diffuse cutaneous systemic sclerosis 
Arthritis and rheumatism  2009;60(2):578-583.
Rationale
This study was designed to determine safety, to provide preliminary data regarding potential efficacy and to investigate the effects on autoimmunity and fibrosis of rituximab in patients with diffuse cutaneous systemic sclerosis (dcSSc).
Methods
Fifteen patients with dcSSc, having their first non-Raynaud's associated disease manifestation within 18 months of trial entry, were recruited to receive two intravenous doses of 1000 mg rituximab two weeks apart. Safety, clinical, and exploratory outcomes were evaluated at baseline and at 6 months. The primary outcome was the change in modified Rodnan skin score (mRSS) at 6 months compared to baseline.
Results
Adverse events included frequent infusion reactions and rare infections (urinary tract infection and dental abscess, each in one patient). Mean mRSS change was not significantly different between baseline (20.6) and 6-months (20.2). Pulmonary function tests and other measures of major organ involvement were stable. Modest B cell infiltrates present in most skin biopsies at baseline were completely depleted at 6-months in most subjects. Autoantibody titers showed only modest and variable changes after treatment.
Conclusions
In this pilot study, treatment with rituximab appeared to be safe and well-tolerated among patients with dcSSc. Rituximab resulted in both depletion of circulating B cells and depletion of dermal B cells but had little effect on levels of SSc-associated autoantibodies. Rituximab did not appear to result in a significant beneficial effect on skin disease; the potential efficacy in other organs such as the lung could not be clearly evaluated in this small open label trial.
doi:10.1002/art.24249
PMCID: PMC2637937  PMID: 19180481
3.  Rpp25 is a major target of autoantibodies to the Th/To complex as measured by a novel chemiluminescent assay 
Introduction
Autoantibodies to the Th/To antigen have been described in systemic sclerosis (SSc) and several proteins of the macromolecular Th/To complex have been reported to react with anti-Th/To antibodies. However, anti-Th/To has not been clinically utilized due to unavailability of commercial tests. The objective of the present study is to evaluate the newly developed ELISA and chemiluminescent immunoassay (CLIA) to measure autoantibodies to Rpp25 (a component of the Th/To complex) using immunoprecipitation (IP) as the reference method.
Methods
The first cohort consisted of 123 SSc patients including 7 anti-Th/To positive samples confirmed by IP. Additional seven anti-Th/To positive samples from non-SSc patients were also tested. For evaluation of the QUANTA Flash Rpp25 CLIA (research use only), 8 anti-Th/To IP positives, a cohort of 70 unselected SSc patients and sera from various disease controls (n = 357) and random healthy individuals (n = 10) were studied.
Results
Anti-Rpp25 antibodies determined by ELISA were found in 11/14 anti-Th/To IP positive but only in 1/156 (0.6%) negative samples resulting in a positive percent agreement of 78.6% (95% confidence interval [CI] 49.2, 95.3%) and a negative percent agreement of 99.4% (95% CI 96.4, 100.0%). To verify the results using a second method, 53 samples were tested by ELISA and CLIA for anti-Rpp25 reactivity and the results were highly correlated (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001). To define the cutoff of the CLIA, anti-Th/To IP positive and negative sera were tested using the anti-Rpp25 CLIA. At the cutoff selected by receiver operating characteristic (ROC) analysis 8/8 (100.0%) of the anti-Th/To positive sera but only 2/367 (0.5%) of the controls were positive for anti-Rpp25 antibodies. The positive and negative percent agreements were 100.0% (95% CI 63.1, 100.0%) and 99.5% (95% CI 98.0, 99.9%), respectively. In the disease cohorts 2/70 (2.9%) of the SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). ROC analysis showed discrimination between SSc patients and controls with an area under the curve value of 0.732 (95% CI 0.655, 0.809).
Conclusion
Rpp25 is a major target of autoantibodies to the Th/To autoantigen complex. Further studies are needed to evaluate the clinical utility of the new assays.
doi:10.1186/ar4210
PMCID: PMC3672760  PMID: 23587095
4.  Clinical associations and potential novel antigenic targets of autoantibodies directed against rods and rings in chronic hepatitis C infection 
BMC Gastroenterology  2013;13:50.
Background
Chronic hepatitis C virus (HCV) infection is frequently associated with extrahepatic autoimmune disorders while interferon (IFN) and ribavirin treatment may exacerbate these conditions. Autoantibodies from HCV patients identify a novel indirect immunofluorescence (IIF) pattern on HEp-2 cells characterized by cytoplasmic rods and rings (RR). Our objectives were to determine the prevalence and clinical associations of RR autoantibodies in HCV patients, and identify related novel autoantibody targets.
Methods
Sera from 315 patients with HCV (301 treatment naive, 14 treated with interferon and/or ribavirin) were analyzed for the presence of RR antibodies by IIF on commercially available HEp-2 cell substrates. Antibodies to inosine monophosphate dehydrogenase 2 (IMPDH2) and cytidine triphosphate synthase 1 (CTPS1) were detected by addressable laser bead assay and other potential targets were identified by immunoscreening a protein microarray. Clinical and demographic data including HCV genotype, mode of infection, prior antiviral therapy, and histological findings were compared between RR antibody positive (RR+) and negative (RR-) patients.
Results
The median age of the HCV cohort was 51 years, 61% were male, and 76% were infected with HCV genotype 1 (G1). Four percent (n=14) had been treated with IFN-based therapy (IFN monotherapy, n=3; IFN/ribavirin, n=11); all had a sustained virologic response. In total, 15 patients (5% of the cohort) were RR+. RR+ and RR- patients had similar demographic and clinical characteristics including age, sex, mode of HCV infection, prevalence of the G1 HCV genotype, and moderate to severe fibrosis. Nevertheless, RR+ patients were significantly more likely than RR- cases to have been treated with IFN-based therapy (33% vs. 3%; adjusted odds ratio 20.5 [95% confidence interval 5.1-83.2]; P<0.0005). Only 1/10 RR positive sera had detectable antibodies to IMPHD2 and none had antibodies to CTPS1. Potentially important autoantibody targets identified on protein arrays included Myc-associated zinc finger protein (MAZI) and ankyrin repeat motif.
Conclusion
The majority of HCV patients with RR autoantibodies previously received IFN/ribavirin antiviral therapy. Further studies are necessary to determine the genesis of intracellular RR and elucidate the clinically relevant autoantigens as well as the clinical and prognostic significance of their cognate autoantibodies.
doi:10.1186/1471-230X-13-50
PMCID: PMC3606316  PMID: 23506439
5.  Clinical Phenotypes of Patients with Anti-DFS70/LEDGF Antibodies in a Routine ANA Referral Cohort 
Objective. To analyze the clinical value of anti-DFS70 antibodies in a cohort of patients undergoing routine antinuclear antibodies (ANAs) testing. Methods. Sera with a dense fine speckled (DFS) indirect immunofluorescence (IIF) pattern from 100 consecutive patients and 100 patients with other IIF patterns were tested for anti-DFS70 antibodies by a novel chemiluminescence immunoassay (CIA) and for ANA by ANA Screen ELISA (both INOVA). Results. Among the 100 patients with a DFS IIF pattern, 91% were anti-DFS70 positive by CIA compared to 3% in the comparator group (P < 0.0001). The CIA and IIF titers of anti-DFS antibodies were highly correlated (rho = 0.89). ANA by ELISA was positive in 35% of patients with the DFS IIF pattern as compared to 67% of patients with other patterns (P < 0.0001). Only 12.0% of patients with DFS pattern and 13.4% with DFS pattern and anti-DFS70 antibodies detected by CIA had systemic autoimmune rheumatic disease (SARD). Only 5/91 (5.5%) patients with anti-DFS70 antibodies had SARD and their sera were negative on the ANA Screen ELISA. Conclusion. Although anti-DFS70 antibodies cannot exclude the presence of SARD, the likelihood is significantly lower than in patients with other IIF patterns and should be included in test algorithms for ANA testing.
doi:10.1155/2013/703759
PMCID: PMC3580898  PMID: 23476678
6.  The Clinical Significance of the Dense Fine Speckled Immunofluorescence Pattern on HEp-2 Cells for the Diagnosis of Systemic Autoimmune Diseases 
Antinuclear antibodies (ANAs) are a serological hallmark in the diagnosis of systemic autoimmune rheumatic diseases (SARD). The indirect immunofluorescence (IIF) assay on HEp-2 cells is a commonly used test for the detection of ANA and has been recently recommended as the screening test of choice by a task force of the American College of Rheumatology. However, up to 20% of apparently healthy individuals (HI) have been reported to have a positive IIF ANA test, primarily related to autoantibodies that target the dense fine speckles 70 (DFS70) antigen. Even more important, the DFS IIF pattern has been reported in up to 33% of ANA positive HI, but not in ANA positive SARD sera. Since the intended use of the ANA HEp-2 test is to aid in the diagnosis and classification of SARD, the detection and reporting of anti-DFS70 antibodies and their associated pattern (DFS) as a positive test significantly reduce the specificity and the positive likelihood of the ANA test. This has significant implications for medical management and diagnostic algorithms involving the detection of ANA. Recently, a novel immunoadsorption method has been developed that specifically blocks anti-DFS70 antibodies and, therefore, significantly increases the specificity of the ANA test for SARD. This immunoadsorption method has the potential to overcome a significant limitation of the ANA HEp-2 assay. The present paper summarizes the current knowledge about anti-DFS70 antibodies and their clinical impact on ANA testing.
doi:10.1155/2012/494356
PMCID: PMC3523143  PMID: 23304189
7.  Anti-Fibrillarin Antibody in African American Patients with Systemic Sclerosis: Immunogenetics, Clinical Features, and Survival Analysis 
The Journal of rheumatology  2011;38(8):1622-1630.
Background
Anti-U3-RNP or anti-fibrillarin antibodies (AFA) are detected more frequently among African American (AA) patients with systemic sclerosis (SSc) compared to other ethnic groups and are associated with distinct clinical features. The current study examines the immunogenetic, clinical, and survival correlates of AFA in a large group of AA patients with SSc.
Methods
Overall, 278 AA SSc patients and 328 unaffected AA controls were enrolled from three North American cohorts. Clinical features, autoantibody profile, and HLA-class-II genotyping were captured. To compare the clinical manifestations, relevant clinical features were adjusted for disease duration. The Cox proportional hazards regression was used to determine the effect of AFA on survival.
Results
Fifty (18.5%) AA patients had AFA. After Bonferroni correction, HLA-DRB1*08:04 was associated with AFA, compared to unaffected AA controls (OR=11.5, p<0.0001) and AFA negative SSc patients (OR=5.2, p=0.0002). AFA positive AA patients had younger age of disease onset, higher frequency of digital ulcers, diarrhea, pericarditis, higher Medsger Perivascular and lower Lung Severity Indices (p=0.004, p=0.014, p=0.019, p=0.092, p=0.006, and p=0.016, respectively). After adjustment for age at enrollment, AFA positive patients did not have different survival compared with patients without AFA (p=0.493).
Conclusion
These findings demonstrate strong association between AFA and HLA-DRB1*08:04 allele in AA patients with SSc. Moreover, AA SSc patients with AFA had younger age of onset, higher frequency of digital ulcers, pericarditis, and severe lower gastrointestinal involvement, but less severe lung involvement compared to AA patients without AFA. However, presence of AFA did not change survival.
doi:10.3899/jrheum.110071
PMCID: PMC3149738  PMID: 21572159
Scleroderma; GENISOS; anti-U3-RNP; digital ulcer; HLA DRB1; and Scleroderma Family Registry
8.  Repression of GW/P body components and the RNAi microprocessor impacts primary ciliogenesis in human astrocytes 
BMC Cell Biology  2011;12:37.
Background
In most cells, the centriolar component of the centrosome can function as a basal body supporting the formation of a primary cilium, a non-motile sensory organelle that monitors information from the extracellular matrix and relays stimuli into the cell via associated signaling pathways. Defects in the formation and function of primary cilia underlie multiple human diseases and are hallmarks of malignancy. The RNA silencing pathway is involved in the post-transcriptional silencing of > 50% of mRNA that occurs within GW/P bodies. GW/P bodies are found throughout the cytoplasm and previously published live cell imaging data suggested that in a malignant cell type (U2OS), two GW/P bodies reside at the centrosome during interphase. This led us to investigate if a similar relationship exists in primary cells and if the inhibition of the miRNA pathway impairs primary cilium formation.
Results
Two GW/P bodies as marked by GW182 and hAgo2 colocalized to the basal body of primary human astrocytes as well as human synoviocytes during interphase and specifically with the distal end of the basal body in the pericentriolar region. Since it is technically challenging to examine the two centrosomal GW/P bodies in isolation, we investigated the potential relationship between the global population of GW/P bodies and primary ciliogenesis. Astrocytes were transfected with siRNA directed to GW182 and hAgo2 and unlike control astrocytes, a primary cilium was no longer associated with the centrosome as detected in indirect immunofluorescence assays. Ultrastructural analysis of siRNA transfected astrocytes revealed that knock down of GW182, hAgo2, Drosha and DGCR8 mRNA did not affect the appearance of the earliest stage of ciliogenesis but did prevent the formation and elongation of the ciliary axoneme.
Conclusions
This study confirms and extends a previously published report that GW/P bodies reside at the centrosome in U2OS cells and documents that GW/P bodies are resident at the centrosome in diverse non-malignant cells. Further, our study demonstrates that repression of key effector proteins in the post-transcriptional miRNA pathway impairs primary cilium formation.
doi:10.1186/1471-2121-12-37
PMCID: PMC3179929  PMID: 21880135
centrosome; centriole; basal body; primary cilia; P-bodies; GW182; Ago2; Drosha; DGCR8; siRNA; miRNA
9.  Divergent GW182 functional domains in the regulation of translational silencing 
Nucleic Acids Research  2010;39(7):2534-2547.
MicroRNA (miRNA)-mediated gene regulation has become a major focus in many biological processes. GW182 and its long isoform TNGW1 are marker proteins of GW/P bodies and bind to Argonaute proteins of the RNA induced silencing complex. The goal of this study is to further define and distinguish the repression domain(s) in human GW182/TNGW1. Two non-overlapping regions, Δ12 (amino acids 896–1219) containing the Ago hook and Δ5 (amino acids 1670–1962) containing the RRM, both induced comparable silencing in a tethering assay. Mapping data showed that the RRM and its flanking sequences in Δ5, but not the Ago hook in Δ12, were important for silencing. Repression mediated by Δ5 or Δ12 was not differentially affected when known endogenous repressors RCK/p54, GW182/TNGW1, TNRC6B were depleted. Transfected Δ5, but not Δ12, enhanced Ago2-mediated repression in a tethering assay. Transfected Δ12, but not Δ5, released endogenous miRNA reporter silencing without affecting siRNA function. Alanine substitution showed that GW/WG motifs in Δ12 (Δ12a, amino acids 896–1045) were important for silencing activity. Although Δ12 appeared to bind PABPC1 more efficiently than Δ5, neither Δ5 nor Δ12 significantly enhanced reporter mRNA degradation. These different functional characteristics of Δ5 and Δ12 suggest that their roles are distinct, and possibly dynamic, in human GW182-mediated silencing.
doi:10.1093/nar/gkq1099
PMCID: PMC3074120  PMID: 21131274
10.  The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells 
PLoS ONE  2010;5(10):e13445.
Background
GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells.
Methodology/Principal Findings
RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells.
Conclusions/Significance
The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.
doi:10.1371/journal.pone.0013445
PMCID: PMC2956662  PMID: 20976148
11.  Primary Biliary Cirrhosis (PBC), PBC Autoantibodies, and Hepatic Parameter Abnormalities in a Large Population of Systemic Sclerosis Patients 
The Journal of rheumatology  2009;36(10):2250-2256.
Objective
To investigate the diagnostic accuracy of antimitochondrial antibodies (AMA), sp100, and gp210 antibodies for primary biliary cirrhosis (PBC) in a large population of patients with systemic sclerosis (SSc); to examine concordance of these antibodies with subsets of SSc. Further, to assess the association of SSc-related antibodies with hepatic parameter abnormalities.
Methods
We obtained medical records to verify the diagnoses of SSc and PBC. Sera from all participants were examined for the presence of SSc- and PBC-related antibodies, as well as for abnormalities in hepatic parameters.
Results
We examined 817 patients with SSc, of whom 16 (2%) had confirmed PBC. The sensitivity and specificity of AMA by a MIT3 ELISA for PBC were 81.3% and 94.6%, respectively. Sp100 had a sensitivity and specificity of 31.3% and 97.4%, respectively, while gp210 had an even lower sensitivity. We were able to detect all PBC cases using AMA(MIT3) and sp100 as a combined marker, resulting in a significantly improved sensitivity of 100% (p = 0.042) with an incremental decrease in specificity to 92.6%. Independent of AMA or sp100 status, there was an association of anticentromere B (CENP-B) and anti-topoisomerase antibodies (ATA) with higher alkaline phosphatase levels (p = 0.051 and p = 0.003, respectively) while anti-RNA polymerase III (anti-RNAP) was associated with lower alkaline phosphatase levels (p = 0.019) among the patients with SSc.
Conclusion
Utilization of AMA(MIT3) and sp100 antibodies as a combined diagnostic marker leads to an improved detection of PBC in patients with SSc. CENP-B and ATA are associated with alkaline phosphatase elevation.
doi:10.3899/jrheum.090340
PMCID: PMC2885441  PMID: 19723904
LIVER CIRRHOSIS; BILIARY; SCLERODERMA; SYSTEMIC SCLEROSIS
12.  Major histocompatibility complex (MHC) class II alleles, haplotypes and epitopes which confer susceptibility or protection in systemic sclerosis: analyses in 1300 Caucasian, African-American and Hispanic cases and 1000 controls 
Annals of the rheumatic diseases  2009;69(5):822-827.
Objective
To determine human leucocyte antigen-class II (HLA-class II) (DRB1, DQB1, DQA1 and DPB1) alleles, haplotypes and shared epitopes associated with scleroderma (systemic sclerosis (SSc)) and its subphenotypes in a large multi-ethnic US cohort by a case–control association study.
Patients and methods
1300 SSc cases (961 white, 178 black and 161 Hispanic subjects) characterised for clinical skin forms (limited vs diffuse), SSc-specific autoantibodies (anticentromere (ACA), anti-topoisomerase I (ATA), anti-RNA polymerase III (ARA), anti-U3 ribonucleoprotein (fibrillarin)) and others were studied using molecular genotyping. Statistical analyses in SSc itself by ethnicity, gender, skin type and autoantibodies were performed using exact logistic regression modelling for dominant, additive and recessive effects from HLA.
Results
The strongest positive class II associations with SSc in white and Hispanic subjects were the DRB1*1104, DQA1*0501, DQB1*0301 haplotype and DQB1 alleles encoding a non-leucine residue at position 26 (DQB1 26 epi), while the DRB1*0701, DQA1*0201, DQB1*0202 haplotype and DRB1*1501 haplotype were negatively correlated and possibly protective in dominant and recessive models, respectively. These associations did not discriminate between limited and diffuse SSc. SSc in black subjects was associated with DRB1*0804, DQA1*0501, DQB1*0301 alleles. DPB1*1301 showed the highest odds ratio for ATA (OR = 14). Moreover, it showed no linkage disequilibrium or gene interaction with DR/DQ. ACA was best explained by DQB1*0501 and DQB1*26 epi alleles and ARA by DRB1*0404, DRB1*11 and DQB1*03 alleles in white and Hispanic subjects but DRB1*08 in black subjects.
Conclusion
These data indicate unique and multiple HLA-class II effects in SSc, especially on autoantibody markers of different subphenotypes.
doi:10.1136/ard.2009.111906
PMCID: PMC2916702  PMID: 19596691
13.  Co-clustering of Golgi complex and other cytoplasmic organelles to crescentic region of half-moon nuclei during apoptosis 
Cell biology international  2008;33(2):148-157.
Early apoptosis is defined by stereotypic morphological changes, especially evident in the nucleus, where chromatin condenses and compacts, and assumes a globular, half-moon or crescent-shaped morphology. Accumulating evidence suggests that cytoplasmic organelles such as mitochondria and the Golgi complex are major sites of integration of pro-apoptotic signaling. In this study, cytoplasmic organelles including Golgi complex, mitochondria, endosomes, lysosomes, and peroxisomes were shown to condense at the same unique region adjacent to the crescentic nucleus during a relatively early stage of apoptosis induced by staurosporine or other agents. The co-clustering phenomenon may be caused by shrinkage of cytoplasm during apoptosis although cytoskeletal markers actin and tubulin were not condensed and appeared excluded. These data suggest the co-clustering of cytoplasmic organelles plays an interesting role during the progression of the apoptotic process. It is possible that modification of pro-apoptotic proteins may arise as a result of the interplay of these cytoplasmic organelles.
doi:10.1016/j.cellbi.2008.10.016
PMCID: PMC2667205  PMID: 19000931
Apoptosis; Golgi complex; Staurosporine
14.  Optimization of immunoprecipitation–western blot analysis in detecting GW182-associated components of GW/P bodies 
Nature protocols  2009;4(5):674-685.
Characterizing the components of GW/processing bodies is key to elucidating RNA interference and messenger RNA processing pathways. This protocol addresses challenges in isolating a low-abundance protein GW182 and GW body (GWB)-associated proteins by building on previous reports that used polyclonal sera containing autoantibodies to GW/P body components. This protocol uses commercially available monoclonal antibodies to GW182 that are covalently coupled to Protein A or G sepharose beads and then used to immunoprecipitate GW182 and associated proteins from cell extracts. Immunoprecipitates are separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and probed by western blot with antibodies directed to proteins of interest. This protocol, which is expected to take 4–5 d, provides a biochemical approach for detecting GW182 and associated proteins in biological samples and thus facilitates the elucidation of the diverse functions of GWBs. It is expected that this protocol can be adapted to the detection of other RNA-binding complexes.
doi:10.1038/nprot.2009.34
PMCID: PMC2797048  PMID: 19373232
15.  Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells 
BMC Cancer  2009;9:448.
Background
Primary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression. Defects in the formation and/or function of these structures underlie a variety of human diseases such as Alström, Bardet-Biedl, Joubert, Meckel-Gruber and oral-facial-digital type 1 syndromes. The expression and function of primary cilia in cancer cells has now become a focus of attention but has not been studied in astrocytomas/glioblastomas. To begin to address this issue, we compared the structure and expression of primary cilia in a normal human astrocyte cell line with five human astrocytoma/glioblastoma cell lines.
Methods
Cultured normal human astrocytes and five human astrocytoma/glioblastoma cell lines were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. Monospecific antibodies were used to detect primary cilia and map the relationship between the primary cilia region and sites of endocytosis.
Results
We show that expression of primary cilia in normal astrocytes is cell cycle related and the primary cilium extends through the cell within a unique structure which we show to be a site of endocytosis. Importantly, we document that in each of the five astrocytoma/glioblastoma cell lines fully formed primary cilia are either expressed at a very low level, are completely absent or have aberrant forms, due to incomplete ciliogenesis.
Conclusions
The recent discovery of the importance of primary cilia in a variety of cell functions raises the possibility that this structure may have a role in a variety of cancers. Our finding that the formation of the primary cilium is disrupted in cells derived from astrocytoma/glioblastoma tumors provides the first evidence that altered primary cilium expression and function may be part of some malignant phenotypes. Further, we provide the first evidence that ciliogenesis is not an all or none process; rather defects can arrest this process at various points, particularly at the stage subsequent to basal body association with the plasma membrane.
doi:10.1186/1471-2407-9-448
PMCID: PMC2806408  PMID: 20017937
17.  The changing landscape of the clinical value of the PM/Scl autoantibody system 
Autoantibodies to the polymyositis/scleroderma (PM/Scl) complex have been associated with systemic sclerosis and PM/Scl overlap syndrome. The report of Hanke and colleagues in a recent issue of Arthritis Research and Therapy is the first to describe the separate evaluation of anti-PM/Scl-75c and PM/Scl-100 autoantibodies and their relationship to clinical manifestations of systemic sclerosis. Several observations are of paramount interest, but are not in general agreement with earlier studies. These include the prevalence of anti-PM/Scl antibodies in systemic sclerosis, the association with certain clinical manifestations and prognosis of patients. This report will hopefully trigger systematic multi-centre studies to confirm and/or elucidate the novel line immunoassay and clinical associations.
doi:10.1186/ar2646
PMCID: PMC2688186  PMID: 19351430
19.  Clinical and Serological Features of Patients with Autoantibodies to GW/P Bodies 
Clinical immunology (Orlando, Fla.)  2007;125(3):247-256.
GW bodies (GWBs) are unique cytoplasmic structures involved in messenger RNA (mRNA) processing and RNA interference (RNAi). GWBs contain, mRNA, components of the RNA-induced silencing complex (RISC), microRNA (miRNA), Argonaute proteins (i.e. Ago2), the Ge-1/Hedls protein and other enzymes involving mRNA degradation. The objective of this study was to identify the target GWB autoantigens reactive with 55 sera from patients with anti-GWB autoantibodies and to identify clinical features associated with these antibodies. Analysis by addressable laser bead immunoassay (ALBIA) and immunoprecipitation of recombinant proteins indicated that autoantibodies in this cohort of anti-GWB sera were directed against Ge-1/Hedls (58%), GW182 (40%) and Ago2 (9%). GWB autoantibodies targeted epitopes that included the N-terminus of Ago2 and the nuclear localization signal (NLS) containing region of Ge-1/Hedls. Clinical data was available on 42 patients of which 39 were female and the mean age was 61 years. The most common clinical presentations were neurological symptoms (i.e. ataxia, motor and sensory neuropathy) (33%), Sjögren’s syndrome (SjS) (31%) and the remainder had a variety of other diagnoses that included systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and primary biliary cirrhosis (PBC). Moreover, 44% of patients with anti-GWB antibodies had reactivity to Ro52. These studies indicate that Ge-1 is a common target of anti-GWB sera and the majority of patients in a GWB cohort had SjS and neurological disease.
doi:10.1016/j.clim.2007.07.016
PMCID: PMC2147044  PMID: 17870671
autoantibodies; P bodies GW182; Ago2; Ge-1
20.  Limited reliability of the indirect immunofluorescence technique for the detection of anti-Rib-P antibodies 
Arthritis Research & Therapy  2008;10(6):R131.
Introduction
Autoantibodies to the ribosomal P proteins represent a highly specific marker for the diagnosis of systemic lupus erythematosus, where they have been associated with certain clinical manifestations. Historically, autoantibodies against ribosomal P proteins have been detected by indirect immunofluorescence, immunodiffusion, immunoblot, and other immunoassays. More recently, enzyme-linked immunosorbent assays and line and addressable laser bead immunoassays have become more widely used. The primary goal of this study was to determine the sensitivity of indirect immunofluorescence using conventional HEp-2 substrates in the detection of sera with ribosomal P antibodies as detected by other immunoassays.
Methods
Anti-ribosomal P-positive sera (n = 345) as detected by an addressable laser bead immunoassay were collected between 2003 and 2007 and analysed by indirect immunofluorescence. Furthermore, 51 anti-ribosomal P-positive samples from an unselected systemic lupus erythematosus cohort (n = 100) and the Centers for Disease Control and Prevention (CDC) anti-nuclear antibody (ANA) reference sera were tested for anti-ribosomal P reactivity.
Results
In the cohort of 345 anti-ribosomal P-positive samples identified by addressable laser bead immunoassay, a low sensitivity (<30%) of indirect immunofluorescence on HEp-2 cell substrates was observed. Although the degree of sensitivity varied among different manufacturers, all immunofluorescence substrates exhibited limited sensitivity and false-negative results were not restricted to samples with low anti-ribosomal P titers. Even the anti-ribosomal P reactivity of CDC ANA reference serum number 12 was not clearly predictable by indirect immunofluorescence. Comparison of five different methods for the detection of anti-ribosomal P found moderate qualitative agreements.
Conclusions
Based on our data, we conclude that indirect immunofluorescence on HEp-2 cells is not a reliable screening test for the prediction of ribosomal P antibodies. As this method is widely used as a first-line screening test for anti-nuclear and other autoantibodies, special considerations for the detection of ribosomal P antibodies are needed. As with many other autoantibodies, further effort is required for the standardisation of ribosomal P immunoassays.
doi:10.1186/ar2548
PMCID: PMC2656233  PMID: 19000323
21.  Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies 
Molecular Biology of the Cell  2007;18(9):3375-3387.
Gene silencing using small interfering RNA (siRNA) is a valuable laboratory tool and a promising approach to therapeutics for a variety of human diseases. Recently, RNA interference (RNAi) has been linked to cytoplasmic GW bodies (GWB). However, the correlation between RNAi and the formation of GWB, also known as mammalian processing bodies, remains unclear. In this report, we show that transfection of functional siRNA induced larger and greater numbers of GWB. This siRNA-induced increase of GWB depended on the endogenous expression of the target mRNA. Knockdown of GW182 or Ago2 demonstrated that the siRNA-induced increase of GWB required these two proteins and correlated with RNAi. Furthermore, knockdown of rck/p54 or LSm1 did not prevent the reassembly of GWB that were induced by and correlated with siRNA-mediated RNA silencing. We propose that RNAi is a key regulatory mechanism for the assembly of GWB, and in some cases, GWB may serve as markers for RNAi in mammalian cells.
doi:10.1091/mbc.E07-01-0070
PMCID: PMC1951753  PMID: 17596515
22.  Heterogeneity of autoantibodies in 100 patients with autoimmune myositis: insights into clinical features and outcomes 
The objective of this study was to determine the prevalence, mutual associations, clinical manifestations, and diagnoses associated with serum autoantibodies, as detected using recently available immunoassays, in patients with autoimmune myositis (AIM). Sera and clinical data were collected from 100 patients with AIM followed longitudinally. Sera were screened cross-sectionally for 21 autoantibodies by multiplex addressable laser bead immunoassay, line blot immunoassay, immunoprecipitation of in vitro translated recombinant protein, protein A assisted immunoprecipitation, and enzyme-linked immunosorbent assay. Diagnoses were determined using the Bohan and Peter classification as well as recently proposed classifications. Relationships between autoantibodies and clinical manifestations were analyzed by multiple logistic regression. One or more autoantibodies encompassing 19 specificities were present in 80% of the patients. The most common autoantibodies were anti-Ro52 (30% of patients), anti-Ku (23%), anti-synthetases (22%), anti-U1RNP (15%), and anti-fibrillarin (14%). In the presence of autoantibodies to Ku, synthetases, U1RNP, fibrillarin, PM-Scl, or scleroderma autoantigens, at least one more autoantibody was detected in the majority of sera and at least two more autoantibodies in over one-third of sera. The largest number of concurrent autoantibodies was six autoantibodies. Overall, 44 distinct combinations of autoantibodies were counted. Most autoantibodies were unrestricted to any AIM diagnostic category. Distinct clinical syndromes and therapeutic responses were associated with anti-Jo-1, anti-fibrillarin, anti-U1RNP, anti-Ro, anti-Ro52, and autoantibodies to scleroderma autoantigens. We conclude that a significant proportion of AIM patients are characterized by complex associations of autoantibodies. Certain myositis autoantibodies are markers for distinct overlap syndromes and predict therapeutic outcomes. The ultimate clinical features, disease course, and response to therapy in a given AIM patient may be linked to the particular set of associated autoantibodies. These results provide a rationale for patient profiling and its application to therapeutics, because it cannot be assumed that the B-cell response is the same even in the majority of patients in a given diagnostic category.
doi:10.1186/ar2276
PMCID: PMC2206383  PMID: 17688695
23.  Effect of dsDNA binding to SmD-derived peptides on clinical accuracy in the diagnosis of systemic lupus erythematosus 
Systemic lupus erythematosus is characterized by antibodies to a variety of intracellular self-antigens, such as dsDNA and Sm, and these serve as hallmarks in the diagnosis of systemic autoimmune diseases. Several studies have shown that SmD1 and SmD3 synthetic peptides represent highly functional antigens for autoantibody detection and thus for diagnostic applications. The present study analysed the technical and clinical accuracy of an anti-SmD1 (amino acids 83–119) and an anti-SmD3 (amino acids 108–122) ELISA for the detection of anti-Sm antibodies. Depending on the cut-off value of the SmD1 ELISA, we found a high degree of concordance between the two tests. At an optimized cut-off value of 100 units for SmD1 we found the same clinical sensitivity (12.5%) and specificity (100%) in a group of systemic lupus erythematosus patients (n = 48) and in controls (n = 99). The concordance at this cut-off value was 100% (P < 0.0001; χ2 = 127.61). Using a second panel of sera (n = 65) preselected based on positive anti-Sm results, we confirmed the high degree of concordance between the two assays. Using dsDNA-coated ELISA plates and biotinylated peptides we confirmed the high dsDNA binding properties for SmD1, which were significantly higher than the SmD3-derived peptide. However, no cross-linking of anti-dsDNA antibodies to SmD1 was observed after adding increasing amounts of dsDNA to anti-dsDNA positive, anti-SmD1 negative serum. We therefore conclude that the reported difference in the sensitivity is related to the different cut-off levels and not to the detection of anti-dsDNA antibodies bridged via dsDNA to the SmD1 peptide. Moreover, we found that a subpopulation of anti-Sm antibodies cross-reacted with SmD1 and SmD3. Taken together, the data indicate that both SmD peptide ELISAs represent accurate assays and may be used as important standards for the detection of anti-Sm antibodies.
doi:10.1186/ar2266
PMCID: PMC2206372  PMID: 17640359
24.  Detection of the Argonaute Protein Ago2 and microRNAs in the RNA Induced Silencing Complex (RISC) Using a Monoclonal Antibody 
Journal of immunological methods  2006;317(1-2):38-44.
MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.
doi:10.1016/j.jim.2006.09.010
PMCID: PMC1913063  PMID: 17054975
Argonaut protein; GW182; GW bodies; Monoclonal antibody; MicroRNA; P body; RNA interference
25.  Autoimmune targeting of key components of RNA interference 
RNA interference (RNAi) is an evolutionarily conserved mechanism that is involved in the post-transcriptional silencing of genes. This process elicits the degradation or translational inhibition of mRNAs based on the complementarity with short interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently, differential expression of specific miRNAs and disruption of the miRNA synthetic pathway have been implicated in cancer; however, their role in autoimmune disease remains largely unknown. Here, we report that anti-Su autoantibodies from human patients with rheumatic diseases and in a mouse model of autoimmunity recognize the human Argonaute (Ago) protein, hAgo2, the catalytic core enzyme in the RNAi pathway. More specifically, 91% (20/22) of the human anti-Su sera were shown to immunoprecipitate the full-length recombinant hAgo2 protein. Indirect immunofluorescence studies in HEp-2 cells demonstrated that anti-Su autoantibodies target cytoplasmic foci identified as GW bodies (GWBs) or mammalian P bodies, structures recently linked to RNAi function. Furthermore, anti-Su sera were also capable of immunoprecipitating additional key components of the RNAi pathway, including hAgo1, -3, -4, and Dicer. Together, these results demonstrate an autoimmune response to components of the RNAi pathway which could potentially implicate the involvement of an innate anti-viral response in the pathogenesis of autoantibody production.
doi:10.1186/ar1959
PMCID: PMC1779426  PMID: 16684366

Results 1-25 (40)