Marvin J. Fritzler

Stress granules and processing bodies are dynamically linked sites of mRNP remodeling

Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2α phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.

Disruption of GW bodies impairs mammalian RNA interference.

The GW182 RNA-binding protein was initially shown to associate with a specific subset of mRNAs and to reside within discrete cytoplasmic foci named GW bodies (GWBs). GWBs are enriched in proteins that are involved in mRNA degradation. Recent reports have shown that exogenously introduced human Argonaute-2 (Ago2) is also enriched in GWBs, indicating that RNA interference function may be somehow linked to these structures. In this report, we demonstrate that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2. Disruption of these cytoplasmic foci in HeLa cells interferes with the silencing capability of a siRNA that is specific to lamin-A/C. Our data support a model in which GW182 and/or the microenvironment of the cytoplasmic GWBs contribute to the RNA-induced silencing complex and to RNA silencing.

Autoantibodies and microvascular damage are independent predictive factors for the progression of Raynaud's phenomenon to systemic sclerosis: A twenty‐year prospective study of 586 patients, with validation of proposed criteria for early systemic sclerosis

OBJECTIVE To identify in patients with Raynauds phenomenon (RP) independent markers that predict progression to definite systemic sclerosis (SSc) and to determine in patients with progression to SSc the type and sequence of microvascular damage and its relationship to SSc-specific autoantibodies. METHODS Consecutive patients referred for evaluation of RP who had no definite connective tissue disease were evaluated for microvascular damage by nailfold capillary microscopy (NCM) and for anticentromere (anti-CENP-B), anti-Th/To, anti-topoisomerase I, and anti-RNA polymerase III (anti-RNAP III) autoantibodies by specific assays. Patients were studied prospectively. RESULTS Of the 586 patients who were followed up for 3,197 person-years, 74 (12.6%) developed definite SSc. A characteristic sequence of microvascular damage was identified, starting with enlarged capillaries, followed by capillary loss, and then by capillary telangiectases. Definite SSc was diagnosed in close temporal relationship to capillary loss. Enlarged capillaries, capillary loss, and SSc-specific autoantibodies independently predicted definite SSc. Anti-CENP-B and anti-Th/To antibodies predicted enlarged capillaries; these autoantibodies and anti-RNAP III predicted capillary loss. Each autoantibody was associated with a distinct time course of microvascular damage. At followup, 79.5% of patients with 1 of these autoantibodies and abnormal findings on NCM at baseline had developed definite SSc. Patients with both baseline predictors were 60 times more likely to develop definite SSc. The data validated the proposed criteria for early SSc. CONCLUSION In RP evolving to definite SSc, microvascular damage is dynamic and sequential, while SSc-specific autoantibodies are associated with the course and type of capillary abnormalities. Abnormal findings on NCM at baseline together with an SSc-specific autoantibody indicate a very high probability of developing definite SSc, whereas their absence rules out this outcome.

The CREST syndrome: A distinct serologic entity with anticentromere antibodies

The CREST syndrome is a variant of systemic sclerosis characterized by the presence of calcinosis. Raynauds phenomenon, esophageal motility abnormalities, sclerodactyly and telangiectasia. The serums of 27 patients with the CREST syndrome have been examined for the presence of antinuclear antibodies. Twenty-six of 27 (98 percent) serums contained high titers (> one:80) of an antibody that produces a discrete speckled pattern of immunofluorescence on a human laryngeal carcinoma cell line (HEp-2). The antibody has been shown to react with the centromeric region of metaphase chromosomes. This antibody was also found in three of 14 patients with Raynauds disease, in one of 60 patients with systemic lupus erythematosus, in three of 26 patients with systemic sclerosis with diffuse scleroderma and in one of 15 patients with mixed connective tissue disease. The antibody was not detected in the serums of patients with rheumatoid arthritis. Sjögrens sicca complex or linear scleroderma. Patients with osteoarthritis who were age- and sex-matched to the group with the CREST syndrome did not have anticentromere antibodies. Autoantibodies found in other connective tissue diseases (anti-DNA, anti-RNP, Sjögrens syndrome antigen B (anti-SS-B) were not found in serums from patients with the CREST syndrome. A case report illustrating the appearance of the anticentromere antibody at a time when Raynauds phenomenon antedated the clinical diagnosis of CREST syndrome is presented.

International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies

Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1–13), anti-double stranded DNA antibodies (14–18), specific antibodies (19–23) and validation of methods (24–25) were created. Significant differences between experts were observed regarding recommendations 24–25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

B cell depletion with rituximab in patients with diffuse cutaneous systemic sclerosis

OBJECTIVE To determine the safety of rituximab, to provide preliminary data regarding the potential efficacy of rituximab, and to investigate the effects of rituximab on autoimmunity and fibrosis in patients with diffuse cutaneous systemic sclerosis (dcSSc). METHODS Fifteen patients with dcSSc, all of whom experienced their first non-Raynauds disease-associated disease manifestation within 18 months of trial entry, were recruited to receive 2 intravenous doses of rituximab (1,000 mg), administered 2 weeks apart. Safety, clinical, and exploratory outcomes were evaluated at baseline and at 6 months. The primary outcome was the change in the modified Rodnan skin thickness score (MRSS) at 6 months compared with baseline. RESULTS Adverse events included frequent infusion reactions and rare infections (urinary tract infection and dental abscess occurred in 1 patient each). The mean change in the MRSS between baseline and 6 months was not significant. Results of pulmonary function tests and other measures of major organ involvement were stable. The modest B cell infiltrates that were present in most skin biopsy specimens at baseline were completely depleted at 6 months in most patients. Autoantibody titers showed only modest and variable changes after treatment. CONCLUSION In this pilot study, treatment with rituximab appeared to be safe and well tolerated among patients with dcSSc. Rituximab treatment resulted in both depletion of circulating B cells and depletion of dermal B cells but had little effect on the levels of SSc-associated autoantibodies. Rituximab treatment did not appear to result in a significant beneficial effect on skin disease. The potential efficacy of rituximab in other organs such as the lung could not be clearly evaluated in this small open-label trial.

GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation

A novel cytoplasmic compartment referred to as GW bodies was initially identified using human autoantibodies to a 182 kDa protein named GW182. GW bodies are small, generally spherical, cytoplasmic domains that vary in number and size in several mammalian cell types examined to date. Based on our earlier studies, GW bodies were proposed to be cytoplasmic sites for mRNA storage and/or degradation. In the present study, immunogold electron microscopy identified electron dense structures of 100-300 nm diameter devoid of a lipid bilayer membrane. These structures appeared to comprise clusters of electron dense strands of 8-10 nm in diameter. By costaining with CENP-F and PCNA, and employing a double-thymidine block to synchronize HeLa cells, GW bodies were observed to be small in early S phase and larger during late S and G2 phases of the cell cycle. The majority of GW bodies disassembled prior to mitosis and small GW bodies reassembled in early G1. The analysis of GW bodies in two experimental models of cell proliferation using reversal of 3T3/serum-starvation and concanavalin A stimulation of mouse splenocytes and T cells, revealed that proliferating cells contained larger, brighter, and more numerous GW bodies as well as up to a fivefold more total GW182 protein than quiescent cells. In vitro gene knockdown of GW182 led to the disappearance of GW bodies demonstrating that GW182 is a critical component of GW bodies. The incremental expression of the GW182 protein in cells induced to proliferate and the cyclic formation and breakdown of GW bodies during mitosis are intriguing in view of the notion that GW bodies are specialized centers involved in maintaining stability and/or controlling degradation of mRNA.

Addendum to the International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies Quality Control Guidelines, Comments, and Recommendations for Testing in Other Autoimmune Diseases

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in Wegener granulomatosis, microscopic polyangiitis and its renal-limited variant (pauci-immune crescentic glomerulonephritis), and Churg-Strauss syndrome. The International Consensus Statement on testing and reporting of ANCA states that ANCA are demonstrated most readily in these conditions by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 or myeloperoxidase. The group that produced the International Consensus Statement has developed guidelines for the corresponding quality control activities, examples of comments for various IIF patterns and ELISA results, and recommendations for ANCA testing when inflammatory bowel disease and other nonvasculitic ANCA-associated autoimmune diseases are suspected.

Marked differences in fine specificity and isotype usage of the anti–citrullinated protein antibody in health and disease

OBJECTIVE Anti-citrullinated protein antibodies (ACPAs) display high association with rheumatoid arthritis (RA) and are implicated in its pathogenesis. The presence of ACPAs is known to precede the onset of RA. In order to identify the features that could confer its pathogenicity, we extensively characterized this antibody response in a unique North American native population of patients with RA and their unaffected relatives. METHODS The levels of IgA, IgM, and IgG ACPAs, as well as IgM and IgA rheumatoid factor (RF), were measured in serum samples obtained from 81 patients with RA and 195 of their unaffected relatives. The isotype distribution, the fine specificity of the ACPA response, and its association with RF were compared in health and disease. RESULTS ACPA positivity was observed in 19% of the healthy relatives and approximately 91% of the patients with RA. ACPA isotype usage was strikingly lower in unaffected relatives than in patients with RA (1-2 versus 5-6 isotypes). Fine specificity studies showed that reactivity to citrullinated fibrinogen and vimentin was present in sera from patients with RA, while it was virtually absent in their unaffected relatives. Finally, the ACPA and RF responses were associated in patients with RA but were discordant in their healthy relatives. Extended analyses revealed that the presence of ACPAs was associated with RA irrespective of RF status, while the association of RF with disease relied on its interaction with ACPAs. CONCLUSION The fine specificity and isotype usage of the ACPA response are qualitatively different in health and disease. Epitope spreading and expansion of the isotype repertoire might be necessary for development of RA, and this could be facilitated by the presence of RF antibodies.

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.