Complex genetic association of 6q23 with autoimmune rheumatic conditions

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.     

Anti-PM/Scl antibodies are found in Japanese patients with various systemic autoimmune conditions besides myositis and scleroderma

IntroductionAnti-PM/Scl antibodies are associated with polymyositis (PM)/systemic scleroderma (SSc) overlap syndromes and are also found in other systemic autoimmune diseases. Although anti-PM/Scl reactivity is found in 3-11% of PM or SSc patients and in approximately 25% of PM/SSc overlap patients, previous large studies of Japanese patients with scleroderma reported that anti-PM/Scl are not found in Japanese patients at all. The PM/Scl autoantigen complex comprises 11–16 different polypeptides; ELISA with PM1-α peptide, which is a major epitope of the PM/Scl complex, has frequently been used for the detection of these antibodies in recent studies. However, no ELISA kit is commercially available in Japan.MethodsIn this study, we developed an immunoassay for measuring antibodies against recombinant PM/Scl-100 and PM/Scl-75 polypeptides, which are the two major targets of the complex, and we investigated their presence in 600 Japanese patients with various systemic autoimmune conditions. Immunoprecipitation analysis using the recombinants in addition to traditional radiolabeled cell extracts were also applied to ELISA-positive sera.ResultsIn ELISA, 11 patients were positive for anti-PM/Scl-100 antibodies and 7 of these 11 patients were also positive for anti-PM/Scl-75 antibodies. Immunoprecipitation analysis using the recombinants in addition to traditional radiolabeled cell extracts confirmed that 9 out of these 11 patients immunoprecipitated the typical sets of PM/Scl proteins. In total, 4/16 (25%) undifferentiated connective tissue disease (UCTD) patients, 3/126 (2.4%) dermatomyositis patients, 1/223 (0.4%) SSc patients, 1/88 (1.1%) Sjögren’s syndrome patients, 0/123 patients with systemic lupus erythematosus, 0/17 patients with overlap syndrome and 0/7 patients with PM were judged to be positive for anti-PM/Scl antibodies.ConclusionsThis is the first report of Japanese autoimmune patients with anti-PM/Scl antibodies. In Japanese patients, anti-PM/Scl antibodies are only very rarely found, and they are not always specific for dermatomyositis (DM) or SSc; they are also present in various autoimmune conditions with the highest prevalence being in UCTD. All anti-PM/Scl-positive DM cases are complicated with interstitial lung disease and/or cancer, while no life-threatening involvement was found in other anti-PM/Scl-positive cases. Further studies on larger cohorts are necessary to define the clinical significance of anti-PM/Scl antibodies in autoimmune diseases.     

Clinical evaluation of autoantibodies to a novel PM/Scl peptide antigen

Anti-PM/Scl antibodies represent a specific serological marker for a subset of patients with scleroderma (Scl) and polymyositis (PM), and especially with the PM/Scl overlap syndrome (PM/Scl). Anti-PM/Scl reactivity is found in 24% of PM/Scl patients and is found in 3–10% of Scl and PM patients. The PM/Scl autoantigen complex comprises 11–16 different polypeptides. Many of those proteins can serve as targets of the anti-PM/Scl B-cell response, but most frequently the PM/Scl-100 and PM/Scl-75 polypeptides are targeted. In the present study we investigated the clinical relevance of a major alpha helical PM/Scl-100 epitope (PM1-α) using a newly developed peptide-based immunoassay and compared the immunological properties of this peptide with native and recombinant PM/Scl antigens. In a technical comparison, we showed that an ELISA based on the PM1-α peptide is more sensitive than common techniques to detect anti-PM/Scl antibodies such as immunoblot, indirect immunofluorescence on HEp-2 cells and ELISA with recombinant PM/Scl polypeptides. We found no statistical evidence of a positive association between anti-PM1-α and other antibodies, with the exception of known PM/Scl components. In our cohort a negative correlation could be found with anti-Scl-70 (topoisomerase I), anti-Jo-1 (histidyl tRNA synthetase) and anti-centromere proteins. In a multicenter evaluation we demonstrated that the PM1-α peptide represents a sensitive and reliable substrate for the detection of a subclass of anti-PM/Scl antibodies. In total, 22/40 (55%) PM/Scl patients, 27/205 (13.2%) Scl patients and 3/40 (7.5%) PM patients, but only 5/288 (1.7%) unrelated controls, tested positive for the anti-PM1-α peptide antibodies. These data indicate that anti-PM1-α antibodies appear to be exclusively present in sera from PM/Scl patients, from Scl patients and, to a lesser extent, from PM patients. The anti-PM1-α ELISA thus offers a new serological marker to diagnose and discriminate different systemic autoimmune disorders.     

Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

The human exosome: an autoantigenic complex of exoribonucleases in myositis and scleroderma

The anti-PM/Scl autoantibodies are known to characterize a subset of autoimmune patients with myositis, scleroderma (Scl), and the PM/Scl overlap syndrome. The major autoantigens that are recognized by anti-PM/Scl autoantibodies are designated PM/Scl-100 and PM/Scl-75. These autoantigens have been reported to associate into a large complex consisting of 11 to 16 proteins and to play a role in ribosome synthesis. Recently, it was discovered that the PM/Scl complex is the human counterpart of the yeast (Saccharomyces cerevisiae) exosome, which is an RNA-processing complex consisting of 11 3' → 5' exoribonucleases. To date, 10 human exosome components have been identified, although only some of these were studied in more detail. In this review, we discuss some recent advances in the characterization of the PM/Scl complex.     

Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

Clinical significance of antibodies to Ro52/TRIM21 in systemic sclerosis

Introduction

Autoantibodies to Ro52 recently identified as TRIM21 are among the most common autoantibodies in systemic autoimmune rheumatic diseases, but their clinical association remains poorly understood. We undertook this study to determine the clinical and serologic associations of anti-Ro52/TRIM21 antibodies in patients with systemic sclerosis (SSc).

Methods

Detailed clinical data and sera from 963 patients with SSc enrolled in a multicenter cohort study were collected and entered into a central database. Antibodies to Ro52/TRIM21 and other autoantibodies were detected with an addressable laser-bead immunoassay and different enzyme-linked immunosorbent assay (ELISA) systems. Associations between anti-Ro52/TRIM21 antibodies and clinical and other serologic manifestations of SSc were investigated.

Results

Anti-Ro52/TRIM21 antibodies were present in 20% of SSc patients and overlapped with other main SSc-related antibodies, including anti-centromere (by immunofluorescence and centromere protein (CENP)-A and CENP-B ELISA), anti-topoisomerase I, anti-RNA polymerase III, and anti-Pm/Scl antibodies. Anti-Ro52/TRIM21 antibodies were strongly associated with interstitial lung disease (odds ratio (OR), 1.53; 95% confidence interval (CI), 1.11 to 2.12; P = 0.0091) and overlap syndrome (OR, 2.06; 95% CI, 1.01 to 4.19; P = 0.0059).

Conclusions

Anti-Ro52/TRIM21 antibodies were the second most common autoantibodies in this SSc cohort. In SSc, anti-Ro52/TRIM21 antibodies may be a marker of interstitial lung disease and overlap syndrome.     

The association of the human PM/Scl-75 autoantigen with the exosome is dependent on a newly identified N terminus

The exosome is a complex of 3' --> 5' exoribonucleases that functions in a variety of cellular processes, all concerning the processing or degradation of RNA. Paradoxically, the previously described cDNA for the human autoantigenic exosome subunit PM/Scl-75 (Alderuccio, F., Chan, E. K., and Tan, E. M. (1991) J. Exp. Med. 173, 941-952) encodes a polypeptide that failed to interact with the exosome complex. Here, we describe the cloning of a more complete cDNA for PM/Scl-75 encoding 84 additional amino acids at its N terminus. We show that only the longer polypeptide is able to associate with the exosome complex. This interaction is most likely mediated by protein-protein interactions with two other exosome subunits, hRrp46p and hRrp41p, one of which was confirmed in a mammalian two-hybrid system. In addition we show that the putative nuclear localization signal present in the C-terminal region of PM/Scl-75 is sufficient, although not essential for nuclear localization of the protein. Moreover, the deletion of this element abrogated the nucleolar accumulation of PM/Scl-75, although its association with the exosome was not disturbed. This suggests that this basic element of PM/Scl-75 plays a role in targeting the exosome to the nucleolus.     

C1D and hMtr4p associate with the human exosome subunit PM/Scl-100 and are involved in pre-rRNA processing

The exosome is a complex of 3'-5' exoribonucleases and RNA-binding proteins, which is involved in processing or degradation of different classes of RNA. Previously, the characterization of purified exosome complexes from yeast and human cells suggested that C1D and KIAA0052/hMtr4p are associated with the exosome and thus might regulate its functional activities. Subcellular localization experiments demonstrated that C1D and KIAA0052/hMtr4p co-localize with exosome subunit PM/Scl-100 in the nucleoli of HEp-2 cells. Additionally, the nucleolar accumulation of C1D appeared to be dependent on PM/Scl-100. Protein-protein interaction studies showed that C1D binds to PM/Scl-100, whereas KIAA0052/hMtr4p was found to interact with MPP6, a previously identified exosome-associated protein. Moreover, we demonstrate that C1D, MPP6 and PM/Scl-100 form a stable trimeric complex in vitro. Knock-down of C1D, MPP6 and KIAA0052/hMtr4p by RNAi resulted in the accumulation of 3'-extended 5.8S rRNA precursors, showing that these proteins are required for rRNA processing. Interestingly, C1D appeared to contain RNA-binding activity with a potential preference for structured RNAs. Taken together, our results are consistent with a role for the exosome-associated proteins C1D, MPP6 and KIAA052/hMtr4p in the recruitment of the exosome to pre-rRNA to mediate the 3' end processing of the 5.8S rRNA.     

Human cell growth requires a functional cytoplasmic exosome, which is involved in various mRNA decay pathways

The human exosome is a 3'-5' exoribonuclease complex that functions both in the nucleus and in the cytoplasm to either degrade or process RNA. Little is known yet about potential differences among core exosome complexes in these different cellular compartments and the roles of the individual subunits in maintaining a stable and functional complex. Glycerol gradient sedimentation analyses indicated that a significant subset of nuclear exosomes is present in much larger complexes (60-80S) than the cytoplasmic exosomes ( approximately 10S). Interestingly, siRNA-mediated knock-down experiments indicated that the cytoplasmic exosome is down-regulated much more efficiently than the nuclear exosome. In addition, we observed that knock-down of hRrp41p or hRrp4p but not PM/Scl-100 or PM/Scl-75 leads to codepletion of other subunits. Nevertheless, PM/Scl-100 and PM/Scl-75 are required to maintain normal levels of three different mRNA reporters: a wild-type beta-globin mRNA, a beta-globin mRNA containing an AU-rich (ARE) instability element, and a beta-globin mRNA bearing a premature termination codon (PTC). The increased levels of ARE- and the PTC-containing mRNAs upon down-regulation of the different exosome subunits, in particular PM/Scl-100, appeared to be due to decreased turnover rates. These results indicate that, although not required for exosome stability, PM/Scl-100 and PM/Scl-75 are involved in mRNA degradation, either as essential subunits of a functional exosome complex or as exosome-independent proteins.     

The peritrophic matrix limits the rate of digestion in adult Anopheles stephensi and Aedes aegypti mosquitoes

The peritrophic matrix (PM) is a chitin-containing acellular sheath that surrounds the blood meal and separates the food bolus from the midgut epithelium. Intense molecular traffic through the PM occurs during digestion. Digestive enzymes secreted by the midgut epithelium must traverse the PM to reach their substrates in the food bolus, and digestion products must cross the PM in the opposite direction to be absorbed by the epithelial cells. Here we report that the PM limits the rate of digestion. PM disruption by two independent means (chitinase and anti-PM antibodies) consistently increases the rate of blood digestion. The significance of these results in relation to PM function is discussed.     

Evaluation of Current Knowledge, Awareness and Practice of Spirometry among Hospital -based Nigerian Doctors

Background

The association between systemic sclerosis and pulmonary arterial hypertension (PAH) is well recognized. Vascular endothelial growth factor (VEGF) has been reported to play an important role in pulmonary hypertension. The aim of the present study was to examine the relationship between systolic pulmonary artery pressure, clinical and functional manifestations of the disease and serum VEGF levels in systemic sclerosis.

Methods

Serum VEGF levels were measured in 40 patients with systemic sclerosis and 13 control subjects. All patients underwent clinical examination, pulmonary function tests and echocardiography.

Results

Serum VEGF levels were higher in systemic sclerosis patients with sPAP ≥ 35 mmHg than in those with sPAP < 35 mmHg (352 (266, 462 pg/ml)) vs (240 (201, 275 pg/ml)) (p < 0.01), while they did not differ between systemic sclerosis patients with sPAP < 35 mmHg and controls. Serum VEGF levels correlated to systolic pulmonary artery pressure, to diffusing capacity for carbon monoxide and to MRC dyspnea score. In multiple linear regression analysis, serum VEGF levels, MRC dyspnea score, and D_LCO were independent predictors of systolic pulmonary artery pressure.

Conclusion

Serum VEGF levels are increased in systemic sclerosis patients with sPAP ≥ 35 mmHg. The correlation between VEGF levels and systolic pulmonary artery pressure may suggest a possible role of VEGF in the pathogenesis of PAH in systemic sclerosis.     

The Crystal Structure of the Streptococcal Collagen-like Protein 2 Globular Domain from Invasive M3-type Group A Streptococcus Shows Significant Similarity to Immunomodulatory HIV Protein gp41

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.     

Implications in the difference of anti-Mi-2 and -p155/140 autoantibody prevalence in two dermatomyositis cohorts from Mexico City and Guadalajara

Introduction

Autoantibodies and clinical manifestations in polymyositis/dermatomyositis (PM/DM) are affected by both genetic and environmental factors. The high prevalence of DM and anti-Mi-2 in Central America is thought to be associated with the high UV index of the area. The prevalences of autoantibodies and the clinical manifestations of PM/DM were evaluated comparing two cohorts in Mexico.

Methods

Ninety-five Mexican patients with PM/DM (66 DM, 29 PM; 67 Mexico City, 28 Guadalajara) were studied. Autoantibodies were characterized by immunoprecipitation using ³⁵S-methionine labeled K562 cell extract. Clinical information was obtained from medical records.

Results

DM represented 69% of PM/DM and anti-Mi-2 was the most common autoantibody (35%), followed by anti-p155/140 (11%); however, anti-Jo-1 was only 4%. The autoantibody profile in adult-onset DM in Mexico City versus Guadalajara showed striking differences: anti-Mi-2 was 59% versus 12% (P = 0.0012) whereas anti-p155/140 was 9% versus 35% (P = 0.02), respectively. A strong association of anti-Mi-2 with DM was confirmed and when clinical features of anti-Mi-2 (+) DM (n = 30) versus anti-Mi-2 (-) DM (n = 36) were compared, the shawl sign (86% versus 64%, P < 0.05) was more common in the anti-Mi-2 (+) group (P = 0.0001). Levels of creatine phosphokinase (CPK) were higher in those who were anti-Mi-2 (+) but they responded well to therapy.

Conclusions

Anti-Mi-2 has a high prevalence in Mexican DM and is associated with the shawl sign and high CPK. The prevalence of anti-Mi-2 and anti-p155/140 was significantly different in Mexico City versus Guadalajara, which have a similar UV index. This suggests roles of factors other than UV in anti-Mi-2 antibody production.     

Peritrophic membrane structure and formation in the larva of a moth, Heliothis

The peritrophic membrane (PM) in tobacco budworm larvae (Heliothis virescens, Lepidoptera: Noctuidae), is a continuous sac which encloses the food bolus in the midgut and hindgut. The PM is a single-walled structure 3-5 mum thick which is comprised of two main layers or laminae. The laminae may be fused into a single structure or remain separated by a space which may contain additional thin strands of matrix. Staining with an anti-PM antibody and wheat germ agglutinin (WGA) illustrate the laminar nature of the PM and suggest that protein and chitin have co-incident spatial distributions within the matrix. By transmission electron microscopy, the PM is composed of a loose network of fibrils and small granules, the only structural difference among laminae being a compaction of the matrix along the edges of the two limiting laminae facing the endoperitrophic and ectoperitrophic spaces. By scanning electron microscopy, the PM surface has a wrinkled, felt-like texture without pores or slits. Contrary to the classical view that lepidopterans are Type I insects with respect to PM formation in which the PM forms along the full length of the midgut, the PM in the tobacco budworm forms primarily from secretions of specialized midgut epithelial cells at the junction of the foregut and midgut. The secretory cells, their secretions and the nascent PM stain intensely with the anti-PM antibody but not with WGA suggesting that chitin is added more posteriorly. The PM may be supplemented by the addition of minor amounts of matrix material along the length of the midgut. PM synthesis begins during embryogenesis prior to the initiation of feeding. The PM in neonates is only about 0.1 mum thick but otherwise is structurally similar to that in older larvae.     

Angiogenic and angiostatic factors in systemic sclerosis: increased levels of vascular endothelial growth factor are a feature of the earliest disease stages and are associated with the absence of fingertip ulcers

To examine whether the lack of sufficient neoangiogenesis in systemic sclerosis (SSc) is caused by a decrease in angiogenic factors and/or an increase in angiostatic factors, the potent proangiogenic molecules vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, and the angiostatic factor endostatin were determined in patients with SSc and in healthy controls. Forty-three patients with established SSc and nine patients with pre-SSc were included in the study. Serum levels of VEGF, basic fibroblast growth factor and endostatin were measured by ELISA. Age-matched and sex-matched healthy volunteers were used as controls. Highly significant differences were found in serum levels of VEGF between SSc patients and healthy controls, whereas no differences could be detected for endostatin and basic fibroblast growth factor. Significantly higher levels of VEGF were detected in patients with Scl-70 autoantibodies and in patients with diffuse SSc. Patients with pre-SSc and short disease duration showed significant higher levels of VEGF than healthy controls, indicating that elevated serum levels of VEGF are a feature of the earliest disease stages. Patients without fingertip ulcers were found to have higher levels of VEGF than patients with fingertip ulcers. Levels of endostatin were associated with the presence of giant capillaries in nailfold capillaroscopy, but not with any other clinical parameter. The results show that the concentration of VEGF is already increased in the serum of SSc patients at the earliest stages of the disease. VEGF appears to be protective against ischemic manifestations when concentrations of VEGF exceed a certain threshold level.     

Treatment of autoimmune disease with extracorporeal photochemotherapy: progressive systemic sclerosis.

In this report, we describe the use of extracorporeal photochemotherapy in n the treatment of two patients with rapidly advancing progressive systemic sclerosis. Both patients experienced improvement in the cutaneous as well as the systemic manifestations of their disease while undergoing therapy. The potential therapeutic mechanisms are discussed.