Clinical significance of autoantibodies in dermatomyositis and systemic sclerosis.

Autoimmune connective tissue diseases, including dermatomyositis and systemic sclerosis, have a heterogeneous clinical presentation and prognosis; moreover, their clinical features are often incomplete and overlap with other rheumatic disorders, which can make diagnosis and prognostic stratification challenging. Specific autoantibodies have been associated with certain clinical findings as well as prognostic implications, and many new associations have been made over the last decade. Although patient populations manifest considerable heterogeneity, autoantibodies can be used to help predict clinical features, prognosis, and response to therapy. In this review, the clinical and prognostic implications associated with disease-specific autoantibodies in dermatomyositis and scleroderma are summarized with an emphasis on how the clinician can use this information for patient care.

The Clinical Relevance of Antifibrillarin (anti-U3-RNP) Autoantibodies in Systemic Sclerosis.

Systemic sclerosis (SSc) is a heterogeneous autoimmune disease associated with several antinuclear autoantibodies useful to diagnosis and prognosis. The aim of the present multicentric study was to determine the clinical relevance of antifibrillarin autoantibodies (AFA) in patients with SSc. The clinical features of 37 patients with SSc positive for AFA (AFA+) and 139 SSc patients without AFA (AFA-) were collected retrospectively from medical records to enable a comparison between AFA- and AFA+ patients. Antifibrillarin autoantibodies were screened by an indirect immunofluorescence technique using HEp2 cells and identified by an in-house Western blot technique and/or an EliA test. Comparing AFA+ and AFA- patients, AFA+ patients were significantly younger at disease onset (36.9 versus 42.9; P = 0.02), more frequently male (P = 0.02) and of Afro-Caribbean descent (65% versus 7.7%; P < 0.001). At diagnosis, the Rodnan skin score evaluating the cutaneous manifestations was higher (13.3 versus 8.7; P = 0.01) and myositis was also more common in the AFA+ group (31.4% versus 12.2%; P < 0.01). Patients with AFA+ were not associated with diffuse cutaneous SSc or with lung involvement and no difference in survival was observed. Antifibrillarin autoantibodies are associated with patients of Afro-Caribbean origin and can identify patients with SSc who are younger at disease onset and display a higher prevalence of myositis.

Anti-U3 ribonucleoprotein antibody-positive inflammatory myopathy: a case report.

BACKGROUND: The discovery of myositis-specific autoantibodies and myositis-associated autoantibodies has led to a new serological classification. Human U3 RNP, which consists of the U3 small nucleolar RNA and anti-U3 RNP antibody, is directed against one of the subunits. Anti-U3 RNP antibodies have been detected in 5-8 % of patients with systemic sclerosis, and antibody-positive patients with systemic sclerosis have shown more frequent skeletal muscle involvement than that of antibody-negative patients with systemic sclerosis. CASE PRESENTATION: A 74-year-old Japanese man positive for anti-U3 RNP antibody was referred to our hospital because of gait disturbance and dysphagia. His serum myoglobin and creatine kinase levels were elevated, and myopathic changes were observed in his proximal legs by needle electromyography. A muscle biopsy was performed at the quadriceps femoris muscle, which showed high signal intensity on fat-suppressed and T2-weighted magnetic resonance images. The patient was diagnosed with probable polymyositis because CD8-positive lymphocytes had invaded only the endomysium and not into the muscle fibers. Severe proliferation of the interstitial connective tissue and edematous changes were observed. Oral prednisolone therapy was started, and the patient's muscle weakness of the proximal limbs improved remarkably within 1 month. Dysphagia caused by incomplete function of the cricopharyngeal muscle persisted for 5 years. CONCLUSIONS: Our findings indicate that mild muscle weakness with steroid-resistant dysphagia may be a clinical feature of patients with anti-U3 RNP antibody-positive inflammatory myopathy.

Detection of anti-U3-RNP/fibrillarin IgG antibodies by line immunoblot assay has comparable clinical significance to immunoprecipitation testing in systemic sclerosis.

The aim of this study was to evaluate the performance and clinical relevance of a commercially available line immunoblot assay (LIA) for detecting anti-U3-RNP/fibrillarin (anti-U3-RNP), against immunoprecipitation (gold standard). This study involved a multi-ethnic cohort of 1000 American systemic sclerosis (SSc) patients and 50 healthy controls. Antinuclear antibodies and centromere antibodies were detected by indirect immunofluorescent antibody test, anti-topo I by immunodiffusion and anti-RNAP III by ELISA. The presence of anti-U3-RNP in select serum samples was detected by immunoprecipitation (IP) and LIA. By IP, U3-RNP antibody was detected in 75 (7.5 %) patients with SSc. Overall agreement between LIA and IP was very good (κ = 0.966). Analytic sensitivity and specificity of the U3-RNP LIA was 100 and 94.7 %, respectively. Clinical features associated with positivity for the anti-U3-RNP antibody include diffuse cutaneous SSc and increased prevalence of renal crisis, consistent with previous studies that used IP. Testing for U3-RNP antibodies is only performed by a small number of laboratories due to the complexity of both performance and interpretation of the IP. LIA is faster and less complex than IP. Excellent agreement between IP and LIA demonstrates that LIA is an acceptable and attractive alternative to IP for anti-U3-RNP detection.

Gender and ethnicity differences in the prevalence of scleroderma-related autoantibodies.

Autoantibodies to topoisomerase I (topo I), RNA polymerase III (RNAPIII), centromere, U3RNP/fibrillarin, Th, PM-Scl, and U1RNP found in scleroderma (SSc) are associated with unique clinical subsets. The effects of race and gender on autoantibody prevalence and clinical manifestations were examined. Autoantibodies in sera from 105 SSc (include 75 Caucasian, 24 African-American, 6 others; 89 females and 16 males) were analyzed by immunofluorescence and immunoprecipitation. Clinical information was from database. SSc-related autoantibodies seldom coexist except for anti-topo I and anti-U1RNP. Anti-topo I (35% vs 15%), anti-U3RNP (30% vs 3%, p = 0.0005), and anti-U1RNP (30% vs 13%) were more common in African-Americans vs Caucasians. Anti-centromere (17%) and anti-PM-Scl (only in 8% of female) were found only in Caucasians. In race/gender combination, all three African-American males had anti-topo I (p = 0.04). Anti-U3RNP (35% vs 3%, p = 0.0005) and anti-U1RNP were common in African-American females. In African-American, all nucleolar dominant staining sera had anti-U3RNP; nuclear pattern was topo I (50%), U1RNP (19%), and RNAPIII (13%). In Caucasian, nucleolar was anti-Th (43%) and PM-Scl (29%); nuclear pattern was RNAPIII (29%), topo I (24%), and U1RNP (18%). Anti-topo I, anti-RNAPIII, and anti-U3RNP were associated with diffuse SSc while anti-centromere, anti-Th, and anti-U1 with limited disease. Proximal scleroderma was less common in African-American with anti-topo I (38% vs 91% in Caucasian, p = 0.04). The production of SSc-related autoantibodies is gender and race dependent, and this can be highly relevant in understanding their clinical significance.

Dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inheritable bone marrow failure syndrome characterized by reticulated hyperpigmentation, dystrophic nails and oral leukoplakia. Another name for the condition is Zinsser-Cole-Engman syndrome. Hematologic manifestations usually do not appear in childhood but later in early adulthood. Patients are also prone to carcinomas, particularly of the head and neck. The disease has X-linked or autosomal dominant/recessive inheritance. Early childhood variants (Hoyeraal-Hreidarsson syndrome) are associated with immunological abnormalities in the form of low T- and B-cell numbers. Four genes, namely DKC1 (codes for dyskerin), TERC and TERT (code for telomerase) and NOP10, have been implicated in the pathogenesis; the short telomeres provide a marker for genetic linkage studies. Androgens, with or without granulocyte colony stimulating factor, have been tried in the treatment of the conditions with variable results. Stem cell transplantation from matched sibling donor is currently the treatment of choice. It requires modified nonmyeloablative conditioning protocols, since the patients with DC are prone to pulmonary and hepatic complications.

Anti-U3 RNP autoantibodies in systemic sclerosis.

OBJECTIVE: To describe the classification, demographic and clinical features, and survival in anti-U3 RNP autoantibody-positive patients with systemic sclerosis (SSc). METHODS: Medical records of 108 anti-U3 RNP-positive and 2,471 anti-U3 RNP-negative SSc patients first evaluated during 1985-2003 were reviewed. Anti-U3 RNP antibody was detected by protein and RNA immunoprecipitation. Disease classification, demographic and clinical features, organ system involvement, and survival were compared between the 2 patient groups, by Student's t-test, chi-square analysis, and Mantel-Haenszel test. RESULTS: The anti-U3 RNP-positive group had a higher proportion of African American patients (27% versus 5%; P < 0.001) and male patients (29% versus 19%; P = 0.021), and was younger at the time of first physician diagnosis (mean age 42.8 years versus 47.4 years; P = 0.001). The 2 groups had similar proportions of patients with diffuse cutaneous involvement (47% and 45% in those with and those without anti-U3 RNP, respectively). However, among patients with diffuse cutaneous involvement, the mean maximum modified Rodnan skin score was significantly lower in the anti-U3 RNP group (22.3 versus 27.9; P < 0.001). Skeletal muscle involvement was more frequent in anti-U3 RNP-positive patients (25% versus 14%; P = 0.002), as was "intrinsic" pulmonary arterial hypertension (PAH) (31% versus 13%; P < 0.001). The frequency of gastrointestinal involvement, cardiac involvement, pulmonary fibrosis, and "renal crisis" did not differ significantly between the 2 groups. Survival was worse in the anti-U3 RNP-positive group (hazard ratio 1.38 [95% confidence interval 1.05-1.82]). PAH was the most common known cause of death in patients with anti-U3 RNP (30%, versus 10% in the anti-U3 RNP-negative group; P < 0.001). CONCLUSION: The present findings demonstrate that the frequencies of African American race and male sex are greater among SSc patients with anti-U3 RNP antibody than those without, and the former group is younger at SSc diagnosis. Anti-U3 RNP-positive patients have more frequent skeletal muscle involvement and PAH, the latter being the most common cause of death.

[Preparation and characterization of anti-Drosophila Dnop5 antibody].

AIM: To prepare the rabbit antibody against Dnop5 and identify its specificity. METHODS: Dnop5 cDNA was amplified by RT-PCR, and then was subcloned into the fusion expression vectors pET28a(+). After being expressed in E.coli BL21, the truncated Dnop5 protein was purified and used to immunize rabbit. Purified antibody was obtained through affinity chromatography column with the expressed Dnop5. The specificity of the purified antibody was characterized by Western blot and immunohistochemical staining. RESULTS: The Dnop5 gene was successfully inserted into pET28a(+). After induction, the fusion protein was expressed in the form of inclusion body. The purified fusion protein was obtained by affinity chromatography. After immunization of rabbits, the antibody against Dnop5 was obtained. Western blot analysis and immunohistochemical staining showed that the antibody had a good specificity. CONCLUSION: The rabbit antibody against Dnop5 has been successfully prepared, which lays the foundation for further study on the Dnop5 function.

Human scleroderma sera contain autoantibodies to protein components specific to the U3 small nucleolar RNP complex.

OBJECTIVE: To determine whether antifibrillarin autoantibodies in scleroderma patients are associated with autoantibodies to protein components specific for U3 small nucleolar RNP (U3 snoRNP). METHODS: Sera from 220 scleroderma patients were examined for antinucleolar autoantibodies (ANoA) and for antibodies to fibrillarin and the U3 snoRNP-specific proteins Mpp10 and hU3-55K. Clinical correlates were determined for the different autoantibody specificities. RESULTS: Fifty-nine of the 220 patients were positive for ANoA, and 31 of these patients were antifibrillarin positive. Anti-hU3-55K was found in 10 patients, all of whom were antifibrillarin positive. Twenty-nine patients had anti-Mpp10 antibodies; 23 of these were antifibrillarin positive and 6 were antifibrillarin negative. ANoA, including antifibrillarin, anti-hU3-55K, and anti-Mpp10, were associated with diffuse, rather than limited, systemic or localized scleroderma. Esophageal and lung involvement were more common in patients with antifibrillarin and anti-Mpp10 antibodies, and the highest frequency was in patients with anti-Mpp10 alone. CONCLUSION: Antifibrillarin autoantibodies are associated with autoantibodies to protein components specific to U3 snoRNP, particularly Mpp10. The prevalence of anti-Mpp10 antibodies in antifibrillarin-positive patients suggests that the U3 snoRNP particle is a source of immunogenic/antigenic material for the anti-snoRNP response in scleroderma. Autoantibodies to snoRNP components were more frequent in patients with diffuse scleroderma than in those with either the limited systemic or localized forms. The increased expression of these antibodies in patients with the more severe form of scleroderma, coupled with the observations that fibrillarin expression is positively linked to collagen expression in fibroblasts and that fibrillarin is overexpressed in scleroderma fibroblasts, suggests a source of snoRNP to initiate and maintain these autoantibody responses.

Characterization of the sequences encoding for Xenopus laevis box C/D snoRNP Nop56 protein.

Nop56p was initially identified in yeast as the third common component of the ribonucleoprotein particles (snoRNPs) assembled on box C/D small nucleolar RNAs (snoRNAs). Thereafter, the characterization of Nop56p homologs in Archaea and in several eukaryotes pointed to the highly conserved structure of this factor. Studies in yeast indicate that Nop56 is not required for the stability of box C/D snoRNAs. Through the isolation of a Xenopus laevis Nop56 cDNA clone, we have been able to characterize the X. laevis Nop56 protein (XNop56p). We showed that it is a common component of X. laevis box C/D snoRNPs and that it displays the same electrophoretic mobility of p62 protein that we previously characterized as a box C/D snoRNP component, not essential for snoRNA stability in X. laevis. Mapping the 5' end of X. laevis Nop56 transcript indicates that it starts with a pyrimidine tract and the analysis of genomic clones revealed a snoRNA encoded in one of NOP56 introns. Although these two characteristics could suggest that XNOP56 is a TOP gene, it is not translationally controlled in a growth-dependent manner.

Prevalence in myositis of antibodies recognizing anti-U3 RNA probably in a novel complex with 22/25 kD protein and not fibrillarin.

New antibodies against a U3 snRNP, which were named anti-Myo 22/25 antibodies, were detected in four (8%) of 53 serum samples from patients with polymyositis/dermatomyositis (PM/DM) by RNA immunoprecipitation. In the protein immunoprecipitation analysis, all four serum samples precipitated 22 kDa and 25 kDa proteins, which were not precipitated by normal serum or serum positive for antifibrillarin antibodies. Three of the four PM/DM patients had other identified autoantibodies including anti-PL-12 antibodies, antihistone antibodies (AHA), anti-SS-A antibodies and anti-SS-B antibodies defined by double immunodiffusion, ELISA or RNA immunoprecipitation, although there were no significant correlations between anti-Myo 22/25 antibodies and clinical or laboratory findings. There may be a subgroup of PM/DM patients whose sera are positive for anti-Myo 22/25 antibodies.

Fibrillarin and other snoRNP proteins are targets of autoantibodies in xenobiotic-induced autoimmunity.

Exposure of SJL/J mice to mercury induces an anti-nucleolar autoantibody response. The predominant target is fibrillarin, a 34-kDa component of the small nucleolar ribonucleoprotein particles (snoRNP), but other proteins are also recognized. To characterize these proteins, monoclonal IgG anti-nucleolar antibodies were produced from HgC12-treated SJL/J mice. One monoclonal, 17C12, recognized fibrillarin, while two others, 7G3 and 6G10, were found to immunoprecipitate snoRNP particles but not fibrillarin. Antibody 6G10 gave a nucleolar immunofluorescence pattern in human, murine, and amphibian cells, but was negative in immunoblot. The 7G3 monoclone reacted with a 60-kDa protein conserved in human and murine, but not amphibian, cell lines. The 7G3 and 6G10 antigens and fibrillarin colocalized to the nucleolus and Cajal bodies in interphase cells and decorated metaphase chromosomes. These studies suggest that the mercury-induced anti-nucleolar antibody response targets other protein components of the snoRNP particles in addition to fibrillarin.