Report on the second International Consensus on ANA Pattern (ICAP) workshop in Dresden 2015.

The second meeting for the International Consensus on Antinuclear antibody (ANA) Pattern (ICAP) was held on 22 September 2015, one day prior to the opening of the 12th Dresden Symposium on Autoantibodies in Dresden, Germany. The ultimate goal of ICAP is to promote harmonization and understanding of autoantibody nomenclature, and thereby optimizing ANA usage in patient care. The newly developed ICAP website www.ANApatterns.org was introduced to the more than 50 participants. This was followed by several presentations and discussions focusing on key issues including the two-tier classification of ANA patterns into competent-level versus expert-level, the consideration of how to report composite versus mixed ANA patterns, and the necessity for developing a consensus on how ANA results should be reported. The need to establish on-line training modules to help users gain competency in identifying ANA patterns was discussed as a future addition to the website. To advance the ICAP goal of promoting wider international participation, it was agreed that there should be a consolidated plan to translate consensus documents into other languages by recruiting help from members of the respective communities.

Safety and efficacy of ocrelizumab in patients with rheumatoid arthritis and an inadequate response to methotrexate: results of a forty-eight-week randomized, double-blind, placebo-controlled, parallel-group phase III trial.

OBJECTIVE: To evaluate the efficacy and safety of treatment with ocrelizumab plus methotrexate (MTX) in patients with active rheumatoid arthritis (RA) and an inadequate response to MTX. METHODS: STAGE was a phase III randomized, double-blind, parallel-group international study to evaluate the safety and efficacy of ocrelizumab compared with placebo in patients with active RA continuing MTX treatment. Patients receiving stable doses of MTX were randomized to receive 2 infusions of placebo (n = 320), ocrelizumab 200 mg (n = 343), or ocrelizumab 500 mg (n = 343) on days 1 and 15 as well as weeks 24 and 26. Coprimary end points were the proportion of patients with an American College of Rheumatology 20% improvement criteria (ACR20) response at weeks 24 and 48. Secondary end points included the change from baseline in the modified Sharp/van der Heijde score (SHS) and the ACR50/70 responses. RESULTS: The ACR20 response rates were 35.7% in the placebo group, 56.9% in the ocrelizumab 200 mg group, and 54.5% in the ocrelizumab 500 mg group at 24 weeks, and 27.6%, 58.3%, and 62.1%, respectively, at 48 weeks (P < 0.0001 versus placebo for each dose at both time points). At week 48, both of the ocrelizumab doses improved the ACR50 and ACR70 response rates 3-fold as compared with placebo and showed a statistically significant (P < 0.0001) reduction in joint damage progression relative to placebo (mean change in SHS reduced by 85% and 100% for the 200-mg and 500-mg doses, respectively). Rates of serious infection were comparable in the placebo (3.48 per 100 patient-years) and ocrelizumab 200 mg (3.54 per 100 patient-years) groups but were elevated in the ocrelizumab 500 mg group (8.66 per 100 patient-years). CONCLUSION: With both ocrelizumab doses, the primary end point was met, and the signs and symptoms of RA were significantly improved at weeks 24 and 48. Ocrelizumab also significantly inhibited the progression of joint damage. A higher rate of serious infections was observed with 500 mg of ocrelizumab as compared with ocrelizumab 200 mg or placebo.

Diverse humoral autoimmunity to the ribosomal P proteins in systemic lupus erythematosus and hepatitis C virus infection.

Autoantibodies to the three ribosomal P proteins (Rib-P) are specifically found in 10% to 40% of systemic lupus erythematosus (SLE) patients. Most anti-Rib-P autoantibodies bind to a C-terminal epitope shared by all three Rib-P proteins P0, P1 and P2. In the present study, we shed more light on the humoral autoimmune response to the Rib-P antigen as it occurs in autoimmunity and infectious disease. In a mutational analysis of the major C-terminal epitope, we verified the key role of phenylalanine residues Phe ( 111 ) and Phe ( 114 ) for binding of most anti-Rib-P serum autoantibodies present in SLE sera (n = 28). By nuclear magnetic resonance (NMR) investigation of a peptide comprising the C-terminal 22 amino acids, we observed hallmarks for alpha-helical secondary structure of the Rib-P epitope core (GFGLFD). Based on NMR data and on SPOT epitope analysis, we propose a structural model of the Rib-P major epitope, which displays Phe ( 111 ) and Phe ( 114 ) on one side of the helix. Apart from that, two sera from the hepatitis C virus (HCV) control group (n = 68) were found to contain antibodies specific for P2, but not for the other Rib-P proteins. Using a SPOT peptide array scanning the P2 amino acid sequence, we identified reactivity with two distinct epitopes (residues 21-35 and 41-55 of Rib-P2) shared by both HCV sera. We conclude that anti-Rib-P autoreactivity occurs in SLE, Chagas' disease (CD) and-as firstly described here-during HCV infection. Anti-Rib-P reactivity in SLE sera primarily depends on Phe ( 111 ) and Phe ( 114 ) of the alpha-helical C-terminal epitope. In contrast, anti-Rib-P autoantibodies in HCV infection mainly recognize epitopes within the N-terminal half of ribosomal P2.

Anti-CCP antibodies have more diagnostic impact than rheumatoid factor (RF) in a population tested for RF.

To compare the diagnostic powers of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) in a population selected for its high statistical relevance, over a 6-month period, an informed consent to test for anti-CCP was obtained from 1,025 consecutive patients for whom RF was ordered at a University laboratory. Within 1 year, a diagnosis was obtained without informing the physician about the anti-CCP result. Extensive statistical analyses were performed. A total of 768 patients satisfied the inclusion criteria, and 132 were classified as having RA, yielding a pre-test probability of RA of 17%. The sensitivities for anti-CCP and RF were 62 and 64% (P = 0.83), and the specificities were 97 and 90% (P < 0.001), respectively. The positive predictive value (PPV) was 79% for anti-CCP and 56% for RF (P < 0.001), whereas the negative predictive value was 92% for both. The likelihood ratio (LR) was 17.9 for anti-CCP and 6.2 for RF (P < 0.005). Forty RA patients were diagnosed with RA of less than 2 years length, and the same significant statistic differences between anti-CCP and RF were observed. Placing the results of both tests together, or using different cutoff points, increased the diagnostic utility of the tests. The anti-CCP test has statistically shown significant higher specificity, PPV, and LR for RA than the RF test in a clinically diverse population. If new criteria are to be devised to help diagnose early RA, anti-CCP should be included because it has a greater diagnostic impact than RF.

Autoantibodies in the diagnosis of systemic rheumatic diseases.

Distinct profiles of autoantibodies directed to intracellular antigens can be detected in the systemic connective tissue diseases. They aid in establishing the correct diagnosis and are included in many sets of diagnostic criteria, such as the ones developed for systemic lupus erythematosus (anti-Smith antigen and anti-double-strand DNA antibodies), mixed connective tissue disease (anti-U1-nuclear ribonucleoprotein antibodies), and Sjögren's syndrome (SS) (anti-SS-A/Ro and anti-SS-B/La antibodies). They are useful prognostic markers in some situations and facilitate clinical and treatment follow-up. Autoantibodies have also been used as probes to gain insights into cell biology, helping to isolate and purify intracellular proteins involved in key cellular functions. We give detailed information on two of the most useful techniques for the detection of autoantibodies in the clinical and research laboratory settings, indirect immunofluorescence and immunoblotting. We also discuss several of the antigen-autoantibody systems found in systemic lupus erythematosus (Smith antigen, U1-nuclear ribonucleoprotein, SS-A/Ro, SS-B/La, proliferating cell nuclear antigen ribosomal ribonucleoprotein, double-strand DNA, histones, antiphospholipids, Ku, Ki/SL), systemic sclerosis (centromere, topo I, RNA polymerases, fibrillarin, polymyositis-Scl, Th/To), polymyositis/dermatomyositis (transferRNA synthetases, signal recognition particle, and others), and SS (SS-A/Ro, SS-B/La, nucleolar organizing region-90, p80-coilin), addressing their clinical significance, common detection methods, immunogenetic associations, and the molecular and cellular biology of the cognate antigens.

Evaluation of immune parameters in depressed patients.

The association between depression and altered immunological activities has repeatedly been suggested, but experimental data show contradictory results. In this work, cellular and humoral immunological activities were evaluated in patients presenting major depression, unipolar subtype. Natural killer cell activity (NKCA) was significantly reduced in patients as compared to healthy controls (p < 0.001). However, lymphocyte mitogenic responses and immunoglobulin titers (IgG, IgM, and IgA) were similar for all samples. Hematological, hormonal, and nutritional variables presented normal values in patients and in controls. A familial history of depression was related to lower NKCA and higher phytohemagglutinin responses (p < 0.01). These data suggest possible differential inhibition of cellular immune responses in depressed patients.

Immunoglobulin allotype Gm(1,2;21) in ankylosing spondylitis with peripheral arthritis.

Frequencies of immunoglobulin G (Gm) allotypes were determined in 240 patients with ankylosing spondylitis (AS). The uncommon phenotype Gm(1,2;21) was increased in frequency in 55 patients with AS and peripheral arthritis (14.5% v 3.5% of healthy blood donors; p less than 0.05). In 16 patients with arthritis only of wrist/hand or ankle/forefoot, or both, the Gm(1,2;21) frequency was even higher (31.3%; p less than 0.0005). Patients with AS negative for the HLA antigen B27 (n = 28) differed from the B27 positive patients (n = 205) with regard to the frequency of the Gm(1,2,3;5,21) phenotype (39.3% v 9.3%; p less than 0.0005). These findings support the notion of genetic heterogeneity among patients with AS.

[Scintigraphic diagnosis of esophageal involvement in systemic sclerosis (scleroderma)]. / Szintigraphische Diagnostik der Osophagusbeteiligung bei systemischer Sklerose (Sklerodermie).

Motor function disturbances (MFD) of the esophagus occur in the majority (90%) of patients with systemic sclerosis. We have tested the diagnostic value of esophagus scintigraphy in 20 patients with systemic sclerosis (12 CREST and 8 with diffuse form) and 18 controls (11 normals and 7 with pyrosis and/or regurgitation), in order to establish sensitivity, specificity, effects of body posture, and use of a liquid or solid meal in the evaluation of MFD of the esophagus. The patients drank 4 ml of fruit juice marked with 500 microCi of 99mTc-sulfur-colloid and afterwards 10 g of an equally marked and scrambled egg. Pictures were made with a Gamma-Camera until 180 s. Radioactivity was plotted against time and separately analysed for the upper, middle and distal esophagus. 65% of the systemic sclerosis patients had dysphagia and 70% had abnormal barium meal transit in the esophagus. The scintigrams were altered in all the patients with systemic sclerosis (p less than 0.001). Sensitivity using fluids was 95%, and specificity was 89%; with solid food 100% and 50%, respectively. Tests done in the upright position showed a better clearance of the esophagus in patients with systemic sclerosis and control patients, with loss of sensitivity. The qualitative analysis yielded little benefit to the reported results. The quantitative analysis under use of 2 indices (total transit time and clearance rate) permitted a clear distinction among patients and control individuals with and without upper digestive symptoms in the first 15 s of the test. The esophagus scintigraphy is a simple, quantitative and very sensitive method for the diagnosis of MFD of the esophagus in patients with systemic sclerosis.

Non-muscle myosin as target antigen for human autoantibodies in patients with hepatitis C virus-associated chronic liver diseases.

Three patients with hepatitis C virus (HCV)-related chronic liver disease were shown to have autoantibodies strongly reacting with cytoskeletal fibres of non-muscle cells. The heavy chain of non-muscle myosin microfilament was the main target for those autoantibodies, as determined by (i) cell and tissue immunofluorescence studies showing colocalization with an anti-myosin antibody prototype; (ii) primary reactivity in immunoblotting with a 200-kD protein, using either MOLT-4 cells, human platelets, or affinity-purified non-muscle myosin as antigen extract; and (iii) immunoblotting of similar immunoreactive fragments in papain-digested MOLT-4 cell extracts, by using those human sera and antibody prototype. Autoantibodies to non-muscle myosin heavy chain were not previously reported in patients with chronic liver diseases, especially in those associated with HCV infection.

Autoantibodies to the nucleolar organizer antigen NOR-90 in children with systemic rheumatic diseases.

OBJECTIVE: To determine the frequency of autoantibodies directed against the nucleolar organizer antigen NOR-90 [human upstream binding factor (hUBF)] in pediatric patients with systemic rheumatic diseases. Two children with antibodies to NOR-90 are reported. METHODS: Two hundred thirty-eight sera from children with systemic rheumatic diseases were screened for autoantibodies directed against hUBF by indirect immunofluorescence and immunoblotting using MOLT-4 cell extracts. Reactivity with hUBF was confirmed by immunoprecipitation of the recombinant hUBF protein. RESULTS: Two sera out of 238 (< 1.0%) with reactivity against hUBF were identified. One patient had recurrent abdominal pain, headache, and Raynaud's phenomenon. There has been no evidence of an active systemic rheumatic disease in more than 8 years of followup. The 2nd patient had Raynaud's phenomenon and systemic lupus erythematosus. CONCLUSION: Antibodies to hUBF are rare in children with systemic rheumatic diseases. Raynaud's phenomenon was a clinical feature common to both patients.

Diversity of antinuclear antibody responses in hepatocellular carcinoma.

BACKGROUND/AIMS: The development of antinuclear antibodies (ANA) in malignancies has been described but its mechanism is still not understood. The aim of this study was to examine ANA specificities in hepatocellular carcinoma to further understand autoimmunity in cancer. METHODS: Two hundred and four hepatocellular carcinoma patients were compared with 68 chronic hepatitis C, with 126 chronic hepatitis B and with 30 alcoholic liver cirrhosis patients, as well as with 87 healthy donors. Indirect immunofluorescence, immunoblotting, and immunoprecipitation were used to study ANA reactivities. RESULTS: Hepatocellular carcinoma had a significantly higher frequency of ANA using HEp-2 cells as substrate (31%) than chronic hepatitis C (10%), chronic hepatitis B (9.5%), alcoholic liver cirrhosis (10%) or healthy donors (4.5%). A great diversity of ANA specificities was found in hepatocellular carcinoma. Three hepatoma sera had antibodies that co-localized with non-snRNP splicing factor SC35, suggesting that the antigenic targets might be involved in mRNA splicing. We identified antibodies to two known nuclear autoantigens: fibrillarin and p330d/CENP-F. These autoantigens are involved in the 5' processing of precursor ribosomal RNA transcripts and in mitotic functions, respectively. CONCLUSIONS: Diversity was found in the autoantibody specificity, in contrast to the specific subsets of autoantibodies seen in several systemic rheumatic autoimmune diseases. Our data suggest that immune response in hepatocellular carcinoma targets important proteins involved in cellular biosynthetic or proliferative functions.

Motor activity of the gallbladder in systemic sclerosis.

OBJECTIVE: We sought to measure gallbladder emptying in scleroderma patients, when stimulated by exogenous cholecystokinin. METHODS: Twenty-eight consecutive scleroderma patients were evaluated. Ten were excluded for the presence of gallstones. Gallbladder motor function was studied in 18 patients and 18 controls, using specific parameters for the quantification of gallbladder emptying dynamics. Resting gallbladder volumes were compared using the Dodds method with real-time ultrasound. Cholecystokinin (CCK)-stimulated gallbladder function (0.02 microg/kg CCK intravenous infusion/30 min) was assessed by a scintigraphic technique using 99mTc-DISIDA. Five patients presented with CREST syndrome, 13 with the diffuse form of scleroderma. Four were men, 14 women (average age = 46.6+/-15.4 yr). Patients and controls were paired by gender, age, and weight. RESULTS: Resting gallbladder volumes were larger in the four men with scleroderma than in the women with this disease (p < 0.03, Mann-Whitney). The mean gallbladder resting volume in scleroderma patients was not different from the mean volume detected among controls (p = 0.25), even when controlling for gender (p = 0.78 for women, p = 0.08 for men), scleroderma disease subtype (p = 0.50), or disease duration (p = 0.48). Latency period, ejection period, ejection rate, or ejection fraction as measured during cholecystokinin-stimulated scintigraphic studies were not significantly different between patients and controls. A trend was detected for reduction of the ejection period in scleroderma women (p = 0.70) when compared with scleroderma men. More than 35% of the scleroderma patients presented biliary lithiasis. CONCLUSIONS: There was no significant difference in gallbladder dynamics measured by a scintigraphic technique in scleroderma patients, compared with controls, when gallbladder motor function was evaluated by intravenous CCK.

Immunogenetic associations of scleroderma-related antinuclear antibodies.

Patients selected for the presence of scleroderma-related antibodies (anti-DNA-topoisomerase I [anti-topo I; n = 43], anticentromere antibody [ACA; n = 63], or anti-Pm-Scl [n = 12]) were studied for class I and class II major histocompatibility complex antigens, as well as for Gm and Km allotypes. Anti-topo I was associated with HLA-DR5 (70% of patients versus 30.6% of controls; Pcorr = 0.0018, relative risk [RR] = 5.3). All patients with anti-Pm-Scl were positive for HLA-DR3 (versus 23.5% of controls; Pcorr less than 0.001); 6 of these patients were DR3/4 heterozygous (50% versus 3.5% of controls; Pcorr less than 0.001, RR = 27.3). Patients with ACA were frequently positive for HLA-DR1, DR4, or DRw8, with 73.7% demonstrating at least 1 of these alleles (versus 41.2% of controls; Pcorr = 0.0152, RR = 4.0). This group of ACA-positive patients who had DR1, DR4, and/or DRw8 consisted mainly of a subgroup of patients with rheumatoid arthritis. We conclude that different class II major histocompatibility complex antigens influence the formation of anti-topo I and anti-Pm-Scl. Important clinical differences between these patient groups and the immunogenetic heterogeneity support the notion of different antibody-defined scleroderma subsets.

Valor da imunofluorescencia em biopsia de pele normal no diagnostico do lupus eritematoso sistemico. / Value of inmunofluorescence in normal skin biopsy in the diagnosis of systemic lupus erythematosus

Foram estudadas por meio de imunofluorescencia direta trinta e nove biopsias de pele normal de pacientes portadores de colagenoses. Procuramos observar, em nosso meio a correlacao entre depositos de imunoglobulinas e/ou complemento na juncao dermo-epidermica e o diagnostico de Lupus Eritematoso Sistemico. Foi possivel verificar a alta especificidade e sensibilidade do metodo no diagnostico diferencial dessa colagenose. Concluem os autores ressaltando o importante auxilio no diagnostico que esse procedimento pode proporcionar