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.

FAS -670A>G promoter polymorphism is associated with soluble Fas levels in primary Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease characterized by lymphocytic infiltration of exocrine glands. Soluble Fas receptor (sFas) has been suggested as a Fas-mediated apoptosis blocker that could impair clonal deletion in infiltrated autoreactive cells. The FAS -670A>G promoter polymorphism has been studied in pSS. However, a relationship between FAS -670A>G promoter polymorphism and sFas levels in pSS had not been found. We examined this relationship in 77 Mexican pSS patients and 84 healthy subjects were included. Genotypes were identified by PCR-RFLP, and Fas soluble levels were quantified by ELISA. No significant differences between allele and genotype frequencies were found between these two groups. The sFas levels in the serum of pSS patients were significantly higher than in controls (9961 vs 8840 pg/mL, respectively). In addition, AA genotype carriers had significantly higher levels of sFas than GG carriers (pSS: 10,763 and 9422 pg/mL; controls: 9712 and 8305 pg/mL, respectively). An additive model analysis between genotypes (AG+GG vs AA) in both groups, demonstrated a significant association between carriers of the A allele and high sFas levels. In conclusion, carrying the double dose of A allele of FAS -670A>G polymorphism is associated with high levels of sFas in pSS, but it is not a susceptibility marker for pSS.

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.

Oxidized low-density lipoprotein and beta2-glycoprotein I in patients with systemic lupus erythematosus and increased carotid intima-media thickness: implications in autoimmune-mediated atherosclerosis.

Oxidative stress and LDL modification (oxLDL) are early pro-atherogenic events. OxLDL binds beta2GPI producing immunogenic oxLDL/beta2GPI complexes. Antibodies to these complexes have been associated with arterial thrombosis in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). Circulating oxLDL/beta2GPI complexes, IgG and IgM antibodies to these complexes were measured by ELISA in 30 SLE patients asymptomatic for cardiovascular disease (mean age 31 years) and 27 age/sex matched healthy controls. Carotid intima-media thickness (IMT) was measured by ultrasound in all patients and controls. Forty-seven percent of SLE presented plaques (median IMT of 0.65 +/- 0.12 mm) while only 7% of the controls had plaques (median IMT of 0.50 +/- 0.04 mm, P < 0.001). Median optical density (OD450nm) for oxLDL/beta2GPI complexes in SLE was 0.244 +/- 0.07, higher than controls (0.174 +/- 0.09, P < 0.001). Median OD for IgG anti-oxLDL/beta2GPI antibodies was also higher in SLE (0.297 +/- 0.26) compared to controls (0.194 +/- 0.07, P < 0.001) while the median OD for IgM antibodies in SLE (0.444 +/- 0.46) was not different than controls (0.326 +/- 0.22, P = 0.267). There was no correlation between IMT and oxLDL/beta2GPI complexes, IgG or IgM antibodies, possibly reflecting the complex interrelationship between these serologic elements and tissue factors in the arterial wall. These results support the hypothesis that oxLDL/beta2GPI complexes and IgG (not IgM) anti-oxLDL/beta2GPI antibodies contribute to the development of autoimmune-mediated atherosclerosis.

Therapy with intermittent pulse cyclophosphamide for pulmonary hypertension associated with systemic lupus erythematosus.

The aim of this study was to compare the efficacy of intravenous cyclophosphamide (IVCYC) versus oral enalapril in mild or moderate pulmonary hypertension (PH) in systemic lupus erythematosus (SLE). Thirty-four patients with SLE who had systolic pulmonary artery pressure (SPAP) > 30 mmHg by Doppler echocardiography were randomized to receive IVCYC (0.5 g/mt2 body surface area, monthly), or oral enalapril (10 mg/day) for six months. The primary outcome was the significant decrease in SPAP. An additional outcome measure included the improvement in the heart functional class (NYHA). Sixteen patients received cyclophosphamide and 18 enalapril. IVCYC decreased the median values of SPAP from 41 to 28 mmHg (P < 0.001), and enalapril from 35 to 27 mmHg (P = 0.02). IVCYC reduced more than twice as much SPAP than enalapril (P = 0.04). In those patients with SPAP > or = 35 mmHg, cyclophosphamide decreased from 43 to 27 mmHg (P = 0.003), but enalapril was not effective (P = 0.14). The NYHA functional class improved only in those with cyclophosphamide (P = 0.021). Also IVCYC had a higher frequency of side effects including infections (RR = 1.6; 95% CI, 1.001-2.47), and gastrointestinal side effects (RR = 14.6; 95% CI, 2.15-99.68). We concluded that IVCYC was effective in mild and moderate PH associated with SLE. Further research is needed to evaluate its long-term efficacy.

Autoantibodies to receptor induced neoepitopes of fibrinolytic proteins in rheumatic and vascular diseases.

OBJECTIVE: Abnormal plasminogen activation has been implicated in vascular and rheumatic diseases. The development of an autoimmune response to neoepitopes of plasminogen and its activator (tissue-type plasminogen activator, t-PA) was explored in sera from patients with rheumatoid arthritis (RA, n = 30), Behcet's disease (n = 20), primary antiphospholipid syndrome (APS, n = 23), and idiopathic arterial (n = 33) or venous thrombosis (n = 16). METHODS: Sera diluted 1/50 were incubated with either plasminogen or t-PA bound to their natural receptors (immobilized fibrin or monocytic cells), and bound immunoglobulins were detected using a sheep peroxidase labeled anti-human Fab IgG. Controls included plates coated with fibrin or cells alone or plasminogen passively adsorbed to the plastic. Sera were considered positive when the absorbance at 405/490 nm was above the mean + 2 SD of normal sera. RESULTS: Reactivity of sera against plasminogen bound to cells (28%) or to fibrin (22%) was a predominant feature in patients with RA compared with other patient groups and controls. However, some patients with primary APS had reactivity against cell and fibrin bound plasminogen (9 and 13%, respectively). Autoantibodies against fibrin bound t-PA were detected in only 8% of patients with arterial or venous thrombosis. CONCLUSION: Conformational changes induced by molecular assembly of plasminogen on cell or fibrin surfaces result in the expression of neoepitopes recognized by autoantibodies. These autoantibodies could be markers of the proteolytic events associated with plasminogen activation in autoimmune diseases.

Factitious disorders mimicking systemic lupus erythematosus.

Factitious disorders are one of the most difficult challenges to the sagacity of the physician. Self-inflicted injuries and diseases have been recognized since Biblical times. In the Middle Ages, hysterics were known to place leeches in their mouths to simulate hemoptysis and to abrade their skin to reproduce skin conditions. Münchausen syndrome, originally described in 1951, is the term applied to persons who seek medical care by feigning illness in the absence of any organic medical or surgical disease. Since the first report of the syndrome, many case reports have documented the performance of unneeded operations and the administration of dangerous drugs to these patients. Rheumatologic manifestations in this syndrome are rare, and include septic arthritis, osteomyelitis, destructive arthropathy, reflex sympathetic dystrophy and systemic lupus erythematosus (SLE). We reviewed the English literature for the past 29 years and found only 8 patients with Münchausen syndrome who simulated SLE. It is interesting to consider the damage mechanisms (some of the patients satisfied 4 or more of the criteria for the classification of SLE) and how a complex disease with a broad spectrum of manifestations such as lupus can be simulated, the only limits being the patient's knowledge and imagination.

Clinical, serologic, and immunogenetic features of familial idiopathic inflammatory myopathy.

OBJECTIVE: To describe the clinical, serologic, and immunogenetic features of familial idiopathic inflammatory myopathy (IIM) and to compare these with the features of sporadic IIM. METHODS: Clinical signs and symptoms, autoantibodies, HLA-DRB1 and DQA1 alleles, and GM/KM phenotypes were compared among 36 affected and 28 unaffected members of 16 unrelated families in which 2 or more blood relatives developed an IIM. In addition, findings in patients with familial IIM were compared with those in 181 patients with sporadic IIM. The families included 3 pairs of monozygotic twins with juvenile dermatomyositis, 11 families with other siblings or relatives with polymyositis or dermatomyositis, and 2 families with inclusion body myositis. RESULTS: The clinical features of familial IIM were similar to those of sporadic IIM, although the frequency of myositis-specific autoantibodies was lower in familial than in sporadic IIM. DRB1*0301 was a common genetic risk factor for familial and sporadic IIM, but contributed less to the genetic risk of familial IIM (etiologic fraction 0.35 versus 0.51 in sporadic IIM). Homozygosity at the HLA-DQA1 locus was found to be a genetic risk factor unique to familial IIM (57% versus 24% of controls; odds ratio 4.2, corrected P = 0.002). CONCLUSION: These findings emphasize that 1) familial muscle weakness is not always due to inherited metabolic defects or dystrophies, but may be the result of the development of IIM in several members of the same family, and 2) multiple genetic factors are likely important in the etiology and disease expression of familial IIM, as is also the case for sporadic myositis, but DQA1 homozygosity is a distinct risk factor for familial IIM.

Diffuse alveolar hemorrhage in limited cutaneous systemic sclerosis with positive perinuclear antineutrophil cytoplasmic antibodies.

A 54-year-old woman with limited cutaneous systemic sclerosis (SSc) developed repeated diffuse alveolar hemorrhage with positive perinuclear antineutrophil cytoplasmic antibodies (pANCA). Reports of diffuse alveolar hemorrhage in SSc have been rarely described only in the diffuse clinical variant. To our knowledge, this is the first report of pANCA positive diffuse alveolar hemorrhage in the limited form of SSc. Diffuse alveolar hemorrhage is a rare, but potentially life threatening cause of respiratory distress in the limited form of SSc that should be recognized and treated effectively.

Antibodies to fibrin-bound tissue-type plasminogen activator in systemic lupus erythematosus are associated with Raynaud's phenomenon and thrombosis.

Fibrinolysis triggered by t-PA bound to fibrin is one of the main antithrombotic mechanisms. Defects in the fibrinolytic system-decreased tissue-type plasminogen activator (t-PA) activity and elevated levels of plasminogen activator inhibitor (PAI-1), in patients with SLE have been associated with an increased tendency to thrombosis. In the present study, 43 patients with SLE fulfilling the ACR criteria for the disease, were studied for the presence of autoantibodies to fibrin-bound t-PA, i.e. the physiological active form of this plasminogen activator. A solution of 200 IU/ml of t-PA was incubated with solid-phase fibrin prepared as previously described (Anal Biochem 1986; 153; 201-210). Sera diluted 1:50 were incubated with fibrin-bound t-PA, the plates were then washed, and bound immunoglobulins were detected using a polyvalent peroxidase-labeled goat anti-human Ig. Plates coated with fibrin alone were used as controls. Sera were considered positive when A490/630 obtained with normal human sera in two independent test was greater than the mean plus 2 SD. Eleven of 43 (26%) SLE sera demonstrated antibody reactivity against fibrin-bound t-PA. Within the anti-t-PA positive group there was a higher proportion of SLE patients with severe Raynaud's phenomenon and thrombotic events when compared to the anti-t-PA negative group: 36% vs 6% and 18% vs 6% respectively. These results suggest that autoantibodies to fibrin-bound t-PA could play a role in the pathogenesis of vascular disease in some SLE patients.

Mixed connective tissue disease. A clinico-serological study of 17 cases.

Mixed connective tissue disease (MCTD) was described as a distinct clinical syndrome in 1972. Since then many cases have been reported in the literature worldwide. In this study we present our experience with a group of 17 Mexican patients with this syndrome, and we analyze their clinical and serological features, as well as the causes of death in these patients. The patients are Mexican mestizos living in Guadalajara and most of them have been followed-up at Hospital General de Occidente for a period of 1-10 years. The female/male ratio was 16:1, and their age ranged from 14-55 years with a mean of 29 years. The disease duration has ranged from 1-17 years, with a mean of 6 years. Among the clinical manifestations we have found a high frequency of lymphadenopathy when compared with published series (13/17 or 76%), and the laboratory findings in our patients included a very high polyclonal increase of gammaglobulins (93%), lymphopenia (76%), direct immunofluorescence speckled nuclear epidermal deposits in skin biopsies (75%) and positive rheumatoid factor (65%). Other clinical and serological features were similar to those reported in other series of patients with MCTD. Six of the 17 patients have died (35%), and in 3 of them (17.5%) the cause of death was due to an infectious disease that suddenly presented, and apparently was not related to a concomitant high dose of steroids or malnutrition in the patients. It seems that in addition to the already well known autoimmune abnormalities that occur in MCTD, there are other features like the presence of lymphadenopathy, the high polyclonal increase of gammaglobulins, and the lymphopenia, that reflect the profound disturbance of the immune system in this syndrome, possibly contributing to the sudden appearance of a severe infectious disease in some of our patients.

Antibodies to fibrin bound tissue type plasminogen activator in systemic sclerosis.

OBJECTIVE: Abnormalities of tissue type plasminogen activator (tPA) and plasminogen activator inhibitor have been described in some patients with systemic sclerosis (SSc). We studied 128 unselected SSc sera for the presence of autoantibodies to fibrin bound tPA. METHODS: A solid phase fibrin-tPA immunoassay utilized 500 IU/ml tPA bound to solid phase fibrin. Sera diluted 1/50 were incubated with the fibrin bound tPA, the plates were washed, and bound immunoglobulins were detected using polyvalent peroxidase labelled goat antihuman immunoglobulins. Controls included plates coated with fibrin alone or tPA passively adsorbed to the plastic. Sera were considered positive when the A490/630 was above the mean + 2 SD (> 0.055) obtained with normal human serum in 2 independent tests. RESULTS: 25/128 (20%) SSc sera demonstrated antibody reactivity with fibrin bound tPA (mean A490/630 = 0.112). Detailed clinical data were available on 117/128 patients with SSc and on 21/25 anti-tPA positive patients. The mean age of the anti-tPA positive group was 51 yrs and of the anti-tPA negative group 49.6 yrs. Within the anti-tPA positive group there was a significantly higher proportion (p > 0.05) of patients with the CREST (calcinosis, Raynaud's esophageal dysmotility, sclerodactyly, telangiectasias) variant of SSc (7/25 = 28% vs 11/103 = 11%) and pulmonary hypertension (5/21 = 24% vs 6/96 = 6%). CONCLUSION: Our study demonstrates that 20% of unselected patients with SSc have anti-tPA antibodies and that there is a higher representation of patients with CREST syndrome in this subgroup. The high frequency of pulmonary hypertension in the anti-tPA positive group suggests that these autoantibodies may play a pathogenic role in certain patients with SSc.

Autoantibodies in Parry-Romberg syndrome: a serologic study of 14 patients.

OBJECTIVE: To determine autoantibody profiles of patients with Parry-Romberg syndrome (PRS). METHODS: Antinuclear antibodies (ANA) in 14 patients with PRS were studied by indirect immunofluorescence (IIF), immunodiffusion and immunoblotting. Antinative DNA antibodies and rheumatoid factor (RF) were also analyzed. RESULTS: ANA were positive in 8 patients (57%). The patterns of staining included nucleolar, nuclear speckled and homogeneous. Anticentromere antibodies were observed in 2 and antihistone antibodies in 3 sera. Rheumatoid factor was found in 5 (36%) sera. Antinative DNA or antibodies that precipitated rabbit thymus extract were not found in any patients. CONCLUSION: The serologic abnormalities observed in this study suggests that autoimmunity could play a pathogenic role in PRS.

Autoimmune phenomena in leprosy, particularly antinuclear antibodies and rheumatoid factor.

Recent reports have emphasized the importance of the rheumatic manifestations in leprosy, especially the lepromatous form. Lepromatous leprosy has been associated with the production of autoantibodies of various types. The incidence and potential pathogenetic significance of autoantibodies, especially rheumatoid factor and antinuclear antibodies in patients with leprosy is reviewed.

Systemic lupus erythematosus induced by isoniazid.

The case is described of a 73 year old man who presented with a lupus-like syndrome related to treatment with isoniazid and had IgG antinuclear antibodies against the nucleo-histone complex (H2A-H2B)-DNA. After a short course of treatment with prednisone and discontinuation of isoniazid the patient's lupus symptoms resolved and a gradual decrease in antibodies to (H2A-H2B)-DNA occurred. This case suggests that isoniazid is capable of inducing an autoantibody specificity associated with drug related lupus.