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.

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.

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.

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.

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.

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.

Studies of antinuclear antibodies in rheumatoid arthritis.

Antinuclear antibodies (ANA) by the indirect immunofluorescence method and their immunospecificities were investigated in 60 patients with rheumatoid arthritis using HEp-2 cells as substrates. ANA were detected in 35% of the patients. The patterns of nuclear staining observed included: diffuse or patchy homogeneous (20%), speckled (8.3%), centromere (3.3%), nucleolar (1.6%) and in 1 patient a mixed pattern (1.6%). The presence of antihistone antibodies (20%) correlated with the presence of an homogeneous pattern on the HEp-2 cells.

Acute familial myositis with a common autoimmune response.

In 1986, we diagnosed and treated 4 patients, all members of 1 rural family, who presented simultaneously with a polymyositis-like syndrome, anticytoplasmic antibodies (on HEp-2 cells), and precipitating antibodies of anti-Jo-1 and anti-PM-Scl specificities. Serum samples from these patients reacted by immunodiffusion against serum from a rodent that had been caught in the patients' house. The precipitin line showed complete immunologic identity with anti-Jo-1. To study the specificity of this serum-serum reaction, 2 mice (Mus musculus) and 1 rat (Rattus rattus) from the neighboring areas of the patients' house were caught and killed. Sections of the liver, kidney, spleen, and skeletal muscle were processed for histopathologic examination and for direct and indirect immunofluorescence studies of the patients' sera. The rodents' sera were tested by immunodiffusion against serum samples from 25 patients with systemic lupus erythematosus, 11 with rheumatoid arthritis, 6 with mixed connective tissue disease, and 11 with various other rheumatic diseases, as well as 16 healthy controls. A serum-serum precipitin reaction was noted between the rodents' sera and sera from 2 of the patients with polymyositis-like syndrome. A distinct, but weaker, reaction was found with 2 lupus patients' sera (8%), and with 1 healthy control serum (6%). The rodents' sera were antinuclear antibody positive by indirect immunofluorescence on HEp-2 cells. These results, suggest that some rodents could be carriers of an as-yet-unknown transmissible agent that, in susceptible individuals, induces clinical and serologic manifestations similar to those of polymyositis.

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.

Evidence of a new antigen-antibody system (anti-Mic-1) in patients with systemic lupus erythematosus and hyperthyroidism.

Autoimmune thyroid diseases may occur in association with systemic rheumatic disorders, and usually they show a high prevalence of antithyroglobulin and antimicrosomal antibodies. We report 6 patients with the clinical association of systemic lupus erythematosus (SLE) and hyperthyroidism. Of interest, in 5 of the 6 patients (83%), we found an antibody directed against a microsomal extract of human thyroid gland which was different than previous microsomal antibodies in that it was a precipitating antibody; we have called it anti-Mic-1 antibody. We investigated the prevalence of this specific autoimmune reaction in 58 patients with idiopathic SLE, 30 with hyperthyroidism, 15 with Hashimoto's thyroiditis, 25 with insulin dependent diabetes mellitus, 45 with rheumatoid arthritis, and 25 healthy controls. No control had anti-Mic-1 antibody. In addition, this antibody was shown to be organ specific. We suggest that patients with the combined association of SLE and hyperthyroidism may represent a different subset in the spectrum of SLE. The high prevalence of this antigen-antibody reaction in these cases may serve as a serological marker of this association.

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.

Prevalence of antinuclear antibodies in patients with habitual abortion and in normal and toxemic pregnancies.

The prevalence of antinuclear antibodies (ANA) was investigated in the sera from patients with habitual abortion (HA) and in normal (NP) and toxemic pregnancies (TP). ANAs were more prevalent in patients with HA (30%) as compared with TP (15%) and the control group (6.6%). Only one patient, from the HA group, fulfilled four criteria for systemic lupus erythematosus (SLE). Most other patients with positive ANAs had some of the criteria for SLE, but none of the ANA negative patients had any of them. These studies suggest that there is a high prevalence of ANA in our group of women with HA and their identification may help to identify those patients who may eventually develop SLE which has been reported to be preceded in many women by repeated spontaneous abortions. In addition, immunological factors, at least in some cases, could play a role in the pathogenesis of HA.

Study of rheumatic manifestations and serologic abnormalities in patients with lepromatous leprosy.

We investigated the rheumatic and laboratory features associated with rheumatic syndromes in 32 patients with lepromatous leprosy. Twenty-seven (84%) developed a broad range of rheumatic manifestations, the most common being the presence of arthritis which was symmetric and polyarticular, resembling rheumatoid arthritis. The laboratory abnormalities included an elevated sedimentation rate in 32 cases (100%), a positive rheumatoid factor in 6 (18.7%), and antinuclear antibodies in one (3.1%). A careful history and the recognition of rheumatic manifestations will help in the identification of this type of leprosy.

Prevalence of anti-SSA (Ro) antibodies in a Mexican population of patients with various systemic rheumatic diseases.

The prevalence of anti-SSA (Ro) antibodies was investigated in 126 patients with various systemic rheumatic diseases and in 77 normal subjects. Also, we studied the relationship between these autoantibodies and the clinical features of our patients with SLE. Anti-SSA (Ro) occurred with a frequency of 14% in SLE but was not associated with specific clinical features of the disease. In Sjögren's syndrome the frequency was 25%, in rheumatoid arthritis 20.3% and in mixed connective tissue disease 15.4%. Finally, in the control group anti-SSA (Ro) antibodies were not present. We believe that the presence of this antibody is not specific to any rheumatic disease in our Mexican population, and should not be used as a "marker antibody" of some clinical features in patients with SLE.

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.

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.

Trichinosis with severe myopathic involvement mimicking polymyositis. Report of a family outbreak.

We describe an outbreak of trichinosis in 3 members of a rural family. In the 3 patients eating raw pork was the source of infection. They presented with myalgias and severe proximal muscle weakness mimicking polymyositis. The diagnosis was made by demonstration of larvae of Trichinella spiralis in the muscle biopsy and also by the presence of anti-Trichinella antibodies detected by double immunodiffusion in their sera. We call attention to the unusual clinical presentation of trichinosis in our patients that was manifested by severe muscle weakness that may be confused with polymyositis.