Serum Autoantibody Profiles Research Articles

Association of Fibrosing Myopathy in Systemic Sclerosis and Higher Mortality.

To determine if a unique subtype of scleroderma muscle disease exists by comparing the clinical features of systemic sclerosis (SSc; scleroderma) patients with predominant fibrosis on muscle biopsy to those with inflammatory muscle histopathology. This retrospective, cross-sectional study included SSc patients with muscle weakness and an available muscle biopsy. Biopsies with fibrosis but without inflammation/necrosis were designated as "fibrosing myopathy," and those with inflammation and/or necrosis were assigned a category of "inflammatory myopathy." Clinical data, including features of SSc, serum creatine kinase (CK) levels, electromyography, autoantibody profile, and survival, were compared between the 2 groups. The study population consisted of 37 weak SSc patients, 8 with fibrosing myopathy and 29 with inflammatory myopathy. Compared to those with inflammatory myopathy, patients with fibrosing myopathy were more likely to have diffuse SSc skin subtype (87% versus 62%; P = 0.18), African American race (62.5% versus 37.9%; P = 0.20), and a lower mean ± SD forced vital capacity (55.5 ± 31.9 versus 66.4 ± 17.6; P = 0.23). They also had lower mean ± SD CK values (516 ± 391 versus 2,477 ± 3,511 IU/liter; P = 0.007) and lower aldolase values (13.8 ± 4.7 versus 27.3 ± 4.7; P = 0.01). Patients with fibrosing myopathy had a significantly higher mortality (5 of 8 [62.5%] versus 4 of 29 [14.3%]; P = 0.005). Fibrosing myopathy is a unique histologic subtype of muscle disease among weak patients with SSc and is associated with significantly worse mortality compared to those with inflammation and/or necrosis on muscle biopsy.

Elevated serum autoantibodies against co-inhibitory PD-1 facilitate T cell proliferation and correlate with disease activity in new-onset systemic lupus erythematosus patients

BackgroundProgrammed cell death protein 1 (PD-1) plays an important role in immune response regulation as a co-inhibitory signal during T cell activation. However, there is little known about the serum autoantibody profile of PD-1 in systemic lupus erythematosus (SLE), a disease characterized by the breakdown of immune tolerance to self-antigens and an excessive production of autoantibodies. Thus, we aim to investigate the serum levels and function of anti-PD-1 in patients with new-onset SLE.MethodsSerum levels of anti-PD-1 IgG and IgM isotypes were detected in new-onset SLE patients (n = 90), rheumatoid arthritis (n = 50), primary Sjogren’s syndrome (n = 50), ankylosing spondylitis (n = 25), and healthy controls (HC) (n = 80) using an enzyme-linked immunosorbent assay (ELISA). The correlation of anti-PD-1 with clinical characteristics and laboratory parameters of patients with new-onset SLE was analyzed. The effects of purified anti-PD-1 IgG from SLE patients on T cell proliferation were measured using flow cytometry.ResultsThe data revealed increased levels of anti-PD-1 IgG, but not IgM, especially in new-onset SLE patients, and the positive rate of anti-PD-1 IgG was 30 (33.3%). The level of anti-PD-1 IgG was closely associated with malar rash (OR = 15.773), arthritis (OR = 22.937), serositis (OR = 16.008), hematological (OR = 35.187), renal (OR = 8.306), and neurological involvement (OR = 37.282). Moreover, the serum levels of anti-PD-1 IgG were positively correlated with the SLE disease activity index (SLEDAI) score (r = 0.296, p = 0.0046) and the erythrocyte sedimentation rate (ESR) (r = 0.2446, p = 0.0201). In vitro examination showed that purified anti-PD-1 IgG obtained from SLE patients enhanced T cell proliferation when co-cultured with dendritic cells (DCs).ConclusionsThe current study indicates, for the first time, that the serum levels of co-inhibitor autoantibodies against PD-1 are elevated in new-onset SLE patients and are associated with disease activity in SLE. Autoantibodies against PD-1, facilitating T cell proliferation, revealed a new insight into the function of negative regulation signals involved in the pathogenesis of SLE.

Detection of Early Cancer: Genetics or Immunology? Serum Autoantibody Profiles as Markers of Malignancy

The search for effective methods for detecting cancers at very early stages is currently a top priority of cancer research. While numerous oncogenes have been identified in and associated with human cancers, the last 50 years of molecular and genetic studies have not led to a breakthrough in either the diagnosis or the treatment of cancers. Therefore, the role of oncogenes in carcinogenesis is still unclear, as is their usefulness in the diagnosis of human cancers. In the present review, we discuss the concept of oncogenes and summarize the current approaches for the early detection of human cancers based on antibodies arrays.

Autoantigen microarrays reveal autoantibodies associated with proliferative nephritis and active disease in pediatric systemic lupus erythematosus.

IntroductionPediatric systemic lupus erythematosus (pSLE) patients often initially present with more active and severe disease than adults, including a higher frequency of lupus nephritis. Specific autoantibodies, including anti-C1q, anti-DNA and anti-alpha-actinin, have been associated with kidney involvement in SLE, and DNA antibodies are capable of initiating early-stage lupus nephritis in severe combined immunodeficiency (SCID) mice. Over 100 different autoantibodies have been described in SLE patients, highlighting the need for comprehensive autoantibody profiling. Knowledge of the antibodies associated with pSLE and proliferative nephritis will increase the understanding of SLE pathogenesis, and may aid in monitoring patients for renal flare.MethodsWe used autoantigen microarrays composed of 140 recombinant or purified antigens to compare the serum autoantibody profiles of new-onset pSLE patients (n = 45) to healthy controls (n = 17). We also compared pSLE patients with biopsy-confirmed class III or IV proliferative nephritis (n = 23) and without significant renal involvement (n = 18). We performed ELISA with selected autoantigens to validate the microarray findings. We created a multiple logistic regression model, based on the ELISA and clinical information, to predict whether a patient had proliferative nephritis, and used a validation cohort (n = 23) and longitudinal samples (88 patient visits) to test its accuracy.ResultsFifty autoantibodies were at significantly higher levels in the sera of pSLE patients compared to healthy controls, including anti-B cell-activating factor (BAFF). High levels of anti-BAFF were associated with active disease. Thirteen serum autoantibodies were present at significantly higher levels in pSLE patients with proliferative nephritis than those without, and we confirmed five autoantigens (dsDNA, C1q, collagens IV and X and aggrecan) by ELISA. Our model, based on ELISA measurements and clinical variables, correctly identified patients with proliferative nephritis with 91 % accuracy.ConclusionsAutoantigen microarrays are an ideal platform for identifying autoantibodies associated with both pSLE and specific clinical manifestations of pSLE. Using multiple regression analysis to integrate autoantibody and clinical data permits accurate prediction of clinical manifestations with complex etiologies in pSLE.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0682-6) contains supplementary material, which is available to authorized users.

DNA-methylation and autoantibodies based cancer diagnosis from body fluids

Special focus and aim of our research activities at AIT, the Austrian Institute of Technology, is to define reliable biomarkers suitable for early and non-invasive disease diagnosis from body fluids such as serum/plasma and saliva. Along a selection of research projects, which are described in more detail underneath, we will present and introduce the broad portfolio of high throughput technologies we successfully apply for diagnostic biomarker discovery and validation. As a first show case of successful non-invasive disease biomarker discovery we will present a study where we investigated and compared the genome wide methylation levels of lung cancer patients, patients suffering from lung fibrosis, patients with COPD (chronic obstructive pulmonary disease), and DNA samples derived from healthy lungs. Along this study we could identify specific methylation patterns for each of these lung diseases. After quantitative PCR validation of 240 disease specific methylation markers in the discovery sample set, the 90 top markers were picked and applied for serum testing (n=204). When we applied gradient boosting classification for differential diagnosis of tested lung diseases and healthy controls an AUC value of 0.95 was reached here to separate cancer from all other non-cancer samples whereas in differential diagnosis of healthy-, COPD and fibrosis patients AUC values of 0.71 and 0.49 were obtained for fibrosis, respectively COPD. Thus in case of COPD the presented method may be used to monitor cancer risk within COPD patients. Our second show case comprises a study where we screened cancer patients’ sera for tumor-specific antibody profiles using an in-house developed 16k protein-microarray. This methodology, which will be described in detail, enabled us to define different tumor-associated antigen (TAA) classifier panels for the big 4 cancer entities (breast, colon, prostate and lung cancer) which all showed very promising classification successes in distinction of patients versus controls. We will further present preliminary data obtained when comparing serum and saliva autoantibody profiles of breast-cancer patients and healthy controls.

Multiple Slowly Growing Nodular Lesions on the Lower Legs in a 78-Year-Old Obese Woman

A 78-year-old woman presented with a 3-year history of slowly growing painless nodular lesions on both legs that were refractory to topically applied corticosteroids. On physical examination there were approximately 20 erythematous waxy plaques and nodules on edematous lower legs (Figure 1). Twenty years prior, the patient had undergone hysterectomy and adnexectomy followed by radiation for treatment of endometrial cancer. Thereafter, she experienced chronic lymphedema of both legs. Furthermore, the patient has chronic heart failure (NYHA class III), obesity (body mass index [BMI], 32 [calculated as weight in kilograms divided by height in meters squared]), arterial hypertension, and type II diabetes mellitus. For 60 years she had been receiving thyroid hormone therapy with levothyroxine sodium (currently 100 μg/d) for treatment of hypothyroidism. She did not have a history of Graves disease. Results of laboratory investigations, including routine blood tests, tests of liver enzymes, renal function, and thyroid profile, serum protein analysis, immunoglobulin levels, and autoantibody profile were within normal limits. Venous duplex ultrasonography excluded a relevant chronic venous insufficiency. A punch biopsy sample of the lower leg was obtained (Figure 2 and Figure 3). What is your diagnosis?

Significance of Anti-retinal Autoantibodies in Cancer-associated Retinopathy with Gynecological Cancers

BackgroundThe presence of autoantibodies (AAbs) is the primary serological indicator of autoimmunity. Cancer-associated retinopathy (CAR) is associated with AAbs and different types of cancer. The goal of the study was to examine the profile of serum autoantibodies in women with gynecological cancers with and without paraneoplastic visual manifestation.MethodsRetrospective studies of a cohort of 46 women with symptoms of CAR and gynecological tumors, including endometrial, cervical, ovarian, and fallopian tubes, 111 women with similar tumors without symptoms of CAR, and 60 age-matched healthy controls. Presence of serum AAbs and the identity of targeted antigens were performed by western blotting and their significance was evaluated using an Fisher’s exact test.ResultsThe patients with gynecological CAR had the highest proportion of seropositivity (80%), followed by patients with gynecological cancers without CAR (61%) and healthy controls (58%). Differences in recognition frequencies were found for 17 antigens and 5 retinal antigens were frequently targeted: enolase, aldolase C, carbonic anhydrase II, recoverin and GAPDH. The occurrence of anti-glycolytic enzymes was 2-3 times more frequent in CAR and cancer patients than healthy controls. Anti-recoverin AAbs were prevalent in endometrial CAR. Anti-CAII antibodies were not significantly different between groups of women. In this cohort, cancer was diagnosed before the onset of retinopathy with latency from 2 months to 30 years. The discovery of the ovarian and endometrial cancers and manifestation of visual problems often coincided but Fallopian tube carcinoma was found after visual onset.ConclusionNew retinal targets were identified for gynecological CAR. Each gynecological-CAR has its own autoantibody profile different from non-CAR profile, implying that a complex autoantibody signature may be more predictable for diagnosis than a singular AAb. Specific anti-retinal AAbs were most prevalent in women with CAR but their profiles were not fully distinguished from cancer controls.

Abstract A37: Low-density protein array reveals inflammation associated serum-autoantibody profile in prostate cancer.

Abstract Background: Chronic prostatic inflammation of the prostate is one of the main reasons for false positive serum PSA tests in prostate cancer screening. In addition there is accumulating evidence that an inflammatory microenvironment may support the development of malignancy and the progression to metastatic disease. Easy accessible and non-invasive indicators for prostate inflammation would be very helpful for a biopsy decision after an elevated PSA test result. However, at present, the histological examination of prostate biopsies remains the only way for identification of prostatic inflammation. The finding that cancer is immunogenic and that patients are able to elicit and potentially amplify an immune response against their tumor antigens suggests that autoantibodies serve as non-invasive serum biomarkers for prostate cancer diagnosis. Consequently, autoantibodies may also be capable of detecting differential disease progression grades -e.g. inflammatory states- enabling a more specific and individualized treatment in cancer therapy. In the present study, we aimed to find new non-invasive biomarkers for the diagnosis of prostatic inflammation in prostate cancer. Methods: Seventy prostate cancer tissue samples derived from radical prostatectomy were classified into two groups (high/low inflammation) according to the amount of tissue infiltrating lymphocytes. Clinical parameters including PSA, fPSA, GSC, prostate weight, prostate volume, tumor size, age and CRP were controlled for equal distribution amongst the two groups. The corresponding blood serum samples were analyzed using a low density protein array (microarray) containing 4012 prostate cancer associated and inflammation related recombinant proteins. Statistical comparison of the serum autoantibody profile in the two groups was used to identify differentially expressed biomarkers. Finally, specificity of candidate autoantibody markers was validated by immunohistochemical detection of corresponding autoantigens in prostate tissue. Results: Autoantibodies against 165 antigens were found to be differentially expressed in the high compared to the low inflammation group. 30 out of these proteins have recently been identified to be associated with prostate cancer. The identified autoantibody profile discriminated between prostate cancer patients bearing high or low inflammation in prostate tissue with a ROC curve AUC of 0.71. Out of the top ranked autoantibodies, three candidate proteins were selected due to their differential expression and significant up-regulation in serum samples of the high inflammation group: SPAST (Spastin), STX18 (Syntaxin 18) and SPOP (speckle-type POZ protein). Immunohistochemical analysis showed that targets of all three autoantibody candidates are expressed in epithelial prostate cells. Remarkably, the highest staining intensity was detected in tissue infiltrating lymphocytes in both high and low inflammation samples. Conclusion: The presented data evaluates the possible use of serum autoantibody markers as non-invasive indicators for the diagnosis of chronic inflammation in prostate cancer. The polyclonal immune response to aberrant proteins in heterogeneous diseases such as cancer and autoimmunity favors the development of autoantibody markers with potential specificities to detect disease subtypes and disease progression grades. We provide evidence for an inflammation specific antibody profile and verify the expression of three biomarker candidates in prostate tissue. Citation Format: Bettina Schlick, Petra Massoner, Angelika Luecking, Christof Saifarth, Georg Schaefer, Klaus Marquart, Peter Amersdorfer, Peter Schulz-Knappe, Helmut Klocker. Low-density protein array reveals inflammation associated serum-autoantibody profile in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A37.

Myositis specific and myositis associated autoantibodies in idiopathic inflammatory myopathies: a serologic study of 46 patients

To characterize serum autoantibody profiles of patients with idiopathic inflammatory myopathies (IIM) by searching for myositis-specific (MSA) and myositis-associated (MAA) antibodies with sensitive and specific laboratory tests. We tested the sera from 46 Caucasian patients diagnosed as affected with IIM at the Division of Rheumatology of Padova University (21 polimyositis, PM; 22 dermatomyositis, DM; 3 myositis overlap syndrome). All patients had definite IIM according to the criteria of Bohan-Peter. MSA including anti-tRNA synthetase (anti-Jo-1 and others) and anti-Mi-2 were determined by RNA immunoprecipitation and a modified immunoblot test, respectively. MAA (-U1RNP, -U2RNP, RoRNP, PM/Scl, Ku) were detected by counterimmunoelectrophoresis and immunoblot. Serum MSA and/or MAA were found in 30/46 (65%) patients with IIM. Twenty-three patients (50%) were positive for at least one MSA: anti-Jo-1 in 15 (33%), anti-Mi-2 in 6 (13%), and other anti-tRNA synthetase in 3 (6%). One patient was anti-Jo-1/Mi-2 positive. Moreover, 18 patients (39%) were positive for at least one MAA: anti-Ro/SSA in 13 (28%), anti-U1RNP in 4 (9%), anti-PM/Scl in 1 (2%) and anti-Ku in 1 (2%). Coexisting MSA and MAA were observed in 8 patients (17%), anti-Jo-1/SSA positive in most cases. Anti-Jo-1 was predominantly associated with PM (57% in PM vs 14% in DM), whereas anti-Mi-2 was exclusively found in DM patients (27%). Anti-synthetase antibodies were closely associated with interstitial lung disease and polyarthritis; anti-Mi-2 positive DM patients did not have lung involvement. Notably, anti-Ro/SSA antibody was frequently observed and almost equally detected in either PM or DM (about 30%): in more than 50% of cases the antibody was associated with one MSA. By means of analytically reliable methods, MSA was detected in 50% of our IIM patients. Searching for MSA in patients with IIM is recommended because of its diagnostic and prognostic value.

AIRE gene mutations and autoantibodies to interferon omega in patients with chronic hypoparathyroidism without APECED

To assess autoimmune regulator (AIRE) gene mutations, class II HLA haplotypes, and organ- or non-organ-specific autoantibodies in patients with chronic hypoparathyroidism (CH) without associated Addison's disease (AD) or chronic candidiasis (CC). Twenty-four patients who had CH without AD or CC were included in the study. AIRE gene mutations in all 14 exons were studied using PCR in 24 patients, 105 healthy controls and 15 first-degree relatives of CH patients with AIRE mutations. Human leucocyte antigens (HLA) were determined for all 24 patients and 105 healthy controls. Autoantibodies to a range of antigens including NACHT leucine-rich-repeat protein-5 (NALP5) and interferon omega (IFNω) were tested in all 24 patients. AIRE gene mutations were found in 6 of 24 (25%) patients, all females, and this was significantly higher (P < 0·001) compared with AIRE mutations found in healthy controls (2/105). Three patients (12·5%) had homozygous AIRE mutations characteristic of Autoimmune-Poly-Endocrinopathy-Candidiasis-Ectodermal-Dystrophy and all three were also positive for IFNω-autoantibodies. Three patients (12·5%) had heterozygous AIRE mutations; two of these were novel mutations. One of the patients with heterozygous AIRE mutations was positive for both NACHT leucine-rich-repeat protein 5 and IFNω autoantibodies. Heterozygous AIRE mutations were found in 10 of 15 first-degree relatives of CH patients with AIRE mutations, although none was affected by CH. Class II HLA haplotypes were not statistically different in patients with CH compared to healthy controls. Analysis of AIRE gene mutations together with serum autoantibody profile should be helpful in the assessment of patients with CH, in particular young women with associated autoimmune diseases.

Brain-reactive autoantibodies are nearly ubiquitous in human sera and may be linked to pathology in the context of blood–brain barrier breakdown

Previous studies have reported antibodies bound to cells in postmortem Alzheimer's disease (AD) brains, which are only rarely observed in the brains of healthy, age-matched controls. This implies that brain-reactive autoantibodies exist in the sera of AD individuals and can gain access to the brain interstitium. To investigate this possibility, we determined the prevalence of brain-reactive antibodies in sera from AD patients, patients with other neurodegenerative diseases, age-matched, non-demented controls and healthy younger individuals via immunohistochemistry and western blot analysis. Surprisingly, western analyses revealed that 92% of all human sera tested contain brain-reactive autoantibodies. When sera were used to probe western blots of human, pig, or rat brain membrane proteins, a number of comparably-sized protein targets were detected, suggesting cross-species reactivity. While the presence of brain-reactive autoantibodies was nearly ubiquitous in human sera, some autoantibodies appeared to be associated with age or disease. Furthermore, the intensity of antibody binding to brain tissue elements, especially the surfaces of neurons, correlated more closely to the serum's autoantibody profile than to age or the presence of neurodegenerative disease. However, while the blood–brain barrier (BBB) in control brains remained intact, BBB breakdown was common in AD brains. Results suggest a high prevalence of brain-reactive antibodies in human sera which, in the common context of BBB compromise, leads us to propose that these antibodies may contribute to the initiation and/or pathogenesis of AD and other neurodegenerative diseases.

Multiple serological biomarkers for colorectal cancer detection

The aim of this study was to initiate a survey of human autoantibody responses to a panel of select colorectal tumor-associated antigens identified by previous serological analysis of a cDNA expression library and to subsequently identify multiple serological biomarkers for the detection of colorectal cancer. For screening of autoantibodies against colorectal tumor-associated antigens, sera from 94 colorectal cancer patients and 54 normal controls were analyzed by enzyme-linked immunosorbent assay using recombinant rCCCAP, rHDAC5, rP53, rNMDAR and rNY-CO-16 proteins as coating antigens. Seropositivity among colorectal cancer patients to the 5 individual coating antigens varied from 18.1% to 35.1%. Seropositivity to any of the 5 coating antigens was 58.5% and combining this analysis with evaluation of serum carcinoembryonic antigen (> or =5 ng/ml) significantly increased the seropositivity to 77.6%. Seropositivity of early-stage (Dukes' Stages A and B) colorectal cancer patients to CEA was 21.9%, and seropositivity to any of the 5 colorectal cancer-associated antigens was 53.7%, and the combination of these 2 measurements resulted in a higher diagnostic capacity (65.9%) than either marker alone. In conclusion, these results collectively indicated that combined detection of serum autoantibody profiles against our panel of colorectal tumor-associated antigens and the analysis of carcinoembryonic antigen provides a promising diagnostic biomarker for colorectal cancer, particularly among early-stage patients.

A comprehensive evaluation of serum autoantibodies in primary biliary cirrhosis

In primary biliary cirrhosis (PBC) serum markers other than anti-mitochondrial antibodies (AMA) are promising in terms of disease severity and comorbidities, as well represented by anti-nuclear antibodies (ANA). The aim of the present study was thus to evaluate the prevalence and clinical significance of a large profile of serum autoantibodies in PBC sera. We utilized 69 sera from European patients with PBC (including 20 AMA-negative) and 297 sera from geographically and sex-matched healthy controls. All sera were tested for the presence of ANA and autoantibodies associated with thrombophilia, vasculitis, and gastrointestinal disease. Autoantibodies other than AMA were detected in 53/69 (76%) PBC sera vs. 105/297 (35%) among controls. The prevalence of ANA (targeting dsDNA, Sm, chromatin, ribosomal-P, RNP, SmRNP, SSA, SSB, and centromere) and thrombophilia-associated autoantibodies (i.e. anti-β2GPI, phosphatydilserine, prothrombin) was common among patients with PBC. When clinical features were compared, the presence of anti-prothrombin IgM was associated with a worse prognosis as represented by a higher Mayo score. We demonstrate an increased prevalence of ANA and thrombophilia-associated autoantibodies in PBC sera and an association between the latter autoantibodies and PBC stage. The role of thrombophilia-associated antibodies will warrant further studies, based in particular on the incidence of portal hypertension at early stages of PBC.

Anti-cyclic citrullinated peptide and anti-keratin antibodies in patients with idiopathic inflammatory myopathy

To investigate the prevalence of anti-cyclic citrullinated peptide (anti-CCP) and anti-keratin antibodies (AKAs) in a cohort of patients with idiopathic inflammatory myopathy. In a cross-sectional study, we determined the presence of anti-CCP and AKAs by ELISA and IIF, respectively, in a cohort of 90 consecutive patients with idiopathic inflammatory myopathy. Associations between anti-CCP and clinical manifestations or other autoantibodies were determined with the chi-square and Mann-Whitney U-tests. Radiographs of hands were retrospectively evaluated. Serum autoantibody profile was determined in all patients. Twelve patients were positive to anti-CCP (13.3%); in eight cases values were moderate-high. AKAs were not detected in any patient. Comparison between patients positive and negative to anti-CCP did not show clinical or biological differences. Arthritis joint erosions or positive status to anti-synthetase antibodies were not more frequent in patients with anti-CCP antibodies. Prevalence of RF was the only variable significantly associated with the presence of these antibodies (P = 0.043). High titres of anti-CCP can occasionally be found in patients with inflammatory myopathy. Therefore, a possible diagnosis of RA should be considered with caution in these patients.

Obstructive sleep apnea in patients with inflammatory myopathies

The purpose of this study was to determine the frequency of obstructive sleep apnea in patients with inflammatory myopathy. An observational and prospective study was performed on a cohort of adult patients with inflammatory myopathy followed at a specialized outpatient clinic. Sixteen consecutive adult patients were evaluated by the Epworth Sleepiness Scale (ESS) and by complete polysomnography study. Disease activity and severity were assessed using the Myositis Disease Activity Assessment Tool (MDAAT) and Myositis Damage Index (MDI), respectively. Associations between sleep parameters and other factors were calculated using the chi-square test, Fisher's exact test, Mann-Whitney U-test, and Wilcoxon's test. A serum autoantibody profile was determined for all patients. The mean apnea-hypopnea index was 28.7 (23.8), and 14 patients (87%) had an apnea-hypopnea index >5. The mean frequency of respiratory arousals was 20.1 (12.5). Eleven (68%) patients reported frequently-always snoring, and 3 (19%) had excessive daytime sleepiness (ESS >10). Seven patients were offered continuous positive airway pressure (CPAP) therapy; 4 tolerated the procedure well and reported a clear improvement in daytime sleepiness and/or sleep quality. No significant association was observed between the apnea-hypopnea index and clinical or immunological groups. Dysphagia, disease activity, and disease severity were not significantly associated with any sleep parameters. The frequency of obstructive sleep apnea in adult patients with inflammatory myopathy is high. The possibility that these alterations play a role in persistent fatigue in these patients cannot be ruled out.

Serologic Profiles Aiding the Diagnosis of Autoimmune Gastrointestinal Dysmotility

Autoimmune gastrointestinal dysmotility is a limited autoimmune dysautonomia occurring idiopathically or in the context of an anatomically remote neoplasm, previously documented or unsuspected. Here we report 24 Mayo Clinic patients in whom the profile of serum autoantibodies aided this diagnosis. All patients were ascertained serologically in the course of service evaluation for autoantibodies consistent with neurologic autoimmunity. Review of their histories, motility studies, and laboratory findings revealed that all had presented with subacute gastrointestinal dysmotility. Recorded motility abnormalities included esophageal dysmotility 8 (6 had achalasia), delayed gastric emptying 12, slow small intestinal transit 7, slow colonic transit 4, and pelvic floor dyssynergia 4. Four patients underwent abdominal surgery; 2 commenced total parenteral nutrition. Plasma membrane cation channel autoantibodies were detected in 23 patients: neuronal voltage-gated calcium channel (5 N-type and 1 P/Q-type), acetylcholine receptor (11 ganglionic-type and 4 muscle-type), and neuronal voltage-gated potassium channel autoantibodies (4). Two patients had antineuronal nuclear autoantibodies, type 1. Approximately half of the patients had neural autoantibodies (including skeletal muscle striational and glutamic acid decarboxylase, 65kd isoform) or other antibody markers of organ-specific autoimmunity (thyroid or gastric parietal cell specificities). Neoplasia was diagnosed in 11 patients (9 recent, 2 remote): lung, breast and endometrial, gastrointestinal and thymoma. Moderate to dramatic improvement in gastrointestinal symptoms was reported after immunotherapy in 4 of 4 patients treated and after pyridostigmine treatment in 2 of 2 patients treated. Autoimmune serology aids the diagnosis of autoimmune gastrointestinal dysmotility, both paraneoplastic and idiopathic, and might guide management.

Abstract 5158: Serological autoantibody profiling in bladder cancer using protein arrays

Abstract Cancer cells might release at least part of their contents into the blood stream directly or through extracellular fluids upon cell damage or death. Cancer proteins not present in non-neoplastic cell subpopulations might then elicit a host immune response. The serum autoantibody repertoire from cancer patients might therefore, be exploited for the identification of tumor-associated antigens (TAAs). The recognition that human tumors stimulate the production of autoantibodies against such autologous cellular proteins (TAAs) has opened the door to the possibility that autoantibodies could be translated into serological tools for early diagnosis and/or disease management. In this report, we attempt at exploring the serum proteome of patients with bladder cancer using protein arrays. The serum autoantibody profiles of 18 patients with bladder cancer and 6 control specimens including cases affected with other neoplasias, benign urological diseases and healthy individuals was investigated using protein arrays containing more than 12,000 proteins. Data analyses included T-test as well as clustering strategies using the Prospector and CIMMiner softwares. Supervised clustered heatmaps served to highlight identified biological differences among autoantibody profiles arrays of bladder cancer cases and controls. Moreover, protein expression patterns of the identified immunogenic TAAs were assessed by immunohistochemistry on tissue microarrays (n=92). Supervised clustering analysis segregated bladder cancer patients from controls. A panel of 171 autoantibodies was identified to be differentially expressed in bladder cancer patients as compared to control individuals in a significant manner. Bladder cancer TAAs included proteins involved in cell proliferation, signal transduction, apoptosis, DNA-binding and transcription factors, among others. Immunohistochemical analyses on tissue arrays served to validate the increased protein expression of identified TAAs in bladder tumors. Moreover they revealed the relevance of expression patterns of proteins such as clusterin for bladder cancer diagnostics, staging and outcome prognosis. Protein expression patterns of clusterin were significantly associated with clinico-pathological variables such as tumor size (P=0.034), T1 substaging (P=0.013) or progression into muscle invasive disease (P=0.020). Thus, the novel application of high-throughput protein arrays for serum autoantibody profiling is providing critical information not only to identify TAAs candidate biomarkers for bladder cancer diagnostics and potential immunological therapeutic targets, such as clusterin, but also to further characterize bladder cancer progression.

Serum autoantibody profile of patients with age‐related macular degeneration

Age‐related macular degeneration (AMD) is the leading cause of irreversible blindness in industrial countries. Recent findings indicate that both genetic and immunological factors are involved in the pathogenesis of AMD. Autoantibodies in particular have been implicated in AMD pathogenesis, yet only a few antibodies have been identified in sera from patients with AMD. In order to reveal an autoantibody profile for AMD and identify biomarkers for progression of this disease, we incubated an antigen microarray with serum samples from patients with AMD and healthy controls. Sera from the AMD group contained high levels of IgG autoantibodies to some antigens including cyclic nucleotide phosphodiesterase and proliferating cell nuclear antigen, when compared to the normal group. Moreover, the IgG/IgM ratio for antibodies to some systemic antigens correlated with the stage of AMD. Our data suggest that these IgG autoantibodies and the ratio may be used as biomarkers for AMD. Further studies are needed to clarify whether the autoantibodies directly cause AMD or are secondary products in the progression of AMD. This work is supported by the Dobbs Foundation, Special Trustees of Moorfields Eye Hospital and the NIH.

Bullous pemphigoid antigen II (BP180) and its soluble extracellular domains are major autoantigens in mucous membrane pemphigoid: the pathogenic relevance to HLA class II alleles and disease severity

Mucous membrane pemphigoid (MMP), a chronic autoimmune subepithelial blistering disease, is associated with circulating IgG and/or IgA autoantibodies against several basement membrane zone antigens. The heterogeneity of clinical presentation and diversity of target autoantigens have contributed to difficulties in characterizing this condition immunologically. To analyse serum autoantibody profile and HLA class II alleles in MMP patients and to correlate this with the clinical presentation of disease. Well-defined subgroups consisting of 124 patients with MMP were examined for IgG and IgA reactivity with immunoblotting using human epidermal, dermal and placental amnion proteins. The results were further analysed on the basis of detailed clinical (sites of involvement and disease severity) and immunopathological criteria (immunofluorescence study and HLA class II alleles). Immunoblot assay revealed that the majority of MMP patients had IgG (93 of 124, 75%) and/or IgA autoantibodies (63 of 124, 51%) to BP180 (including its soluble ectodomains, 120-kDa LAD-1 and 97-kDa LABD97 antigens). Other antigens targeted predominantly by IgG autoantibodies included: BP230 in 34 (27%), beta4 integrin in 26 (21%), and laminin 5 in three (2%). All the BP230+ sera and 23 (88%) beta4 integrin+ sera also reacted with at least one of the BP180 antigens. Over 85% of patients with reactivity to beta4 integrin had ocular involvement. In most cases of MMP, more severe clinical features were associated with antibody reactivity to multiple basement membrane zone antigens, as well as reactivity to multiple BP180 component antigens. Dual BP180/LAD-1 reactivity with IgG and IgA was associated with a more severe phenotype. In addition, the subset-dependent autoantibody reactivity correlated well with specific HLA class II alleles, DQB1*0301, DRB1*04 and DRB1*11. Our results confirmed that BP180 is a major autoantigen targeted by the sera of patients with MMP. The disease-prevalent HLA class II alleles and humoral autoimmune response against the particular subsets of antigenic epitope(s) within BP180 ectodomain may contribute to the clinicopathological significance and disease severity of MMP.

Autoantibodies to extracellular matrix protein 1 in lichen sclerosus

Lichen sclerosus is a common acquired inflammatory disorder of skin and mucous membranes. The aetiology is unknown, although HLA-subtype susceptibility and high rates of other autoimmune disorders suggest that autoantibodies to specific mucocutaneous antigens are involved. The clinicopathological similarities between lichen sclerosus and lipoid proteinosis, which results from mutations in extracellular matrix protein 1 (ECM1), suggest this protein as an autoantigen. We analysed serum autoantibody profiles in 171 individuals (86 with lichen sclerosus, 85 healthy controls) by immunoblotting of extracts from normal human skin and lipoid proteinosis skin (lacking ECM1). We generated a full-length glutathione-S-transferase fusion protein for ECM1 to confirm specific immunoreactivity. We affinity-purified serum from patients with lichen sclerosus and did indirect immunofluorescence microscopy on normal skin with or without preabsorption with recombinant ECM1. By immunoblotting, IgG autoantibodies were found in 20 (67% [95% CI 45-84]) of 30 lichen sclerosus serum samples. The highest titre was 1 in 20. The bands were not detected in ECM1-deficient substrate. These samples, and those from 56 other patients with lichen sclerosus, showed immunoreactivity to the recombinant ECM1 protein (64 of 86 positive; 74% [65-84]). Only six (7% [2-13]) of 85 control serum samples were positive. Affinity-purified IgG from serum of patients with lichen sclerosus labelled skin similarly to a polyclonal antibody to ECM1. The positive staining was blocked by preabsorption with excess recombinant ECM1 protein. These findings provide evidence for a specific humoral immune response to ECM1 in lichen sclerosus and offer insight into disease diagnosis, monitoring, and approaches to treatment.