Non-organ-specific autoimmunity in adult 47,XXY Klinefelter patients and higher-grade X-chromosome aneuploidies.

Current literature regarding systemic autoimmune diseases in X-chromosome aneuploidies is scarce and limited to case reports. Our aim was to evaluate the frequency of anti-nuclear (ANAs), extractable nuclear (ENA), anti-double-stranded DNA (dsDNAs), anti-smooth muscle (ASMAs) and anti-mitochondrial (AMAs) antibodies in a large cohort of adults with Klinefelter's syndrome (KS, 47,XXY) and rare higher-grade sex chromosome aneuploidies (HGAs) for the first time. Sera from 138 X-chromosome aneuploid patients [124 adult patients with 47,XXY KS and 14 patients with HGA (six children, eight adults)] and 50 age-matched 46,XY controls were recruited from the Sapienza University of Rome (2007-17) and tested for ANAs, ENAs, anti-dsDNAs, ASMAs and AMAs. Non-organ-specific immunoreactivity was found to be significantly higher in patients with 47,XXY KS (14%) than in the controls (2%, p = 0.002). Among all the antibodies investigated, only ANAs were observed significantly more frequently in patients with 47,XXY KS (12.1%) than in the controls (2%, p = 0.004). No anti-dsDNA immunoreactivity was found. Stratifying by testosterone replacement therapy (TRT), non-organ-specific autoantibody frequencies were higher in TRT-naive (p = 0.01) and TRT-treated groups than in controls. No patients with HGA were found positive for the various autoantibodies. Non-organ-specific autoantibodies were significantly present in 47,XXY adult patients. Conversely, HGAs did not appear to be target of non-organ-specific immunoreactivity, suggesting that KS and HGAs should be considered as two distinct conditions. The classification and diagnosis of systemic autoimmune diseases is frequently difficult. To support a correct clinical evaluation of KS disease and to prevent eventual secondary irreversible immune-mediated damages, we highlight the importance of screening for non-organ-specific autoimmunity in Klinefelter's syndrome.

Acromegaly and joint pain: is there something more? A cross-sectional study to evaluate rheumatic disorders in growth hormone secreting tumor patients.

PURPOSE: The aim of the present study was to evaluate the rheumatic profile in acromegalic patients to better characterize joint pain. METHODS: The immunological pattern (rheumatoid factor; antinuclear antibodies-ANA, extractable nuclear antigens-ENA-Ab; anti-citrullinated protein antibodies; erythrocyte sedimentation rate) was evaluated in 20 acromegaly subjects (AS) and 20 control subjects (CS). Bilateral joint ultrasound of hands/wrists and nail capillaroscopy were also performed. RESULTS: Articular pain was more frequent in AS than in CS (p = 0.027). No difference was detected in immunological parameters. ANA and ENA-Ab were positive in only 10% of AS and in 5% of CS, while no difference was found in anti-citrullinated protein antibodies. No difference was detected between rheumatoid factor positivity, but threefold higher IgG were detected in AS compared to CS. The erythrocyte sedimentation rate was significantly higher in AS than CS (p = 0.040), while in AS, there was a trend in increased Power Doppler (PWD) articular uptake. The capillaroscopic evaluation showed a significant difference in almost each parameter (presence and number of tortuous capillaries, capillary enlargements, and hemorrhages), showing a moderate-to-severe microangiopathy in AS. CONCLUSION: The results of our study suggest that joint damage in acromegaly has not an autoimmune etiology. Increased erythrocyte sedimentation rate levels and PWD alteration in acromegalic population reflect a possible inflammatory nature, while the capillaroscopic findings suggest a moderate-to-severe microangiopathy that could help to identify patients with a greater macroangiopathic risk.

Detection of Anti-Extractable Nuclear Antigens in Patients with Systemic Rheumatic Disease via Fluorescence Enzyme Immunoassay and Its Clinical Utility.

PURPOSE: Testing for autoantibodies to extractable nuclear antigens (ENAs) plays an important role in the diagnosis and management of systemic rheumatic disease. Currently, no gold standard tests are available for detecting anti-ENAs. To address this gap, we aimed to identify an assay that exhibits satisfactory diagnostic performance in the detection of five common anti-ENAs by comparing two commonly used assays, an automated fluorescent enzyme immunoassay (FEIA) and a microplate ELISA assay. MATERIALS AND METHODS: Sera from 100 patients with systemic rheumatic disease were collected and assayed with FEIA and microplate ELISA to detect anti-ENAs. Statistical analyses were performed to check the agreement rate between the two platforms using kappa coefficients. Analytical sensitivity and specificity for each assay were calculated. RESULTS: The concordance rates between ELISA and FEIA ranged from 89% for anti-RNP to 97% for anti-Scl-70, and the kappa coefficients of the two assays were in the range of 0.44 to 0.82. Between the two assays, a significant difference in sensitivity and specificity was seen only for anti-Sm and anti-RNP, respectively. CONCLUSION: In this study, FEIA and ELISA showed comparable efficiency for detecting anti-ENAs.

From HDL-cholesterol to HDL-function: cholesterol efflux capacity determinants.

PURPOSE OF REVIEW: The validity of HDL-cholesterol (HDL-C) elevation as a therapeutic target has been questioned, in comparison to enhancing HDL functionality. Cholesterol efflux capacity (CEC) is an in-vitro assay that measures the ability of an individual's HDL to promote cholesterol efflux from cholesterol donor cells such as macrophages. CEC of HDL is a predictor of cardiovascular risk independent of HDL-C levels. However, molecular determinants of CEC and the effects of diseases and therapeutic interventions on CEC have not been completely defined. RECENT FINDINGS: We review here recent findings on elevated HDL-C and disease risk, as well as determinants of CEC, from genetics and proteomics to pathophysiology and therapeutic interventions that contribute to our understanding of CEC as a biomarker of HDL functionality. SUMMARY: Elevated HDL-C levels are not always protective against cardiovascular disease and mortality. CEC is a heritable trait, and genetic polymorphisms in genes involved in HDL and triglycerides metabolism are associated with CEC. Multiple HDL proteins correlate positively with CEC levels and inversely with noncalcified plaque burden. Differences in CEC assays that make comparisons between studies difficult are also emphasized. CEC should be measured in clinical trials of lipid-modifying and anti-inflammatory therapies to determine whether increases are cardioprotective.

Analytical and clinical comparison of two fully automated immunoassay systems for the detection of autoantibodies to extractable nuclear antigens.

BACKGROUND: Detection of antinuclear antibodies (ANA) by indirect immunofluorescence assay (IIFA) is increasingly substituted by fully automated solid phase immunoassays. This study evaluated the performance of an automated chemiluminescence immunoassay (CIA) and fluorescence enzyme immunoassay (FEIA) and compared their performance to that of IIFA. METHODS: The study included an unselected prospective study population suspected of systemic autoimmune rheumatic disease. ANA were measured by IIFA, while in parallel sera were tested by CIA QUANTA Flash CTD Screen Plus on the BIO-FLASH® and FEIA EliA CTD Screen on the Phadia® 250 system. As validation, retrospective cohorts of patients with ANA-associated rheumatic disease (AARD) and healthy controls were tested. RESULTS: Prospectively, sensitivity of IIFA, CIA and FEIA was 90%, 99% and 92%, respectively. Specificity was 76%, 76% and 84%, respectively. Total percent agreements between the three methods were 75.2% (IIFA vs. CIA), 79.2% (IIFA vs. FEIA) and 85.4% (FEIA vs. CIA). The AUC values were 0.95 for CIA and 0.93 for FEIA and did not significantly differ. Retrospectively in individual AARD cohorts, similar results were obtained comparing both CTD screens. CONCLUSIONS: Both FEIA and CIA CTD screen significantly outperformed IIFA, with a higher specificity for FEIA and higher sensitivity for CIA. Based on ROC analysis, major contributor to the difference between the two solid phase immunoassays was the cut-off.

Apoptosis of keratinocytes and serum DNase I activity in patients with cutaneous lupus erythematosus: relationship with clinical and immunoserological parameters.

BACKGROUND: Dysregulation of apoptosis has an important role in the induction of autoimmunity. OBJECTIVE: To evaluate the influence of keratinocyte apoptosis and deoxyribonuclease I (DNase I) activity on the clinical and immunoserological parameters of cutaneous lupus erythematosus (CLE). METHODS: We studied 69 CLE patients (39 with discoid LE (DLE), 12 with subacute CLE (SCLE), 12 with acute and 6 with intermittent CLE). Thirty of sixty-nine patients fulfilled criteria for systemic LE (SLE). Apoptotic index (AI) was evaluated immunohistochemically in lesional and non-lesional, photoprotected skin. Serum DNase I activity, antichromatin and anti-ENA antibodies were measured by ELISA. Disease activity was determined by SLEDAI-2K, SLICC/ACR, CLASI and RCLASI. RESULTS: AI in lesions was higher than in non-lesional skin (P < 0.001). There was no difference in AI between CLE and SLE patients. Patients with SCLE had higher lesional AI than patients with DLE (P < 0.05). We found a positive correlation between the lesional AI with CLASI A (P < 0.05) and RCLASI D (P < 0.05). CLE and SLE patients had significantly lower DNase I activity than healthy controls (P < 0.001). Patients with normal DNase I activity and low AI had significantly lower CLASI A than patients with decreased DNase I activity and/or elevated AI (P < 0.05). CONCLUSIONS: Increased keratinocyte apoptosis characterizes lesions of all CLE forms, especially of SCLE. AI correlates with CLE markers of acute and chronic inflammation. Normal level of apoptosis and DNase I activity simultaneously reduce the level of acute inflammation in CLE. Serum DNase I activity and AI might be important biomarkers in the evaluation of CLE patients.

Serum antinuclear and extractable nuclear antigen antibody prevalence and associated morbidity and mortality in the general population over 15 years.

The prevalence of ANA and anti-ENA in the general population is not well established, especially their clinical significance in healthy subjects. We herein determined the prevalence and predictive value of serum ANA and anti-ENA for connective tissue diseases (CTD), cancer, and mortality. We took advantage of a randomly selected sample of the 1998 general population (Isola I) consisting of 2828 subjects (53% women, age 43±13 years) from a well-defined Northern Italian area. Serum ANA and anti-ENA were tested on the 2690 samples available in 2012 (Isola II, 50% women, age 58±13 years). Administrative databases were searched for CTD, cancer diagnosis, and death cases occurring between enrollment and December 31, 2013. The hazard ratio (HR) was calculated for incident cases. Serum ANA is positive in 18.1% for any titer and 6.1% for titers ≥1:160, 23% in subjects over 50 years and 13.1% and 6.1% for any titer and titers ≥1:160, respectively, in women. The HR for CTD development was significantly high for all ANA titers, with the highest for ANA ≥1:160 (HR 14.19, 95% CI 3.07-65.68). ANA positivity was not associated with cancer (HR 1.03; 95% CI 0.75-1.43), or with mortality (HR adjusted for age and sex 1.40; 95% CI 0.94-2.09). Serum anti-ENA is positive in a minority of subjects with highest figures for anti-nucleosome (1.9%), -histone (1.6%) and -PM/Scl (1.5%). In conclusion, serum ANA prevalence in the general population is highest in senior subjects and in women, while the female predominance is significantly lower compared to overt CTD. Serum ANA is associated with an increased probability of CTD development over time, but does not influence survival or cancer risk.

Ultrasound of the salivary glands is a strong predictor of labial gland biopsy histopathology in patients with sicca symptoms.

BACKGROUND: The international classification criteria for Sjögren's syndrome necessitate the presence of either extractable nuclear antibody or a characteristic focal inflammatory infiltrate in a minor salivary gland. Thus, patients who are extractable nuclear antibody-negative will need to have a labial salivary gland biopsy, which is an invasive procedure associated with morbidity. The aim of this study was to evaluate the viability of ultrasound imaging of the major salivary glands as a predictor of the histology to explore whether ultrasound can help in stratifying Sjögren's patients and reduce the need for biopsy. METHODS: The records of 85 patients suspected of having Sjögren's syndrome and who have had biopsy and ultrasound were analysed retrospectively. The histology and the ultrasound were reported by experts independently. The reporting was impartial as the examiners were blinded to the results of the other investigations and to the diagnosis. RESULTS: Out of the 85 patients, 34 had positive ultrasound, 29 of whom also had positive histology. Fifty-one patients had negative ultrasound, of whom 49 were also negative for histological features of Sjögren's syndrome. The results show that the ultrasound had a positive predictive value of 85% and a striking negative predicative value of 96% of the histology results. The overall concordance between the ultrasound and the histology was 91% (Kappa = 0.826). CONCLUSIONS: Our study shows that potentially the ultrasound has a role in stratifying patients who are extractable nuclear antibody-negative and can help to prioritize the biopsy for those who have sonographic evidence of SS.

Comparison of the clinical utility of the Elia CTD Screen to indirect immunofluorescence on Hep-2 cells.

BACKGROUND: We compared the Elia CTD Screen (ECS), a fluoroenzymeimmunoassay incorporating 17 human antinuclear antigens (ANA), with indirect immunofluorescence (IIF) on Hep-2 cells in order to determine the clinical utility of the ECS in additon to or without IIF. METHODS: We examined 1708 consecutive serum samples submitted for ANA testing using the ECS and IIF in parallel. Positive screen results were further examined by quantitative fluoroenzymeimmunoassays and/or immunoblots for antibody identification. The medical records were evaluated for systemic rheumatic disorders. RESULTS: Concordance between ECS and IIF was observed in 1344 (78.8%) samples. ECS had a better detection rate for anti-dsDNA, -SSA/Ro, -SSB/La, -U1RNP and -Jo-1 antibodies, whereas IIF was superior in the detection of anti-CENP-B antibodies as well as anti-histone, -nucleosome and -Pl-12 antibodies, which are not included in the ECS antigen panel. ECS had a 100% sensitivity for Sjögren's syndrome, systemic sclerosis and Sharp syndrome. The sensitivity for Sjögren's syndrome was slightly higher for ESC than for IIF (94%). IIF had a higher diagnostic sensitivity for systemic lupus erythematosus, indeterminated connective tissue disease, Raynaud's syndrome and limited scleroderma, compared to ESC (100% vs. 80%, 100 vs. 75%, 89 vs. 57%, 100 vs. 88.9%). CONCLUSIONS: Our results suggest that the ECS represents an appropriate diagnostic tool for ANA screening. However, since some antigens are not incorporated in the ECS panel, and some ANA can also be missed by IIF, sequential or parallel screening with ECS and IIF may be reasonable when the clinical suspicion for connective tissue disease is high.

A functional variant that affects exon-skipping and protein expression of SP140 as genetic mechanism predisposing to multiple sclerosis.

Several variants in strong linkage disequilibrium (LD) at the SP140 locus have been associated with multiple sclerosis (MS), Crohn's disease (CD) and chronic lymphocytic leukemia (CLL). To determine the causal polymorphism, we have integrated high-density data sets of expression quantitative trait loci (eQTL), using GEUVADIS RNA sequences and 1000 Genomes genotypes, with MS-risk variants of the high-density Immunochip array performed by the International Multiple Sclerosis Genetic Consortium (IMSGC). The variants most associated with MS were also correlated with a decreased expression of the full-length RNA isoform of SP140 and an increase of an isoform lacking exon 7. By exon splicing assay, we have demonstrated that the rs28445040 variant was the causal factor for skipping of exon 7. Western blots of peripheral blood mononuclear cells from MS patients showed a significant allele-dependent reduction of the SP140 protein expression. To confirm the association of this functional variant with MS and to compare it with the best-associated variant previously reported by GWAS (rs10201872), a case-control study including 4384 MS patients and 3197 controls was performed. Both variants, in strong LD (r(2) = 0.93), were found similarly associated with MS [P-values, odds ratios: 1.9E-9, OR = 1.35 (1.22-1.49) and 4.9E-10, OR = 1.37 (1.24-1.51), respectively]. In conclusion, our data uncover the causal variant for the SP140 locus and the molecular mechanism associated with MS risk. In addition, this study and others previously reported strongly suggest that this functional variant may be shared with other immune-mediated diseases as CD and CLL.

[Frequency of antibodies against surface and nuclear antigens of hepatitis B virus in population of St. Petersburg in 2013].

AIM: Determine the frequency of antibodies to surface (anti-HBs) and core (anti-HBc) antigens of hepatitis B in population of St. Petersburg of various age for evaluation of protective herd immunity against hepatitis B virus (HB). MATERIALS AND METHODS: Blood sera of 970 individuals (491 males, 479 females) of 10 age groups from 0 to 50 years and older were examined for the presence of anti-HBs and anti-HBc IgG by enzyme immunoassay using commercial diagnostic test-systems. RESULTS: In general anti-HBs at the level of 5 mIU/ml and above were detected in 603 of the examined individuals (62.2%). Anti-HBs at the level of 10 mIU/ml and above were detected in 53.9%. The frequency of anti-HBs in protective titers in males and females in general turned out to be similar (52.6% and 55.2%, respectively). Juxtaposition of age-specific parameters of seroprotection and acute HB morbidity in St. Petersburg revealed an inverse correlation of medium strength (r = - 0.54). CONCLUSION: Results of the study confirm high effectiveness of the program of HB vaccine prophylaxis in St. Petersburg and emphasize the necessity of further implementation of broad measures of population immunization against HB.

Antinuclear antibody test.

The antinuclear antibody (ANA) test is widely used as a serological marker of autoimmune disease. Antinuclear antibodies are immunoglobulins or antibodies that bind to one or more antigens expressed within the nucleus of human cells. Used selectively, the ANA test can be a useful laboratory tool to help confirm or exclude the diagnosis of systemic rheumatic disease. However, the relatively high prevalence of ANAs in other inflammatory conditions, as well as healthy individuals, can make a positive result difficult to interpret.

Clinical performance evaluation of a novel rapid response chemiluminescent immunoassay for the detection of autoantibodies to extractable nuclear antigens.

BACKGROUND: We analyzed the performance of a novel ENA screening chemiluminescent immunoassay (CIA) and the confirmation QUANTA Flash tests. METHODS: Sera (n=1079) from patients referred to a rheumatology clinic were screened by QUANTA Flash ENA7 (INOVA Diagnostics). All positive (n=89) and a matched control group (n=90) were reflexed for autoantibodies to the individual antigens. Moreover, sera from patients with systemic lupus erythematosus (SLE, n=252), systemic sclerosis (SSc, n=64), polymyositis/dermatomyositis (PM/DM, n=72), Sjögren's syndrome (SjS, n=39) as well as disease controls (n=605) were tested by ENA7 CIA and by Quanta Lite ENA6 ELISA (INOVA). RESULTS: 89/1079 (8.3%) samples were ENA7 CIA positive with the following reactivity profile: RNP (36.0%), Sm (13.5%), Scl-70 (9.0%), Jo-1 (0.0%), Ro60 (44.9%), Ro52 (39.3%) and SS-B (24.7%). In the negative group, the reactivity profile was: RNP (1.1%), Sm (1.1%), Scl-70 (2.2%) and 0.0% for Jo-1, Ro60, Ro52 and SS-B. The positive/negative/total agreements (ENA7 CIA vs. confirmation assays) were 95.3%/91.5%/93.3%. The sensitivity of the ENA7 CIA was 62.3% in SLE, 54.7% in SSc, 92.3% in SjS, 50.0% in PM/DM, and 61.8% in the total systemic autoimmune rheumatic disease (SARD) population (specificity 95.0%). CONCLUSION: The QUANTA Flash ENA7 CIA is a reliable screening test.

An evaluation of autoimmune antibody testing patterns in a Canadian health region and an evaluation of a laboratory algorithm aimed at reducing unnecessary testing.

Autoantibody tests are often ordered inappropriately. We aimed to evaluate the ordering patterns of these tests in our local health region and to develop a laboratory algorithm aimed at reducing unnecessary tests. Laboratory data including the number and sequence of tests, ordering physician specialties and results for antinuclear (ANA), extractable nuclear antigen (ENA) and anti-double stranded DNA (anti-dsDNA) antibody tests from 2007 to 2009 were evaluated. Based on this information and a clinical consensus meeting, an algorithm was developed and applied retrospectively to 1 year of inpatient laboratory data to simulate potential cost savings. We identified a large volume of these autoantibody tests performed, equating to testing costs of $862,706.72, where less than 17 % of each were positive. Repeated ANA tests were mostly ordered after a previously negative result, and 1 % of patients with negative results changed to ≥1:160 on repeat testing. Close to half of all ENA and anti-dsDNA tests that were ordered were done so simultaneously with ANA, suggesting their use as screening tests. This was done more frequently in the inpatient setting. An algorithm was developed where ENA and anti-dsDNA tests would be cancelled if ANA was negative in the same sample. ANA repeated within 1 year would be cancelled and the prior result provided. Application of the algorithm retrospectively simulated a 30 % cost savings. Repeat testing and simultaneous ordering of multiple tests contributed to the excessive ordering of autoantibody tests in our health region. Our proposed algorithm would reduce testing costs and should be accompanied by appropriate educational information for physicians.