Association of Apolipoprotein E (APOE) Polymorphisms With Serological Lipid and Inflammatory Markers.

Background  The study aims to assess the association of apolipoprotein E (APOE) gene polymorphisms with serological lipid and inflammatory markers to determine their potential role in predicting the risk of cardiovascular diseases (CVDs) and Alzheimer's disease (AD).  Methodology  A total of 915 individuals underwent testing for lipid and inflammatory biomarkers at Vibrant America Clinical Laboratory. Clinical data, blood lipid and inflammatory profiles, and APOE genotyping were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).  Results Compared to the E3/E3 genotype, individuals with E2/E3 genotypes showed higher levels of high-density lipoprotein (HDL), triglycerides, apolipoprotein A (APOA), high-sensitivity C-reactive protein (hs-CRP), and myeloperoxidase (MPO). E2/E4 genotype carriers had higher levels of HDL, triglycerides, Lp(a), and N-terminal pro b-type natriuretic peptide (BNPNT). E3/E4 genotypes were associated with elevated levels of total cholesterol, LDL, Lp(a), hs-CRP, small-density low-density lipoprotein (SDLDL), oxidized LDL (OXLDL), MPO, LDL-CAL, PLAC, and APOB. The E4/E4 group displayed higher concentrations of total cholesterol, LDL, APOB, Lp(a), hs-CRP, SDLDL, OXLDL, MPO, LDLCAL, and PLAC compared to E3/E3 carriers. These findings highlight the potential atherogenic effect of the ε4 allele and the protective effect of the ε2 allele based on lipid and inflammatory marker profiles.  Conclusions This study provides strong evidence linking APOE gene polymorphism to abnormal serum lipid and inflammatory profiles. Individuals carrying the ε4 alleles exhibited dysregulated lipid metabolism and abnormal inflammatory markers, increasing their risk of CVD and AD. Early detection and prompt diagnosis are crucial for implementing therapeutic, dietary, and lifestyle interventions to mitigate risks and prevent or delay lipid and inflammation-related disorders.

Gut commensals and their metabolites in health and disease.

Purpose of review: This review comprehensively discusses the role of the gut microbiome and its metabolites in health and disease and sheds light on the importance of a holistic approach in assessing the gut. Recent findings: The gut microbiome consisting of the bacteriome, mycobiome, archaeome, and virome has a profound effect on human health. Gut dysbiosis which is characterized by perturbations in the microbial population not only results in gastrointestinal (GI) symptoms or conditions but can also give rise to extra-GI manifestations. Gut microorganisms also produce metabolites (short-chain fatty acids, trimethylamine, hydrogen sulfide, methane, and so on) that are important for several interkingdom microbial interactions and functions. They also participate in various host metabolic processes. An alteration in the microbial species can affect their respective metabolite concentrations which can have serious health implications. Effective assessment of the gut microbiome and its metabolites is crucial as it can provide insights into one's overall health. Summary: Emerging evidence highlights the role of the gut microbiome and its metabolites in health and disease. As it is implicated in GI as well as extra-GI symptoms, the gut microbiome plays a crucial role in the overall well-being of the host. Effective assessment of the gut microbiome may provide insights into one's health status leading to more holistic care.

Influence of genetic polymorphisms on serum biomarkers of cardiac health.

Cardiovascular diseases (CVDs) are a leading cause of death worldwide which is why early risk prediction is crucial. Discrete Polygenic risk score (PRS) measurement using saliva or dried blood spot samples collected at home poses a convenient means for early CVD risk assessment. The present study assessed the effects of 28 disease-associated single nucleotide polymorphisms (SNPs) on 16 serological cardiac markers and also aggregated the risk alleles into a PRS to evaluate its applicability in CVD-risk prediction. The study assessed genetic and serological markers in 184 individuals. The association between serological markers and individual genetic variants was evaluated using a two-tailed t test while the associations of serum markers with the PRS was analyzed using the Pearson correlation. The comparative analysis of genotypes revealed statistically significant associations between serum markers and CVD-associated SNPs with Apo B: Apo A-1, LDL Direct, Apo B, sdLDL, hsCRP, Lp(a), NT-proBNP, and PLAC levels being significantly associated with the risk alleles of the SNPs, rs12526453, rs5186, rs10911021, rs1801131, rs670, rs10757274, and rs10757278. Increased PLAC levels were associated with rs10757274 and rs10757278 (P < .05). The SNPs, rs1801133, rs1549758, rs1799983, rs5082, and rs5186 were significantly associated with an increase in the cardioprotective markers, HDL and ApoA1 (P < .05). Furthermore, the PRS was associated with increasing levels of several serum cardiac markers (r2 > 0.6). Significant correlations were observed between high PRSs and NT-proBNP and ox-LDL levels with the r2 values being 0.82 (95% CI = 0.13-0.99; P = .03) and 0.94 (95% CI = 0.63-0.99; P = .005), respectively. The present study reports that SNPs have differential effects on serum markers with rs12526453, rs5186, rs10911021, rs1801131, rs670, rs10757274, and rs10757278 showing significant associations with elevated marker levels, which are indicators of deteriorating cardiac health. A unified PRS using several SNPs was also associated with an increase in serum markers levels, especially, NT-proBNP and ox-LDL. Genetic assessment via a convenient at-home collection to calculate the PRS can serve as an effective predictive tool for early CVD-risk assessment. This may help identify the risk groups that may require increased serological monitoring.

Preliminary Study on the Association of Serum Branched-Chain Amino Acids With Lipid and Hepatic Markers.

Background: Serum levels of branched-chain amino acids (BCAAs) are associated with various vital physiological functions and thus elevation in circulating levels results in several metabolic disturbances. Serum levels of BCAAs are strong predictors of various metabolic disorders. Their association with cardiovascular health is uncertain. The study aimed to investigate the association of BCAAs with circulating levels of vital cardiovascular and hepatic markers. Methods: The study population of 714 individuals was included from the population tested for the vital cardio and hepatic biomarkers at the Vibrant America Clinical Laboratories. The subjects were stratified into four quartiles based on the serum levels of BCAAs, and their association with vital markers was studied using the Kruskal-Wallis test. Pearson's correlation analyzed the univariant relationship of BCAAs with selected cardio and hepatic markers. Results: BCAAs exhibited a strong negative correlation with serum HDL. Serum triglycerides were found to have a positive correlation with serum levels of leucine and valine. Univariant analysis exhibited a strong negative correlation between serum levels of BCAAs and HDL, and a positive correlation was observed between triglycerides and amino acids isoleucine and leucine. Among analyzed hepatic markers, alanine transaminase exhibited a considerable association with BCAAs. Conclusions: The elevated levels of serum BCAAs are strongly associated with serum HDL and triglycerides. Consumption of these supplements must be in coordination with healthcare providers to avoid metabolic and cardiovascular risk.

Evaluation of renal markers in systemic autoimmune diseases.

Renal involvement is a common occurrence in subjects with systemic autoimmune diseases. The renal manifestation and its severity depend on the underlying condition and may reversely complicate the clinical course of autoimmune diseases. Renal function markers have been widely used in the assessment of normal functioning of kidneys including glomerular filtration rate and concentrating and diluting capacity of the kidney. An increase or decrease in the values of these markers may indicate kidney dysfunction. In this study, a number of critical renal markers were examined in seropositive autoimmune diseases including systemic lupus erythematosus (SLE), connective tissue disorder (CTD), and rheumatoid arthritis (RA). The data from three cohorts of subjects enrolled in renal markers and autoimmune antibody testing between January 2015 to August 2019 were retrospectively studied. The prevalence of renal markers that were out of the reference range and their average levels in female and male subgroups across SLE, CTD, and RA cohorts were compared and analyzed. The levels of renal markers are significantly affected by the presence of autoantibodies, in particular eGFR, cystatin C, and albumin. Autoantibodies were also more frequent in subjects with severe renal function damage. Close follow-up of both renal markers and autoantibodies may potentially assist in the early diagnosis of kidney diseases and improve the survival and life expectancy of autoimmune patients.

A multiplex chemiluminescent immunoassay for serological profiling of COVID-19-positive symptomatic and asymptomatic patients.

The COVID-19 pandemic affects more than 81 million people worldwide with over 1.7 million deaths. As the population returns to work, it is critical to develop tests that reliably detect SARS-CoV-2-specific antibodies. Here we present results from a multiplex serology test for assessing the antibody responses to COVID-19. In an initial large cohort, this test shows greater than 99% agreement with COVID-19 PCR test. In a second outpatient cohort consisting of adults and children in Colorado, the IgG responses are more robust in positive/symptomatic participants than in positive/asymptomatic participants, the IgM responses in symptomatic participants are transient and largely fall below the detection limit 30 days after symptom onset, and the levels of IgA against SARS-CoV-2 receptor binding domain are significantly increased in participants with moderate-to-severe symptoms compared to those with mild-to-moderate symptoms or asymptomatic individuals. Our results thus provide insight into serology profiling and the immune response to COVID-19.

Effect of Micronutrients on Thyroid Parameters.

Micronutrients are involved in various vital cellular metabolic processes including thyroid hormone metabolism. This study aimed to investigate the correlation between serum levels of micronutrients and their effects on thyroid parameters. The correlation of serum levels of micronutrients and thyroid markers was studied in a group of 387 healthy individuals tested for thyroid markers (T4, T3, FT4, FT3, TSH, anti-TPO, RT3, and anti-Tg) and their micronutrient profile at Vibrant America Clinical Laboratory. The subjects were rationalized into three groups (deficient, normal, or excess levels of micronutrients), and the levels of their thyroid markers were compared. According to our results, deficiency of vitamin B2, B12, B9 and Vit-D25[OH] (p < 0.05) significantly affected thyroid functioning. Other elemental micronutrients such as calcium, copper, choline, iron, and zinc (p < 0.05) have a significant correlation with serum levels of free T3. Amino acids asparagine (r = 0.1765, p < 0.001) and serine (r = 0.1186, p < 0.05) were found to have a strong positive correlation with TSH. Valine, leucine, and arginine (p < 0.05) also exhibited a significant positive correlation with serum levels of T4 and FT4. No other significant correlations were observed with other micronutrients. Our study suggests strong evidence for the association of the levels of micronutrients with thyroid markers with a special note on the effect of serum levels of certain amino acids.

Antibody profiling and prevalence in US patients during the SARS-CoV2 pandemic.

BACKGROUND: Antibody diagnostics play an important role in disease detection and can potentially aid in monitoring of the immune responses to see if an individual has developed immunity. Developing high throughput diagnostics which does not involve handling of infectious material becomes imperative in the case of pandemics such as the recent outbreak of SARS-CoV2. METHODS: A protein microarray technology was used to detect the plurality of antibody response to four novel antigens namely S1 glycoprotein, Receptor binding domain (RBD), S2 glycoprotein and Nucleoprotein of the novel coronavirus named SARS-CoV2 using serum samples. A DBS card was additionally used to compare its performance with a venipuncture-based serum separator tube (SST) draw. RESULTS: The three main subclasses of antibodies IgM, IgA and IgG were analyzed to see the variations in immune responses in the affected population and compared to their microbial RT-PCR based NP swab results. The clinical sensitivity and specificity were determined to be 99.67% and 99.77%. In the matrix comparison study, which would enable patients to test without risk of transmitting the virus, DBS (Dried Blood Spot) matched with higher than 98% accuracy to a venipuncture-based SST collection. CONCLUSION: Multiplex testing enables higher sensitivity and specificity which is essential while establishing exposure on a population scale. This flexible platform along with a discrete collection methodology would be crucial and broadly useful to scale up testing in current and future pandemics. Minimum sample volume that can be collected using DBS cards can be processed in this multiplex pillar plate format enabling the capacity to provide the reliability of high throughput analyzers while having the ease of collection similar to rapid tests.

An ultra-high-density protein microarray for high throughput single-tier serological detection of Lyme disease.

Current serological immunoassays have inherent limitations for certain infectious diseases such as Lyme disease, a bacterial infection caused by Borrelia burgdorferi in North America. Here we report a novel method of manufacturing high-density multiplexed protein microarrays with the capacity to detect low levels of antibodies accurately from small blood volumes in a fully automated system. A panel of multiple serological markers for Lyme disease are measured using a protein microarray system, Lyme Immunochip, in a single step but interpreted adhering to the standard two-tiered testing algorithm (enzyme immunoassay followed by Western blot). Furthermore, an enhanced IgM assay was supplemented to improve the test's detection sensitivity for early Lyme disease. With a training cohort (n = 40) and a blinded validation cohort (n = 90) acquired from CDC, the Lyme Immunochip identified a higher proportion of Lyme disease patients than the two-tiered testing (82.4% vs 70.6% in the training set, 66.7% vs 60.0% in the validation set, respectively). Additionally, the Immunochip improved sensitivity to 100% while having a lower specificity of 95.2% using a set of investigational antigens which are being further evaluated with a large cohort of blinded samples from the CDC and Columbia University. This universal microarray platform provides an unprecedented opportunity to resolve a broad range of issues with diagnostic tests, including multiplexing, workflow simplicity, and reduced turnaround time and cost.

Evaluation of the Vibrant DNA microarray for the high-throughput multiplex detection of enteric pathogens in clinical samples.

BACKGROUND: Rapid detection of a wide range of etiologic agents is essential for appropriate treatment and control of gastrointestinal (GI) infections. A variety of microbial species including bacteria, viruses, parasites, and fungi have been recognized as diarrheagenic enteric pathogens. However, multiplex testing of various targets in a single reaction needs further improvement because of its limitation in species and throughput. RESULTS: This study aims at developing and evaluating a DNA microarray-based qualitative multiplexed polymerase chain reaction (PCR) assay, Vibrant GI pathogen panel (GPP), for simultaneous detection of 27 enteric GI pathogenic targets (16 bacteria, 5 viruses, 4 parasites, and 2 fungi) directly from stool specimens. Limits of detection ranged from 102 to 104 cells/mL for bacteria, 102 to 103 cells/mL for parasites, 102 to 103 RNA copies/mL for viruses, and 102 to 103 cells/mL for fungi. Performance characteristics were determined using 27 Quantitative Genomic DNAs, 212 spiked stool specimens, 1067 clinical and archived stool specimens. Overall sensitivity was 95.9% (95% CI 92.4-98.1) and specificity was 100% (95% CI 99.9-100). Polymicrobial detections contained either two or three organisms was 20.2% (35/173) of positive clinical specimens and 3.3% (35/1055) of all clinical specimens. CONCLUSION: The Vibrant GPP is a comprehensive, high-throughput, and rapid DNA microarray to provide etiologic diagnosis of GI infections in the laboratory setting.

Significance of Anti-TPO as an Early Predictive Marker in Thyroid Disease.

Even though most thyroid subjects are undiagnosed due to nonspecific symptoms, universal screening for thyroid disease is not recommended for the general population. In this study, our motive is to showcase the early appearance of thyroid autoantibody, anti-TPO, prior to the onset of thyroid hormone disruption; hence the addition of anti-TPO in conjunction with traditional thyroid markers TSH and FT4 would aid to reduce the long-term morbidity and associated health concerns. Here, a total of 4581 subjects were tested multiple times for TSH, FT4, anti-TPO, and anti-Tg and followed up for 2 years. We streamlined our subjects into two groups, A1 (euthyroid at first visit, but converted to subclinical/overt hypothyroidism in follow-up visits) and A2 (euthyroid at first visit, but converted to hyperthyroidism in follow-up visits). According to our results, 73% of hypothyroid subjects (from group A1) and 68.6% of hyperthyroid subjects (from group A2) had anti-TPO 252 (±33) and 277 (±151) days prior to the onset of the thyroid dysfunction, respectively. Both subclinical/overt hypothyroidism and hyperthyroidism showed a significantly higher percentage of subjects who had anti-TPO prior to the onset of thyroid dysfunction compared to the combined control group. However, there was no significant difference in the subjects who had anti-Tg earlier than the control group. Further assessment showed that only anti-TPO could be used as a standalone marker but not anti-Tg. Our results showcase that anti-TPO appear prior to the onset of thyroid hormone dysfunction; hence testing anti-TPO in conjunction with TSH would greatly aid to identify potentially risk individuals and prevent long-term morbidity.

Overlap of Characteristic Serological Antibodies in Rheumatoid Arthritis and Wheat-Related Disorders.

BACKGROUND AND AIMS: Rheumatoid arthritis (RA) and celiac disease (CD) are members of the autoimmune disease family while they have been shown to share multiple aspects in epidemiology and clinical manifestations. The aim of this study was to assess the presence of wheat protein antibodies in RA seropositive subjects and the presence of RA diagnostic markers in subjects with seropositive wheat-related disorders including CD. METHODS: Serum samples were collected from 844 subjects with joint pain and/or gastrointestinal symptoms and tested by a CD panel (anti-tTG and anti-DGP), a Wheat Zoomer (WZ) antibody panel (IgG/IgA to 14 wheat proteins), and a RA panel (anti-CCP and anti-RF). Retrospective analysis was completed using de-identified clinical data and test results. RESULTS: The prevalence of RA markers was first investigated in CD- or WZ-positive subjects and negative controls. 49 subjects were seropositive in the CD panel with 10 (20%) RA positivity. 605 subjects were seropositive in the WZ panel with 106 (18%) RA positivity. 222 subjects were seronegative in either panels with 12 (6%) RA positivity. Next, the frequency of the CD markers and the clinically relevant wheat protein antibodies were investigated in the RA-positive subjects and negative controls. 128 subjects in this cohort were seropositive in the RA panel with 10 (8%) CD positivity and 106 (83%) WZ positivity, compared to 716 RA seronegative controls with 39 (5%) CD positivity and 499 (70%) WZ positivity. CONCLUSIONS: Our data presents an apparent trend of overlapped serological antibody biomarker positivity in RA and wheat-related disorders.

Insights into cardiovascular risk and nutritional status in subjects with wheat-related disorders.

Objective: Wheat-related disorders are a spectrum of disorders associated with different autoimmune and non-autoimmune diseases. However, it is unclear whether these wheat-related disorders lead to adverse health effects such as cardiovascular risk, nutritional deficiencies etc. The objective of the study was to explore the lipid profiles and the nutritional status of subjects with wheat-related disorders to understand the potential threat of wheat on cardiovascular risk and nutritional deficiency. Method: A total of 1041 subjects who showed wheat-related symptoms were initially tested for the wheat protein antibody panel (Wheat Zoomer (WZ) panel and Coeliac Disease (CD) panel), then for cardiovascular panel and the micronutrient panel at Vibrant America Clinical Laboratory. Results: Subjects with both Wheat Zoomer positivity (WZ+) and Coeliac Disease positivity (CD+) had significantly low levels of high-density lipoproteins (HDL) (279/483(57.8%) and 29/47(61.7%) respectively), but only subjects with WZ + had low levels of Apo A1 (44/424(9.5%)), and high levels of Omega 6 fatty acids (53/334(15.9%)). None of the micronutrients tested showed a significant imbalance in WZ + subjects. Conclusion: Subjects with positive serology for WZ have deranged blood lipid profiles but did not show any significant micronutrient deficiency. Hence, our results showcase a significant association of wheat-related disorders to cardiovascular risk.

Synthetic Neoepitopes of the Transglutaminase-Deamidated Gliadin Complex as Biomarkers for Diagnosing and Monitoring Celiac Disease.

BACKGROUND & AIMS: Celiac disease (CeD) has characteristics of an autoimmune disease, such as increased antibody levels to tissue transglutaminase (tTG). However, assays to measure these biomarkers in blood samples do not identify patients with sufficient accuracy for diagnosis or monitoring of CeD. We aimed to discover biomarkers of CeD derived from neoepitopes of deamidated gliadin peptides (DGP) and tTG fragments and to determine if immune reactivity against these epitopes can identify patients with CeD with mucosal healing. METHODS: We analyzed serum samples from 90 patients with biopsy-proven CeD and 79 healthy individuals (controls) for immune reactivity against the tTG-DGP complex (discovery cohort). A fluorescent peptide microarray platform was used to estimate the antibody-binding intensity of each synthesized tTG-DGP epitope. We validated our findings in 82 patients with newly diagnosed CeD and 217 controls. We tested the ability of our peptide panel to identify patients with mucosal healing (based on the histologic analysis) using serum samples from patients with treated and healed CeD (n = 85), patients with treated but unhealed CeD (n = 81; villous atrophy despite a adhering a gluten-free diet), patients with untreated CeD (n = 82) and disease controls (n = 27), villous atrophy without CeD), and healthy controls (n = 217). Data were analyzed using principal component analysis followed by machine learning and support vector machine modeling. RESULTS: We identified 172 immunogenic epitopes of the tTG-DGP complex. We found significantly increased immune reactivity against these epitopes vs controls. In the both cohort, the set of neoepitopes derived from the tTG-DGP complex identified patients with CeD with 99% sensitivity and 100% specificity. Serum samples from patients with untreated CeD had the greatest mean antibody-binding intensity against the tTG-DGP complex (32.5 ± 16.4). The average antibody-binding intensity was significantly higher in serum from patients with treated but unhealed CeD mucosa (15.1 ± 7.5) than in patients with treated and healed CeD mucosa (5.5 ± 3.4) (P < .001). The assay identified patients with mucosa healing status with 84% sensitivity and 95% specificity. CONCLUSIONS: We identified immunogenic epitopes of the tTG-DGP complex, and found that an assay to measure the immune response to epitopes accurately identified patients with CeD, as well as patients with mucosal healing. This biomarker assay might be used in detection and monitoring of patients with CeD.

High Frequency of Extractable Nuclear Autoantibodies in Wheat-Related Disorders.

BACKGROUND AND AIMS: There has been broad interest to explore the presence of autoimmunity among wheat-sensitive individuals, but neither the pathogenesis nor the relevance has been established. In this study, we evaluated the frequencies and levels of autoantibodies, which are important biomarkers of autoimmunity, in subjects with wheat-related disorders and controls. Anti-nuclear antibodies (ANA) and the specific ones against extractable nuclear antigens (ENA) were investigated. METHODS: A total of 713 subjects who showed symptoms related to wheat ingestion were addressed to Vibrant America Clinical Laboratory from December 2015 to November 2017. Serum samples were collected from all subjects and tested with a wheat protein antibody panel (IgG and IgA to 18 proteins at the peptide level) and an autoantibody panel (ANA by immunofluorescence analysis and 10 ENA antibodies). Retrospective analysis was completed using de-identified clinical data and test results. RESULTS: In the retrospective analysis, 38 (5%) were seropositive in a Celiac Disease panel, 491 (83%) were seropositive in a wheat protein antibody panel "Wheat Zoomer," and 84 (12%) were seronegative in both panels. Anti-nuclear antibodies were detected in similar portions of the celiac disease subjects (13%), the Wheat Zoomer-positive subjects (12%), and seronegative controls (15%), which is also very close to the reported occurrence of ANA positivity (15%) in the healthy population. The prevalence of anti-ENA was reported to be less than 2% in the general population; however, our study found it to be much higher in the celiac disease subjects (29%) and the wheat-sensitive subjects (27%), compared with a smaller proportion of seronegative controls (19%). The prevalence of anti-histone was especially prominent among the celiac disease subjects (73%) and the Wheat Zoomer-positive subjects (60%). CONCLUSIONS: High proportions of wheat-related disease subjects carry ENA antibodies that are important specific biomarkers of autoimmunity.

Exploring Systemic Autoimmunity in Thyroid Disease Subjects.

INTRODUCTION: Individuals with one autoimmune disease are at risk of developing a second autoimmune disease, but the pathogenesis or the sequential occurrence of multiple autoimmune diseases has not been established yet. In this study, we explored the association and sequential occurrence of antibodies in thyroid disease and systemic autoimmune disease subjects. We evaluated thyroid hormones, thyroid-stimulating hormone (TSH), free thyroxine (FT4), thyroid autoantibodies, anti-thyroperoxidase (anti-TPO), and anti-thyroglobulin (Tg) to comprehend the association with systemic autoimmune autoantibodies, anti-nuclear antibodies (ANA), and autoantibodies to extractable nuclear antigens (ENA) in subjects with thyroid-related symptoms. METHODS: A total of 14825 subjects with thyroid-related symptoms were tested at Vibrant America Clinical Laboratory for thyroid markers (TSH, FT4, anti-TPO, and anti-Tg) and an autoimmune panel (ANA panel and ENA-11 profile) from March 2016 to May 2018. Thyroid-positive (based on TSH and FT4 levels), anti-TPO-positive, and anti-Tg-positive subjects were assessed for the prevalence of ANA and anti-ENA antibodies. A 2-year follow-up study was conducted to assess the sequential order of appearance of autoimmune markers in thyroid and systemic autoimmune diseases. RESULTS: In the retrospective analysis, 343/1671 (20.5%), 2037/11235 (18.1%), and 1658/9349 (17.7%) of thyroid+, anti-TPO+, and anti-Tg+ subjects were found to be seropositive for ANA. Anti-ENA was detected in a higher prevalence than ANA with 475/1671 (28.4%), 3063/11235 (27.3%), and 2511/9349 (26.9%) in the same groups of subjects, respectively. Our results are found to be much higher than the reported prevalence of anti-ENA in general population. During the 2-year follow-up study, anti-TPO appeared significantly earlier than ANA and anti-ENA in an average of 253 (±139) and 227 (±127) days, respectively. CONCLUSIONS: A high prevalence of anti-ENA and ANA was found to be coexisting with autoimmune thyroid disease subjects, with anti-TPO occurring prior to the onset of ANA and anti-ENA. Therefore, frequent follow-ups and evaluation of ANA and anti-ENA in subjects with anti-TPO positivity would be beneficial in early detection of other systemic autoimmune diseases.

Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays.

BACKGROUND: Most antibodies recognize conformational or discontinuous epitopes that have a specific 3-dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD) by using a silicon-based peptide array and computational methods. METHODS: Using a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes. RESULTS: Using a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity. CONCLUSIONS: These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultra-high-density peptide microarray described here would be broadly useful to develop high-fidelity diagnostic tests and explore pathogenesis.