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

A Potential Peptide Therapeutic Derived from the Juxtamembrane Domain of the Epidermal Growth Factor Receptor

<div><p>The epidermal growth factor receptor (EGFR) is involved in many cancers and EGFR has been heavily pursued as a drug target. Drugs targeting EGFR have shown promising clinical results for several cancer types. However, resistance to EGFR inhibitors often occurs, such as with KRAS mutant cancers, therefore new methods of targeting EGFR are needed. The juxtamembrane (JXM) domain of EGFR is critical for receptor activation and targeting this region could potentially be a new method of inhibiting EGFR. We hypothesized that the structural role of the JXM region could be mimicked by peptides encoding a JXM amino acid sequence, which could interfere with EGFR signaling and consequently could have anti-cancer activity. A peptide encoding EGFR 645–662 conjugated to the Tat sequence (TE-64562) displayed anti-cancer activity in multiple human cancer cell types with diminished activity in non-EGFR expressing cells and non-cancerous cells. In nude mice, TE-64562 delayed MDA-MB-231 tumor growth and prolonged survival, without inducing toxicity. TE-64562 induced non-apoptotic cell death after several hours and caspase-3-mediated apoptotic cell death with longer treatment. Mechanistically, TE-64562 bound to EGFR, inhibited its dimerization and caused its down-regulation. TE-64562 reduced phosphorylated and total EGFR levels but did not inhibit kinase activity and instead prolonged it. Our analysis of patient data from The Cancer Genome Atlas supported the hypothesis that down-regulation of EGFR is a potential therapeutic strategy, since phospho- and total-EGFR levels were strongly correlated in a large majority of patient tumor samples, indicating that lower EGFR levels are associated with lower phospho-EGFR levels and presumably less proliferative signals in breast cancer. Akt and Erk were inhibited by TE-64562 and this inhibition was observed <em>in vivo</em> in tumor tissue upon treatment with TE-64562. These results are the first to indicate that the JXM domain of EGFR is a viable drug target for several cancer types.</p> </div

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

TE-64562 interacts with EGFR and inhibits dimerization.

<p>(<b>A</b>) SK-N-MC cells were transfected with the intracellular domain (ICD) of EGFR (645–1186) or the ICD of EGFR lacking the entire JXM region (ΔJM) or the JMA region (ΔJMA). Biotinylated peptides at a concentration of 0.1 µM (+) or 0.5 µM (++) were incubated with SK-N-MC cells for 2 hours and precipitated from cellular lysates with streptavidin-coated beads. The resulting bead-precipitates were analyzed by Western blot for the presence of the EGFR constructs. Results are representative of three independent experiments. (<b>B</b>) Streptavidin beads were pre-bound with biotinylated peptides and incubated with transfected SK-N-MC lysates. The non-biotinylated version of TE-64562 was added to compete for binding in lanes 3 and 4. The resulting bead-precipitates and lysates were analyzed by Western blot for the presence of the EGFR constructs. Results are representative of two independent experiments. (<b>C</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (12.5 and 25.0 µM), an EGFR specific tyrosine kinase inhibitor (TKI, 1.0 µM), Tat (25.0 µM) or vehicle for 30 minutes, followed by EGF treatment (25 ng/mL) for 10 minutes. Cellular proteins were cross-linked using bis(sulfosuccinimidyl) suberate (BS3), cells were lysed and lysates analyzed by Western blot for EGFR. The quantification of the dimer band is shown. The EGFR dimer band 25.0 µM TE-64562 was not detectable (N.D.). Results are representative of three independent experiments.</p

TE-64562 causes EGFR down-regulation and prolongs EGFR phosphorylation.

<p>(<b>A</b>) Serum starved MDA-MB-231 cells were treated with EGF for 2 minutes followed by TE-64562 (10 µM) treatment for the indicated amounts of time. The intensity of the EGFR bands was quantified with respect to the intensity of the respective α-tubulin bands. Mean intensity values are plotted from two independent experiments and error bars represent the standard error of the mean. Significance was assessed by a two-tailed, unpaired t test (*P<0.026). (<b>B</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (2.5, 5.0, 10.0 and 20.0 µM), Tat (20 µM), an EGFR specific tyrosine kinase inhibitor (TKI, 2.0 µM) or vehicle for 30 minutes, followed by EGF treatment (10 ng/mL) for 10 minutes. Results are representative of three independent experiments. The intensity of the EGFR (N = 3 to 7, depending on concentration) or phospho-EGFR (N = 2) bands were quantified with respect to the intensity of the respective α-tubulin band. Mean intensity values are plotted from two (phospho) or three or more (total) independent experiments and error bars represent the standard error of the mean. Significant differences (Mann-Whitney test) were assessed between each treatment condition and untreated control (*P<0.03). (<b>C</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (5.0, 10.0 and 20.0 µM) or T-Poly-Ala control peptide (5.0, 10.0 and 20.0 µM) or vehicle for 30 minutes, followed by EGF treatment (10 ng/mL) for 10 minutes. Results represent one of three independent experiments. (<b>D</b>) Correlation plot of breast cancer data from The Cancer Genome Atlas. The normalized EGFR protein expression data from antibody array data was plotted for each individual in the study. Shown in red are the 320 individuals who showed a positive correlation between EGFR expression and phospho-EGFR Y1173 levels. Shown in blue are the 32 individuals who did not show a correlation. Plotting and linear regression were performed in Prism 5.0 (GraphPad Software, Inc., USA). (<b>E</b>) Serum starved MDA-MB-231 cells were treated with TE-64562 (10 µM) or vehicle for 30 minutes followed by EGF (10 ng/mL) for 5, 10, 30, 60 120 or 240 minutes. Cell lysates were collected and analyzed by Western blot for phospho-EGFR (Y1173) and EGFR. Blots were stripped and re-probed for α-tubulin. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s005" target="_blank">Figure S5</a>.</p

TE-64562 treatment reduces Akt and Erk phosphorylation in MDA-MB-231 xenograft tumor tissue.

<p>(<b>A–B</b>) Nude mice bearing subcutaneous, MDA-MB-231 xenographic tumors were injected with the TE-64562 peptide (40 mg/kg; 7 µmol/kg), Tat-peptide (20 mg/kg; 7 µmol/kg) or vehicle (saline), intraperitoneally for four days, once per day. On the last day, the mice were injected 30 minutes prior to extracting the tumor. Frozen tumor sections were stained for (<b>A</b>) phospho-Akt (S473) or (<b>B</b>) phospho-Erk and counterstained with DAPI. Representative stained tumor sections are shown with the area in the box enlarged in the images below each section. Large scale bars = 500 µm and small scale bars = 50 µm. (<b>C</b>) A ∼1–2 mm cross-sectional slice of the tumor was lysed in RIPA buffer by sonication and the resulting lysates were analyzed by Western blot. Each lane represents a tumor from a different mouse. (<b>D</b>) Western blot data is quantified and plotted. Each treatment group was compared statistically (*P≤0.0364). Error bars represent the standard error of the mean. Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s006" target="_blank">Figure S6</a>.</p

Effect of Tat-645-662 on cell viability and colony growth of various human cancer and normal cell lines.

<p>(<b>A–B</b>) The indicated cell line was plated overnight, then serum starved overnight and treated with TE-64562 for 24 hours. For the HMEC and MDA-MB-231 cells were treated in HMEC media. Representative dose response curves are shown from one experiment run in triplicate with error bars representing the standard error of the mean. In the legend, the mean EC₅₀ values (± standard deviation from two to three independent experiments) derived from the nonlinear fit of dose response curves are shown (generated and fitted in Prism 5.0 GraphPad Software, Inc., USA). Also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049702#pone.0049702.s002" target="_blank">Figure S2</a>. (<b>C</b>) MDA-MB-231 (breast), A-549 (non-small cell lung), DLD-1 (colo-rectal) MIA-PaCa-2 (pancreatic) and SK-N-MC (neuroepithelioma, EGFR-null) cells were grown in soft agar containing 5% serum alone or treated with Tat peptide (20 µM) or Tat-645-62 peptide (10 or 20 µM) and allowed to grow for 2 weeks with addition of medium or medium containing peptide every 2 days. Means of the counts of four or more plates from at least two independent experiments are plotted. The significantly difference between the mean counts (*P<0.04) for TE-64562 treatment was assessed by comparison to untreated control.</p