Clinical Utility of an Enzyme-Linked Immunosorbent Assay for Detecting Anti-Melanoma Differentiation-Associated Gene 5 Autoantibodies

ObjectiveAutoantibodies to melanoma differentiation-associated gene 5 (MDA5) are specifically expressed in patients with dermatomyositis (DM) and are associated with a subset of DM patients with rapidly progressive interstitial lung disease (RP-ILD). Here, we examined the clinical utility of a newly developed enzyme-linked immunosorbent assay (ELISA) system for detecting these antibodies.MethodsHere we developed an improved ELISA for detecting anti-MDA5 antibodies. We then performed a multicenter clinical study involving 8 medical centers and enrolled 242 adult patients with polymyositis (PM)/DM, 190 with non-PM/DM connective tissue disease (CTD), 154 with idiopathic interstitial pneumonia (IIP), and 123 healthy controls. Anti-MDA5 antibodies in the patients’ serum samples were quantified using our newly developed ELISA, and the results were compared to those obtained using the gold-standard immunoprecipitation (IP) assay. In addition, correlations between the ELISA-quantified anti-MDA5 antibodies and clinical characteristics were evaluated.ResultsIn patients with PM/DM, the anti-MDA5 antibody measurements obtained from the ELISA and IP assay were highly concordant; the ELISA exhibited an analytical sensitivity of 98.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.5% (compared to the IP assay). Anti-MDA5 antibodies were detected in 22.7% of the DM patients, but not in any of the patients with PM, non-PM/DM CTD, or IIP. Clinically amyopathic DM, RP-ILD, arthritis, and fever were more prevalent in DM patients who were anti-MDA5 antibody-positive than in those who were antibody-negative (P ≤ 0.0002 for all comparisons). In addition, anti-MDA5 antibody-positive patients with RP-ILD exhibited higher antibody levels than those without RP-ILD (P = 0.006).ConclusionOur newly developed ELISA can detect anti-MDA5 antibodies as efficiently as the gold standard IP assay and has the potential to facilitate the routine clinical measurement of anti-MDA5 antibodies in patients who suspected to have DM.     

Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies

The homeobox protein, PEPP2 (RHOXF2), has been suggested as a cancer/testis (CT) antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs) is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2^271-279). The CTLs induced by PEPP2^271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2’-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2’-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.     

Quantitative and antioxidative behavior of Trolox in rats’ blood and brain by HPLC‐UV and SMFIA‐CL methods

Trolox, a water‐soluble vitamin E analogue has been used as a positive control in Trolox equivalent antioxidant capacity and oxygen radical antioxidant capacity assays due to its high antioxidative effect. In this study, the ex vivo antioxidative effects of Trolox and its concentration in blood and brain microdialysates from rat after administration were evaluated by newly established semi‐microflow injection analysis, chemiluminescence detection and HPLC‐UV. In the administration test, the antioxidative effect of Trolox in blood and brain microdialysates after a single administration of 200 mg/kg of Trolox to rats could be monitored. The antioxidative effects in blood (12.0 ± 2.1) and brain (8.4 ± 2.1, × 10³ antioxidative effect % × min) also increased. Additionally, the areas under the curve (AUC)s_0–360 (n = 3) for blood and brain calculated with quantitative data were 10.5 ± 1.2 and 9.7 ± 2.5 mg/mL × min, respectively. This result indicates that Trolox transferability through the blood–brain barrier is high. The increase in the antioxidative effects caused by Trolox in the blood and brain could be confirmed because good correlations between concentration and antioxidative effects (r ≥ 0.702) were obtained. The fact that Trolox can produce an antioxidative effect in rat brain was clarified. Copyright © 2015 John Wiley & Sons, Ltd.     

Induction and differentiation of adipose-derived stem cells from human buccal fat pads into salivary gland cells

Atrophy or hypofunction of the salivary gland because of aging or disease leads to hyposalivation that affects patient quality of life by causing dry mouth, deterioration of mastication/deglutition, and poor oral hygiene status. Current therapy for atrophy or hypofunction of the salivary gland in clinical practice focuses on symptom relief using drugs and artificial saliva; therefore, there is still a need to develop new therapies. To investigate potential novel therapeutic targets, we induced the differentiation of salivary gland cells by co-culturing human adipose-derived stem cells isolated from buccal fat pads (hBFP-ASCs) with human salivary-gland-derived fibroblasts (hSG-fibros). We examined their potential for transplantation and tissue neogenesis. Following the culture of hBFP-ASCs and hSG-fibros, differentiated cells were transplanted into the submandibular glands of SCID mice, and their degree of differentiation in tissues was determined. We also examined their potential for functional tissue reconstitution using a three-dimensional (3D) culture system. Co-cultured cells expressed salivary-glandrelated markers and generated new tissues following transplantation in vivo. Moreover, cell reconstituted glandular structures in the 3D culture system. In conclusion, coculture of hSG-fibros with hBFP-ASCs led to successful differentiation into salivary gland cells that could be transplanted to generate new tissues.     

Tunicamycin-induced inhibition of protein secretion into culture medium of Arabidopsis T87 suspension cells through mRNA degradation on the endoplasmic reticulum

The N-glycosylation inhibitor tunicamycin triggers endoplasmic reticulum stress response and inhibits efficient protein secretion in eukaryotes. Using Arabidopsis suspension cells, we showed that the reduced secretion of mannose-binding lectin 1 (MBL1) protein by tunicamycin is accompanied by a significant decrease in MBL1 mRNA, suggesting that mRNA destabilization is the major cause of the inhibition of protein secretion in plants.     

Improved enantioselectivity of thermostable esterase ST0071 from archaeon Sulfolobus tokodaii by site-saturation mutagenesis

An archaeon GGG(A)X-type esterase (ST0071) can catalyze the hydrolysis of various acetates of secondary alcohols, but shows low enantioselectivity. Using structure-guided site-saturation mutagenesis, we successfully identified a G274W variant that has excellent selectivity compared with that of wild-type ST0071.     

Comparison of patient-specific intensity modulated radiation therapy quality assurance for the prostate across multiple institutions

AimTo evaluate the success of a patient-specific intensity modulated radiation therapy (IMRT) quality assurance (QA) practice for prostate cancer patients across multiple institutions using a questionnaire survey.BackgroundThe IMRT QA practice involves different methods of dose distribution verification and analysis at different institutions.Materials and MethodsTwo full-arc volumetric modulated arc therapy (VMAT) plan and 7 fixed-gantry IMRT plan with DMLC were used for patient specific QA across 22 institutions. The same computed tomography image and structure set were used for all plans. Each institution recalculated the dose distribution with fixed monitor units and without any modification. Single-point dose measurement with a cylindrical ionization chamber and dose distribution verification with a multi-detector or radiochromic film were performed, according to the QA process at each institution.ResultsTwenty-two institutions performed the patient-specific IMRT QA verifications. With a single-point dose measurement at the isocenter, the average difference between the calculated and measured doses was 0.5 ± 1.9%. For the comparison of dose distributions, 18 institutions used a two or three-dimensional array detector, while the others used Gafchromic film. In the γ test with dose difference/distance-to-agreement criteria of 3%?3 mm and 2%?2 mm with a 30% dose threshold, the median gamma pass rates were 99.3% (range: 41.7%–100.0%) and 96.4% (range: 29.4%–100.0%), respectively.ConclusionThis survey was an informative trial to understand the verification status of patient-specific IMRT QA measurements for prostate cancer. In most institutions, the point dose measurement and dose distribution differences met the desired criteria.