Nobuhide Hayashi

A Novel Serum Metabolomics-Based Diagnostic Approach for Colorectal Cancer

Background To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer. Methodology/Principal Findings We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0–2 colorectal cancer (82.8%). Conclusions/Significance Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer.

Oxidative stress mediates cell surface expression of SS-A/Ro antigen on keratinocytes.

Exposure to ultraviolet radiation exacerbates the skin lesions of autoimmune diseases, and is known to induce cell surface expression of SS-A/Ro antigen on keratinocytes in vitro. Following up on recent reports on ultraviolet-B (UVB)-induced oxidative stress, we examined the role of oxidative stress in the surface expression of SS-A/Ro antigen on human keratinocytes. First, the exclusive induction by UVB irradiation of the 52-kDa protein (Ro52) but not of the 60-kDa protein (Ro60) of SS-A/Ro antigen was demonstrated by means of indirect immunofluorescence. The surface expression of Ro52 induced by UVB irradiation was concentration-dependently inhibited by N-acetyl-L-cysteine, an antioxidant. Furthermore, surface expression of Ro52 was similarly induced by diamide, a chemical oxidant. We next used Hoechst 33342 staining and the TUNEL assay to demonstrate that a low dose (20 mJ/cm(2)) of UVB did not induce apoptosis but induced the surface expression of Ro52. Moreover, zVAD-fmk, a pan-caspase inhibitor, did not inhibit UVB-induced surface expression of Ro52 even at a high dose (200 mJ/cm(2)) of UVB, which was sufficient to induce apoptosis in keratinocytes in the absence of zVAD-fmk. Taken together, we concluded that UVB-induced surface expression of Ro52 on keratinocytes is mediated by oxidative stress through a pathway other than apoptosis.

Utility of transmural myocardial strain profile for prediction of early left ventricular dysfunction in patients with Duchenne muscular dystrophy.

Myocardial damage in Duchenne muscular dystrophy (DMD) has lethal outcomes, making early detection of myocardial changes in patients with DMD vital, because early treatment can help prevent the development of myocardial fibrosis. The aim of the present study was, therefore, to test the hypothesis that transmural strain profile (TMSP) analysis can predict future left ventricular (LV) dysfunction in patients with DMD with preserved ejection fraction. We studied 82 consecutive patients with DMD without LV wall motion abnormality, with an ejection fraction of 60 ± 5% (all ≥55%) and age 11 ± 3 years. Echocardiography was performed at baseline and 1 year of follow-up. TMSP in the posterior wall was evaluated from the mid-LV short-axis view. A normal TMSP pattern (1 peak in the endocardium, group 1) was seen in 44 patients, and TMSP with a notch (2 peaks in the endocardium, group 2) in the remaining 38 (46%). Wall motion abnormality in the posterior wall was observed in 16 patients (42%) in group 2 at 1 year of follow-up but in none of the patients in group 1 (42% vs 0%; p <0.001). Importantly, multivariate analysis showed that only TMSP with a notch (odds ratios 1.524, p <0.001) was an independent determinant of the presence of LV posterior wall motion abnormality at 1 year of follow-up. In conclusion, subclinical LV dysfunction can be detected by evaluation of TMSP in patients with DMD who do not have wall motion abnormalities by conventional echocardiography. TMSP with a notch proved effective for evaluating subtle early changes in patients with DMD and might be useful for predicting LV dysfunction.

2-Chloroadenosine but not adenosine induces apoptosis in rheumatoid fibroblasts independently of cell surface adenosine receptor signalling.

The apoptotic effect of adenosine and its analogues was studied in fibroblast‐like synoviocytes derived from rheumatoid arthritis patients (RA‐FLSs). Evoked cell death was quantitatively examined by assessing DNA fragmentation using an enzyme‐liked immunosorbent assay and by measuring phosphatidylserine exposure through flow cytometric analysis of annexin V binding. Exposing cells for 24 h to 2‐chloroadenosine (2‐CADO), a nonspecific, adenosine deaminase (ADA)‐resistant, adenosine receptor (AdoR) agonist, induced DNA fragmentation, and thus apoptosis, in RA‐FLSs at concentrations 50 μM. By contrast, incubation with adenosine for up to 72 h did not evoke DNA fragmentation, even in the presence of ADA inhibitor coformycin and nucleoside transporter inhibitor nitrobenzylmercaptopurin (NBMPR). Transcription of all four AdoR isoforms was detected in RA‐FLSs; nevertheless selective AdoR agonists similarly failed to induce DNA fragmentation. DNA fragmentation evoked by 2‐CADO was inhibited by NBMPR and by 5′‐iodotubercidin, an adenosine kinase inhibitor, but not by xanthine amine congener, an A1 and A2 receptor antagonist, or by selective AdoR antagonists. The nonspecific caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp fluoromethyl ketone abolished the apoptotic effect of 2‐CADO. These results suggest that 2‐CADO induces apoptosis in RA‐FLSs independently of AdoR‐mediated signalling. Instead, 2‐CADO, but not adenosine, is taken up into RA‐FLSs via human equilibrative nucleoside transporter‐1, where it is phosphorylated by adenosine kinase. The resultant phospho‐2‐CADO induces DNA fragmentation by activating a caspase pathway.

Association of the TAP2*Bky2 allele with presence of SS-A/Ro and other autoantibodies in Japanese patients with systemic lupus erythematosus

We previously reported that a new allele of transporter associated with antigen processing (TAP) 2 gene, TAP2*Bky2 (Val577), was significantly increased in Japanese patients with Sjögren’s syndrome (SS) and had a strong association with SS-A=Ro antibody production. In the present study, it was investigated whether the association of TAP2*Bky2 with SS-A=Ro antibody production was also found in Japanese patients with systemic lupus erythematosus (SLE). Polymorphisms of the TAP1 and TAP2 genes were determined in 114 Japanese SLE patients by the polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) method. The allele frequencies of the TAP1 and TAP2 genes in SLE patients were not significantly different from those in controls, although the allele frequency of TAP2*Bky2 was slightly higher in SLE patients than in healthy control subjects (9.2% vs 5.5%, P = 0.126). The allele frequency of TAP2*Bky2 was significantly higher in SLE patients with oral ulcers than in those without. It was noteworthy that TAP2*Bky2 was significantly associated with the appearance of not only SS-A=Ro antibody but also SS-B=La, nRNP, and Sm antibodies in the patients. The association of TAP2*Bky2 was found with the antibody production to both 60 and 52 kDa SS-A=Ro antigens. As TAP2*Bky2 had a strong linkage disequilibrium with DRB1*08032, TAP2*Bky2 or its haplotype with DRB1*08032 may be involved in SS-A=Ro antibody production not only in SS but also SLE patients, indicating that TAP2*Bky2 may be a susceptible gene not only to the disease of SS but also to the SS-A=Ro autoantibody production.

Very early stage left ventricular endocardial dysfunction of patients with hypereosinophilic syndrome

Cardiac involvement in hypereosinophilic syndrome (HES) patients entails significant morbidity and mortality. Left ventricular (LV) endocardial damage is important for the development of cardiac involvement in HES patients. However very early stage LV endocardial damage, such as prior to the first stage of an acute necrotic stage, remains uncertain. We studied 32 HES patients, all with normal conventional echocardiographic findings. Global radial and circumferential strain (GRS and GCS) were determined for each peak global strain curve from the mid-LV short-axis view, and global longitudinal strain (GLS) was averaged each peak global strain curve from standard apical views by means of two-dimensional speckle-tracking method. Thirty-one age-, gender-, LV ejection fraction-matched normal subjects were studied for comparison. GRS and GRS were similar for HES patients and normal controls, but GLS for HES patients was significantly lower than that for normal controls (16.2 ± 3.3 % vs 19.3 ± 2.9 %, p < 0.001). Furthermore, receiver operating characteristic curve analysis identified GLS ≤17.0 % as the best predictor of LV endocardial dysfunction with a sensitivity of 66 %, specificity of 78 %, and area under the curve of 0.781 (p = 0.0001). In conclusions, LV endocardial dysfunction pre-existed even in HES patients without apparent cardiac involvement. GLS as assessed with the two-dimensional speckle-tracking method is a promising tool for the better management of very early stage of HES patients.

Predicting scores for left ventricular dysfunction in Duchenne muscular dystrophy

Background:  The aim of this study was to predict left ventricular (LV) dysfunction and the timing to perform echocardiography in patients with Duchenne muscular dystrophy (DMD). We developed a scoring system using clinical parameters and examined its efficacy. It is indispensable to utilize echocardiogram for evaluating myocardial damage of DMD patients, but there is no established guideline for determining the clinical conditions which require echocardiographic examination.

Cardiac involvement in Fukuyama muscular dystrophy is less severe than in Duchenne muscular dystrophy

BACKGROUND One of the main complications in patients with muscular dystrophies is cardiac dysfunction. The literature on cardiac involvement in patients with Fukuyama congenital muscular dystrophy (FCMD) is limited. AIM To compare cardiac involvement between patients with FCMD and Duchenne muscular dystrophy (DMD). METHODS We compared cardiac involvement between 30 patients with FCMD and 181 patients with DMD using echocardiography and serum biomarkers. All patients were receiving regular checkups at Kobe University Hospital. We used single regression analysis to compare echocardiographic parameters, age, and serum biomarkers. RESULTS Almost all clinical and echocardiographic parameters were lower in patients with FCMD than DMD. The brain natriuretic peptide concentration in patients with FCMD showed no correlation with age or left ventricular ejection fraction (r=0.231, p=0.22 and r=0.058, p=0.76, respectively). A log-rank test revealed that the risk of left ventricular systolic dysfunction was lower in patients with FCMD than DMD (p=0.046, hazard ratio=0.348). CONCLUSION The clinical progression of cardiac dysfunction is significantly milder in patients with FCMD than DMD, while skeletal muscle involvement is significantly worse in patients with FCMD. These data suggest that the pathophysiological findings of FCMD can be explained by less severe cardiac dysfunction in FCMD than DMD.

Combining passive leg-lifting with transmural myocardial strain profile for enhanced predictive capability for subclinical left ventricular dysfunction in Duchenne muscular dystrophy

BACKGROUND We previously reported that the transmural myocardial strain profile (TMSP) was an effective predictor for subclinical left ventricular (LV) dysfunction in patients with Duchenne muscular dystrophy (DMD) with preserved LV ejection fraction (LVEF), but its predictive power when used alone proved to be limited. METHODS A total of 95 DMD patients with LVEF of 59±5% (all ≥55%) and age 11.3±3.0 years were analyzed retrospectively. Echocardiography was performed at baseline and 1-year follow-up, and all baseline measurements were repeated during a passive leg-lifting maneuver with legs elevated to approximately 45° from the horizontal position. TMSP of the posterior wall was evaluated from the mid-LV short-axis view. On the basis of our previous findings, TMSP with a notch was adopted as a predictor for evaluation of subclinical LV dysfunction in DMD patients whose LVEF remains preserved. RESULTS At baseline, normal TMSP comprised 35 patients (37%), and the remaining 60 (63%) were classified as TMSP with a notch. Twenty-nine patients (48%) had developed LV wall motion abnormality at the 1-year follow-up, but this was observed only in the group of patients with TMSP with a notch at rest and also during passive leg-lifting. Furthermore, this group showed significantly more frequent development of LV wall motion abnormality at 1-year follow-up, with better sensitivity, specificity, and positive and negative predictive values for prediction of this abnormality than for other sub-groups. CONCLUSIONS Most DMD patients suffer from progressive skeletal muscle weakness, so that combining TMSP with passive leg-lifting may make TMSP even more effective as a simple and non-invasive predictor of LV subclinical dysfunction.

Claviform aspergillus‐related vegetation in the left ventricle of a patient with systemic lupus erythematosus

A 38‐year‐old woman was diagnosed with systemic lupus erythematosus and received immunosuppressive therapy. After 6 months of treatment, workup for low‐grade fever yielded elevated enzyme‐linked immunosorbent assay titers for Aspergillus antigen in serum and ascites, leading to the diagnosis of disseminated aspergillosis. Transthoracic echocardiography revealed a claviform vegetation attached to the left ventricular anterior septum. Two days after the start of antifungal Amphotericin‐B therapy, the patient suffered from several neurologic disorders. A second transthoracic echocardiography revealed that the vegetation decreased in size. Two weeks later, the vegetation increased again. Combination therapy of Amphotericin‐B and Voriconazole was initiated, and the vegetation eventually disappeared completely.