Diagnosis of Pancreatic Neoplasms Using a Novel Method of DNA Methylation Analysis of Mucin Expression in Pancreatic Juice

Mucins (MUC) play crucial roles in carcinogenesis and tumor invasion in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasms (IPMNs). Our immunohistochemistry (IHC) studies have shown a consensus position on mucin expression profiles in pancreatic neoplasms as follows: MUC1-positive but MUC2-negative expression in PDACs; MUC1-negative but MUC2-positive expression in intestinal-type IPMNs (dangerous type); MUC1-negative and MUC2-negative expression in gastric-type IPMNs (safe type); High MUC4 expression in PDAC patients with a poor outcome; and MUC4-positive expression in intestinal-type IPMNs. We also showed that three mucin genes (MUC1, MUC2 and MUC4) expression in cancer cell line was regulated by DNA methylation. We have developed a novel ‘methylation-specific electrophoresis (MSE)’ method to analyze the DNA methylation status of mucin genes by high sensitivity and resolution. By using the MSE method, we evaluated pancreatic juice samples from 45 patients with various pancreatic lesions. The results were compared with final diagnosis of the pancreatic lesions including IHC of mucin expression in the paired pancreatic tissues. The results indicated that the DNA methylation status of MUC1, MUC2 and MUC4 in pancreatic juice matched with the mucin expression in tissue. Analyses of the DNA methylation status of MUC1, MUC2 and MUC4 were useful for differential diagnosis of human pancreatic neoplasms, with specificity and sensitivity of 87% and 80% for PDAC; 100% and 88% for intestinal-type IPMN; and 88% and 77% for gastric-type IPMN, respectively. In conclusion, MSE analysis of human pancreatic juice may provide useful information for selection of treatment for pancreatic neoplasms.     

Stereoscopic Analysis of Optic Nerve Head Parameters in Primary Open Angle Glaucoma: The Glaucoma Stereo Analysis Study

PurposeThe Glaucoma Stereo Analysis Study (GSAS), a cross sectional multicenter collaborative study, used a stereo fundus camera to assess various morphological parameters of the optic nerve head (ONH) in glaucoma patients and investigated the relationships between these parameters and patient characteristics.ResultsPatient characteristics included refractive error of −3.38±3.75 diopters, intraocular pressure (IOP) of 13.6±2.6 mmHg, and visual field mean deviation (MD) of −4.71±3.26 dB. Representative ONH parameters included a horizontal disc width of 1.66±0.28 mm, vertical disc width of 1.86±0.23 mm, disc area of 2.42±0.63 mm², cup area of 1.45±0.57 mm², and cup volume of 0.31±0.22 mm³. Correlation analysis revealed significant negative associations between vertical cup-to-disc ratio (0.82±0.08) and MD (r = −0.40, P<0.01) and between disc tilt angle (10.5±12.5 degrees) and refractive error (r = −0.36, P<0.01). Seventy-five percent of the eyes had a positive value for rim decentering (0.30±0.42), indicating that rim thinning manifested more often as an inferior lesion than a superior lesion.ConclusionWe used stereoscopic analysis to establish a database of ONH parameters, which may facilitate future studies of glaucomatous changes in ONH morphology.     

Overtime Work and Prevalence of Diabetes in Japanese Employees: Japan Epidemiology Collaboration on Occupational Health Study

Objective

Epidemiologic evidence on long working hour and diabetes has been conflicting. We examined the association between overtime work and prevalence of diabetes among Japanese workers.

Methods

The subjects were 40,861 employees (35,170 men and 5,691 women), aged 16 to 83 years, of 4 companies in Japan. Hours of overtime were assessed using self-reported questionnaires. Diabetes was defined as a fasting plasma glucose ≥126 mg/dl (7.0 mmol/l), hemoglobin A1c ≥6.5% (48 mmol/mol), or current use of anti-diabetic drug. Multiple logistic regression analysis was used to calculate odds ratio of diabetes for each category of overtime.

Results

After adjustment for age, sex, company, smoking, and BMI, there was a suggestion of U-shaped relationship between overtime work and prevalence of diabetes (P for quadratic trend = 0.07). Compared with those who worked <45 hours of overtime per month, the adjusted odds ratios (95% confidence interval) of diabetes were 0.86 (0.77–0.94), 0.69 (0.53–0.89), and 1.03 (0.72–1.46) for those who worked 45–79, 80–99, and ≥100 hours of overtime per month, respectively. In one company (n = 33,807), where other potential confounders including shift work, job position, type of department, alcohol consumption, sleep duration, leisure time physical activity, and family history of diabetes was additionally adjusted for, similar result was obtained (P for quadratic trend = 0.05).

Conclusions

Long hours of overtime work may not be associated with increased prevalence of diabetes among Japanese workers.     

Evaluation of Monoclonal Antibody-Based Sandwich Direct ELISA (MSD-ELISA) for Antigen Detection of Foot-and-Mouth Disease Virus Using Clinical Samples

A monoclonal antibody-based sandwich direct ELISA (MSD-ELISA) method was previously developed for foot-and-mouth disease (FMD) viral antigen detection. Here we evaluated the sensitivity and specificity of two FMD viral antigen detection MSD-ELISAs and compared them with conventional indirect sandwich (IS)-ELISA. The MSD-ELISAs were able to detect the antigen in saliva samples of experimentally-infected pigs for a longer term compared to the IS-ELISA. We also used 178 RT-PCR-positive field samples from cattle and pigs affected by the 2010 type-O FMD outbreak in Japan, and we found that the sensitivities of both MSD-ELISAs were about 7 times higher than that of the IS-ELISA against each sample (P<0.01). In terms of the FMD-positive farm detection rate, the sensitivities of the MSD-ELISAs were about 6 times higher than that of the IS-ELISA against each farm (P<0.01). Although it is necessary to conduct further validation study using the other virus strains, MSD-ELISAs could be appropriate as a method to replace IS-ELISA for FMD antigen detection.     

Effects of Aprepitant on the Pharmacokinetics of Controlled-Release Oral Oxycodone in Cancer Patients

Purpose

Oxycodone is a µ-opioid receptor agonist widely used in the treatment of cancer pain. The predominant metabolic pathway of oxycodone is CYP3A4-mediated N-demethylation to noroxycodone, while a minor proportion undergoes 3-O-demethylation to oxymorphone by CYP2D6. The aim of this study was to investigate the effects of the mild CYP3A4 inhibitor aprepitant on the pharmacokinetics of orally administered controlled-release (CR) oxycodone.

Method

This study design was an open-label, single-sequence with two phases in cancer patients with pain who continued to be administered orally with multiple doses of CR oxycodone every 8 or 12 hours. Plasma concentration of oxycodone and its metabolites were measured up to 8 hours after administration as follows: on day 1, CR oxycodone was administered alone; on day 2, CR oxycodone was administered with aprepitant (125 mg, at the same time of oxycodone dosing in the morning). The steady-state trough concentrations (Css) were measured from day 1 to day 3.

Results

Aprepitant increased the area under the plasma concentration-time curve (AUC_0–8) of oxycodone by 25% (p<0.001) and of oxymorphone by 34% (p<0.001), as well as decreased the AUC_0–8 of noroxycodone by 14% (p<0.001). Moreover, aprepitant increased Css of oxycodone by 57% (p = 0.001) and of oxymorphone by 36% (p<0.001) and decreased Css of noroxycodone by 24% (p = 0.02) at day 3 compared to day 1.

Conclusions

The clinical use of aprepitant in patients receiving multiple doses of CR oxycodone for cancer pain significantly altered plasma concentration levels, but would not appear to need modification of the CR oxycodone dose.

Trial Registration

UMIN.ac.jp UMIN000003580.     

Associations between Diurnal 24-Hour Rhythm in Ambulatory Heart Rate Variability and the Timing and Amount of Meals during the Day Shift in Rotating Shift Workers

It has not hitherto been clarified whether there is an association between dietary behavior and circadian variation in autonomic nervous system activity among shift workers. This study examines diurnal 24-h rhythm in heart rate variability (HRV) and dietary behavior among rotating shift workers, while taking into account the sleep-wake cycle and physical activity. The subjects were 11 female and 2 male nurses or caregivers working in a rotating 2-shift system at a health care facility. All the subjects were asked to undergo 24-h electrocardiograph and step count recordings, and to record the time of each meal and the amounts of each food and beverage consumed. Coarse graining spectral analysis was used for approximately 10-min segments of HRV to derive the total power (TOT: >0.04 Hz) of the periodic components and the integrated power of periodic components in the low-frequency (LF: 0.04–0.15 Hz) and high-frequency (HF: >0.15 Hz) ranges. Then the ratio of HF power to TOT (HF nu) and the ratio of LF power to HF power (LF/HF) were calculated to assess cardiac vagal tone and cardiac sympathovagal balance, respectively. Single cosinor analysis was used to obtain 24-h period variations in both variables of HRV. Acrophases of HF nu and LF/HF expressed in time since awakening were significantly (p<0.05) delayed for subjects having breakfast at a later time after awakening. Multivariable regression analysis indicated that the timing of breakfast, the ratio of energy intake at dinner to total energy intake, and total energy intake were correlated to the acrophases of HF nu and/or LF/HF. These results suggest that the phase angle between circadian variation in cardiac autonomic nervous system activity and the sleep-wake cycle may be associated with dietary behavior in shift workers.     

Angiopoietin-Like Protein 2 Induced by Mechanical Stress Accelerates Degeneration and Hypertrophy of the Ligamentum Flavum in Lumbar Spinal Canal Stenosis

Chronic inflammation and subsequent fibrosis induced by mechanical stress play an important role in ligamentum flavum (LF) hypertrophy and degeneration in patients with lumbar spinal canal stenosis (LSCS). Angiopoietin-like protein 2 (Angptl2) is a chronic inflammatory mediator induced under various pathological conditions and increases the expression of TGF-β1, which is a well-characterized mediator in LF hypertrophy. We investigated whether Angptl2 is induced by mechanical stress, and whether it contributes to LF hypertrophy and degeneration by activating the TGF-β1 signaling cascade. In this study, we investigated human LF tissue and LF fibroblasts isolated from patients who underwent lumbar surgery. We found that Angptl2 was abundantly expressed in fibroblasts of hypertrophied LF tissues at both the mRNA and protein levels. This expression was not only positively correlated with LF thickness and degeneration but also positively correlated with lumbar segmental motion. Our in vitro experiments with fibroblasts from hypertrophied LF tissue revealed that mechanical stretching stress increases the expression and secretion of Angptl2 via activation of calcineurin/NFAT pathways. In hypertrophied LF tissue, expression of TGF-β1 mRNA was also increased and TGF-β1/Smad signaling was activated. Angptl2 expression in LF tissue was positively correlated with the expression of TGF-β1 mRNA, suggesting cooperation between Angptl2 and TGF-β1 in the pathogenesis of LF hypertrophy. In vitro experiments revealed that Angptl2 increased levels of TGF-β1 and its receptors, and also activated TGF-β1/Smad signaling. Mechanical stretching stress increased TGF-β1 mRNA expression, which was partially attenuated by treatment with a calcineurin/NFAT inhibitor or Angptl2 siRNA, indicating that induction of TGF-β1 expression by mechanical stretching stress is partially mediated by Angptl2. We conclude that expression of Angptl2 induced by mechanical stress in LF fibroblasts promotes LF tissue degeneration by activation of TGF-β1/Smad signaling, which results in LF hypertrophy in patients with LSCS.     

Genetic Control of Startle Behavior in Medaka Fish

Genetic polymorphisms are thought to generate intraspecific behavioral diversities, both within and among populations. The mechanisms underlying genetic control of behavioral properties, however, remain unclear in wild-type vertebrates, including humans. To explore this issue, we used diverse inbred strains of medaka fish (Oryzias latipes) established from the same and different local populations. Medaka exhibit a startle response to a visual stimulus (extinction of illumination) by rapidly bending their bodies (C-start) 20-ms after the stimulus presentation. We measured the rates of the response to repeated stimuli (1-s interval, 40 times) among four inbred strains, HNI-I, HNI-II, HO5, and Hd-rR-II1, and quantified two properties of the startle response: sensitivity (response rate to the first stimulus) and attenuation of the response probability with repeated stimulus presentation. Among the four strains, the greatest differences in these properties were detected between HNI-II and Hd-rR-II1. HNI-II exhibited high sensitivity (approximately 80%) and no attenuation, while Hd-rR-II1 exhibited low sensitivity (approximately 50%) and almost complete attenuation after only five stimulus presentations. Our findings suggested behavioral diversity of the startle response within a local population as well as among different populations. Linkage analysis with F2 progeny between HNI-II and Hd-rR-II1 detected quantitative trait loci (QTL) highly related to attenuation, but not to sensitivity, with a maximum logarithm of odds score of 11.82 on linkage group 16. The three genotypes (homozygous for HNI-II and Hd-rR-II1 alleles, and heterozygous) at the marker nearest the QTL correlated with attenuation. Our findings are the first to suggest that a single genomic region might be sufficient to generate individual differences in startle behavior between wild-type strains. Further identification of genetic polymorphisms that define the behavioral trait will contribute to our understanding of the neural mechanisms underlying behavioral diversity, allowing us to investigate the adaptive significance of intraspecific behavioral polymorphisms of the startle response.     

Metabolomics Evaluation of Serum Markers for Cachexia and Their Intra-Day Variation in Patients with Advanced Pancreatic Cancer

Purpose

Cancer cachexia is a multifactorial syndrome characterized by progressive loss of weight and muscle atrophy. Using metabolomics, we investigated serum markers and their intra-day variation in advanced pancreatic cancer patients with cachexia.

Methods

Patients were enrolled in two groups: those with or without cachexia. Blood samples collected at 6:30 AM, 11:30 AM, 4:30 PM, and 9:30 PM were analyzed using metabolomics, and serum levels of IL-6, TNF-α, and leptin were measured and compared between the two groups. Intra-day variation was then evaluated.

Results

Twenty-one patients were enrolled in total. In the cachexia group (n = 9), median body weight loss rate over 6 months was greater, performance status was poorer, and anorexia was more severe than in the non-cachexia group (n = 12). Each metabolites level showed substantial intra-day variation, and some of them displayed significant differences between the two groups. Levels of paraxanthine remained markedly lower in the cohort with cachexia at all measurement points. Besides, median IL-6 and TNF-α levels appeared higher and leptin concentration appeared lower in the cachexia group, albeit without statistical significance.

Conclusion

Some metabolites and some serological marker levels were affected by cancer cachexia. Although paraxanthine levels were consistently lower in patients with cachexia, we identified that many metabolites indicated large intra- and inter-day variation and that it might be necessary to pay attention to intra-day variation in metabolomics research.