Quantitative analysis of p16 methylation in Barrett's carcinogenesis.

p16 hypermethylation in Barrett's carcinogenesis has been evaluated in studies which did not take into account sample heterogeneity and yielded qualitative (methylated/unmethylated) instead of accurate quantitative (percentage of CpG methylation) data. We aimed to measure the degree of p16 methylation in pure samples representing all the steps of Barrett's tumorogenesis and to evaluate the influence of sample heterogeneity in methylation analysis. METHODS: 77 paraffin-embedded human esophageal samples were analyzed. Histological grading was established by two pathologists in: negative for dysplasia, indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia and adenocarcinoma. Areas of interest were selected by laser-capture microdissection. p16 methylation was quantified by pyrosequencing. An adjacent section of the whole sample was also analyzed to compare methylation data. RESULTS: After microdissection, we obtained 15 samples of squamous epithelium, 36 non-dysplastic Barrett's esophagus, 3 indefinite for dysplasia, 24 low-grade dysplasia, 4 high-grade dysplasia and 12 adenocarcinoma. Squamous epithelium showed the lowest methylation rates: 6% (IQR 5-11) vs. 11%(7-39.50) in negative/indefinite for dysplasia, p<0.01; 10.60%(6-24) in low-grade dysplasia, p<0.05; and 44.50%(9-66.75) in high-grade dysplasia/adenocarcinoma, p<0.01. This latter group also exhibited higher methylation rates than Barrett's epithelium with and without low-grade dysplasia (p<0.05). p16 methylation rates of microdissected and non-microdissected samples did not correlate unless the considered histological alteration comprised >71% of the sample. CONCLUSIONS: p16 methylation is an early event in Barrett's carcinogenesis which increases with the severity of histological alteration. p16 methylation rates are profoundly influenced by sample heterogeneity, so selection of samples is crucial in order to detect differences.

The CD14 (-159 C/T) SNP is associated with sCD14 levels and allergic asthma, but not with CD14 expression on monocytes.

LPS-ligation to CD14/TLR-4 on monocytes/macrophages triggers the production of IL-12-family cytokines. IL12/18 promote TH1-differentiation, counteracting the TH2-driven asthma. Therefore, CD14 modulation could alter the TH2-differentiation and should be taken into account when studying asthma. To analyse the alteration in CD14 levels and its association with CD14 (-159 C/T) SNP (rs2569190) in Caucasian adults with stable allergic asthma, we performed a cross-sectional study (277 healthy subjects vs. 277 patients) where clinical parameters, CD14 values and the CD14 (-159 C/T) SNP were studied. Apart from typical biomarkers, we found an increment of neuron-specific enolase (NSE) in allergic asthma, probably linked to monocyte activity. Indeed, we evidenced increased monocyte numbers, but lower CD14 expression and normalised sCD14 values in patients. Moreover, we noticed an association of the T allele (P = 0.0162) and TT genotype (P = 0.0196) of the CD14 SNP with a decreased risk of allergic asthma and augmented sCD14 levels. In conclusion, monocyte CD14 expression and normalized sCD14 values were reduced in stable state asthmatics, and this could be related to the presence of an expanded CD14low monocyte subset. This study also demonstrates that the CD14 (-159 C/T) polymorphism is a risk factor for moderate-severe allergic asthma in adult Caucasians.

Expansion of a CD26low Effector TH Subset and Reduction in Circulating Levels of sCD26 in Stable Allergic Asthma in Adults.

BACKGROUND AND OBJETIVE: The pathogenesis of asthma is dependent on the balance between regulatory and effector T cells, which display differential expression of CD25 and CD26. Therefore, alteration of circulating levels of sCD25 and sCD26 during allergic asthma could be conditioned by changes in leukocyte phenotype. Objectives: To analyze expression of CD25 and CD26 on T lymphocytes and their soluble derivatives (sCD25, sCD26) during stable phases of moderate-severe allergic asthma. METHODS: Cross-sectional study with 2 adult cohorts of allergic asthmatics. Clinical, anthropometric, pulmonary, hematological, and biochemical parameters were measured. Phenotyping was performed with flow cytometry in both circulating and cultured leukocytes. Dipeptidyl peptidase 4 (DPP4) activity was assayed in culture supernatants. RESULTS: In vitro studies revealed upregulation of CD26 on human T lymphocytes upon activation, especially under TH17-favoring conditions, and a correlation with soluble DPP4 activity (rs=0.641; P<.001). CD26 expression on lymphocytes was higher in asthmatics, while serum sCD26 was lower in women and patients. The latter finding could be associated with an expanded CD25low/CD26low/CD127low subset of effector CD4+ T cells in allergic asthma, with no changes in Treg percentages. However, women showed an increased Teff/Treg ratio, which could explain their greater susceptibility to asthma. CONCLUSIONS: Allergic asthma causes an increment in CD25lowCD26low helper T cells detected in stable stages. These changes are mirrored in serum and should be considered in the light of the downmodulating role of CD26 in major chemokines related to the pathogenesis of asthma such as CCL11 (eotaxin), CCL5 (RANTES), and CXCL12a (SDF-1α).

A methodology to quantify the differences between alternative methods of heart rate variability measurement.

This work proposes a systematic procedure to report the differences between heart rate variability time series obtained from alternative measurements reporting the spread and mean of the differences as well as the agreement between measuring procedures and quantifying how stationary, random and normal the differences between alternative measurements are. A description of the complete automatic procedure to obtain a differences time series (DTS) from two alternative methods, a proposal of a battery of statistical tests, and a set of statistical indicators to better describe the differences in RR interval estimation are also provided. Results show that the spread and agreement depend on the choice of alternative measurements and that the DTS cannot be considered generally as a white or as a normally distributed process. Nevertheless, in controlled measurements the DTS can be considered as a stationary process.

Accuracy of heart rate variability estimation by photoplethysmography using an smartphone: Processing optimization and fiducial point selection.

This work compares several fiducial points to detect the arrival of a new pulse in a photoplethysmographic signal using the built-in camera of smartphones or a photoplethysmograph. Also, an optimization process for the signal preprocessing stage has been done. Finally we characterize the error produced when we use the best cutoff frequencies and fiducial point for smartphones and photopletysmograph and compare if the error of smartphones can be reasonably be explained by variations in pulse transit time. The results have revealed that the peak of the first derivative and the minimum of the second derivative of the pulse wave have the lowest error. Moreover, for these points, high pass filtering the signal between 0.1 to 0.8 Hz and low pass around 2.7 Hz or 3.5 Hz are the best cutoff frequencies. Finally, the error in smartphones is slightly higher than in a photoplethysmograph.

Real time heart rate variability assessment from Android smartphone camera photoplethysmography: Postural and device influences.

The aim of this paper is to present a smartphone based system for real-time pulse-to-pulse (PP) interval time series acquisition by frame-to-frame camera image processing. The developed smartphone application acquires image frames from built-in rear-camera at the maximum available rate (30 Hz) and the smartphone GPU has been used by Renderscript API for high performance frame-by-frame image acquisition and computing in order to obtain PPG signal and PP interval time series. The relative error of mean heart rate is negligible. In addition, measurement posture and the employed smartphone model influences on the beat-to-beat error measurement of heart rate and HRV indices have been analyzed. Then, the standard deviation of the beat-to-beat error (SDE) was 7.81 ± 3.81 ms in the worst case. Furthermore, in supine measurement posture, significant device influence on the SDE has been found and the SDE is lower with Samsung S5 than Motorola X. This study can be applied to analyze the reliability of different smartphone models for HRV assessment from real-time Android camera frames processing.

In vitro vitamin K3 effect on conjunctival fibroblast migration and proliferation.

PURPOSE: To evaluate the dose effect of vitamin K3 on wound healing mechanisms. METHODS: Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3 to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [(3)H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K3 1 mg/L, group 4/vitamin K3 2 mg/L, group 5/vitamin K3 4 mg/L, and group 6/vitamin K3 6 mg/L. Each experiment was carried out in triplicate and 4 times. RESULTS: There were no differences among groups at the initial time. In vitro wound repair was slower in groups 4, 5, and 6. There were no differences between control and ethanol groups and between control and vitamin K3 1 mg/L groups. Fibroblast mitogenic activity was statistically decreased in all vitamin K groups; statistical differences were found among vitamin K3 1 mg/mL and higher doses too. In groups 5 and 6, cellular toxicity was presented. CONCLUSIONS: Vitamin K3 is able to inhibit fibroblast proliferation. Vitamin K3 2 mg/L or higher doses inhibit wound healing repair, exhibiting cellular toxicity at 4 and 6 mg/L.

Heart rate variability analysis using a seismocardiogram signal.

Seismocardiography is a simple and non invasive method of recording cardiac activity from the movements of the body caused by heart pumping. In this preliminary study we use a smartphone to record this acceleration and estimate the heart rate. We compare the heart rate variability parameters from the seismocardiogram and ECG reference signal. The results show a great similarity and are strongly influenced by the instability in the sampling frequency of the device. The differences between RR series are lower than 10 ms.

Characterization of the prostaglandin E2 pathway in a rat model of esophageal adenocarcinoma.

UNLABELLED: Accumulating evidence indicates that the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway plays a key role in esophageal carcinogenesis. A better understanding of the pathway downstream of COX-2 may reveal novel targets for the prevention of esophageal adenocarcinoma (EAC). The objective of this study was to characterize the profile of genes involved in PGE2 metabolism and signaling in an experimental model of EAC. Esophagojejunostomy with gastric preservation was performed in wistar rats to induce gastroduodenal reflux. Rats were sacrificed 2 or 4 months after surgery. Nine non-operated rats were used to obtain normal (control) esophageal tissues. RESULTS: All rats that underwent esophagojejunostomy developed inflammation. In addition, 90% of the animals showed intestinal metaplasia; of those, 40% progressed to AC. This process was accompanied by a significant increase in esophageal PGE2 levels and the induction of both mRNA and protein levels of COX-2, COX-1, prostaglandin E synthase, 15-hydroxyprostaglandin dehydrogenase, and PGE2 receptors EP3, EP4 and especially EP2, which rose to particularly high levels in experimental rats. In addition, exposure to a selective COX-2 inhibitor (SC58125) or an EP1/EP2 antagonist (AH6809), but not an EP4 antagonist (AH23848B), significantly reduced cell proliferation of esophageal explants in 24 hour-organ culture experiments. Our data suggest that, in addition to COX-2, other components of the PGE2 pathway, including COX-1, may play important roles in the development of EAC induced by gastroduodenal reflux in the rat. Although it must be confirmed in vivo, the EP2 receptor may represent a promising selective target in the prevention of Barrett's associated AC.

New indices for quantification of the power spectrum of heart rate variability time series without the need of any frequency band definition.

This paper presents a new family of indices for the frequency domain analysis of heart rate variability time series that do not need any frequency band definition. After proper detrending of the time series, a cumulated power spectrum is obtained and frequencies that contain a certain percentage of the power below them are identified, so median frequency, bandwidth and a measure of the power spectrum asymmetry are proposed to complement or improve the classical spectral indices as the ratio of the powers of LF and HF bands (LF/HF). In normal conditions the median frequency provides similar information as the classical indices, while the bandwidth and asymmetry can be complementary measures of the physiological state of the tested subject. The proposed indices seem to be a good choice for tracking changes in the power spectrum in exercise stress, and they can guide in the determination of frequency band limits in other animal species.

Algorithm for phase extraction from a set of interferograms with arbitrary phase shifts.

The generalized analytical quadrature filter from a set of interferograms with arbitrary phase shifts is obtained. Both symmetrical and non symmetrical algorithms for any order are reported. The analytic expression is obtained through the convolution of a set of two-frame algorithms and expressed in terms of the combinatorial theory. Finally, the solution is applied to obtain several generalized tunable quadrature filters.

The effect of electrocardiographic lead choice on RR time series.

Results of heart rate variability analysis depend on the quality of the initial RR time series that is measured only in one lead of the ECG. This work shows that RR time series can subtly change from lead to lead so the choice of the analyzed lead is another source of uncertainty. The standard deviation of the differences of two RR time series obtained from different leads can change from 0.5 ms to more than 20 ms depending on the amount of noise, the morphological changes of the QRS complexes, the strategies of fiducial point determination and the measured subject. This source of uncertainty is in healthy subjects greater than that associated to the sampling frequency of the ECG for sampling frequencies greater than 400 Hz.

Drowsiness detection by thoracic effort signal analysis in real driving environments.

Detection of drowsiness while driving is a leading objective in advanced driver assistance systems. This work presents a new index to assess the alertness state of drivers based on the respiratory dynamics derived from an inductive band. More than 100 hours of driving in real environments from 13 healthy subjects were analyzed. The proposed method has a sensitivity of 93.7% and specificity of 86.3% in detecting full awake drivers while it has a sensitivity of 83.1% and specificity of 95.3% in detecting drowsy drivers. The results show that the proposed index may be promising to assess the alertness state of real drivers.

Prostaglandin EP2 receptor expression is increased in Barrett's oesophagus and oesophageal adenocarcinoma.

BACKGROUND: Accumulating evidence suggests that cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is involved in oesophageal adenocarcinogenesis. PGE2 exerts its biological action by binding to specific receptors (EP1, EP2, EP3 and EP4). AIM: To investigate which PGE2 receptor subtypes regulate PGE2 signals in the oesophageal adenocarcinoma sequence. METHODS: Expression was determined in oesophageal biopsies from 85 patients with oesophagitis, Barrett's metaplasia, intraepithelial neoplasia, oesophageal adenocarcinoma and normal oesophagus. Levels of mRNA and protein expression were determined by quantitative PCR, immunohistochemistry and western-blot. Expression of EP receptors was also determined in response to acid and bile exposure in the Barrett's adenocarcinoma cell line OE33. RESULTS: All four EP receptors subtypes were expressed in human oesophageal tissues. COX-2 and, especially, EP2 were increased in the Barrett's metaplasia-intraepithelial neoplasia-adenocarcinoma sequence. Expression of the EP4 receptor protein was increased in oesophageal adenocarcinoma. In contrast, expression levels of COX-1 and EP3 receptor were decreased along the sequence. No differences in EP1 expression were found. Treatment with the bile acid deoxycholate increased COX-2, EP1, EP2 and EP4 expression in OE33 cells. CONCLUSIONS: Our data suggest that in addition to COX-2, EP2 and EP4 receptors could be a selective target in the prevention and/or treatment of the Barrett's-associated adenocarcinoma.

Investigación translacional en patología digestiva: del laboratorio a la clínica y viceversa beneficiario: el paciente / No disponible

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Thoracic versus whole body bioimpedance measurements: the relation to hydration status and hypertension in peritoneal dialysis patients.

The whole body bioimpedance technique is a highly promising non-invasive, reproducible, fast and inexpensive bed-side method for monitoring hydration status. Using segmental bioimpedance measurements, it is possible to obtain information about the fluid change in each body segment (Song, Lee, Kim and Kim 1999 Perit. Dial. Int. 19 386-90). In this pilot study we have measured 25 male patients (30-65 yr, BMI 20-32 kg m(-2)) undergoing continuous ambulatory peritoneal dialysis (CAPD). Tetrapolar impedance measurements were obtained using the right-side technique (whole body), and a segmental impedance method focused in the thorax region. Blood pressure (BP) measurements were taken manually with a sphygmomanometer. Patients were classified as either stable (group 0) or unstable (group 1) using clinical parameters of overall cardiovascular risk. The Mahalanobis distance (dM2) was calculated for the mean blood pressure (BP(mean)), and the impedance parameter R normalized by body height H for the right-side (R(RS)/H) and the thorax segment (R(TH)/H). Differences between groups were significant (p < 0.0001) for R(TH)/H and for BP(mean), and less significant (p = 0.016) for R(RS)/H. Group 1 patients showed a small dM2 as compared with a reference patient (a critical patient with acute lung edema) with high BP(mean) and low values of R(TH)/H and R(RS)/H. Moreover, Group 0 patients showed a larger dM2 with respect to the reference patient, with lower BP(mean) and higher values of R(TH)/H and R(RS)/H. All patients classified as unstable by clinical assessment were correctly classified using R(TH)/H in conjunction with BP(mean) using dM2. Segmental-monofrequency non-invasive bioimpedance of the thoracic region could provide a simple, objective non-invasive method of support for facilitating the clinical assessment of CAPD patients.