This study had been conducted to confirm the protective role of propolis extract on CCl4-induced hepatorenal oxidative stress and resultant injury. Propolis extracts collected from Gwalior district and 24 female Sprague Dawley rats were used for experiment. Animals were exposed to CCl4  for 12 weeks (5 days/week) followed by treatment with propolis extract  for consecutive 2 weeks. CCl4 exposure significantly depleted blood sugar and hemoglobin level and raised the level of transaminases, alkaline phosphatase, lactate dehydrogenase, protein, urea, albumin, bilirubin, creatinine, triglycerides, and cholesterol in serum. Lipid peroxidation was enhanced, whereas GSH was decreased significantly in liver and kidney in CCl4-intoxicated group. Ethanolic extract of propolis successfully prevented these alterations in experimental animals. Activities of catalase, adenosine triphosphatase, glucose-6-phosphatase, acid, and alkaline phosphatase were also maintained towards normal with propolis therapy. Light microscopical studies showed considerable protection in liver and kidney with propolis treatment, thus, substantiated biochemical observations. This study confirmed hepatoprotective potential of propolis extract against chronic injury induced by CCl4 by regulating antioxidative defense activities.Carbon tetrachloride (CCl The chronic liver diseases are common worldwide and are characterized by a progressive evolution from steatosis to chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma , 2. Free50 higher than 7 g/kg for mice [4 [Propolis is an adhesive, resinous substance collected, transformed and used by honeybees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of intruders. Honeybees collect the resin from cracks in the bark of trees and leaf buds. They bring propolis back to the hive, where it is modified and mixed with other substances, including bees' own wax and salivary secretions . Propolifor mice , 15. Prefor mice –21 and s mice [4  and acet mice [4 . In the ad libitum. Experiments were conducted in accordance with the guidelines set by the Institutional Animal Ethics Committee (501/01/A/CPCSEA) of Jiwaji University. Hepatorenal injury was induced by CCl4  that was administered for 12 weeks (5 days/week) [Female Sprague Dawley rats (130 ± 10 g) were kept (3/cage: 6/group) in the animal house under standard husbandry conditions . The animals were fed on pelleted diet  and drinking water ys/week) .Apis mellifera was collected from apiaries nearby Gwalior district (MP) by Professor O. P. Agrawal, Senior Entomologist, School of Studies in Zoology, Jiwaji University, Gwalior (India). Propolis is a plant-derived product, and it has been proved that bees do not change its chemical composition [Crude propolis from the hive of position . It is rposition . A 90% eposition , 17, yieposition , 23. Sil4 for 12 weeks (5 days/week) and group 2 served as experimental control. Group 3 and 4 received treatments of propolis and silymarin, respectively, for 2 weeks (5 days/week). Blood was collected from retro-orbital venous plexus after 48 h of the last administration. The animals of entire groups were euthanized; liver and kidney were immediately excised and processed for biochemical analyses and histological preparations. Scheme of different treatments is given in Animals of group 1 received vehicle only and served as normal control. Animals of group 2–4 received CClSerum was isolated after keeping the blood for 1 h at room temperature followed by centrifugation at 1000 g for 15 min and stored at −20°C until analyzed. Tissue samples of liver and kidney were homogenized with ice-cold 150 mM KCl for the determination of TBARS and CAT activity and in 1% sucrose for GSH determination. Homogenates of liver and kidney were prepared in chilled hypotonic solution (10% w/v) for other biochemical assays, that is, total protein, cholesterol, adenosine triphosphatase (ATPase), acid, and alkaline phosphatase (ACPase and ALPase). Blood was immediately used for determination of hemoglobin  and bloon moles TBARS/g tissue using an extinction coefficient of 1.56 × 105/M/cm. The GSH measurement was performed using dithionitrobenzoic acid [μ moles GSH/g tissue. Catalase activity was determined as per method of Aebi [2O2 was monitored by decrease in the absorbance at λ 240 nm. H2O2 concentration was calculated using extinction coefficient of 0.0394/mM/cm and activity was expressed as n moles H2O2/min/mg protein.Lipid peroxidation (LPO) was measured using thiobarbituric acid (TBA)  and exproic acid . The GSH of Aebi . DecompoFresh tissues of liver and kidney were immediately processed to determine glycogen by anthrone reagent method . Total pμm thickness were stained with hematoxylin and eosin (H&E) and examined under compound light microscope.For histological studies, samples from liver and kidney were fixed in Bouin's fixative and processed routinely for embedding in paraffin. Tissue sections of 5 P ≤ 0.05 followed by Student's t-test [Data are expressed as mean ± SE of six animals used in each group. Statistical analysis was carried out by one way analysis of variance (ANOVA) considered significant at s t-test . 4 exhibited elevation in the leakage of AST, ALT, LDH, and SALP significantly (P ≤ 0.05) (P ≤ 0.05), thereby, confirming its protective effect in chronic injury. Chronic administration of CCl4 decreased hemoglobin level, whereas significant rise was observed in blood sugar, serum triglycerides and cholesterol (P ≤ 0.05) (P ≤ 0.05). CCl4 exposure significantly increased serum albumin, bilirubin, urea, and creatinine (P ≤ 0.05) (P ≤ 0.05). Efficacy of propolis was well compared with positive control silymarin, and percent protection clearly showed that propolis possesses almost equal degree of protection in all biochemical endpoints as silymarin-treated positive control.Chronic exposure to CCl ≤ 0.05) . Treatme ≤ 0.05) . Propoli ≤ 0.05) . Treatme4 administration enhanced the formation of TBARS in hepatic and renal tissues after its chronic exposure (P ≤ 0.05). Therapy with propolis extract diminished the production of TBARS (P ≤ 0.05), which ultimately reduced peroxidative stress up-to a considerable extent. The level of GSH and catalase was significantly decreased in liver and kidney after administration of CCl4 (P ≤ 0.05). Propolis extract was therapeutically effective in restoring GSH level and in maintaining CAT enzyme in liver and kidney in a significant manner (P ≤ 0.05), which could help in mitigating oxidative stress.Peroxidative stress and antioxidant status both in liver and kidney tissues were determined in terms of LPO, GSH, and catalase Figures . CCl4 ad4 exposure . Significant fall was noticed in activities of ALPase and ATPase in liver and kidney after 12-week administration of CCl4 (P ≤ 0.05). Therapeutic effect of propolis extract was found to be significant in both organs, and altered enzymatic activities were reversed towards control (P ≤ 0.05). Significant fall in total protein and glycogen contents was observed after chronic exposure to CCl4, whereas triglycerides were increased significantly in liver and kidney . Propolis was found to be equally effective as ilymarin on the basis of percent protection.Significantly enhanced enzymatic activity of ACPase was found in liver and kidney after CCl4 toxicity showed degeneration so liver cells were seen swollen. It is encountered simultaneously with ballooning degeneration and steatosis. Focal necrosis was seen clearly. Nuclear changes such as karyopyknosis and degeneration of the cell membrane indicated necrosis. These liver showed presence of fibrous septa with heterogenous population of nonparenchymal cells, foamy degeneration due to plenty of vacuolation and disturbed hepatic cord array  are formed from the metabolic conversion of CCl4 by cytochrome P-450 [2 tension rises, greater fraction of •CCl3 present in the system reacts rapidly with O2, and many orders of magnitude of more reactive free radicals CCl3OO• are generated [Various environmental toxicants and clinically useful drugs can cause severe cellular damages in different organs through their metabolic activation. CClve drugs . The reame P-450 . As O2 tenerated . These fenerated  and covaenerated  resultinenerated .4 enhanced LPO in liver and kidney. Treatment of propolis extract inhibited the generation of TBARS in both tissues that confirmed its antilipid peroxidative effects in chronic condition of hepatorenal injury. GSH constitutes the first line of defense against free radicals [4 administration decreased the activity of CAT and reduced GSH concentration in the tissues, which is in agreement with earlier reports [4. Flavonoids and their esters in propolis are pharmacologically active molecules and have been hypothesized to influence the antioxidant activity of propolis [Lipid peroxidations as well as altered levels of some endogenous scavengers are taken as reliable indices for oxidative stress , 48. In radicals . Another reports , 20. Orapropolis , 50. ASTGSH depletion increases the sensitivity of cells to various aggressions and also has several metabolic effects, for example, a decrease in the rate of gluconeogenesis or an increase in glycogenolysis . Fall in4 exposure [Activities of membranes enzymes ATPase and ALPase were decreased considerably in liver and kidney after CClexposure , 23, 24.exposure . Consideexposure . EfficacSince, liver and kidneys are closely related organs, which take part in different metabolic and excretion events. Any abnormality in liver may also lead to impairment in kidney functions. That is why both the organs had been taken into consideration in this study. In conclusion, results of this study validate the folklore use of propolis against various ailments. Propolis is interestingly effective in ameliorating acute, subchronic, and chronic injury to liver. It also has wider therapeutic index, and thus it may serve as clinically useful hepatoprotective natural product in future.


C-glucosides in the leaves of different species of bamboo was developed. Firstly, the flavone C-glycosides were extracted from the bamboo leaves (51 species in 17 genera) with methanol and chromatographed on silica gel 60 plates in automatic developing chamber (ADC2), and a qualitative survey using simple derivatization steps with the NP reagent was carried out. The flavone C-glycosides were found in 40 of 51 species of bamboo examined. Secondly, an HPLC method with photodiode array and multiple wavelength detector was optimized and validated for the simultaneous determination of flavone C-glycosides, including isoorientin, isovitexin, orientin, and vitexin in the leaves of three species of bamboo and the flavone C-glycosides were confirmed by LC/MS. The optimized HPLC method proved to be linear in the concentration range tested , precise (RSD ≤ 1.56%), and accurate (88–106%). The concentration ranges of isoorientin, isovitexin, orientin, and vitexin in three bamboo leaves samples were 1.00–2.78, 0–0.31, 0–0.07, and 0.20–0.68 mg/g, respectively. The proposed method was validated to be simple and reliable and can be a tool for quality control of bamboo leaf extract or its commercial products. A strategy for analyzing flavone  C-glycosides including isoorientin, isovitexin, orientin, and vitexin  chemical screening is simple, fast, and of low cost in identification of chemical ingredient from plant extracts , 8. Manying HPLC , HPCE [1ing HPLC , HPTLC [ing HPLC , 14, anding HPLC , have beC-glycosides and to optimize and validate an HPLC method for the simultaneous quantitation of marker compounds in different bamboo leaves and its commercial products.The work aimed to develop a two-step procedure to quickly survey a large number of bamboo leave samples (51 species in 17 genera) for the presence of flavone The leaves of fifty-one bamboo species were collected from five regions in China in 2007 and authenticated by Professor Jiusheng Peng from Jiangxi Academy of Forestry, Professor Yulong Ding from Nanjing Forestry University, and Professor Qirong Guo from International Centre for Bamboo and Rattan, respectively. Their family, species, and plant sources are shown in The HPLC-grade acetonitrile and methanol were purchased from fisher scientific (USA). Glacial acetic acid (≥99.7%) was purchased from Sigma-Aldrich (USA). Water was purified with an ultrapure water system . Analytical-grade chemical was obtained from Beijing Chemical Works (China). Commercial products of bamboo-leaf flavonoids labeled with total content of flavonoids (30%) and antioxidant potency were obtained from Jinhua Beikang Biochemical Development Co., Ltd. . The standard compounds of isoorientin, orientin, isovitexin and vitexin were purchased from Shanghai Winherb Medical S & T Development Co., Ltd. .μg/mL and 480 μg/mL. Orientin and vitexin stock solutions were prepared in methanol/water  at the concentration of 480 μg/mL and 440 μg/mL. The standard solutions were prepared by transferring appropriate volumes of stock solutions of each compound to a 10 mL volumetric flask and diluting to volume with methanol/water . A series of standard solutions with concentrations 0.01–100 μg/mL were prepared.Isoorientin and isovitexin stock solutions were prepared in methanol at the concentration of 370 μL) were applied to the plate as bands of 8 mm wide by means of a Camag (Switzerland) Linomat V sample applicator. The plate was developed in an automatic developing chamber (ADC2)  using ethyl acetate : formic acid : water  as the mobile phase. The developing distance was 80 mm. After development, plates were dried and derivatisation was carried out by immersion of the plates in a solution of NP reagent (diphenylborinic acid 2-aminoethyl ester (1.0 g) in ethyl acetate (200 mL) and PEG 400 (20 g) in dichloromethane (400 mL) [The dried bamboo leaf powder (1.0 g) was extracted by percolation with 20 mL of methanol and then sonicated for 30 min at room temperature. Thin-layer chromatography was performed on the plates precoated with silica gel 60 F254 . Samples (4 (400 mL) .μm membrane filter prior to analysis. An aliquot of 20 μL of solution was injected for HPLC and LC/MS analysis.Dried bamboo leaves powder (1.0 g) was accurately weighed and transferred in a 150 mL conical flask with glass stopper, and 60% aqueous methanol (15 mL) was added , 17. The18 column  at 30°C. The solvent system consisted of a mixture of water with 0.5% (v/v) glacial acetic acid and acetonitrile  at a flow rate of 1.0 mL/min.The HPLC-UV/DAD analyses were carried out on a Waters Alliance 2695  system connected to a model 2996 (DAD). The separation was performed using a Cm/z 100 to 1000.The LC-MS analysis was conducted using an Agilent 1100 system equipped with a photodiode array and multiple wavelength detector. The column and the chromatographic condition were the same as those used for the HPLC analysis. The LC effluent was split using a T-splitter to produce a flow of 0.25 mL/min. The identification of isoorientin, isovitexin, orientin, and vitexin was performed on the quadrupole mass spectrometer equipped with electrospray ionization (ESI-MS) in the negative mode. The collision energy was 60 eV. Capillary voltage was 3.5 kV, and the drying gas was set at 350°C. The scan range was set at The HPLC method was validated in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ) precision, specificity, and accuracy according to the International Conference on Harmonization (ICH) guidelines .The calibration curves were constructed by plotting peak area versus concentrations with the help of the Empower software. The standard solutions of the analytes were diluted with 60% methanol to yield a series of appropriate concentrations. LOD and LOQ were estimated experimentally by injecting a series of dilute solutions with known concentrations until the signal-to-noise ratio for the standards reached a 3 : 1 ratio for LOD and 10 : 1 for LOQ.μg/mL) were determined in one day and expressed as relative standard deviation (RSD). For interday precision, the standards were determined for three consecutive days.Intraday and interday variations were chosen to determine the precision of the HPLC method. For intraday precision, three different concentrations of standard solutions . The peaks were considered pure when there was a coincidence between the two spectral sections.The specificity of the method was ascertained by analyzing standard compounds and samples. The peaks for the marker compounds from sample solutions were confirmed by comparing the retention time and UV spectra with the reference standards. The peak purity was checked using DAD .C-glycosides including isoorientin, isovitexin, orientin, and vitexin showed the four characteristic fluorescent bands on the TLC plates with the colour of yellow, green, yellow and green, respectively. A typical TLC profile of extracts from bamboo leaves is shown in To optimize the TLC chromatographic conditions, different mobile phases and derivatisation regents were evaluated. The mobile phase ethyl acetate : formicacid : water  gave a good seperation of the markers. Satisfactory derivatisation efficiency was obtained using diphenylborinic acid 2-aminoethyl ester and PEG 400 as derivatisation regents. After derivatisation, flavone C-glycosides, especially isoorientin, were found in most of these species of bamboo tested. The different species of bamboo showed different major constituents. Identification of isoorientin, isovitexin, orientin, and vitexin in the different species of bamboo was carried out by the same retention factors (Rf) and similar colour to the standards compounds. The results of the flavone C-glycosides survey are presented in C-glycosides were found in 40 of 51 species of bamboo examined. TLC screening method proved to be an efficient strategy for the rapid screening of flavone C-glucosides. Among the fifty-one species of bamboo, B. multiplex cv. Silverstripe, P. heterocycla cv. pubescens, and P. acuta showed strong band intensity as to flavone C-glycosides on the TLC plates, so they were examined further by HPLC.The flavone μm; Waters USA), Zorbax SB-C18 , and YMC-Pack R&D ODS-A  were tested. To enhance the resolution and eliminate the peak tailing of the marker compounds, glacial acetic acid was added to the mobile phase. Different mobile phase compositions and different column temperatures were also optimized. The best results were obtained using a YMC-Pack R&D ODS-A column at 30°C, with acetonitrile and water containing 0.5% (v/v) glacial acetic acid as the mobile phase and isocratic elution (1.0 mL/min) was applied  with given concentration ranges. The parameters of linearity and range were summarized in Standard solutions of isoorientin, orientin, vitexin, and isovitexin were prepared at a concentration range of 0.2–100 The RSD values for both parameters ranged from 0.05 to 1.56% . TherefoC-glycosides are given in  The recovery values of the four flavone C-glycosides in bamboo leaves were identified by comparing their UV/DAD and MS spectra with those of the standards (Figures  The four flavone  Figures  and 4. TC-glycosides from bamboo leaves were detected. The retention time, maximum wavelength, and characteristic fragment ions for individual peaks are presented in The four flavone C-glycosides were found in the leaf of P. heterocycla cv. Pubesceus, which has been used as a raw material to produce commercial products of bamboo-leaf flavonoids. The quantitative data also showed that the content of isoorientin in B. multiplex cv. Silverstripe was higher than that in the two other bamboo species. It could be considered a promising plant source for preparation of isoorientin.The optimized method was applied to determinate the four marker compounds in different samples. These HPLC chromatograms are depicted in C-glucosides in a large number of samples and quantitative analysis of four marker compounds. The proposed procedure proved to be specific and sensitive and can be regarded as an alternative method to detect and quantify flavone C-glucosides in bamboo leaves and its commercial product.In summary, this paper describes a two-step procedure for the rapid screening of flavone


The area under the ROC curve (AUROC) for prediction of cirrhosis was 0.91 (95% CI 0.86–0.96). The index was validated in a separate cohort of 83 patients and the AUROC for this cohort was similar . In conclusion, the new index may complement other methods in diagnosing cirrhosis in patients with chronic HCV infection.Diagnosis of liver cirrhosis is essential in the management of chronic hepatitis C virus (HCV) infection. Liver biopsy is invasive and thus entails a risk of complications as well as a potential risk of sampling error. Therefore, non-invasive diagnostic tools are preferential. The aim of the present study was to create a model for accurate prediction of liver cirrhosis based on patient characteristics and biomarkers of liver fibrosis, including a panel of non-cholesterol sterols reflecting cholesterol synthesis and absorption and secretion. We evaluated variables with potential predictive significance for liver fibrosis in 278 patients originally included in a multicenter phase III treatment trial for chronic HCV infection. A stepwise multivariate logistic model selection was performed with liver cirrhosis, defined as Ishak fibrosis stage 5–6, as the outcome variable. A new index, referred to as Nordic Liver Index (NoLI) in the paper, was based on the model: Log-odds (predicting cirrhosis) = −12.17+ (age×0.11) + (BMI (kg/m Morbidity and mortality in Hepatitis C virus (HCV) infection results mainly from the development of complications of cirrhosis, or hepatocellular carcinoma (HCC) Liver biopsy remains the gold standard for the assessment of liver fibrosis Cholesterol synthesis is a process involving several steps. Lathosterol and desmosterol are intermediates in the cholesterol synthesis pathway and their serum concentration reflects cholesterol synthesis  in order to create a predictive model for HCV-related cirrhosis. This was done within the framework of a phase III treatment trial, with a well-defined cohort of treatment-naïve patients and where pre-treatment liver biopsies were mandatory.2), alcohol consumption during the previous year (g/week) and duration of infection were recorded. Of the 382 patients enrolled, 298 patients had a liver biopsy that fulfilled the criteria for staging and grading and had serum samples analyzed for potential fibrosis markers in accordance with the study protocol. When all patients with missing data in any of the parameters were excluded 278 patients remained. These patients constituted the exploratory set.The exploratory set cohort was derived from a phase III multicenter, investigator initiated, treatment trial for treatment-naïve HCV genotype 2 or 3 infected patients  conducted at 31 centers in Denmark, Finland, Norway and Sweden. Details regarding trial design and outcome have been published previously The validation set was derived from a cohort of 100 chronic HCV infected patients enrolled in a study evaluating the use of liver biopsy, serum fibrosis markers and transient elastography by use of the Fibroscan device Patients with missing data for any of the relevant parameters were excluded from the present study, leaving 83 patients in the validation set.Written informed consent was obtained from each participating patient. Ethics committees in each participating country approved the study , Regional Committee for Ethics in Medical Research, Bergen, Norway , The Scientific Ethical Committee for the Region of Middle Jutland, Viborg, Denmark (Den Videnskabsetiske Komité for Region Midtjylland), The Scientific Ethical Committee for the Region of South Denmark, Vejle, Denmark (Den Videnskabsetiske Komité for Region Syddanmark), and the Ethics Committee, Department of Medicine for the Hospital District of Helsinki and Uusimaa, Finland (Etiska kommittén för invärtesmedicin)). The study has been registered at the NIH trial registry .Written informed consent was obtained from each participating patient. The Regional Ethical Review Board, Gothenburg, Sweden  approved the study.9/L) and Prothrombin complex-INR were analyzed at each center. All other serum samples were stored in −70°C and subsequently analyzed at a central laboratory at Helsinki University Hospital, Finland. Liver function tests and serum fibrosis markers analyzed by standard laboratory methods included normalized AST and ALT, Gammaglutamyl-transferase (GGT) (U/L), Bilirubin (μmol/L), Haptoglobin (g/L), alfa2-macroglobulin (g/L), Hyaluronic acid (HA) (ng/ml), amino-terminal propeptide of type III procollagen (PIIINP) (ug/L), Apolipoprotein A1 (g/L) and carboxy-terminal telopeptide of type I collagen (ICTP) and serum cholesterol (mg/100 ml). The non-cholesterol sterols  were analyzed by gas–liquid chromatography (GLC)  on a 50-m long Ultra 2 capillary column (5% Phenyl-methyl siloxane) , with 5a-cholestane as internal standard In the exploratory cohort, baseline serum samples were drawn within 30 days prior to study entrance. Platelet count  as well as absolute concentrations (μg/100 ml).In the validation set all serum samples were drawn the same day as the liver biopsy and immediately stored in −70°C. Serum sterols were subsequently analyzed as stated above, all other analyzes were performed according to routine laboratory procedures at the Sahlgrenska University Hospital.All liver biopsies in the exploratory and validation cohort were retrieved and re-assessed for necroinflammatory activity and fibrosis stage according to the Ishak protocol The following variables were analyzed in the exploratory set: age, sex, weight, BMI, genotype, platelets, PK-INR, normalized AST and ALT, GGT, Bilirubin, Haptoglobin, alfa2-macroglobulin, HA, PIIINP, Apolipoprotein A1, ICTP, total cholesterol, cholestanol, D8-lathosterol, desmosterol, D7-lathosterol, campesterol, sitosterol, sitostanol, avenasterol and squalene. Classification was made in two groups that encompassed fibrosis stages 0–4 (n = 242) and 5–6 (n = 36), respectively. The suggested classification procedure consisted of two steps: (i) an index that resulted from fitting of a model to the data and (ii) cut-off values for this index that can be used to divide the predictions into two groups.prob) or log-odds (Iodds), which are equivalent. Iodds uses a more natural scale while Iprob may be perceived as more intuitive since it reflects the probability that a certain patient has cirrhosis. Iprob was chosen for further calculations, hereafter referred to as the Nordic Liver Index, NoLI. Cutoff values were chosen in order to minimize the misclassification error. Rather than choosing one cut-off, we suggest applying two, Clow and Chigh. If the index is below Clow, the observation will be classified into group 1. If it is above Chigh it will be classified as group 2. If between Clow and Chigh it will not be classified as either group and further investigations may have to be undertaken to make a correct diagnosis.The data was modeled through logistic regression, with model selection based on Akaike information criterion (AIC). The model selection was done using goodness-of-fit criteria rather than the magnitude of the p-values for each variable. The large number of sometimes highly correlated factors leads to over fitting, i.e. a model that was too large and sensitive to small perturbations in the data. We decided to make the process of model selection more robust by constructing logistic regression for a large number of re-samples of the data and taking only those factors in the final index that appeared in many of these, re-sampled models. The final estimation of the coefficients was performed using the original dataset. The new index based on the model above can be formulated either in terms of probabilities (Iwww.r-project.org) version 2.15.0 was used for all calculations and the package pROC was used to calculate ROC curves and the corresponding CI. Comparisons of baseline characteristics between groups were analyzed by Mann-Whitney’s U test or Chi-squared test where appropriate. Correlation between transient elastography and NoLI was assessed by Spearman’s r test. These latter analyses were performed using IBM SPSS Statistics version 19.0 software package . All reported p-values are two-sided, and p-values <0.05 were considered significant.Diagnostic performance was analyzed by constructing receiver operator characteristics curves (ROC) for specificity and sensitivity with calculation of the area under the ROC curve (AUROC) and the corresponding confidence interval (CI). The R software package  had cirrhosis; baseline characteristics for cirrhotic and non-cirrhotic patients in the exploratory set are reported in There were no patients with clinically decompensated liver cirrhosis in either exploratory or validation set. Patients in the validation set were older than patients in the exploratory set; median age was 49 (IQR 44–55) vs. 42 years (IQR 34–50) (p<0.001), and had a longer duration of infection; 29 (IQR 24–34) vs. 13.5 years  (p<0.001) in the validation and exploratory set respectively. In the exploratory set, 69% of the patients were infected with HCV genotype 3, whereas in the validation set HCV genotype 1 was most frequent, present in 72% of patients.−5), D7-lathosterol (p = 0.02), platelet count (p = 7.0×10−5) and Prothrombin-INR (p = 0.122). The model characteristics are summarized in Application of the model selection methodology described earlier lead to a final model comprising the following variables: Age (p = 0.0002), BMI (p = 8.0×10odds (predicting cirrhosis) = −12.17+ (age×0.11) + (BMI (kg/m2)×0.23) + (D7-lathosterol (μg/100 mg cholesterol)×(−0.013)) + (Platelet count (x109/L)×(−0.018)) + (Prothrombin-INR×3.69).The regression formula for this final model is: Log-odds; Iprob = exp (log-odds)/(1+exp (log-odds)).Predicted probability; Iprob) for further calculations, referred to as the Nordic Liver Index (NoLI). The relation between NoLI and Ishak fibrosis stage is illustrated in the lower right panel of As stated above, we chose predicted probability  of patients with cirrhosis as non-cirrhotic and 5.0% (12/242) of non-cirrhotic as having cirrhosis . Applying the new index to the validation set, the AUROC for prediction of cirrhosis was 0.90 (95% CI 0.83–0.98). In the validation set, patients were also examined by transient elastography. The Spearman correlation coefficient for the new index and liver stiffness values was 0.54 p<0.001; . One patSince the exploratory set included only patients infected with HCV genotypes 2 and 3 and the validation set mainly genotype 1 infected patients, we combined the exploratory and validation set to evaluate potential genotypic differences. No significant differences were detected and the AUROC for specificity vs. sensitivity for each genotype was 0.91 (95% CI 0.82–1.0), 0.86 (95% CI 0.73–0.98) and 0.93 (95% CI 0.88–0.97) for genotypes 1, 2, and 3 respectively.The primary aim of this study was to develop a reliable model for predicting HCV-related liver cirrhosis. The model created could confidently predict cirrhosis in both the exploratory and the validation sets, although the findings need to be confirmed in larger series. Although there is considerable overlap between adjacent fibrosis stages, this model has a good ability to exclude cirrhosis in patients with less severe stages of fibrosis (Ishak F0-4) as well as to predict cirrhosis in biopsy-verified cirrhotic patients (Ishak F5-6). Like many other biochemical indices, NoLI performs better in excluding than confirming cirrhosis.The new index combines age, BMI, platelet count and prothrombin index, i.e. factors that previously have been consistently associated with fibrosis Several studies on the prediction of cirrhosis have been reported during the past 10 years Our study had some potential limitations. Measurement of serum lathosterol is not a standard biochemical test. Although it is measured using gas-chromatography standard patent-free laboratory technique, commonly used for analysis of other compounds, it may be slightly cumbersome to use. Still, the presence of D7-lathosterol improves the diagnostic performance of the NoLI index why we choose to keep it in the model.The prevalence of cirrhosis in the exploratory and validation sets was 13% and 10% respectively, which is low compared with some other studies on non-invasive fibrosis markers using liver biopsy as reference In the exploratory set, liver biopsies and serum samples were not taken at the same time point, although both were sampled prior to initiation of therapy. The index, however, performed well in the validation set where blood samples were drawn on the day of the liver biopsy. As with all non-invasive tests, the use of liver biopsy as comparison poses a problem since liver biopsy is prone to sampling error and thus can both over- and underestimate the true liver fibrosis stage In summary, our results indicate that an index combining well-known predictors of liver fibrosis in combination with measurement of a non-cholesterol sterol can be useful in predicting cirrhosis. These new findings could be of value as a supplement to already existing non-invasive methods and the application of our model potentially could aid clinical decision-making, e.g. on which patients require continued monitoring in spite of successful antiviral treatment.Table S1Summary of the final model for prediction of cirrhosis.(DOCX)Click here for additional data file.


Interactions between Th1 and Th2 immune responses are of importance to the onset and development of allergic disorders. A Toll-like receptor 7 agonist such as AZD8848 may have potential as a treatment for allergic airway disease by skewing the immune system away from a Th2 profile.To evaluate the efficacy and safety of intranasal AZD8848.single ascending dose study, AZD8848 (0.3-600 μg) was given intranasally to 48 healthy subjects and 12 patients with allergic rhinitis (NCT00688779). In a placebo-controlled repeat challenge/treatment study, AZD8848 (30 and 60 μg) was given once weekly for five weeks to 74 patients with allergic rhinitis out of season: starting 24 hours after the final dose, daily allergen challenges were given for seven days (NCT00770003). Safety, tolerability, pharmacokinetics, and biomarkers were monitored. During the allergen challenge series, nasal symptoms and lavage fluid levels of tryptase and α2-macroglobulin, reflecting mast cell activity and plasma exudation, were monitored.In a placebo-controlled 2-macroglobulin were also reduced by AZD8848.AZD8848 produced reversible blood lymphocyte reductions and dose-dependent flu-like symptoms: 30–100 μg produced consistent yet tolerable effects. Plasma interleukin-1 receptor antagonist was elevated after administration of AZD8848, reflecting interferon production secondary to TLR7 stimulation. At repeat challenge/treatment, AZD8848 reduced nasal symptoms recorded ten minutes after allergen challenge up to eight days after the final dose. Tryptase and αRepeated intranasal stimulation of Toll-like receptor 7 by AZD8848 was safe and produced a sustained reduction in the responsiveness to allergen in allergic rhinitis.NCT00688779 and NCT00770003 as indicated above. As suggested by the hygiene hypothesis, infections are of importance to the maturation of the immune system . Th1-medTLRs are receptors of the innate immune system that recognise conserved microbial components known as pathogen-associated molecular patterns (PAMPs) . PAMPs iet al. [In a murine model of allergic asthma, a TLR7 ligand (S28463) administered systemically exerted anti-allergic effects resulting in attenuated airway eosinophilia, normalized airway responsiveness, and prevention of airway remodelling ,12. In aet al.  demonstret al. . Similaret al. -18.et al. [AZD8848 is a selective TLR7 agonist optimised for topical airway treatment through rapid metabolism by plasma esterases, thereby reducing systemic exposure . Observaet al.  reportedHere, we report the results of two studies. In the first study, increasing single doses of AZD8848 were administered intranasally to healthy subjects and patients with allergic rhinitis. Indices of efficacy and tolerance were monitored. In the second study, AZD8848 was administered intranasally once weekly for five weeks to patients with allergic rhinitis: these individuals were then subjected to repeat allergen exposure and disease activity was monitored focusing on symptoms to establish proof of principle.This report comprises two studies evaluating safety and efficacy of single and repeated doses of AZD8848 administered to the nasal airway, both approved by the Regional Ethics Committee and the Swedish Medical Product Agency. They were conducted according to the Declaration of Helsinki and in compliance with Good Clinical Practice, and informed consent was obtained. The studies were of randomized, placebo-controlled, double-blinded, and parallel group designs. Reprotoxicology data was lacking for AZD8848 at the time of these studies and men only were recruited.Eight single ascending intranasal doses of AZD8848 (0.3-600 μg) were given to healthy subjects , in groups each involving four individuals receiving AZD8848 and two receiving placebo. Once a maximum tolerated dose was determined, patients with allergic rhinitis  received single doses of AZD8848 (30 and 100 μg) and placebo.Exclusion criteria for healthy individuals were: any relevant disease including seasonal and perennial allergic rhinitis, asthma, clinically relevant structural nasal abnormalities, and upper respiratory tract infection within two weeks prior to the start of the study.Inclusion criteria for patients were: men with seasonal allergic rhinitis for at least two years, positive skin prick test to birch or grass pollen allergen, asymptomatic condition outside the pollen season, and need for treatment at seasonal allergen exposure.Exclusion criteria for patients were: any relevant disease including perennial allergic rhinitis, asthma, clinically relevant structural nasal abnormalities, upper respiratory tract infection within two weeks prior to the start of the study, use of topical corticosteroids within four weeks prior to the study and use of antihistamines within one week, and immunotherapy.20), to functionally close the connection between the nasal and bronchial airways, while the study drug was administered.The study product was a solution of AZD8848 (60 mg/g), diluted with sterile buffered saline to concentrations required for each dose level. It was administered using a nasal spray device delivering 50 μL per actuation. The placebo product was isotonic saline. The study product was provided in 10 mL amber glass vials fitted with pump spray devices. In order to assure compliance, study personnel administered the doses. To prevent bronchial airway deposition of the drug, the patients were instructed to exhale against a resistance  was measured, reflecting type-1 IFN generation downstream from TLR7, and blood lymphocyte counts were carried out. An extensive safety investigation was performed with laboratory measurements , inspection of the nose (before and 24 hours after dose), vital signs , and continuous ECG. Finally, AEs were monitored and recorded with information about seriousness, causality, intensity, action taken, recovery, and outcome.(Part A), 18 patients were assessed at two dose levels (30 and 60 μg)  (Table (Part B), 56 patients were examined in an outpatient setting: 28 received 60 μg AZD8848 and 28 received placebo . In Part B, 60 μg AZD8848 was given once per week for five weeks . In order to assure compliance, study personnel administered all doses. To prevent bronchial airway deposition of the drug, the patients were instructed to exhale against a resistance (30 cm H20), to functionally close the connection between the nasal and bronchial airways, while the study drug was administered.The study product was AZD8848 (0.3 and 0.6 mg/mL) in buffered saline. It was administered using a nasal spray device delivering 50 μL per actuation. The placebo product had the same composition except for AZD8848. In Part A (n = 18). AZD8848 was not expected to be detectable in plasma due to rapid and complete metabolism. Therefore, exposure was monitored through analysis of its acid metabolite. Blood samples were obtained before and 15 minutes and 0.5, 1.0, 1.5, 2.0, and 4.0 hours after the first and fifth study drug administration in Part A. Analysis of AZD8848 and its acid metabolite was performed using ethanol extraction followed by liquid chromatography and tandem mass spectrometry.Pharmacokinetic characteristics were explored only in Part A study period prior to and 24 hours following the first, third, and fifth administration of the study drug. In Part A (n = 18), the same safety variables were monitored as in the single ascending dose study. In Part B, safety monitoring was reduced to vital signs, clinical chemistry, and ECG at pre-dose, prior to the third dose, 24 hours after the fifth dose, and at follow-up. In addition, a flow-cytometric analysis of lymphocyte subsets was performed. AEs were recorded continuously in both groups as described for the single ascending dose study above.Plasma samples for analysis of IL-1Ra were obtained during the In order to establish individually tolerable, repeatable, yet symptom-producing allergen challenge-doses, a nasal titration procedure was performed . In the During the allergen challenge series, the patients scored nasal symptoms every morning and evening. The scores were entered into diary cards and each registration reflected the preceding twelve hours. Nasal secretion and blockage as well as the most severe of sneezing and itching were scored separately on a four-grade scale: 0 = no, 1 = mild, 2 = moderate, and 3 = severe symptoms. The scores were added to a daily total nasal symptom score (TNSS), with separate morning and evening scores. Nasal symptoms were also scored ten minutes post allergen challenge: secretion and blockage were scored as described above, whereas the number of sneezes were counted and transformed into a sneezing score by the investigators: 0 sneezes = 0, 1–4 sneezes = 1, 5–9 sneezes = 2, and 10 or more sneezes = 3. The scores were added to a daily post challenge TNSS.2-macroglobulin and tryptase, reflecting plasma exudation and mast cell activity, respectively.Nasal saline lavages were obtained at three occasions, i.e. prior to administration of the study drug (baseline observation), 24 hours after the final dose, and 24 hours after the final allergen challenge. Nasal lavages were carried out using a pool-device containing 15 mL fluid and the right nasal cavity was used at all occasions . Each la2-Macroglobulin in nasal lavage fluids was measured using a radioimmunoassay sensitive to 7.8 ng/mL. Tryptase was measured using a radioimmunoassay with detection limit of 0.5 ng/mL . While α2-macroglobulin was analysed in the lavage fluids as they were, samples were concentrated ten times before the analysis of tryptase. In plasma, IL-1Ra was measured using a commercial ELISA .αIn the single ascending dose study, only descriptive statistics were used. Similarly, descriptive statistics only were employed for pharmacokinetic observations and safety variables. In the repeat challenge/treatment study, an ANOVA model was used for comparison between treatments (AZD8848 60 μg vs. placebo): TNSS recorded post challenge and at morning and evening observations were analyzed separately. P-values refer to one-sided hypothesis testing: values <0.05 were considered statistically significant. A multiplicative model was used for biomarkers by log transformation of the response variable and the covariate . If there was a value below limit of quantification (LOQ) in this analysis, it was estimated to LOQ/2. Treatment differences were estimated from respective model and confidence intervals, and p-values were calculated. In the repeat challenge/treatment study, the six patients receiving AZD8848 30 μg per week were not included in the comparative analysis as they were so few.Cmax) was 0.27 and 0.65 nmol/L, time to Cmax (tmax) was 15.0 and 22.5 min, and half-life (t1/2) was 18.4 and 27.6 min in healthy volunteers. Mean Cmax increased with increasing dose levels to a maximum of 1.55 nmol/L with the 600 μg dose. Area under the curve (AUC) increased roughly linearly with ascending dose.AZD8848 was not measurable in plasma, whereas its acid metabolite was readily detected. For the 30 and 100 μg doses, respectively, mean maximum plasma concentration  were subjected to administration of AZD8848 (30 and 100 μg). No differences were observed in the response profile to AZD8848 between these patients and the healthy individuals.Cmax was 0.58 and 0.57 nmol/L, tmax was 17.2 and 20.2 min, and t1/2 was 0.83 and 1.26 hours.AZD8848’s acid metabolite was detected at doses of 30 μg (data not shown) as well as 60 μg . The symptoms subsided within 24 hours, and did not lead to any individual discontinuations. Two individuals did drop out of the study and both received 60 μg AZD8848: one and two doses, respectively. One case was an AE  and the other was an individual found to be wrongfully included.In patients receiving placebo, in agreement with previous observations in this model , increasAZD8848 (60 μg) reduced nasal symptoms recorded ten minutes after allergen challenge from the fourth through the seventh day of the challenge series (i.e. five to eight days after the last dose of AZD8848) with an estimated effect size of 0.74 score units p < 0.05) , but these effects failed to reach statistical significance   Table . Signifi5) Table .In these studies we demonstrate that repeated intranasal TLR7 stimulation is associated with reduced responsiveness to allergen in patients with allergic rhinitis. It is the first observation in man where TLR7 has been successfully evaluated as a therapeutic target for allergic airway inflammation.t1/2 in human plasma is estimated to be 20 seconds [The TLR7 agonist AZD8848 undergoes very rapid enzymatic degradation by butyrylcholinesterase:  seconds . In this seconds , was det seconds ,26. In t2-macroglobulin, reflecting mast cell activity and inflammatory plasma exudation [in vitro and animal reports on anti-allergic effects of AZD8848, indicating that repeated TLR7 stimulation reduces the responsiveness to allergen [In the repeat challenge/treatment study, acute symptoms in response to allergen were consistently reduced from the third day of the allergen challenge series in patients receiving the TLR7 agonist when compared to placebo, indicating a sustained effect of at least one week after the final dose of AZD8848. The same pattern was observed for the low-grade morning and evening symptoms, but these changes failed to reach statistical significance. In agreement with a symptom reducing effect, lavage fluid levels of tryptase and αxudation ,28, wereallergen -23, and The observed reduction in responsiveness to allergen might reflect that the immune system was functionally skewed away from a Th2 response. If so, it did probably not represent repolarisation of T-lymphocytes (i.e. a change from Th2 to Th1 phenotype), as this study involved atopic individuals with established populations of memory T-cells with a life span of at least two to three years . ArguablWhile the repeat challenge/treatment study demonstrated that 60 μg of AZD8848 administered intranasally once weekly for five weeks induced a desired hyporesponsiveness to allergen, likely through activation of TLR7, further studies are warranted to optimize the effect. Preclinical data have indicated that more frequent administration of AZD8848 can produce more marked anti allergic effects (AstraZeneca: data on file). Furthermore, as demonstrated in a Brown Norway rat model of allergic rhinitis/asthma, both nasal and bronchial administration of AZD8848 can reduce the ability of a bronchial allergen challenge to produce bronchial airway eosinophilia and generate IL-13 , suggestIn this study, safety and tolerability of intranasal AZD8848 was evaluated parallel to the exploration of its anti-allergic effects. None of the patients treated with AZD8848 discontinued the study prematurely due to drug-related AE. Furthermore, standard laboratory indices  were unaffected by the treatment, except for the anticipated transient reductions in blood lymphocyte counts. Moreover, vital signs  and continuous ECG were unaffected. However, dose-dependent local side effects were common, albeit of mild intensity. These were dominated by blood-admixed nasal secretions and in these cases nasal inspection revealed superficial mucosal irritations/ulcerations. This effect, and the temporary flu-like symptoms that were experienced by a third of the patients, needs to be further evaluated in order to assess overall tolerability of intranasal AZD8848 as a potential treatment.et al. [The body of knowledge on TLRs is increasing as their distribution and functions are outlined, along with potential associations with specific allergic and airway conditions ,31 and tet al. , which fet al. . Taken tThe natural ligand for TLR7 is single stranded RNA. Accordingly, various respiratory viruses, e.g. influenza, corona, and potentially rhinovirus , may actWe conclude that repeated intranasal stimulation of TLR7 by AZD8848 has a potential to affect the immune system in a way that may result in a sustained reduction in the responsiveness to allergen in allergic rhinitis.The work described in this manuscript was supported and funded through a collaboration with AstraZeneca and Dainippon Sumitomo Pharma. In the past five years, LG and MA have received project related financial support from AstraZeneca, Schering-Plough, Orexo, HealthCap (Biolipox/Orexo/LTB4 Sweden/CC10 Sweden), Bioglan, and Nares. LG and MA are shareholders in Nares . In the past five years, AC has received financial support from AstraZeneca and Mediplast. In the past five years, EH has received financial support from AstraZeneca, Grünenthal, and NovoNordisk. GA and AK are employed by AstraZeneca. LE and PN were employed by AstraZeneca when the study was conducted. CAE, JD, ILS, and HW declare no competing interests.Conception, design and management: LG, GA, AC, LE, EH, PN. Patient examination: LG, CAE, MA, AC, JD, EH, PN, ILS, HW. Data input, drafting, revision and/or approval of manuscript: All. Statistics: LG, AC, AK. All authors read and approved the manuscript.


In the recent years, the use of Doppler-echocardiography has become a standard non-invasive technique in the analysis of cardiac malformations in genetically modified mice. Therefore, normal values have to be established for the most commonly used inbred strains in whose genetic background those mutations are generated. Here we provide reference values for transthoracic echocardiography measurements in juvenile (3 weeks) and adult (8 weeks) 129/Sv mice.Echocardiographic measurements were performed using B-mode, M-mode and Doppler-mode in 15 juvenile (3 weeks) and 15 adult (8 weeks) mice, during isoflurane anesthesia. M-mode measurements variability of left ventricle (LV) was determined.ρ < 0.001. CO was 12.06 ± 4.05 ml/min in juveniles vs 29.71 ± 10.13 ml/min in adults, ρ < 0.001. No difference was found in mitral valve (MV) and tricuspid valve (TV) related parameters between juvenile and adult mice. It was demonstrated that variability of M-mode measurements of LV is minimal.Several echocardiographic measurements significantly differ between juvenile and adult mice. Most of these measurements are related with cardiac dimensions. All B-mode measurements were different between juveniles and adults (higher in the adults), except for fractional area change (FAC). Ejection fraction (EF) and fractional shortening (FS), calculated from M-mode parameters, do not differ between juvenile and adult mice. Stroke volume (SV) and cardiac output (CO) were significantly different between juvenile and adult mice. SV was 31.93 ± 8.67 μl in juveniles vs 70.61 ± 24.66 μl in adults, This study suggests that differences in cardiac dimensions, as wells as in pulmonary and aorta outflow parameters, were found between juvenile and adult mice. However, mitral and tricuspid inflow parameters seem to be similar between 3 weeks and 8 weeks mice. The reference values established in this study would contribute as a basis to future studies in post-natal cardiovascular development and diagnosing cardiovascular disorders in genetically modified mouse mutant lines. It was demonstrated by others that echocardiography is becoming a useful technique for studying cardiovascular development and diagnosing cardiovascular disorders in small animals [Cardiac ultrasound, also known as echocardiography, is one of the most commonly used diagnostic techniques in human cardiology for possible pathology or lesion. This technique uses high frequency ultrasound waves for visualizing the heart and can provide information on the heart anatomy, blood flow pattern and function of heart muscle, vessels and valves. Until recently, echocardiographic application in animals was limited primarily to larger, non-rodent species. Due to advances in ultrasound imaging technology, ultrasound systems have now the spatial and temporal resolution to obtain accurate and reliable images of mouse hearts . As a re animals .Knowledge of findings in healthy animals is important for interpretation of results in genetically modified and surgical animals. Most of the available mouse ES cell lines have been generated using the 129/Sv strain. Therefore, the purpose of this study is to provide reference values for transthoracic echocardiography measurements and calculated parameters using B-mode, M-mode and Doppler-mode in juvenile (3 weeks) and adult (8 weeks) 129/Sv mice, obtained during isoflurane anesthesia.All animal work performed in this study was conducted compliant with the Portuguese law and approved by the Consultive Commission of the Veterinary Agency (Portuguese Ministry of Agriculture), the sole Agency/Committee in Portugal responsible to issue the ethical approval for these type of studies, following the EU guidelines for animal research and welfare.30 wild type male 129/Sv mice (Harlan Laboratories) divided by two groups, 15 juvenile mice (3 weeks) and 15 adult mice (8 weeks), were studied. The mice were housed in our animal facility in a controlled environment, at 22°C with artificial 12 hours of light/dark cycle, standard diet and free access to water were supplied. The body weight (BW) of each mouse was recorded prior to cardiac examination.2 to maintain a light sedation level throughout the procedure. They were immobilized on a heating platform ventral side up to maintain the body temperature at 37°C ± 0.5°C. Heart rate (HR) and respiratory physiology were continuously monitored by ECG electrodes. Mice chests were shaved and warmed ultrasound gel was applied to the area of interest. Transthoracic echocardiography was performed using a Vevo 2100 system  with a 40-MHz transducer. Care was taken to avoid excessive pressure over the sternum, which can distort the signal. Images were captured on cine loops at the time of the study and afterward measurements were done off-line.Mice were continuously anesthetized by 1.5-2% of isoflurane inhalant mixed with 1 L/min 100% OThe heart was first imaged in B-mode in the parasternal long axis view to examine the left ventricle (LV). The measurements included LV endocardial and epicardial length  in diastole and systole. LV lengths were measured from the aortic annulus to the apex level. For long axis view in B-mode, image depth was 11 mm and image width was 12.08 mm. Moreover, parasternal short axis view was obtained at the level of papillary muscles to measure LV endocardial and epicardial area  in diastole and systole. These measurements were obtained by tracing the endocardial and epicardial border of the LV, where the papillary muscles were excluded from the endocardial tracings. In order to estimate LV mass (LVM) and LV volume (LVV), the area-length method was used ,4. LVM wIn order to acquire accurate measurements of cardiac dimensions, M-mode images were obtained from long axis and short axis B-mode images by placing the M-mode sample gate perpendicular to the interventricular septum (IVS) and LV walls, respectively, at the level of papillary muscles. M-mode sample gate depth, length and angle for long axis view were 8–11 mm, 4.6-7.6 mm and 0 dg, respectively. For short axis view, M-mode sample gate depth, length and angle were 8–10 mm, 4.6-7.6 mm and 0 dg, respectively. M-mode from long axis view was performed to measure IVS thickness, while M-mode from short axis view was performed to measure thickness of LV anterior wall (LVAW), LV posterior wall (LVPW) and LV internal diameter (LVID). All M-mode measurements were performed in end-diastole (−d) and end-systole (−s) according to the leading-edge method of the American Society of Echocardiography . End-diaBlood flow was assessed using PW Doppler-mode, by positioning the Doppler sample volume parallel to flow direction, which was assisted by Color Doppler-mode. From a modified short axis view of the pulmonary valve (PV) we measured pulmonary artery diameter (PAD) and PV peak velocity (PVPV). PV peak pressure gradient (PVPPG) was calculated. For pulmonary artery view in B-mode, image depth was 10–11 mm and image width was 9.08 mm. For Doppler mode of pulmonary artery view, Doppler sample volume depth, size and angle were 5–8 mm, 0.22 mm and 5–30 dg, respectively. Ascending aorta valve (AoV) flow was obtained from a suprasternal view to measure AoV peak velocity (AoVPV). The aortic arch view was performed to measure the ascending aorta diameter (AoD) and the descending aorta peak velocity (DAoPV). AoV peak pressure gradient (AoVPPG) was calculated. All arterial diameters were measured in systole, at the time of maximal artery diameter. For aorta view in B-mode, image depth was 8–12 mm and image width was 6–11 mm. Doppler sample volume depth, size and angle for ascending aorta were 6–10 mm, 0.27 mm and 20–55 dg, respectively. For descending aorta, Doppler sample volume depth, size and angle were 6–9 mm, 0.27 mm and 5–30 dg, respectively. Stroke volume (SV) and cardiac output (CO) were calculated using aortic outflow as previously described by others [Mitral valve (MV) and tricuspid valve (TV) inflow were assessed from the apical 4 chamber view. The MV measurements performed were the following: MV early wave peak (MVE), MV atrial wave peak (MVA), no flow time (NFT), aortic ejection time (AET), isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT) and MV ejection time (MVET). The MV peak pressure gradient (MVPPG), LV myocardial performance index (LVMPI) and MVE/A ratio were calculated. For mitral valve view, Doppler sample volume depth, size and angle were 7–11 mm, 0.22 mm and 5–30 dg, respectively. TV measurements included TV early wave peak (TVE) and TV atrial wave peak (TVA). The TV peak pressure gradient (TVPPG) and TVE/A ratio were calculated. For tricuspid valve view, Doppler sample volume depth, size and angle were 6–12 mm, 0.29 mm and 5–50 dg, respectively.All measurements were performed excluding the respiration peaks and obtained in triplicate; the mean or median value was used for data analysis. All calculated parameters were automatically computed by the Vevo 2100 standard measurement package. The equations used by the system are shown in detail . Shapiro-Wilk test was used to assess normality of data. The following parameters were not normally distributed: AoVPV, DAoPV, AoVPPG and RVIDs from juvenile data and LVEndoAs, LVEndoLd and LVEpiLd from adult data. Data are presented as mean ± standard deviation or median with interquartile range (IQR), whenever appropriate. To compare results between the two groups, juvenile and adult mice, Student’s unpaired Satisfactory images could be captured in all animals. Cardiac examination time was around 30 min, in all animals. All echocardiographic measurements performed in juvenile and adult mice were summarized Tables , 2 and 3The RV could only be assessed in 7 juvenile and in 11 adult mice. The TV inflow could only be assessed in 12 juvenile and in 6 adult mice.p < 0.001). In the same way, HR was significantly lower in the juvenile (387.34 ± 44.12 bpm) as compared to adult mice .BW was significantly lower in the juvenile (7.68 ± 1.33 g) as compared to adult mice . In 3 weeks mice, NFT had a moderately strong negative correlation with HR (p < 0.01)  . However, Bland-Altman analysis showed a good agreement of both measurements  and adult (8 weeks) 129/Sv wild-type mice has not been reported. In the present study, cardiac dimensions were significantly different between juvenile and adult mice, as expected. Diastolic function does not differ between juvenile and adult mice. Additionally, we demonstrate that the variability of LV measurements in M-mode is minimal, indicating that this method is reliable.The BW was significantly lower in the juvenile than in the adult mice, as expected. Similar results were observed in other mice strains, as C57BL/6 and CD1 mice ,10. NeveWe observed a significant difference in LV lengths and LV areas in 3 weeks mice vs 8 weeks mice, which could be explained by the significantly higher BW of adult mice, due to the continuous increase of the heart and body weight between these ages.A higher value of EAC was observed in the adult mice, but we couldn’t find any data to correlate with our result. While FAC, a parameter that represents LV systolic function, does not differ between juvenile and adult. According to previous studies in C57BL6 mice, FAC does not differ between these ages, which correlate with our result. However the value found is lower than in our study, around 46% . TogetheAs expected, LVV and LVM were different between 3 weeks mice and 8 weeks mice, since the animals are still in growth. However, when LVM was normalized to BW (LVMi), we noticed that LVMi was higher in the juveniles due to the higher BW of the adult mice. These results are consistent with the literature found for other strains ,12.We observed no significant difference in thickness of IVS and LVPW between juvenile and adult mice. A similar result was obtained in a previous study with other mice strain between 6 and 12 weeks old . LVAW thOur data suggests that EF and FS, parameters of systolic function, does not differ between 3 weeks and 8 weeks mice, which match with previous results found for other mice strains ,8,9. TheConcerning the aorta and pulmonary artery, their respective diameters, peak velocities and peak pressures were different between juvenile and adult mice. The higher arterial diameters in the adults, due to higher body surface in older animals, go along with the literature found . AET wasSV and CO, calculated by the aortic outflow method, were significantly higher in 8 weeks mice. On the other hand, COi was considered the same in the two groups due to BW normalization of CO resulted from higher BW in the adult mice compared to lower BW in the juvenile mice. Parameters that are constant when indexed to BW indicate that these values are related to body size. Our CO result is inconsistent with the data found for other strain , which sWe did not find any significant difference in MV and TV inflow parameters between the two groups, except for IVRT that was significantly lower in the 8 weeks mice. E wave and A wave are diastolic parameters that depend mainly on myocardial relaxation, LV geometry and loading conditions. Therefore during maturation of the LV, from juvenile to adult, these parameters evolve in parallel, keeping the resulting E and A wave constant. IVRT is dependent of LV relaxation, loading conditions and HR. Bearing in mind the constant values of A and E wave, IVRT changes could be dependent on intrinsic myocardial relaxation. A similar result was obtained for MVE ,8, MVA , MVPPG , IVRT 8,8 for otOne limitation of this study is the influence of anesthetic agent as depressor of cardiovascular function. However the use of anesthesia during echocardiography is crucial to facilitate data acquisition, by providing sedation and immobility of animals, avoiding the animal stress during the procedure. We used the isoflurane, since it’s one of the most common inhalant anesthetics, and has many advantages when compared with other anesthetics. It produces minimal cardiac depression and has higher molecular stability . Also, iIt would have been advantageous to consider more than two time points, in order to analyze data during the entire life span of these animals.Also, it would be interesting to analyze, in the future, global and regional strain data for deformations reference values.Since mice are a common model system for studying cardiac development and disease, cardiac characterization of normal/wild type and healthy animals is considered extremely important for interpretation of results in transgenic and surgical animals. And so, this study defines reference echocardiographic measurements and calculated parameters for juvenile (3 weeks) and adult (8 weeks) 129/Sv wild type mice.The work presented here suggests a significant difference of almost all cardiac dimensions as well as outflow-related parameters (pulmonary and aorta flow) of 3 weeks vs 8 weeks 129/Sv wild type mice. Interestingly, inflow-related parameters  do not differ between weeks 3 and 8. In addition, when comparing with the literature, we can detect some differences in the parameters studied, suggesting the great influence of genetic background of each mice strain on the results obtained.The reference values reported in this study could contribute as a basis to further comparative results between strains and to future studies in cardiovascular postnatal development and diagnosing cardiovascular disorders using murine models in the 129Sv genetic background.The authors declare that they have no competing interests.MV performed the image acquisitions and measurements, performed statistical analysis, analyzed and interpreted the ultrasound data and wrote the manuscript. ACA performed the measurements for the inter-observer variability, participated in the interpretation of the results and contributed to the writing of the manuscript. SR participated in the interpretation of the results and contributed to the writing of the manuscript. LBR participated in the interpretation of the results and contributed to the writing of the manuscript. JAB designed the study and wrote the manuscript. All authors have read and approved the final manuscript.Equations automatically computed by the Vevo 2100 system. Table showing equations automatically computed by the Vevo 2100 system.Click here for fileCorrelation analysis between BW or HR and all echocardiographic parameters measured in juvenile mice. Table showing correlation analysis between BW or HR and all echocardiographic parameters measured in juvenile mice.Click here for fileCorrelation analysis between BW or HR and all echocardiographic parameters measured in adult mice. Table showing correlation analysis between BW or HR and all echocardiographic parameters measured in adult mice.Click here for file


Neoadjuvant endocrine therapy with an aromatase inhibitor has shown efficacy comparable to that of neoadjuvant chemotherapy in patients with postmenopausal breast cancer. Preclinical and clinical studies have shown that the antidiabetic drug metformin has anti-tumor activity. This prospective, multicenter, phase II randomized, placebo controlled trial was designed to evaluate the direct anti-tumor effect of metformin in non-diabetic postmenopausal women with estrogen-receptor (ER) positive breast cancer.Patients meeting the inclusion criteria and providing written informed consent will be randomized to 24 weeks of neoadjuvant treatment with letrozole (2.5 mg/day) and either metformin (2000 mg/day) or placebo. Target accrual number is 104 patients per arm. The primary endpoint will be clinical response rate, as measured by calipers. Secondary endpoints include pathologic complete response rate, breast conserving rate, change in Ki67 expression, breast density change, and toxicity profile. Molecular assays will be performed using samples obtained before treatment, at week 4, and postoperatively.This study will provide direct evidence of the anti-tumor effect of metformin in non-diabetic, postmenopausal patients with ER-positive breast cancer.NCT01589367ClinicalTrials.gov Identifier  Metformin, which is commonly used to treat type 2 diabetes, is a relatively safe drug with known pharmacokinetics and manageable toxicities. In addition, numerous experimental, epidemiologic, observational, and clinical studies have shown that metformin has anti-tumor effects . For exaQuestions remain, however, about the clinical benefits of metformin as an anti-cancer agent in patients with breast cancer. Although one large-scale, phase III trial of adjuvant metformin has been initiated in women with breast cancer (NCIC CTG MA.32) , the accThe goal of this study was to evaluate the benefits of combining metformin and letrozole in the neoadjuvant treatment of postmenopausal women with ER-positive breast cancer. The primary end point is the rate of tumor response  at 24 weeks, determined by measurements with calipers; changes in size on ultrasound, mammography, and MRI will be used as secondary efficacy assessments. Clinical response includes complete response (CR) and partial response (PR) by RECIST v1.1 criteria.METEOR is a phase II, prospective, randomized, double-blinded, placebo-controlled multicenter clinical trial. Twenty-one centers belonging to the Korean Breast Cancer Society Study Group are participating in this study (KBCSG-013). Patients will be randomly assigned (1:1) to receive letrozole 2.5 mg/day plus either metformin or placebo for 24 weeks before surgery. The initial dose of metformin will be 1000 mg/day for the first week, followed by 1500 mg/day for the second week, and 2000 mg/day from the third week onward. Patients will be randomized sequentially, stratified by center, with randomization codes. Block randomization will be used (SAS 9.2) in cooperation with the Medical Research Collaborating Center (MRCC) of Seoul National University Hospital. All study personnel will be masked to treatment. This study protocol has been approved by the Korea Food and Drug Administration (KFDA) as well as the institutional review board of each center, and was registered at clinicaltrials.gov (NCT 01589367). Written informed consent was obtained from all participants.Eligible patients are postmenopausal women with histologically confirmed ER-positive, stage II or III, primary breast cancer with palpable and clinically measureable tumors. The target population is 208 women, 104 in each arm. Sample size was calculated based on expected clinical response rates (cRR) of 55% in the letrozole plus placebo arm and 70% in the letrozole plus metformin arm, with α = 0.10 and 80% power and including an estimated 10% drop out rate. ER positivity is defined as ≥10% nuclear staining by immunohistochemistry or Allred score ≥3. Criteria for determining menopause include bilateral oophorectomy, age ≥60 years, or age <60 years and amenorrhea for ≥12 months and FSH >30 mIU/ml. Patients with diabetes (HbA1c ≥6.5% or fasting plasma glucose (FPG) ≥126 mg/dL (≥7 mM)); clinical T4, N3, or M1 disease; bilateral cancer, or inflammatory breast cancer will be excluded, as will patients with a history of lactic acidosis or at high risk of having metformin-induced lactic acidosis, such as those with high alcohol consumption or NYHA class III/lV congestive heart failure.Patients will be randomly assigned 1:1) to 24 weeks of neoadjuvant treatment with metformin plus letrozole or placebo plus letrozole  will be discontinued from the study and scheduled for immediate surgery. Patients with stable disease (SD) will be continued on treatment. Ultrasound-guided core needle biopsy samples will be obtained at the first visit and after four weeks of medication, with Ki67 levels in these samples centrally assessed. It is also recommended that 4-week blood samples be obtained to measure serum biomarker concentrations. Surgery will be scheduled within 2 weeks after completing the 24 weeks of medication. Tissue from surgical specimens will be collected for further planned assessments of biomarkers by paired analysis with the pretreatment and week 4 core needle biopsy specimens. HbA1c, FPG, insulin, c-peptide, and IGF-1 concentrations will be measured at baseline and after 12 and 24 weeks of treatment. Mammography, ultrasonography, and bilateral breast MRI will be performed before starting medication and after completion of treatment just before surgery.http://ctep.cancer.gov/reporting/ctc.html). All parameters will be collected and managed using an e-clinical trial platform (MEBICA™).The primary endpoint is cRR. Tumor response will be assessed by RECIST criteria v1.1. Tumor size will be measured by individual clinicians at monthly visits. The secondary endpoint is pathologic complete response (pCR), defined as the absence of invasive cancer at the primary site and in the axilla. Before treatment, each patients will be categorized by surgeons into one of three groups: 1) marginal for breast conservation, 2) candidate for mastectomy only, and 3) inoperable by standard mastectomy . BaselinIn addition to assaying Ki-67, phosphorylated S6 kinase 1 (p-S6K1) will be analyzed by immunohistochemistry (IHC) in a central laboratory. Fresh frozen tissue of week 4 biopsies and final surgical specimens will be collected from several major hospitals participating in this study. These samples will be used for DNA microarray analysis or whole transcriptome sequencing using next generation sequencing (NGS) technology. Phosphorylation of AMPK(T172), expression of IR(insulin receptor) and OCT1(Organic cation transporter) will be measured. Assessment of apoptosis will be done by Miller-Payne Grading system along with various commercialized kits.We previously reported that breast density reduction after short term adjuvant endocrine therapy was predictive of recurrence-free survival . We inteThe maximum effective dose of metformin to treat hyperglycemia in patients with type 2 diabetes is 1000 mg twice daily. However, the metformin dose that yields the maximal anti-tumor effect is unclear. Results from two xenograft models reported that the human equivalent of 1500–2250 mg/day was needed to inhibit tumorigenesis -23. A prThis study was designed to evaluate the direct anti-tumor effects of metformin in human breast cancer cancers. We expect that metformin will show a synergy with neoadjuvant letrozole in ER-positive breast cancer patients similar to that of the mTOR inhibitor, everolimus. Parallel translational research may provide a better understanding of the mechanism of action of metformin in cancer and may reveal biomarkers predictive of response to metformin.ER: Estrogen receptor; AMPK: Adenosine monophosphate kinase; pCR: Pathologic complete response; mTOR: mammalian target of rapamycin; IGF: Insulin-like growth factor; CR: Complete response; PR: Partial response; RECIST: Response evaluation criteria in solid tumors; cRR: clinical response rate; PD: Progressive disease; SD: Stable disease; MRI: Magnetic resonance image; p-S6K1: phosphorylated S6 kinase 1; NGS: Next generation sequencing.All authors declare that they have no competing interests.JK and WH drafted the manuscript and wrote the original protocol for the study. All authors participated in the design of the study. JK filed for ethical approvals from the KFDA and registered the trial on clinicaltrials.gov. WL performed the statistical analysis. EK designed the molecular study. MK was involved in the pharmaceutical process of SNUH and the process of patient enrollment. DN directed the entire process. All authors read and approved the final manuscript and are proceeding with the study in their respective centers.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/170/prepub


Increased arterial stiffness is a cardiovascular outcome of metabolic syndrome (MetS). The chromosome 9p21 locus has been identified as a major locus for risk of coronary artery disease (CAD). The single nucleotide polymorphism (SNP), rs1333049 on chromosome 9p21.3 has been strongly associated with CAD and myocardial infarction. Increased arterial stiffness could be the link between the 9p21 polymorphism and increased cardiovascular risk. Since the impact of a genetic polymorphism on arterial stiffness especially in Asian populations has not been well defined, we aimed to investigate the association of arterial stiffness with rs 1333049 variant on chromosome 9p21.3 in Thai subjects with and without MetS risk factors.A total of 208 Thai subjects, aged 35–75 years, 135 with and 73 without MetS, according to IDF and NCEP-ATPIII criteria, were included in this study. Aortic-femoral pulse wave velocity (afPWV), brachial-ankle pulse wave velocity (baPWV) and aortic ankle pulse wave velocity (aaPWV) were measured and used as markers of arterial stiffness. The chromosome 9p21.3 locus, represented by the rs 1333049 variant and blood biochemistry were evaluated.Arterial stiffness was elevated in subjects with MetS when compared with nonMetS subjects. PWV, especially afPWV increased progressively with increasing number of MetS risk factors . We also found that the frequency distribution of the rs1333049 genotypes is significantly associated with the afPWV (P <0.05). In multivariate analyses, there was an association between homozygous C allele and afPWV (Odds ratio (OR), 8.16; 95% confidence interval (CI), 1.91 to 34.90; P = 0.005), while the GC genotype was not related to afPWV  when compared with the GG genotype.Our findings demonstrate for the first time that arterial stiffness is associated with genetic polymorphism in 9p21 and metabolic risk factors in a Thai population. Metabolic syndrome (MetS) is defined as a clustering of metabolic abnormalities including central obesity, hyperglycaemia, dyslipidaemia, hypertriglyceridaemia, and elevated blood pressure . MetS isIncreased arterial stiffness, a pathological condition associated with vascular damage and or remodelling, is a cardiovascular outcome of MetS -14. It hA total of 208 subjects, 117 men and 91 women, aged 35–75 years, were recruited from the Outpatient Clinic of Queen Sirikit Heart Center of Northeast Thailand, Faculty of Medicine, Khon Kaen University, Khon Kaen Province, Thailand. MetS was defined using a combination of the International Diabetes Federation (IDF) and National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATPIII) criteria, with modification of waist circumference (WC) for Asians ,20. ThisInformed consent was obtained from each participant. The study protocol was approved by the Ethics Committee for Human Research of Khon Kaen University (HE510414) and the study was conducted in accordance with the Declaration of Helsinki.2. WC was measured midway between the inferior margin of the last rib and the iliac crest. Blood pressure was measured in the left arm of supine subjects after 15 minutes of rest, using a Dinamap Carescape v100 .After 12-hour overnight fasting, blood was drawn from an antecubital vein in the morning, and sent for analysis within 3 hours. Biochemical markers in serum such as fasting glucose, lipid profiles, and uric acid were analysed by a Hitachi 917 automatic analyser  at the Clinical Laboratory Unit of Queen Sirikit Heart Center of Northeastern Thailand. High sensitivity C-reactive protein (hsCRP) concentration was determined by means of high sensitivity particle enhanced immunonephelometry using the BN ProSpec® System . Fasting serum insulin was determined by a sandwich immunoassay with commercial kits . Insulin resistance was estimated using the homeostasis model assessment of insulin resistance (HOMA-IR) index, calculated from the following expression: HOMA-IR = fasting insulin (μU/mL) x fasting plasma glucose (mmol/L)/22.5. Body mass index (BMI) was calculated as weight (kg) divided by height (m)After blood pressure measurement, PWV was measured according to a previously described method  using a The genotyping was performed in 171 subjects and these were taken as a representative sample of the entire study population. DNA was extracted from EDTA treated blood samples using a Genomic DNA mini kit  according to the manufacturer’s protocol. Genotyping for rs1333049 was performed using the TaqMan SNP allelic discrimination genotyping assay . A total of 20 ng purified DNA was amplified in a final volume of 20 μL per well in 96 well plates. The PCR conditions were; initial denaturation at 95°C for 10 min, followed by 40 cycles at 92°C for 15 sec and 60°C for 1 min. Post PCR allelic discrimination was carried out by measuring allele-specific fluorescence on an ABI prism® 7500 Sequence Detection System (Applied Biosystems) using the Sequence Detection System software version 1.1. Genotyping was successful in 96% of rs1333049 polymorphism.t-test or the Mann–Whitney U test as appropriate. Pearson correlation analysis was used to assess relationships between PWV and anthropometric variables, and also between PWV and blood biochemistry. Comparisons among three or more groups were evaluated by one way analysis of variance (ANOVA) followed by Newman-Keuls post-hoc analysis. Multiple logistic regression analysis was employed to identify the risk factors associated with afPWV. Statistical analyses were performed with Stata version 10 . A value of P <0.05 was considered as significant.Statistical comparison between MetS and nonMetS groups was assessed by Student’s The baseline characteristics of subjects are shown in Table PWV values of each arterial segment  were significantly higher in MetS than nonMetS subjects Table . PWV at The polymorphism of rs1333049 G>C has been reported to be associated with increased risk of CVD. We wished to determine whether the SNP could affect arterial elasticity and thus subsequently increase the likelihood of symptomatic CVD. The SNP rs1333049 G>C was analysed in 171 subjects (114 MetS and 57 nonMetS). The frequencies of the rs1333049 “G” and “C” alleles in all subjects were 66.5% and 33.5%, respectively. In MetS subjects, the frequencies of the “G” and “C” alleles were 68.5% and 31.4%, respectively and were not significantly different from nonMetS subjects. In all subjects taken together, the frequency of the genotypes GG, GC and CC were 46%, 41% and 13%, respectively, the distribution being consistent with the Hardy-Weinberg equilibrium, with Chi-squared = 0.769, P = 0.681.The influence of rs1333049 polymorphism on arterial elasticity was analyzed. There was an association of rs1333049 genotypes with afPWV, but not with baPWV or aaPWV. The effect of this was further analysed by assigning subjects to a ‘low’ group if their PWV was less than or equal to 11.0 m/s (based on the mean afPWV of nonMetS subjects) and to a ‘high’ group, if greater than 11.0 m/s. The genotype of rs1333049 distribution in the low and high PWV subjects is shown in the Table Since the effect of age, gender, risk factors and rs1333049 genotypes on PWV was strongest in the aorto-femoral segment, we subjected these data to a more detailed analysis. In univariate tests, afPWV was significantly associated with age, risk factors and genotypes Table . SimilarThis study demonstrates, for the first time, that SNP rs1333049 on chromosome 9p21.3 is an important determinant for the progression of increasing arterial stiffness, especially that of the aorta, in a Thai population. Increased afPWV and aaPWV are associated with increasing number of MetS risk factors, indicating that a clustering of MetS risk may interact synergistically to affect central arterial stiffness. Previous studies have suggested that arterial stiffening in MetS, particularly in diabetic individuals, is associated with elastic lamellae fragmentation and calcification, increased collagen cross-linking, low-grade inflammation, increased smooth muscle basal tone and endothelial dysfunction -25. Theset al. set a cut-off value of cfPWV at ≥ 9 m/s to diagnose arteriosclerosis in patients with essential hypertension [A previous study has reported that central arterial stiffness, as measured by heart-femoral PWV (hfPWV) was more closely associated with CAD, CVD and peripheral arterial disease than peripheral arterial stiffness . hfPWV, rtension  and morertension . In the et al. demonstrated that rs 1333049 polymorphism is an independent determinant of coronary plaque progression in a Chinese Han population [In addition to the aforementioned association, we found increased aortic stiffness in subjects homozygous for the C-allele of rs 1333049 on chromosome 9p21.3, after adjusting for age and the number of MetS risk factors. We suggest the SNP rs1333049 might be involved with the progression of raised aortic stiffness in Thais, although the underlying mechanisms by which SNP rs1333049 on chromosome 9p21.3 contributes to this progression remain unclear. Previous studies have suggested that the chromosome 9p21 locus predicts CAD and involves the initiation or facilitation of atherosclerosis ,33. Wangpulation . Moreovepulation . Taken tCDKN2B-AS) is expressed in several cells and tissues, where a number of SNPs located at the 5’ end including rs1333049 have been suggested to be associated with CAD [p16/CDKN2A and p15/CDKN2B) under both physiological and pathological conditions. A SNP rs1333049 was located in a 190 kb region of the high linkage disequilibrium, upstream both of p16/CDKN2A, p15/CDKN2B and p14/ARF that code for p15 and p16 and p14ARF [A large noncoding RNA gene named ANRIL(with CAD . The ANRd p14ARF . These pd p14ARF . SNP rs1d p14ARF ,39 but ad p14ARF , these dWe have observed that the SNP rs1333049 on chromosome 9p21 locus is associated with increased arterial stiffness in a Thai population. This association is strengthened by the presence of MetS risk. Research on this genetic polymorphism in a large population is required to investigate this association in more detail. Moreover, a mechanistic study is also needed to clarify the effect of the chromosome 9p21 locus on arterial stiffening. Since MetS-associated arterial stiffening increases cardiovascular risk, our findings add to the evidence that management of MetS to prevent advanced cardiovascular complications is clinically important.MetS: Metabolic syndrome; SNP: Single nucleotide polymorphism; IDF: International Diabetes Federation; NCEP-ATPIII: National Cholesterol Education Program Adult Treatment Panel III; afPWV: Aortic-femoral pulse wave velocity; baPWV: Brachial-ankle pulse wave velocity; aaPWV: Aortic-ankle pulse wave velocity; HDL-c: High-density lipoprotein cholesterol; LDL-c: Low-density lipoprotein cholesterol; HOMA-IR: Homeostasis model assessment of insulin resistance; BMI: Body mass index; hsCRP: High sensitivity C-reactive protein.The authors declare that they have no competing interests.UK and SEG designed the study, analyzed data, reviewed and edited the manuscript. VK performed statistical analysis and edited the manuscript. SP wrote the manuscript and performed experiments. PP, LS, CS, NK and NS performed some experiments. PT, PI and VS participated in research design and were responsible for subjects’ recruitment and the clinical report. All authors read and approved the final manuscript.


Objectives. We investigated the relationship between the stage of diabetic retinopathy and pulse wave velocity (PWV). Methods. This was a cross-sectional study of 689 patients (406 men and 283 women) with type 2 diabetes who were admitted to our hospital from 2004 to 2007. Brachial-ankle pulse wave velocity (baPWV) was measured by an arterial pressure measurement device as PWV/ABI. Diagnosis of diabetic retinopathy was made by ophthalmologists based on the Davis classification: no diabetic retinopathy (NDR), simple retinopathy (SDR), pre-proliferative retinopathy (pre-PDR), and proliferative retinopathy (PDR). Results. There was a significant difference in PWV between patients without diabetic retinopathy (1657.0 ± 417.9 m/s (mean ± SD)) and with diabetic retinopathy (1847.1 ± 423.9 m/s) (P < 0.001). In addition, the stage of diabetic retinopathy was associated with aortic PWV (1657.0 ± 417.9 m/s in NDR (n = 420), 1819.4 ± 430.3 m/s in SDR (n = 152), 1862.1 ± 394.0 m/s in pre-PDR (n = 54), and 1901.1 ± 433.5 m/s in PDR (n = 63) (P < 0.001)). Conclusions. In patients with diabetic retinopathy, even in those with SDR, PWV was higher than that in patients without diabetic retinopathy. Physicians should therefore pay attention to the value of PWV and macroangiopathy regardless of the stage of diabetic retinopathy. Pulse wave velocity (PWV) has been used as a noninvasive clinical index of aortic stiffness. It is reported that PWV of patients with diabetes is higher than that of healthy subjects . In a JaRetinal capillary microaneurysms are the hallmark of diabetic retinopathy and its earliest reliable sign, and individual acellular capillaries are usually visible histologically in the earliest stages of diabetic retinopathy. As retinopathy becomes more severe, larger patches of acellular capillaries are seen. When lesions like cotton-wool spots, intraretinal microvascular abnormalities, venous beading, and retinal hemorrhages are prominent, diabetic retinopathy is considered pre-proliferative, and new vessels are likely to appear soon on the surface of the retina or optic disc. When new vessels appear on the surface of the retina or optic disc, diabetic retinopathy is said to have entered the proliferative stage .To our knowledge, no study has compared PWV with the stage of diabetic retinopathy. Therefore, we investigated the relationship between increased PWV and the stage of diabetic retinopathy. From January 2004 to December 2007, 732 Japanese patients with type 2 diabetes who were admitted to Keio University Hospital  were consecutively observed. Among them, 43 patients with acute illness  were excluded from the evaluation. Consequently, a total of 689 patients with type 2 diabetes who were admitted due to having poor glycemic control were enrolled in this study. All of their purposes of admission were to control glucose metabolism and education for diabetes. The study protocol was approved by the ethical committee of the hospital. Informed consent was obtained from all patients. The diagnosis of diabetic retinopathy was made by ophthalmologists based on the Davis classification: no diabetic retinopathy (NDR); simple retinopathy (SDR); pre-proliferative retinopathy (pre-PDR); and proliferative retinopathy (PDR). During hospitalization, fasting plasma glucose (FPG), 2-hour plasma glucose (PG), C-peptide (CPR), hemoglobin A1c (HbA1c), glycated albumin (GA), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen (UN), creatinine (Cr), uric acid (UA) in blood, and 24-hour urine microalbumin were measured. HbA1c was determined by high-performance liquid chromatography  and presented as the equivalent National Glycohemoglobin Standardization Program (NGSP) value . FurtherD1 − D2)/T1, where D1 is the distance from the heart to the left ankle, and D2 is the distance from the heart to the right upper arm. These distances were calculated automatically on the basis of the patient's height. The pressure waveforms obtained at two different sites were simultaneously recorded, and the time interval between the initial rise in the brachial and tibial pressure waveforms was determined as T1. ABI was calculated using the formula ABI = ankle systolic BP/brachial systolic BP.Measurements of brachial-ankle PWV (baPWV) were carried out using an automatic waveform analyzer . Patients lay in the supine position during the test, and occlusion and monitoring cuffs were placed around both the upper and lower extremities. PWV was calculated using the formula: baPWV =  to investigate the relationship between each stage and the value of PWV by the Kruskal-Wallis test. The relationship between the PWV and each factor was evaluated with Spearman's correlation coefficient. The selected variables, which were statistically significant and clinically important, were included in nonparametric multiple regression models to evaluate the association between PWV and each stage of diabetic retinopathy adjusted for some covariates. The purpose of these multivariate analyses was to show the robustness of the results from the univariate analysis.Demographic factors and baseline characteristics were summarized by diabetic retinopathy (DR) and NDR groups. They were compared between the DR and NDR groups using Mann-Whitney Data are presented as mean ± standard deviation (SD) in the text and tables. The significance level for all tests was two-sided, at 5%. All analyses were performed using SPSS 17.0  and SAS 9.2 .Demographic factors and clinical baseline characteristics of patients are shown in P < 0.001). Furthermore, there was a significant positive association between the stage of diabetic retinopathy and PWV. PWV was 1657.0 ± 417.9 m/s in NDR (n = 420), 1819.4 ± 430.3 m/s in SDR (n = 152), 1862.1 ± 394.0 m/s in pre-PDR (n = 54), and 1901.1 ± 433.5 m/s in PDR (n = 63) (P < 0.001) (PWV in patients with diabetic retinopathy (1847.1 ± 423.9 m/s) was significantly higher than that in patients without diabetic retinopathy (1657.0 ± 417.9 m/s) (< 0.001) .P < 0.001).Some sensitivity analyses were performed to evaluate robustness of the results from the univariate analysis. Factors significantly correlated with the PWV by means of Spearman's correlation coefficient were BMI, age, SBP, FPG, and HbA1c . HoweverMeasurement of aortic PWV is considered the gold-standard evaluation of arterial stiffness . Values In the present study, PWV was significantly higher in patients with diabetic retinopathy than in those without. This finding supports the report that diabetic retinopathy is the microvascular complication with the strongest association with increased aortic stiffness . In addiIncreased arterial stiffness is thought to be related to not only hyperglycemia but also to carbonyl and oxidative stress, chronic inflammation, endothelial dysfunction, and formation of advanced glycation end products (AGEs) . It is rSeveral limitations should be taken into account when considering the results of this study. First, the cross-sectional study design and small sample size for each stage of diabetic retinopathy in our study make it difficult to infer the association between PWV and retinopathy. Second, we could not consider the effects of prescribed medication, for instance, anti-platelet agents, which could influence the state of both retinopathy and arterial stiffness. Third, the raw data might have deviated slightly because there was more than one PWV technician and ophthalmologist. However, it was thought that the influence of bias was small because the technicians and ophthalmologists were experts and were not aware of this study when they carried out the examinations. Lastly, the patients with type 1 diabetes were not included in this study. In a recent meta-analysis of observational studies, diabetic retinopathy predicted all-cause mortality and cardiovascular events in patients with type 2 diabetes and also type 1 diabetes . Based oIn conclusion, this study suggested that PWV is significantly higher in patients with diabetic retinopathy than in those without, and that there is a relationship between the stage of diabetic retinopathy and PWV in Japanese patients with type 2 diabetes. Physicians should pay attention to latent macroangiopathy in patients with type 2 diabetes who have diabetic retinopathy, even SDR.