Camellia sinensis L.) leaf contains a large amount of catechins (a group of very active flavonoids) which contribute to major quality attributes of black tea. Based on morphological characters tea plants were classified as Assam, China, and Cambod varieties. The present study is an attempt for biochemical fingerprinting of the tea varieties based on catechin composition in green leaf of cultivars grown in Northeast India. Assam variety cultivars contained the highest level of catechins followed by Cambod and China. The average catechin contents were 231 ± 7 mg g−1, 202 ± 5 mg g−1, and 157 ± 4 mg g−1 of dry weight of green leaf for Assam, Cambod, and China cultivars, respectively. Among the individual catechins the variations in epigallocatechin gallate (EGCG) and epigallocatechin (EGC) were the most prominent among the varieties. High EGC content was found to be a characteristic of Assam variety which was further corroborated through multivariate analysis.Tea ( Camellia assamica, a source of a wide range of the catechins. Tea leaves contain about 180–360 mg g−1 of polyphenols, among which 70–80% are flavanols [Present market is a selective one, and producers with high-quality tea are likely to survive. Quality of made tea of the plains of Northeast (NE) India depends on the quality of raw materials determined primarily by the polyphenolic constituents. It is widely accepted that Crush, Tear, and Curl (CTC) black tea quality attributes depend on flavonol composition (more precisely catechins). Epigallocatechin gallate (EGCG) is an important biochemical marker of Northeast Indian tea as it contributes 50% of total catechins . The stalavanols . Total plavanols . Camellia sinensis and belongs to the family Theaceae. Commercial tea cultivars are recognized under three different taxa, namely, C. sinensis, C. assamica, and C. assamica ssp. lasiocalyx [C. sinensis, C. assamica, or C. assamica ssp. lasiocalyx still exist [Taxonomically tea is known as siocalyx . Howeversiocalyx , and allsiocalyx . Hybridill exist . Based on morphological characteristics, tea is grouped into Assam, China, and Cambod varieties . The claTocklai Experimental Station, Jorhat, Assam, has released 153 germplasms to the tea industry of NE India to be grown in plains. Over 60% of 0.3 million ha of tea growing area of NE Indian plains is covered with these tea varieties. Regional variation of quality within the tea growing region  can be aWidespread cultivation of clonal tea for high yield and uniform quality may diminish the genetic diversity. Conservation of germplasm resources is necessary for sustainability of the tea industry. Tocklai Experimental Station has a field gene bank with over 2000 germplasms which is one of the primary centres of dispersal in the world. In order to ascertain diversity careful study of secondary metabolites, especially those which are major contributors to quality, is essential. Total catechin content could be used to indicate the quality potential of tea, with high content being related to high quality . Earlierin vivo oxidative and hydrolytic processes in presence of mild aeration. The desirable colour and briskness of made tea is dependent on the oxidative polymerization of catechins to TFs and thearubigins (TRs) by the enzymes polyphenol oxidase (PPO) and peroxidase (POD) [The oxidative and hydrolytic enzymes endogenous to the shoots are crucial in triggering of various characteristic quality attributes of black tea. Out of the various stages of black tea processing, the fermentation stage is the most crucial. The mechanical maceration of green tea shoots triggers the enzyme catalyzed oxidative reactions involving catechins as substrates. Upon disruption of the intercellular compartments, catechins present in the cell vacuole undergo se (POD) . The present study was undertaken to assess the variation of catechin  concentrations in extreme and cultivated varieties of Assam, China, and Cambod. The study also took into account the relative expression of individual catechins in cultivars grown in Northeast India. An understanding of catechin profile in different cultivars of tea may provide useful information on plant diversity as well as understanding their role as precursor of quality since type and quantity of catechin significantly influence the formation of two important quality attributes of tea such as theaflavins and thearubigins. This may also support future selection process for improvement of crop quality. −1, 110 kg K ha−1 as K2O and 30 kg P ha−1 as P2O5 was applied per year, pruning and plucking were maintained) where shade applied over the tea bushes contributed to 30–40% light interception. The reference samples represented pure varieties, namely, Assam (Betjan), China , and Cambod (extreme Cambod). Except reference samples, other cultivars representing the three varieties were as follows.Tea shoots comprising apical bud and subtending two leaves were harvested from the experimental garden of Tocklai Experimental Station, Tea Research Association, Jorhat, Assam, India (94°12′E and 26°47′N). A regular 7-day plucking during tea harvesting period was maintained. 7-day plucking interval is a common agricultural practice in tea growing areas of the NE India as it makes the young shoots produce high quality tea . All the3A1, S3A3, Tingamira, TA 17, and T3E3 (where TV stands for Tocklai Vegetative and TA stands for Tin Ali).TV2, TV12, TV13, TV17, TV21, STV7, 14/13/3, 14/100/10, 14/100/16, 14/100/6, 317/1, 317/2, 317/3, 317/4, and P126 (P stands for Panitola).TV9, TV18, TV22, TV23, TV25, TV26, and TV30.−1, total nitrogen: 0.8 ± 0.0001 mg g−1, available P2O5: 0.01 ± 0.001 mg g−1, and available K2O: 0.08 ± 0.0001 mg g−1.The entire harvesting period for the sampling was from March to November for the years 2009 and 2010. Leaf samples were collected from the plots receiving similar agronomical practices. Samples were analyzed fortnightly. Soil samples collected from experimental plots were analyzed following the standard procedure described by Jackson . Averageμg mL−1), ascorbic acid (500 μg mL−1), and acetonitrile (25% v/v) in water. Catechins were quantitatively estimated using waters high-performance liquid chromatography (HPLC) system with Luna 5 μ phenylhexyl phenomenax column (4.5 mm × 250 mm) and UV-Vis detector  set at 278 nm according to the method of International Standard Organisation [μL of the diluted extract was injected into the column through Rheodyne injector. In brief, the elution made was initial 10 min with 100% mobile phase A followed by over 15 min with a linear gradient to 68% mobile phase A and 32% mobile phase B and held at this composition for another 10 min with flow rate 1 mL per min. The mobile phase A consists of 2% acetic acid and 9% acetonitrile and mobile phase B 80% acetonitrile. The chromatographic peaks were identified and estimated by external standard method from response factors (concentration of standards/peak area of standards) determined from different catechin standards procured from Sigma Aldrich, USA (ISO-14502 2005). The solvents used for extraction and analyses were of HPLC grade .About 100 g fresh tea leaf of each sample was deactivated and dried (at 90°C and dryness around 95%) in a microwave convection domestic oven . Microwave drying of the samples did not affect catechin composition of green leaf tea samples . The dried samples were ground finely for analysis. 0.2 g of sample was extracted with 5 mL 70% methanol in a water bath at 70°C over 10 min with intermittent shaking in a vortex mixture. The extract was then cooled and centrifuged at 4000 rpm  for 10 min. The supernatant was decanted into a 10 mL volumetric flask. The extraction was repeated twice and volume was made up with the solvent. 1 mL of the extract was diluted to 5 times with stabilizing agent. The stabilizing agent was prepared from EDTA  as follows:The data were also log-transformed so as to more closely correspond to normal distribution. Further, all the variables were standardized by calculating their standard scores  described by Singh et al. [29 and principal component analysis (PCA) described by Kano et al. . All theBiochemical parameters of green leaf influencing black tea quality of the plains of NE India consist of catechins which are converted to TFs and TRs, the critical parameters of quality of CTC tea , 33. NotIt was observed from this study that the total catechin and some individual catechins could be used as markers to differentiate between the three major varieties. The clear differentiation of China variety from Assam and Cambod could be established using the catechin as marker (see below). Similar observations were reported in Japanese tea . Total g−1, 202 ± 4.58 mg g−1, and 157 ± 3.82 mg g−1 for Assam, Cambod and China varieties, respectively. Large variations in individual catechins and total catechins among the varieties were observed. Assam variety cultivars contained the highest catechins followed by Cambod, and China  among the varieties was between 0.3 and 0.7. The highest CATRAT was found in TV7 of China variety.The second largest contributor to total catechin content was EGC for Assam variety while it was ECG for Cambod and China variety. The variation in EGC content was more prominent between the varieties. The average EGC contents were 51.0 ± 1.0 mg gHierarchical agglomerative cluster analysis (HCA) in the form of dendrogram and principal components analysis (PCA) were used to explore structure and relationships in multivariate data , 28. TheFrom HCA, we could not clearly distinguish the relations among the different varieties of tea samples. Therefore, all the parameters were transformed into three main comprehensive matrices referring to PCA technique. On plotting the first two principal components (Varimax 1 and Varimax 2), they showed two clusters for Assam tea, one cluster for Cambod tea, and three clusters for China tea in PCA Figures . PrincipDifferential display of catechins in cultivars forms a basis for future elucidation of catechin metabolism in tea. Profiling of individual and total catechins was found to be a useful technique to determine genetic diversity in tea germplasms. Among the three pure varieties China variety cultivars contained lower catechins. PCA showed different groupings of catechins for Assam, Cambod, and China teas, and such groupings might be used to differentiate between such varieties.


Although T‐helper type 1 (Th1) cells are considered important in atherosclerosis, the relationships between Th1 and Th2 cells and atherosclerosis have not been examined in population‐based studies.+ cells, representing Th1 and Th2 subpopulations (%Th1 and %Th2), respectively. We found that %CD4 was 1.5% lower per 10 years of age (P<0.0001). Whites had higher %CD4 and lower %Th1 and %Th2 values than other race/ethnic groups. Body mass index (BMI) and blood pressure were associated with %CD4, but no traditional cardiovascular disease (CVD) risk factors were associated with %Th1 or %Th2. In multivariable models, the major independent variable associated with %Th1 was cytomegalovirus (CMV) antibody titer, with minor contributions from age, sex, seasonality, and interleukin‐6. In models with coronary artery calcification level as the outcome, significant independent variables included age, sex, smoking status, and %Th1 . Both %Th1 and %Th2 were associated with common carotid intimal media thickness , as were age, sex, race/ethnicity, blood pressure, and BMI.We measured Th cells as a percentage of lymphocytes by flow cytometry using CD4 staining (%CD4) in 917 participants of the Multi‐Ethnic Study of Atherosclerosis. We also measured interferon gamma–positive and interleukin‐4‐positive CD4Th1 bias is associated with subclinical atherosclerosis in a multiethnic population. The main Th1 correlate was CMV infectious burden. These findings are consistent with a role of Th1 cells in atherosclerosis and suggest the importance of prospective studies of T‐helper cell biasing in CVD. In epidemiological research, systemic biomarkers of chronic low‐level inflammation, such as fibrinogen and C‐reactive protein (CRP), are associated with subclinical atherosclerosis and are independently associated with future clinical CVD events.2 As acute‐phase proteins, these biomarkers are generally nonspecific, although each also plays a role in a specific inflammatory pathway—coagulation and innate immunity, respectively.Although the importance of inflammation in atherosclerosis and cardiovascular disease (CVD) is well accepted,4 aspects of the adaptive immune system also play important roles in atherosclerosis. In particular, T‐helper type 1 (Th1) cells provide important stimulation to macrophage and foam cells in mouse models of atherosclerosis via several mechanisms, including production of interferon gamma (IFN‐γ).5 Th1 bias drives increased atherosclerosis in mice,6 whereas deficiency in IFN‐γ or the Th1‐differentiating transcription factor T‐bet results in reduced lesion development.9 In relatively small clinical studies, subjects with coronary syndromes and advanced atherosclerosis were found to have a bias toward Th1.15 Th1 cells are also critically important in chronic inflammatory disease states such as psoriasis, which may confer an independent risk of myocardial infarction (MI).16 To date, information regarding the relationships between Th1 cells and atherosclerosis have been limited to small‐scale clinical studies in individuals with advanced CVD.As recently reviewed by Hansson et al,17 and by the IL‐4‐mediated suppression of proinflammatory cytokine release.18 Accordingly, Th2 cells have been suggested to be antiatherogenic.6 On the other hand, Th2 cells are proinflammatory in conditions such as asthma19 and have also been reported as proatherogenic.21 Collectively, the role of Th2 cells in atherosclerosis remains unclear.Little is known about the role of the other major T‐helper cell, the Th2 cell, which produces, among other cytokines, interleukin‐4 (IL‐4). Th2 cells can function in anti‐inflammatory capacities by inhibiting Th1 developmentLittle information concerning adaptive immunity and T‐helper function is available from population‐based studies of individuals free of CVD. Therefore, we designed and implemented a study in the setting of the Multi‐Ethnic Study of Atherosclerosis (MESA) called MESA‐Inflammation. We report here the associations between Th1 and Th2 cells and measures of subclinical atherosclerosis in otherwise healthy individuals.22 and are available online at http://www.mesa-nhlbi.org. Briefly, participants include whites, African Americans, Hispanic Americans, and Asian Americans recruited at 6 field centers  during the baseline exam (exam 1) in 2000–2002. Exclusion criteria included: clinical CVD defined as physician‐diagnosed MI, angina, stroke, transient ischemic attack, heart failure, or atrial fibrillation; use of nitroglycerin; or a major procedure related to the treatment of CVD. Subsequently, exams 2, 3, 4, and 5 occurred in 2002–2004, 2004–2005, 2005–2007, and 2010–2012.MESA is a longitudinal epidemiological study of 6814 asymptomatic men and women aged 45 to 84; details have been publishedAs part of exam 1, we selected a random subset of ≈1000 participants for specialized biomarker testing. During exam 4 (2005–2007), 716 participants from this subset plus 201 replacements for those who had died or dropped out were selected for MESA‐Inflammation. Samples were collected at all 6 field centers and sent overnight in specialized containers to the Laboratory for Clinical Biochemistry Research (LCBR) at the University of Vermont. Both the parent MESA study and MESA‐Inflammation received institutional review board approval, and informed consent was obtained.23 Hypertension was classified according to JNC VI.24 Data from exam 4 were used except for variables such as specialized biomarkers only measured at baseline, including soluble intracellular adhesion molucule–1 , soluble interleukin‐2 receptor α , and high‐sensitivity C‐reactive protein, interleukin‐6 (IL‐6), and infectious serologies, which were measured as described previously.26Standardized questionnaires were administered at the baseline exam in 2000–2002 and during each subsequent exam for demographics, medical history, medications, and lifestyle characteristics, anthropomorphic variables, diabetes status, smoking status, blood pressure, and so forth. At each exam fasting blood was drawn, and serum and plasma were prepared and shipped to the MESA Core Laboratory at the University of Vermont. Smoking was defined as never, former (no cigarettes within the past 30 days), or current. Impaired fasting glucose and type 2 diabetes were classified by the 2003 American Diabetes Association (ADA) criteria: fasting glucose between 100 and 125 and ≥126 mg/dL, respectively.27 in plasma specimens having a triglyceride value <400 mg/dL.Lipid and glucose measurements from exam 4 were conducted at the Collaborative Studies Clinical Laboratory at the University of Minnesota Medical Center, Fairview . Serum glucose was measured on a Vitro analyzer ; CV 1.1%. Lipid measurements were performed on EDTA plasma samples collected following an overnight fast. Total cholesterol and triglycerides were measured on a Roche Modular P Chemistry Analyzer , with CVs of 1.6% and 4.0%, respectively. HDL‐cholesterol was measured using the cholesterol oxidase method (Roche Diagnostics) following precipitation of non‐HDL‐cholesterol with magnesium/dextran; CV 2.9%. LDL‐cholesterol was calculated using the formula of Friedwald et al28 have reported the details of the MESA computed tomography scanning and interpretation methods.Scanning centers assessed coronary artery calcification (CAC) by chest computed tomography using either a cardiac‐gated electron‐beam computed tomography scanner or a multidetector computed tomography system. Certified technologists scanned all participants twice with phantoms of known physical calcium concentration. A radiologist or cardiologist read all computed tomography scans at a central reading center . Carr et al29 An ultrasound reading center  measured maximal intimal media thickness (IMT) of the internal carotid (IC) and common carotid (CC) sites as the mean of the maximum IMT of the near and far walls of the right and left sides.Trained technicians in each field center performed B‐mode ultrasonography of the right and left near and far walls of the internal carotid and common carotid arteries, using the Logiq 700 ultrasound device .CAC measurements were made on the full cohort in exam 1 and again in exams 2 and 3  and in exam 4 (about one quarter of the cohort). The CAC data from the exam closest in time to exam 4 was used for these analyses . The common and internal carotid IMTs were measured at baseline and used for this study.+ cells in certain local volunteers on overnight incubation led us to examine blood‐draw tube types. Testing cell levels in blood drawn into heparin, EDTA, citrate, and acid‐citrate‐dextrose tubes that were tested immediately or allowed to sit overnight demonstrated that heparin was the best choice for reproducibility, particularly in those volunteers whose CD4+ cell levels appeared to drop overnight when compared with freshly measured cell levels. In addition, we tested different overnight storage temperatures and found that room temperature was most stable, whereas temperature extremes (4°C and 37°C) showed extensive hemolysis in the plasma, indicating cell damage. Testing was performed to confirm that the shipping boxes and ambient shipping packs were stable to temperature extremes as well as potential movement of packages such as seen with overnight shipping. These results were similar to results published by Nicholson et al30 in their study on stability of blood samples from HIV‐positive and ‐negative individuals, where they reported that heparin‐anticoagulated blood was more stable over time than EDTA and that ambient temperatures were most stable for cellular studies.Once cell populations were decided on, samples were tested for overnight stability. A loss in CD4+ cells, 1.28 for Th1 cells, and 0.55 for Th2 cells, and their respective Pearson correlation coefficients were 0.75, 0.95, and 0.74, indicating that results were similar between the 2 samples.Once a method was developed, we measured our cell populations in freshly drawn versus 24‐hour postdraw samples subjected to standard shipping procedures in a group of 13 to 20 local, apparently healthy volunteers . RegressBecause of the large number of samples that are analyzed in population‐based studies, we examined the ability to fix the cells in paraformaldehyde for extended periods and found that samples could be fixed, stored for at least a week, and analyzed by flow cytometry in batches.7 cells/mL.Heparinized blood  was drawn at the MESA field centers, packaged in Saf‐T‐Pak Styrofoam shipping containers  with 4 gel packs that maintained a temperature of 15°C to 30°C, and shipped via overnight courier to the LCBR at the University of Vermont. Tubes were placed on a shaker for 10 minutes before the start of the protocol. Heparinized blood was centrifuged at 1500 RCF for 15 minutes at room temperature. The cells were resuspended in 35 mL of fully supplemented RPMI‐1640 . Histopaque  was layered under the cell suspension and then centrifuged as above. The peripheral blood mononuclear cell (PBMC) layer was removed and washed with fsRPMI, and the cell pellet was resuspended in fsRPMI. Cells were counted using a Beckman Coulter Z‐2 Counter  and adjusted to a concentration of 1x102. Following stimulation, PBMCs were washed with phosphate‐buffered saline (PBS) containing 1% bovine serum albumin (BSA) and 10 μg/mL Brefeldin A. As negative controls, unstimulated PBMCs that underwent the same protocol without the addition of activators showed no cytokine staining.All reagents and antibodies were from Sigma‐Aldrich  unless otherwise noted. Isolated PBMCs were stimulated with 40 ng/mL phorbol 12‐myristate 13‐acetate (PMA), 1 μg/mL ionomycin, and 10 μg/mL Brefeldin A for 3 hours at 37°C in a humidified atmosphere containing 5% CO+) cells or with PE‐Cy5‐conjugated mouse IgG1 (BD Biosciences) as an isotype control. After washing with PBS/1% BSA/Brefeldin A, cells were fixed using 2% paraformaldehyde  for 15 minutes. Cells were washed and then permeabilized using 0.1% saponin in PBS/1% BSA. Cells were then washed and incubated with fluorescein isothiocyanate (FITC)–conjugated anti‐IFN‐γ and PE‐conjugated anti‐IL‐4 for 20 minutes in the dark, according to the manufacturers’ instructions. FITC‐conjugated mouse IgG2b κ and PE‐conjugated mouse IgG1 κ were analyzed as isotype‐matched controls. Cells were fixed in 2% paraformaldehyde and were maintained at 4°C protected from light until evaluated.The stimulated PBMCs were incubated with Fc Block (BD Biosciences) to block nonspecific antibody‐binding sites, then labeled with R‐phycoerythrin‐cyanine dye Cy5 (PE‐Cy5)‐conjugated anti‐CD4 (BD Biosciences) to surface‐label the CD4‐positive (%CD4+IFN‐γ+) and Th2 cells (CD4+IL‐4+) were expressed as a percentage of CD4+ cells.Cells were analyzed by flow cytometry using an LSR II flow cytometer (BD Biosciences) with a single excitation wavelength (488 nm) and band filters for PE‐Cy5 (670/14 nm), FITC (530/30 nm), and PE (575/26 nm). Single color controls were used to set machine compensation, and background staining was determined using the respective negative isotype control. Data was analyzed using Winlist 6.0 . Lymphocytes (30 000 events) were gated based on their forward and side scatter. T‐helper lymphocytes were gated by positive surface staining for CD4 and were expressed as a percentage of gated lymphocytes; Th1 cells , within subject (CVi), and between subjects (CVg). In cases in which analysis was done only in singletons, no CVa was available. From these data, an index of individuality (II), which is the ratio of the CVi to CVg, was determined. The II is a measure of the ability to detect changes in an individual compared with the total population. In general, assays with an II of <1 have proven useful in population studies. These biovariability data are summarized in 31In an effort to ensure there was no drift in our assay protocol, we enlisted 14 local volunteers who donated samples before the commencement of the study and periodically throughout the study. This also allowed us to assess biovariability. Using nested analysis of variance, as described by Sakkinen et alIn an effort to minimize errors from this protocol, we established a checklist for quality assurance. Using these procedures over a 2‐year period, receiving samples 4 days per week, we lost only 20 overnight samples to late arrival, generally because of weather affecting delivery. A few data points were lost to a backordered reagent, and a corrupt flow‐cytometry data file caused the loss of ≈2% of the data. Together, the assay development and quality assurance plan implemented as part of the MESA‐Inflammation Study allowed us to generate high‐quality cellular data on 917 participants.P>0.05 for elimination). After forcing age, sex, and race/ethnicity into the models, body mass index (BMI), season, cytomegalovirus (CMV) antibody titers, H. pylori index values, hepatitis A index values, HSV antibody titers, C. pneumoniae fluorescence units, CRP, white blood cell count, IL‐6, smoking status , total cholesterol, HDL‐cholesterol, systolic blood pressure, diastolic blood pressure, use of hypertension medication, diabetes status, and education in 3 categories were allowed to enter the model. These same candidate variables were used in analyses of T‐cell indices with subclinical disease. In these analyses only 716 cases were included because CMV antibody titers were only measured in the original sample of 1000 participants.Descriptive displays of means or Pearson correlations were used to describe associations between cell phenotypes and other variables. Linear regression models were constructed with %Th1, %Th2, and %CD4 cell indices as outcomes using backward elimination  were different from those associated with the amount of CAC in those with CAC present, we modeled presence of CAC separately from CAC amount. The former was modeled using relative risk regression and the latter with linear regression. For the linear regression models, robust regression was done to provide protection against the influence of influential outliers, which are potentially present for both CAC and the IMT measures; ln‐transformed Agatston scores were used in these analyses. Relative risk regression was used rather than logistic regression because the parameter of interest is relative risk rather than the odds ratio, and the odds ratio overestimates relative risk when the outcome is common (≈50% of the participants have CAC>0).In the MESA‐Inflammation subgroup, 47% were treated for hypertension, 32% were treated for dyslipidemia, 14% were diabetic, and only 9% were current smokers . The mea34 with highest values in summer and lowest in winter; results were similar for %Th1 but not statistically significant.Unadjusted associations of these phenotypes with CVD risk factors, biomarkers of inflammation, and infectious serologies are shown in We observed significant differences in all 3 cell phenotypes by serologies related to common infections . Higher r2=0.20). No cell phenotype was associated with sIL‐2rα, a purported marker of T‐cell activation,35 but we did observe associations with the soluble form of ICAM‐1, a major endothelial adhesion molecule needed for T‐cell trafficking  after adjustment for age, male sex, race/ethnicity, blood pressure, and BMI. For each 10% unit increase in %Th1, CC IMT increased by 0.02 mm, whereas for each 1% increase in %Th2, CC IMT decreased by 0.02 mm. There was no statistically significant association of the T‐cell phenotypes with the internal carotid IMT. There was no evidence of an interaction with seasonality in either the lnCAC or CC IMT model.The major findings of this population‐based multiethnic study of Th1 and Th2 cells and measures of subclinical atherosclerosis are: (1) T‐helper bias toward Th1 cells is associated with 2 different estimates of subclinical atherosclerosis in a multiethnic healthy population; and (2) a measure of CMV infectious burden is strongly associated with Th1 bias.+ T lymphocytes have been identified in atherosclerotic lesions,36 with evidence that these cells are predominately T‐helper type 1 cells.38 In murine models, Th1 bias has a profound effect on early atherosclerosis,6 and Th1 cells are a main source of IFN‐γ, which has been demonstrated to contribute to murine atherosclerosis39 through multiple mechanisms, including the activation of macrophages and endothelial and smooth muscle cells, the inhibition of macrophage cholesterol efflux, and weakening the fibrous cap.40 However, it is important to note that a cross‐sectional study like ours cannot establish causality, and prospective studies are needed.Our findings of associations of Th1 bias with atherosclerosis are consistent with the substantial evidence in the literature that Th1 cells are important in the initiation and progression of atherosclerosis. In human clinical studies, activated CD46 although there have been inconsistent results.41 We observed a significant negative association of Th2 bias with common carotid IMT, consistent with an antiatherogenic effect. However, human diseases in which Th2 cells have been implicated such as systemic lupus erythematosis and asthma are not atheroprotective and in fact are associated with increased atherosclerotic burden.43 Our current hypothesis is that such diseases increase atherosclerotic burden because of markedly increased inflammation  despite the bias toward Th2 helper function.The role of Th2 cells in atherosclerosis is less certain than that for Th1 cells. In some murine models the key Th2 cytokine IL‐4 has antiatherogenic properties,44 the observations of strong associations in apparently healthy people between CMV serology, including those with latent infection, and CD4 and Th1 lymphocyte subpopulations are novel and suggest a possible role of chronic CMV infection in atherosclerosis.Although it has been established that acute CMV infection invokes a predominant Th1 bias in humans,45 In MESA, no associations were found between baseline measures of atherosclerosis and individual pathogens, including CMV antibody titer, or with overall pathogen burden in a subset of participants.46Previous results regarding CMV infection and atherosclerosis have been inconsistent, especially with regard to the presence of CMV in human plaques.Possible explanations for these discrepancies include the aforementioned possibility of reverse causality; a relatively small sample size for population studies, and CVD measures that do not evaluate the spectrum of disease severity.26 CMV infection may only have an indirect role in the development of atherosclerosis by promoting chronic immune activation (and Th1 biasing) and inflammation , responses that may vary depending on other underlying factors. For example, human immunodeficiency virus (HIV) infection has been reported to enhance the immune response against CMV, and CMV‐specific T cell responses have been independently associated with IMT in HIV‐infected patients.47Alternatively, and in our view more likely, because CMV antibody levels were found to be associated with inflammatory biomarkers in MESA,48 performing such investigations on a larger scale in the context of a multicenter epidemiological study poses new challenges.51To our knowledge, this is the first large‐scale epidemiologic study of T‐helper cell bias in a healthy, multiethnic population. Although smaller cellular immunology studies have been relatively common in specific clinical settings,+ cells in whites compared with the other ethnicities was unexpected and may be a result of unknown differences in infectious agents, other than those studied in MESA, such as helminthic parasites and protozoa, which evoke T‐helper cell responses. Alternatively, race/ethnicity‐specific genetic variation may play a role, which is currently being explored in genome‐wide association studies using data from the NHLBI MESA‐SHARe project (http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000420.v3.p2).The finding that healthy people vary consistently from each other with respect to T‐helper bias, without the influence of overt diseases, strongly suggests that such bias precedes clinical disease and is consistent with the view that T‐helper bias may influence disease occurrence. The observation that a significantly larger fraction of lymphocytes was CD4+ lymphocytes was lower in winter, whereas the fractions of the CD4+ cells that were Th1 and Th2 cells were higher, are consistent with the general thinking regarding increased infectious burden and/or allergic burden in the different seasons.34 The former result is consistent with recent small studies in healthy volunteers with allergic hay fever.52 The latter findings, to our knowledge, have not been observed before.The findings that the fraction of CD451 to determine Th bias, either of which would allow us to exploit nested case–control methodologies. Although we have measured T‐cell subsets in peripheral blood, not within lymph nodes, a recent report suggests that it is unlikely we have missed important features of the immune system.53We note several limitations of the present study. The data are cross‐sectional in nature. The sample size, although the largest assembled for this type of cellular epidemiology work, is too small—and the follow‐up period following MESA exam 4 too short—for meaningful coronary heart disease incident events to accumulate. To address this, we are currently working on both plasma‐based estimates and flow‐cytometric analyses of cryopreserved cells,31 and others54 have shown, regression dilution resulting from analytical and within‐subject variances reduces the apparent strength of most associations, so it is reasonable to assume the actual association of Th bias with atherosclerosis is somewhat greater than we have observed. Because several of the variables were measured earlier than the cell indices , some associations may be greater than we have observed. Finally, at the time we developed the protocol for MESA‐Inflammation, readily available methods did not yet exist for estimating several other T‐cell subsets, such as Th17 cells and Foxp3+ regulatory T cells; we hope to include these cells in future studies.Importantly, we have only measured Th bias at 1 point in the MESA participants (during exam 4). As weIn conclusion, we have demonstrated that CMV is strongly associated with Th1 cell bias and that Th1 bias is associated with subclinical atherosclerosis, independent of known CVD risk factors, in a multiethnic population. These findings are consistent with the role of Th1 cells in atherosclerosis and may provide a candidate mechanism governing the relationships of CMV with cardiovascular disease. They also suggest the importance of prospective studies of T‐helper cell biasing in CVD.


P < 0.001, n = 100; Silkie: r = 0.561, P < 0.001, n = 70; Dongxiang blue-shell: r = 0.619, P < 0.001, n = 30). We also measured the total serum IgY levels in the 3-day-old offspring hatched from the Silkie hens and results were significantly correlated for serum IgY levels  and the yolk IgY levels . The regression analysis showed simple linear regression between IgY levels in hen serum, yolk and offspring serum. Our results suggest that total IgY level could be used as an index for chicken fitness.The correlation between IgY levels of the serum and the yolk has been well documented in wild and domestic birds. The levels of total yolk IgY can be an index of the general health status of birds and may contribute to breeding programs when fitness of the offspring is a concern. We measured the levels of total serum IgY and yolk IgY in three different breeds  using indirect ELISA, and found that there was a significantly positive correlation between the levels of total serum IgY and total yolk IgY in all three breeds (White Leghorn: r = 0.404,  The mechanisms of antibody transmission from hen to yolk and the use of antibodies by offspring in chickens are well documented -6. In chBecause of its low cost, high production efficiency and good biochemical properties, IgY is widely used in immunodiagnostics and immunotherapy . SignifiThe concentration of total serum IgY in the chicken can be indicative of the fitness, health and nutritional state of the birds . Here, wg for 10 min at 4°C. The liquid that remained after blood had clotted was collected and stored as serum. Yolk IgY was isolated using the method reported by Müller et al. [g for 25 min. The supernatants were collected and the pH was adjusted to pH 5.0 ~ 5.2 using 1 mol/L HCl to reach reaction condition. All the serum and yolk IgY samples were kept at -20°C.A total of 70 44-week-old Silkie hens, 30 44-week-old Dongxiang blue-shelled layer hens and 100 44-week-old White Leghorn hens were used in this study. For each breed, the hens were kept in the same conditions and were vaccinated similarly. First, 2 mL blood was collected via the wing vein and three eggs from each hen were collected 1 weeks after bleeding. The blood samples were stored at 4°C for 1 h and centrifuged at 3,000 × r et al. . BrieflyTo analyze the correlation between serum IgY levels of the hens and their offspring, we collected three consecutively laid fertile eggs from Silkie hens and hatched the offspring shortly after for yolk IgY level measurement. All the eggs were hatched together. Next, 1 mL of blood was collected via the jugular vein from 3-day-old chickens and the serum was extracted as described above.Total IgY in both serum and yolk samples were measured using a sandwich ELISA protocol  with minPrior to ELISA analysis, the working solutions of yolk and serum samples were predetermined for all three breeds. For this, we prepared a pool of all yolk and all serum samples as standard solutions, which were diluted into a ten-fold series. The pooled ten-fold series samples were measured by sandwich ELISA to form a standard curve (data not shown). The working dilution was determined by the middle point of the linear part of the standard curve. After the working solutions were determined for each breed, all the samples were diluted by the working dilution, and measured using the sandwich ELISA method.We used the SPSS10.0 statistical software package to perform Pearson correlation coefficient (two-tail) and regression analysis. The correlations of the serum and yolk IgY levels of the hens were analyzed in the three breeds. For the Silkie breed, the IgY levels of the offspring serum, hen serum and the yolk were also analyzed for correlation and regression.405 of the three replications was used as the IgY level.The working dilutions of the samples are shown in Table P < 0.001, n = 30, Figure P < 0.001, n = 100, Figure P < 0.001, n = 70, Figure We found there was a significant correlation between the hen serum IgY and yolk IgY in the three breeds. The correlation of Dongxiang ; y2 = 0.549x2+ 0.479, P2 < 0.05, ; y3 = 0.827x3 – 0.109, P3 < 0.001, ; y4 = 0.350x4 + 1.046, P4 < 0.001, ; y5 = 0.574x5 + 0.476, P5 < 0.05 .The regression equation was made for each breed as follows: yThe transfer of passive immunity from mother to young has long been known to occur in birds as well as mammals . IgY, thWe found a significant correlation among IgY levels in hen serum, yolk and offspring serum in White Leghorn, Silkie and Dongxiang blue-shelled chickens. We have provided a better understanding of the correction of IgY levels among hen serum, yolk and offspring serum. IgY products are widely used nowadays, especially in immunoassays. The total serum IgY concentration could also be used as an index for offspring fitness because of the positive correlation between total IgY concentration and chicken health .Yolk IgY: Immunoglobulin yolk; Serum IgY: Immunoglobulin G; ELISA: Enzyme-linked immunosorbent assay; PBS: Phosphate buffered saline; BSA: Bovine serum albumin; PNPP: Disodium 4-nitrophenyl phosphate.The authors declare that they have no competing interests.The authors’ responsibilities were as follows: LQ designed the study; HS and XC carried out the experiments, participated in the data collection, data analysis, and drafted the manuscript; LQ, GX and SC provided technical expertise and revised the manuscript. All authors have approved the final manuscript.


Behavioral studies with proficient late bilinguals have revealed the existence of orthographic neighborhood density (ND) effects across languages when participants read either in their first (L1) or second (L2) language. Words with many cross-language (CL) neighbors have been found to elicit more negative event-related potentials (ERPs) than words with few CL neighbors (Midgley et al.,  Research over the last 20 years has shown that, within a language, the number of neighbors ; especially those representations from L1 that have some sort of orthographic, phonological, or semantic overlap with L2 input  or early in life ; as for English, seven learned it from birth, two before age 3, three from age 4, and two from age 5. The primary language spoken at home until 2 years of age was Welsh for six participants, a mix of Welsh and English for four participants, and English for four participants. Elementary education was in Welsh for five participants, balanced for one participant, predominantly in Welsh for six participants, and predominantly in English for two participants. Middle school and high school instruction was in both Welsh and English for all early bilinguals. In terms of language proficiency, all early bilingual participants rated themselves as native-like speakers in both languages. All participants rated themselves as native-like in reading English; for Welsh, eleven participants rated themselves as native-like, and three participants as somewhat proficient. Early participants reported speaking Welsh almost half of the time  and reading Welsh for recreational reading 28% of the time (SD = 26.7).Based on self-reports, early bilinguals learned Welsh from birth (n = 11) and high school . When asked to indicate how well they felt they spoke Welsh and English, all participants rated themselves as native-like in English; nine participants rated themselves as native-like in Welsh, four as somewhat proficient, and one between these two levels. In terms of proficiency in reading, all participants rated themselves as native-like in English; eight participants rated themselves as native-like in Welsh, five as somewhat proficient, and one as low proficient. Participants reported to speak Welsh 30% of the time (SD = 22.3) and to read Welsh for recreational reading 22.5% of the time (SD = 14.8).For late bilinguals, the mean age of acquisition for Welsh was 28.3 years (SD = 8.7), and the average number of years of Welsh was 11.9 (SD = 6.9). Four participants held a college degree, and 10 held a post-graduate degree. The primary language spoken at home until 2 years of age was English for 13 participants, and Polish for 1 participant. Elementary education was in English for all participants. Most participants had English as the only language of instruction in both middle school (n = 96). The task was administered at the end of the experimental session before the debriefing. Participants were asked to circle all the familiar Welsh words and, when possible, provide the correct English translation. As expected, early bilinguals translated Welsh words with a higher degree of accuracy (91.15 vs. 80.73%) than late bilinguals and indicated fewer Welsh words as being completely unfamiliar . Welsh words were selected from the Cronfa Electroneg o Gymraeg  between four and five letters in length with half of the items in each language having many orthographic neighbors in the other language and the other half having few neighbors in the other language. English items with high Welsh ND had a mean number of Welsh neighbors of 7.9 . English items with low Welsh ND had 0.23  neighbors on average. The difference between the two means was significant . Stimuli were matched on within-language neighborhood size. The list of stimuli and information about orthographic and lexical characteristics can be found in Grossi et al.  and frequency .The 16 Welsh and 16 English animal names were matched in length . Impedances were kept below 7 kΩ. EEG activity was filtered on-line band pass between 0.1 and 200 Hz and re-filtered off-line with a 30 Hz low pass zero phase shift digital filter. Eye-blinks were detected using the vertical electrooculogram bipolar channel. Potential variations exceeding a threshold of 20% of maximum EEG amplitude over the duration of a complete individual recording session were automatically registered as artifacts and contributed to the computing of a model blink artifact . Artifacts were then individually corrected by subtracting point-by-point amplitudes of the model from signals measured at each channel proportionally to local maximum signal amplitude. Eye movements, drifts, and other artifacts were removed by an algorithm that eliminated all events associated with brain waves that were larger than 75 μV or smaller than −75 μV. The percentage of accepted trials was 89%. Epochs ranged from −500 to 1000 ms after the onset of the critical word. Baseline correction was performed in reference to pre-stimulus activity (500 ms baseline) and individual averages were digitally re-referenced to the left and right mastoid channels offline. Behavioral data were collected simultaneously to ERP data.Analyses were conducted in the following time windows: 175–300 and 300–500 ms . Omnibus analyses were conducted on the following factors: Group (between-subjects), Language , and CLND . In order to describe the scalp distribution of Language and CLND effects, the following repeated measures factors were also included: Hemisphere , Laterality , and Anteriority . Analyses were informed by regions of interest highlighted by Midgley et al.  and condp-values (Geisser–Greenhouse correction) are reported for all within-subject measures with more than one degree of freedom.In late bilinguals, CLND effects started at around 175 ms for Welsh stimuli over central, centroparietal, and parietal sites and continued until approximately 500 ms. In early bilinguals, the largest differences were more frontal. Therefore, for this group, analyses were also carried out over frontal  and frontocentral  sites. The dependent variable was mean ERP amplitude in each of the intervals of interest. Words rated as unfamiliar by the participants were excluded from analysis. Significant interactions involving condition effects were followed up by simple effects analyses. Adjusted p’s < 0.01). Early bilinguals showed no differences in accuracy for the two languages , and were faster in recognizing English  than Welsh  targets (p = 0.008).A detailed discussion of the results can be found in Grossi et al., F = 5.52, p < 0.03; ND × Hemisphere × Group, F = 5.63, p < 0.03; ND × Hemisphere × Laterality × Group, F = 3.94, p < 0.06; ND × Hemisphere × Laterality × Group, F = 12.99, p = 0.001; 300–500 ms: Language × Laterality × Group, F = 3.74, p = 0.06; ND × Hemisphere × Laterality × Group, F = 5.18, p = 0.03; Language × Laterality × Anteriority × Group, F =  5.95, p = 0.006]. Table Figure F,26 = 5.5F = 4.68, p = 0.05]; this effect interacted with Hemisphere and Laterality. Follow-up analyses showed that CLND was significant as a main effect over the left hemisphere sites ; over the right hemisphere sites, ND was significant only over medial sites . Therefore, overall, the CLND effect (more negative ERPs to high than low CLND targets) was more robust over the left hemisphere sites and over the medial sites . These results were confirmed by midline analyses  = 9.25, p = 0.009; no significant interactions between Language and CLND were observed, all p’s > 0.11).Analyses conducted on lateral and medial electrodes showed that ERP amplitudes were more negative for high compared to low CLND targets in this time window ; this effect was qualified by a four-way interaction with Language, Hemisphere, and Anteriority. For English targets, CLND was not significant as a main effect (p = 0.5), but interacted with Hemisphere and Laterality . However, no ND effects were significant in follow-up analyses by Hemisphere and Laterality . In contrast, the ND main effect was significant for Welsh targets . No other interaction between Language and ND reached significance. Analyses over the midline sites revealed a main effect for CLND . This effect did not interact with Language .In hemisphere analyses, ERPs tended to be more negative for high compared to low CLND targets  but not lateral sites . Midline analyses revealed a similar pattern of results: the main effect of Language approached significance  and was qualified by a Language × Anteriority interaction : Welsh targets elicited more negative ERP amplitudes than English targets at CZ and CPZ sites .Event-related potentials were more negative for Welsh than English targets in this time window. In hemisphere analyses, Language interacted with Laterality and with Laterality and Anteriority. Follow-up analyses showed that the Language effect was significant over medial sites . Follow-up analyses carried out on each hemisphere separately did not reveal any significant ND effects. No ND effects were observed over the midline sites . ERP amplitudes were more negative for English than Welsh targets at centroparietal and parietal sites . The distribution of the effect is shown in Figure p’s > 0.22).Hemisphere analyses over central, centroparietal, and parietal sites revealed an interaction between CLND, Hemisphere, and Laterality . The ND effect tended to be significant for English targets . For Welsh targets, ND interacted with Hemisphere . Over the left hemisphere, the ND effect was reversed, in that Welsh words with high CLND tended to elicit more positive ERP amplitudes than Welsh words with low CLND . No ND effects were observed over the right hemisphere sites . The distribution of the effects is shown in Figure Hemisphere analyses over frontal and frontocentral sites revealed a trend for the interaction between Language and CLND . The ND effect was significant for English  but not for Welsh (p = 0.39) targets.Midline analyses revealed a significant interaction between Language and CLND , suggesting distributional differences between high and low CLND targets. However, no ND effects resulted significant in follow-up analyses. No ND or Language effects were detected in this time window in midline analyses .In hemisphere analyses, a trend was found for the interaction between ND, Hemisphere, and Laterality [The present study was aimed at replicating and extending Midgley et al.’s  data on As expected, based on Midgley et al. , targetsp < 0.04, English, p = 0.67 in hemisphere analyses; Welsh, p = 0.005, English, p = 0.16 in midline analyses). We asked whether differences in experience and proficiency with L2 among our participants might have contributed to this pattern of results. Our participants were, as a group, highly proficient, considering their performance in the translation and categorization tasks . However, differences in proficiency and experience existed among them . Furthermore, they reported using Welsh for recreational reading 22.5% of the time (only two participants reported reading Welsh 50% of the time). It is therefore possible that even many years of experience with a second language do not translate in completely symmetrical effects in reading experiments if the first language remains dominant, particularly here in the domain of reading  targets was modulated by the participants’ experience with L2, post hoc analyses based on language proficiency suggest the presence of larger CLND effects for L1 words in more, compared to less, proficient bilinguals, as for the 175–300 ms time window. Overall, these results suggest that, in late bilinguals, electrophysiological CLND effects tend to be asymmetrical, although the level of asymmetry was modulated by experience with the second language, in agreement with behavioral data  automatically activated competing English phonological representations, which would cause inhibition (e.g., Dijkstra et al., Based on the non-selective access hypothesis, it was hypothesized that symmetrical effects would be present for the two languages in early bilinguals. However, this hypothesis was not supported. A frontocentral CLND effect was found at midline sites for English targets in the 175–300 ms time window. For Welsh targets, the effect was mainly localized over the frontal left hemisphere sites and it was reversed. Perhaps high CLND Welsh words (e.g., These results are not entirely consistent with a non-selective model of lexical access, as they seem to contradict behavioral accounts of CL activation in bilinguals. However, most of the available data on CLND effects was gathered in proficient late bilinguals (e.g., Midgley et al., Electrophysiological evidence is mixed. In a letter detection task, Rodriguez-Fornells et al.  asked eaThe functional meaning of the differences in CLND effects between early and late bilinguals is not clear. Differences in proficiency alone are unlikely to explain this pattern, as targets in both languages contributed to the ND main effect in late bilinguals. Therefore, based on proficiency, a more symmetric pattern would be expected in early bilingualsThe results observed in late bilinguals support the recent literature on the modulation of the N400 amplitude by ND (Holcomb et al., One of the limitations of the present study is the relatively small sample size. Future studies should investigate individual differences more systematically, as the present data suggest that the presence of CLND effects for L1 depend on proficiency in L2, at least in late bilinguals. Furthermore, future studies should investigate how different language learning experiences shape aspects of cognition and brain organization in terms of CL interaction, as the differences between early and late bilinguals are not trivially explained by non-selective models of lexical access.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aim. The aim of this study was to investigate the effect of i.v. infusion of magnesium sulphate during spinal anesthesia on duration of spinal block and postoperative pain. Methods. Forty ASA physical status I and status II, aged between 18 and 65, female patients undergoing abdominal hysterectomy under spinal anesthesia were enrolled in this study. Patients in the magnesium group  received magnesium sulphate 65 mg kg−1 infusion in 250 mL 5% dextrose at 3.5 mL/min rate, and control group  received at the same volume of saline during operation in a double-blind randomized manner. Duration of sensory and motor block, systolic, diastolic, and mean arterial blood pressures, heart rates, pain scores , and side effects were recorded for each patient. Blood and CSF samples were taken for analysis of magnesium concentrations. Results. Regression of sensorial block was longer in Group M when compared with that in Group C (175 ± 39 versus 136 ± 32 min) (P < 0.01). The VAS scores were lower in Group M than those in Group C at the 2 time points postoperatively (P < 0.01). Conclusion. 65 mg kg−1 of magnesium sulphate i.v. infusion under spinal anesthesia prolongs spinal sensorial block duration and decreases pain VAS scores without complication in patients undergoing abdominal hysterectomy. Magnesium sulphate has been used as an adjuvant for perioperative analgesia –4. Many The study protocol was approved by the Institutional Ethics Committee and written informed consent was obtained from all patients. Forty ASA physical status I and status II, aged between 18 and 65 female patients undergoing abdominal hysterectomy under spinal anesthesia were enrolled in this study. Exclusion criteria included severe cardiovascular, renal and hepatic dysfunction, neuromuscular diseases, using calcium channel blockers, and inappropriate for spinal anesthesia.−1, patients were transferred to the operating room. After placement of standard monitors included continuous electrocardiogram, pulse oximetry, and noninvasive blood pressure measurements, an 18-gauge IV cannula was placed. Spinal anesthesia was performed through L3-4 or L4-5 interspace in lateral decubitus position. Before the start of the fluid infusion 3 mL of blood sample was taken for analysis of preoperative serum magnesium concentrations, together with 2 mL CSF taken via the subarachnoid puncture. After dural puncture with a 25 G Quincke needle and 2 mL of clear CSF sample was withdrawn, 3 mL of hyperbaric bupivacaine 0.5% solution  was injected into the subarachnoid space over 15 second. The lateral decubitus position was maintained to achieve the estimated level of the block; then patients were turned to supine before surgery started. The level of sensory block was evaluated by the loss of pinprick sensation (20-gauge hypodermic needle). Motor blockade was scored using a modified Bromage scale . Readiness to surgery was defined as the presence of adequate motor block (Bromage score ≥2) and loss of pinprick sensation at T6. The inability to reach a sensory block at T6 within 30 minutes after spinal injection was considered to be a technical block failure and the patient was converted to general anesthesia.After premedication with i.v. midazolam 0.03 mg kgn = 20) received magnesium sulphate 65 mg kg−1 infusion in 250 mL 5% dextroz at 3.5 mL/min rate during operation. In control group  30 mL 0.9% sodium chloride in 250 mL 5% dextrose infusion was administered at 3.5 mL/min rate during operation in a double-blind randomized manner. Age, weight, height, ASA physical status, surgical time, duration of sensory and motor block, systolic, diastolic, and mean arterial blood pressures, heart rates, pain scores , and side effects were recorded for each patient by an investigator blinded of the study groups. Hemodynamic variables were recorded at 10 times . Pain scores were evaluated using a 0–10 cm VAS  at the postoperative periods . When patients were transferred to PACU blood and CSF samples were taken again for analysis of magnesium concentrations.Forty patients were randomly assigned to one of the two groups of 20 patients using sealed and numbered envelopes. Patients in the magnesium group  were compared using Student's t-tests between the two groups. Discrete variables (side effects) were compared using a chi-square test. Repeated-measures ANOVA was used to analyze hemodynamic variables over time between two groups. All data are presented as means ± (SD) or number. A P value ≤ 0.05 was considered as statistically significant.Sample size was estimated using pain scores (VAS) as the primary variable. On the basis of a previous clinical experience and assuming an SD of 1 cm, 17 patients were required in each group to have a There were no significant differences between the two groups with respect to patient characteristic , ASA physical status, and surgery times . The subThree patients in Group M and two patients in Group C developed hypotension, and two patients in Group M and one patient in group C experienced bradycardia during surgery. In accordance with the study protocol, all events were treated. Mean arterial blood pressures and heart rates were similar in the two groups Figures  and 2. TP < 0.01). The VAS scores were lower in Group M than those in Group C at 2 h and 4 h after the surgery (P < 0.001). However, there was no difference at the other time points for VAS scores  when compared with that in Group C (136 ± 32 min)  magnesium concentrations of two groups were shown in This study showed that i.v. magnesium sulphate infusion during abdominal hysterectomy under spinal anesthesia prolonged the duration of sensory block and reduced postoperative pain scores without any notable side effects.−1 bolus of magnesium sulphate followed by a 500 mg h−1 infusion or saline in the same volumes for 24 h, and postoperative analgesic consumption was significantly lower in the magnesium group. But, VAS scores in the two groups during the first 24 h after surgery were similar except at 12 h after surgery, and thus, it was assumed that the dosage of magnesium sulphate used was insufficient for postoperative analgesia. In our study we used total of 4550 mg magnesium sulphate for 70 kg during spinal anesthesia, in Apan et al. [−1 magnesium sulphate for 15 min and then 15 mg kg−1 h−1 by continuous i.v. infusion until the end of surgery during spinal anesthesia reduced postoperative pain and analgesic consumption without complication. Seyhan et al. [−1 of magnesium sulphate reduced postoperative morphine consumption, and when this was followed by a maintenance infusion of 10 mg kg−1 h−1, the effect was enhanced. Moreover, increasing the maintenance infusion to 20 mg kg−1 h−1 provided no additional advantage and induced unwarranted hemodynamic effects.Magnesium sulphate has been used as an adjuvant for perioperative analgesia. Although the basic mechanism of analgesic effect of magnesium sulphate is unclear, it is presumed that its antagonist effect of NMDA receptors prevents the induction of central sensitization due to peripheral nociceptive stimulation and abolishes hypersensitivity. In many studies the effect of magnesium sulphate on postoperative analgesic consumption had been investigated under general anesthesia –9, but rn et al.  study th−1 infusion in 250 mL 5% dextrose at 3.5 mL/min rate during surgery under spinal anesthesia and in the control group patients received saline at the same volumes. We found that the duration of sensory spinal block with 65 mg kg−1 i.v. magnesium sulphate infusion was significantly longer than that in the group control . To our knowledge, this is the first study reporting the prolongation of spinal sensory block duration with i.v. magnesium sulphate infusion under spinal anesthesia in abdominal hysterectomy. Hwang et al. [−1 for 15 min and then 15 mg kg−1 h−1 continuous infusion and saline under spinal anesthesia for total hip replacement. They reported that time to first pain sensation and spinal block height was similar in the two groups. In a previous study Apan et al. [In this study, patients in the magnesium group received magnesium sulphate 65 mg kgg et al.  comparedn et al.  reported−1, and potentially life-threatening complications, primarily involved in the cardiovascular and neuromuscular system, occur when serum magnesium concentrations exceed 5 mmol L−1 [−1, which is below the level of minor side effects. In our study, arterial blood pressure and heart rates were similar in the magnesium and control groups. On the other hand, there are few studies that analyzed CSF characteristics in patients and that have investigated the effect of magnesium levels in CSF on the spinal block. In some studies a direct correlation had been observed between the CSF density and the number of dermatomes blocked under spinal anesthesia [In this study we measured magnesium concentrations both in blood and in cerebrospinal fluid (CSF). Patients in the Group M had a bit higher blood magnesium concentration than those in Group control, but there was no side effect associated with hypermagnesemia. Magnesium causes a dose-dependent negative inotropic effect, and a peripheral vasodilatory effect , 14. Themmol L−1 . In thisesthesia , 17. Echesthesia  investig−1 of magnesium sulphate i.v. infusion under spinal anesthesia prolongs spinal sensorial block duration and decreases pain VAS scores without complication in patients undergoing abdominal hysterectomy. This outcome can investigate with different dosage of magnesium sulphate in further studies.In conclusion, 65 mg kg


Hip fracture in older adults is associated with depression and frailty. This study examined the synergistic effects of depression and hip fracture on physical frailty, and the mediating role of the cortisol:dehydroepiandrosterone sulphate (DHEAS) ratio.This was an observational longitudinal study of patients with a hip fracture carried out in a hospital setting and with follow up in the community.Participants were 101 patients aged 60+ years  with a fractured neck of femur.Measurements of the ability to carry out activities of daily living (ADL), cognitive function, physical frailty and assays for serum cortisol and DHEAS were performed six weeks and six months post-hip fracture. Depressed and non-depressed groups were compared by ANOVA at each time point.Hip fracture patients who developed depression by week six (n = 38) had significantly poorer scores on ADL and walking indices of frailty at both week six and month six, and poorer balance at week six. The association with slower walking speed was mediated by a higher cortisol:DHEAS ratio in the depressed group.Depression following hip fracture is associated with greater physical frailty and poorer long term recovery post-injury. Our data indicate that the underlying mechanisms may include an increased cortisol:DHEAS ratio and suggest that correcting this ratio for example with DHEA supplementation could benefit this patient population. With the ageing of the population, hip fractures are a growing issue, with UK rates predicted to increase to 117,000 per annum by 2016 . The facThe prevalence rate for depression in hip fracture patients across eight US and UK studies ranged from 9–47% . HoweverCortisol levels are often higher in individuals with depression ,10. A reParticipants were 101 older adults  with a mean ± SD age of 83.9 ±7.88 years who were admitted to hospital in-patients with a fractured neck of femur (hip fracture). Participating hospitals were all located in the West Midlands, UK. Inclusion criteria were that patients had sustained a hip fracture, were 60+ years of age and did not meet the exclusion criteria. The latter included existing medical conditions or medications that could affect the immune system (e.g. glucocorticoids and immune suppressants), dementia, taking antidepressants or having a previous diagnosis of depression before the age of 50 years. In this way we aimed to recruit those patients who had likely developed depression post-fracture, rather than those who already had a history of depression. We were thus focusing on those with a first or new episode of depressive symptoms evident post-fracture, and most likely resulting from the trauma of hip fracture. All participants were Caucasian. All participants gave written informed consent prior to the study, which was approved by South Staffordshire Research Ethics Committee (study ref: 09/H1203/80).The study was a longitudinal investigation comparing the physical frailty and cognitive function of depressed and non-depressed older hip fracture patients 6 weeks and 6 months post fracture. Participants completed a questionnaire pack and undertook a range of measures to assess physical frailty. Socio-demographics and health behaviours were recorded to assess for bias between the non-depressed and depressed groups. Participants also provided a blood sample in order to determine serum concentrations of cortisol and DHEAS.The Geriatric Depression Scale (GDS) was specifically developed to screen the older population for depression . A shortThe Oxford Hip Score  (OHS) isPhysical frailty was assessed in part via the OHS but in addition upper body strength was measured as handgrip strength using a hydraulic hand dynamometer, lower body strength using the Timed Up and Go (TUG) test  and the 2 from measured height and weight. Health behaviours were recorded using a questionnaire adapted from the Whitehall study [Data gathered included: date of birth, if suffering from chronic illness or taking ongoing medication, and occupational category of the previous main bread winner using the Registrar General’s classification of occupations . Occupatll study . The timA venous blood sample was taken from each participant between the hours of 0800–1130 am at week 6 and month 6. Serum cortisol and DHEAS were analysed in duplicate using separate ELISA based assays and commercial kits . Intra-assay coefficients were < 10%.Differences between the depressed and non-depressed group on the main demographic, health behaviour, and operation-related variables were tested using chi-square and ANOVA. In order to examine group differences at week 6 and month 6 in the key psychosocial and frailty variables, ANOVA was used. Repeated measures ANOVA and ANCOVA models were run to assess changes in these variables between the two sampling times, and whether trajectory of change was influenced by depression status, respectively. Any socio-demographics or health behaviours which significantly differed between the groups were included as covariates to control for confounding. Week 6 health behaviours were included in analyses predicting week 6 outcomes, and month 6 behaviours for month 6 outcomes. Regression was used to examine the associations between the cortisol: DHEAS ratio and the outcome variables. Where hormone levels were associated with frailty, potential mediation by cortisol: DHEAS was tested by entering depression group at step 1, and cortisol: DHEAS ratio at step 2, and confirmed by Sobel test. Moderation was tested where cortisol:DHEAS was related to depression group but not the outcome variables. Mean-centred variables and interaction terms were created and regressions run with the mean-centred variables entered at step 1, and interaction term at step 2.p = .68) gender (p = .15) or hospital recruitment site (p = .36) bias between those who consented then withdrew and those who remained in the study for the six week testing session. Reasons for withdrawal between week 6 and month 6 included: death or being too unwell to be tested (N = 17), not being able to continue in the study for a variety of reasons including feeling they had too much to cope with, now receiving treatment for depressive symptoms or other medication/illnesses on the list of exclusion criteria, or being non-contactable (N = 18). Again, there was little evidence of selection bias between the sample who withdrew or remained in the study at six months in terms of gender (p = .26), initial depression group status (p = .72), BMI (p = .34), number of medications being taken (p = .09), and hospital recruited at (p = .26). However, those who withdrew were marginally more likely to be from the manual occupational group, (p = .05), were, on average, 3.9 years older (p = .02), and had 0.5 more comorbidities on average (p = .03).Recruitment and withdrawal data are shown in Figure Detailed demographics and health behaviour of the two hip fracture groups (depressed or non-depressed) are shown in Table p < .001, η2 = .666, at week 6. At month 6, this difference remained significant, F = 30.10, p < .001, η2 = .320; those who were classified as depressed at week 6 still had significantly higher depression scores at month 6, with a mean group difference of 3.9. There was no detectable change in GDS depression score between week 6 and month 6. However, six of the non-depressed patients became depressed, as classified by the GDS at month 6, and 11 of the depressed patients became non-depressed, χ2(1) = 11.85, p = .001. The main analysis by group will focus on participants’ depression classification at week 6 in order to examine the effect of a first or new episode of depressive symptoms evident post-fracture, and likely resulting from the hip fracture and its consequences. The difference in depression symptoms between the groups was also confirmed by significant group differences on the HADS depression sub-scale, at week 6, F = 65.02, p < .001, η2 = .399, and month 6, F = 19.46, p < .001, η2 = .233.Mean (SD) for psychosocial variables are reported in Table p < .001, η2 = .161, and month 6, F = 6.22, p = .02, η2 = .098. ADL significantly improved over time, F = 15.97, p < .001, η2 = .258, although this did not vary by depression group.Physical frailty was assessed using four measures. As shown in Table p = .02, η2 = .099, but this deterioration was not influenced by depression status. However, those with depression took significantly longer to complete the TUG test at week 6, F = 4.16, p = .05, η2 = .048, and at month 6, F = 3.90, p = .05, η2 = .067, compared to the non-depressed group. Both groups were far slower than age-related norms at both time-points. Walking speed (TUG) improved over time overall, F = 14.47, p < .001, η2 = .221. Depressed patients also scored significantly worse on the Berg Balance scale at week 6, F = 8.35, p = .005, η2 = .097, compared to the non-depressed. However, there was no significant difference by month 6. Balance also improved over the 6 month period, F = 28.71, p < .001, η2 = .384, in both groups.At both time points, depressed and non-depressed patients did not significantly differ in hand grip strength. Overall, hand grip strength declined over time among those tested at both follow-ups, F = 5.94, p = .008, η2 = .071. This difference remained significant at month 6, F = 5.75, p = .02, η2 = .100. There was no significant change in the cortisol:DHEAS ratio from week 6 to month 6 in either group. Cortisol:DHEAS at week 6 was not associated with the OHS or balance score at either time point or the TUG at week 6. However, it was significantly positively related to TUG test time at month 6, such that the higher the cortisol:DHEAS ratio, the longer it took the participant to walk three metres, β = .44, p = .001, ΔR2 = .20. Cortisol:DHEAS at month 6 was not associated with the month 6 scores for OHS or balance score. However, it was again positively related to TUG test time at month 6, such that the higher the cortisol:DHEAS ratio at month 6, the longer it took the participant to walk three metres β = .39, p = .008, ΔR2 = .15.As shown in Figure p = 0.01), such that a higher cortisol:DHEAS ratio explained the relationship between depression group and longer TUG speed. There was also significant mediation by the cortisol:DHEAS ratio at month 6, p = .04. The regression equation steps with and without adjustment for cortisol:DHEAS are shown in Figure When the regression predicting walking speed (TUG) at month 6 from depression group was rerun with adjustment for week 6 cortisol:DHEAS ratio, the association with depression group became non-significant, with evidence of significant mediation using the Sobel test,  and controls (p = .001), but not between those with hip fracture but no depression and controls (p = .12). This would suggest that the depression and frailty association was not a consequence of changes in the cortisol:DHEAS ratio as a consequence of the hip fracture. Thus, mediation would appear a more compelling explanation than confounding.That the week 6 cortisol:DHEAS ratio was higher among the depressed hip fracture group is supported by previous studies . In the That cortisol:DHEAS did not mediate all of the associations between depression and physical function suggests that other mechanisms also may underlie this relationship, such as reduced motivation to participate in rehabilitation or levels of pro-inflammatory cytokines.This study has several limitations. First, it was primarily designed to investigate the synergistic effect of what we perceive as depressive symptoms that present post-onset and potentially as a result of hip fracture on physical function, through exclusion of patients with a history of depression pre-fracture prior to age 50 years. However, we do realise that in the absence of a pre-fracture measure of depressive symptoms, this is an assumption. Despite excluding those with a previous diagnosis of depression prior to age 50 years and those taking anti-depressants, it is possible that some participants may have had symptoms post-age 50 years but prior to their hip fracture although none were undergoing treatment at the time of fracture. Further, our exclusion of previously depressed patients or those with cognitive decline, or immune-related comorbidities means our findings may not be generalisable to the wider hip fracture population and may have introduced some non-assessable selection bias in the absence of more general socio-demographics of patients not being available prior to informed consent. However, these exclusions do suggest that the associations reported here would be even stronger had patients with poorer physical or mental status been included as these factors are associated with poor recovery . Third, In conclusion, depression emerging post-hip fracture in older adults impairs physical function, including walking speed, balance, and activities of daily living. This is the first time that depressive symptoms in the absence of longer term pre-fracture depression diagnosis have been shown to relate to recovery and physical frailty. Effects on walking speed were mediated by alterations in the cortisol:DHEAS ratio which was heightened among the depressed group. This novel finding implies that in order to speed recovery of physical function and independence following hip fracture, patients should be assessed and treated for depressive symptoms. This is of relevance to surgeons and health professionals alike involved in rehabilitation post-fracture surgery who currently do not screen this patient group for depressive symptoms. Identification and treatment of depression in these patients would improve patient outcomes and quality of life as well as impacting upon health service costs incurred through treatment of those with slower recovery and decreased independence post-fracture. We propose that correcting the cortisol:DHEAS imbalance by oral supplementation with DHEA may be one means of improving depressed mood and contributing to better physical function after hip fracture. However, such an intervention would need to be cautiously informed by the intervention literature in order to determine an effect dosage and regime for an effect in these patients.The authors declare that they have no competing interests.JL and AP conceived of the original research idea and sought and received funding for the study. JU collected the data, NAD conducted laboratory assays. AP and JU conducted the analyses and interpretation. AP was a major contributor in writing the manuscript with JU and also drew the figures. JU constructed the first draft and conducted the literature search. JL, DC, and NAD also contributed to drafts of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2318/13/60/prepub


This study analyses characteristics of lung injuries produced by alveolar overdistension in three animal species. Mechanical ventilation at normal tidal volume (10 mL/Kg) and high tidal volume (50 mL/Kg) was applied for 30 min in each species. Data were gathered on wet/dry weight ratio, histological score, and area of alveolar collapse. Five out of six rabbits with high tidal volume developed tension pneumothorax, and the rabbit results were therefore not included in the histological analysis. Lungs from the pigs and rats showed minimal histological lesions. Pigs ventilated with high tidal volume had significantly greater oedema, higher neutrophil infiltration, and higher percentage area of alveolar collapse than rats ventilated with high tidal volume. We conclude that rabbits are not an appropriate species for in vivo studies of alveolar overdistension due to their fragility. Although some histological lesions are observed in pigs and rats, the lesions do not appear to be relevant. Mechanical ventilation-associated lung injury is a clinically accepted fact and considered one of the main factors for the worsening or persistence of acute lung injury. The mechanism by which alveolar overdistension causes lung injury is not fully understood and is probably multifactorial. A major role is played by disruption of the alveolar-capillary membrane , surfactLung injuries from alveolar overdistension have been reported by several experimental research groups, predominantly in small animals (rats) , 6. ThisThe objective of this study was to determine the histological and morphometric alterations produced by intermittent alveolar overdistension manoeuvre in three animal species frequently used in experimental studies.The study was approved by the ethical committee of our hospital, and the animals were managed according to Spanish norms for the protection of experimental animals .2O and then kept in a 10% formaldehyde bath for at least 72 h for subsequent histological analysis. Both histological and morphometric analyses were conducted by a pathologist blinded to the tidal volume group allocation of the animals.The experiment was conducted on pigs, rabbits, and rats. Two different mechanical ventilation protocols, normal tidal volume (10 mL/Kg) and high volume (50 mL/Kg), were applied in each species, as detailed below. After 30 min of mechanical ventilation, animals were killed by injection with potassium chloride, and the lungs were extracted by sternotomy for histological, morphometric, and gravimetric analyses. The left bronchus was ligated, and this lung was separated for gravimetric analysis. The right lung was infused with a 10% formaldehyde solution to a pressure of 20 cm HTwelve mixed-breed pigs weighing 24–32 Kg were premedicated with intramuscular (i.m.) injection of ketamine (10 mg/Kg) and azaperone (5 mg/Kg). Anaesthesia was induced with intravenous (i.v.) injections of atropine (1 mg), ketamine (2 mg/Kg), and fentanyl (0.15 mg), immediately followed by tracheotomy and intubation with a cuffed tube . Anaesthesia was maintained with continuous infusion of ketamine (20 mg/Kg/h) and atracurium (1 mg/Kg/h), administering supplementary boluses of fentanyl and atracurium when necessary. The animals received a continuous infusion of 0.9% saline solution (3 mL/Kg/h) throughout the experiment.n = 6) or high tidal volume group (50 mL/Kg), respiratory rate of 12 per min (n = 6). The I : E ratio (1 : 2) and FiO2 (100%) were the same for both groups.Pigs were assigned to a normal tidal volume group (10 mL/Kg), respiratory rate of 16 per min . Anaesthesia was induced with i.v. injections of atropine (0.5 mg), ketamine (2 mg/Kg), and fentanyl (0.05 mg), immediately followed by tracheotomy and intubation with a cuffed tube . Anaesthesia was maintained with a continuous infusion of ketamine (20 mg/Kg/h) and atracurium (1 mg/Kg/h), administering supplementary boluses of fentanyl and atracurium when necessary. A 0.9% saline solution infusion of 3 mL/Kg/h was administered throughout the experiment.n = 6) or high tidal volume group (50 mL/Kg), respiratory rate of 20 per min (n = 6). The I : E ratio (1 : 2) and FiO2 (100%) were the same for both groups.Rabbits were assigned to a normal tidal volume group (10 mL/Kg), respiratory rate of 40 per min  and atropine (0.5 mg), immediately followed by tracheotomy and intubation with a 14 G catheter, administering supplementary boluses of ketamine when necessary. n = 6) or high tidal volume group (50 mL/Kg), respiratory rate of 40 per min (n = 6). The I : E ratio (1 : 2) and FiO2 (100%) were the same for both groups.Rats were assigned to a normal tidal volume group (10 mL/Kg), respiratory rate of 75 per min  adapted to a Nikon Labophot-2A microscope . Visilog 3.6  software was used to measure alveolar areas, constructing a routine to permit quantification of alveolar areas, considering areas sized <50 pixels to be those with a tendency to collapse.After separating the left lung of each animal, it was weighed and placed in an oven for ≥72 h at 80°C. After removal from the oven, it was reweighed for calculation of the wet/dry weight ratio.P < 0.05 was considered significant.SPSS 15.0  was used for the data analyses. Quantitative variables were expressed as mean and standard deviation and the Mann-Whitney test was used to compare means of variables with abnormal distribution. Qualitative variables were expressed as percentages (mean percentage ± standard deviation), and the chi-square test was used for comparisons. A All pigs and rats survived in both normal and high tidal volume groups, and no cases of barotrauma were detected. In contrast, five of the six rabbits in the high tidal volume group developed tension pneumothorax and/or pneumoperitoneum, and three died after <30 min of mechanical ventilation. As a consequence, this experiment was halted and the results for rabbits were excluded from the following analyses.P < 0.05).Globally, the histological lesions were minimal, especially in the two rat groups. The total histological score was higher in pigs than in rats , and theP < 0.01). In contrast, this ratio was significantly higher in the rats treated with normal versus high tidal volume   .The percentage area of alveolar collapse was significantly higher in pigs than in rats .P < 0.05) in those treated with high (54.3 ± 8.4%) versus normal tidal volume (43.1 ± 10.5%). Comparing the percentage collapse areas of the upper, middle and lower lobes were 46.3%, 56.4%, and 60.1%, respectively, in the pigs with high tidal volume versus 34.5%, 45.8%, and 48.8%, in the normal tidal volume group; differences between the groups were significant for upper and lower lobes (P < 0.05) but not for middle lobes.In the pigs, the percentage area of collapse was higher (P < 0.05) in those treated with normal (38.5 ± 10.2%) versus high tidal volumes (26.8 ± 4.9%).In the rats, the percentage area of collapse was higher  for 30 min produced an increased tendency to alveolar collapse in intact pigs, but no other major histological lesions were detected. Almost no lesions were observed in rats after the same manoeuvre. In contrast, it caused early alveolar rupture with associated pneumothorax in rabbits, and their lower resistance to alveolar overdistension makes this species inappropriate for this type of experiment.The vulnerability of rabbits to lung injuries has been previously reported. Studies on resistance of pulmonary capillaries, found that a much lower transmural pressure was needed to produce capillary stress failure in rabbits than in dogs or horses , which mThe same aggressive ventilation protocol produced different histological lesions between pigs and rats, with a predominance of oedema and neutrophil infiltration in the pigs and a tendency to haemorrhage in the rats. The gravimetric analysis also revealed differences. In the pigs, lungs were significantly heavier in the high versus normal tidal volume group, whereas in the rats, they were significantly heavier in the normal versus high tidal volume group. However, this surprising result in the rats may be a methodological artefact, since the blood pressure was not measured in this species, and alveolar overdistension in the high volume group may have led to hemodynamic compromise, lung hypoperfusion, and a smaller perfused lung vascular surface , hence rThe most relevant finding was the higher tendency to collapse after ventilation with high tidal volume in the pigs (mostly in lower lobes) than in the rats. It has been reported that the increased alveolar surface area produced by ventilation with high tidal volumes produces surfactant inactivation. Thus, proteoglycan breakdown and glycosaminoglycan fragmentation contribute to an alteration of the extracellular matrix that may produce greater lung collapse .2O and generating a volume of around 90 mL/Kg, almost double the volume in the present study. In rats ventilated with lower volumes (peak pressure of 30 cm H2O), only a moderate interstitial oedema was detected after 60 min of ventilation [2O and with a very high mean volume of around 90 mL/Kg. Other studies in large animals using similar volumes to those in our study required a much longer ventilation time to produce histological lesions or severe lung function impairment [In contrast to our results, other authors found that ventilation with high tidal volume for 30 min caused severe lung injuries in rats  and in dtilation , and othtilation . The tidtilation  observedpairment , 21.We can conclude that animal species respond differently to high tidal volumes. Rabbits are not appropriate animals for this type of experiment, whereas pigs and rats show no important histological lesions after 30 min of ventilation.