Here we describe an efficient micrografting protocol for Nicotiana attenuata, a useful tool for identifying these signals and understanding their functions. Additionally we analyzed transcript accumulation profiles of scions and rootstocks of grafts performed with wild-type and stably transformed N. attenuata. Our results are consistent with the source-to-sink movement of an sRNA silencing signal.To adjust their development to the environment, plants rely on specific signals that travel from shoot to root and  Many studies have shown that plants use long-distance or systemic signals to coordinate and adjust their growth. These signals convey messages throughout the whole plant, from sensor to effector tissues or organs, and they seem to operate with great specificity which can depend not only on the message itself but also on the spatial and temporal scales over which they act . One of de novo biosynthesis of JA in systemic leaves was further determined not to be required for the systemic transmission of the wound signals [Grafting has provided important insights into the study of these systemic wound signals in plants. Grafting a JA biosynthesis mutant with a JA response mutant clearly showed that the production of jasmonic acid (JA) in damaged leaves and the perception of JA by distal leaves are necessary for inducing systemic responses . Moreove signals . This re signals .Nicotiana attenuata has been studied in plant-herbivore interaction in its natural habitat, the Great Basin Desert of Utah. Several local and systemic defense responses are induced in N. attenuata during herbivore attack. Defense traits, which include trypsin proteinase inhibitors (TPI) and specific volatiles, such as trans-α-bergamotene produced by terpene synthases (TPS), are increased in N. attenuata when this plant is attacked by herbivores or elicited by herbivore-specific elicitors [N-methyltransferase (PMT) in the roots for its synthesis and accumulates the alkaloid in leaves and other above-ground parts of tobacco plants [N. attenuata's leaves by its attackers, even by Manduca sexta, a specialist and nicotine-tolerant herbivore [A wild tobacco, licitors -8. A noto plants . This alerbivore . The ideerbivore .Research in plant ecology has been greatly enhanced by the manipulation of gene expression in stably transformed plants. Isogenic lines expressing either low or high levels of transcripts for a particular gene provide a powerful tool to dissect its organism-level function and fitness consequences of the gene under real-world conditions . HoweverN. attenuata, and characterize the influence of the grafting procedure on plant growth. Given that the graft junction is located between the shoots and roots of grafted plants, this technique allows for the independent manipulation of the below- and above-ground parts and the study of their interplay in N. attenuata for defense. Our results represent the first step towards the study of shoot-to-root interplay by micrografting in N. attenuata.Here, we describe a simple and highly efficient micrografting protocol for N. attenuata resulted in low success rate (approximately 10 ~ 15%). Therefore, we tested a method using plants in an earlier phase of development. One-week-old seedlings (approximately 2 mm in height) of different Nicotiana genotypes grown on agar plates were excised below the apical meristem  in which scions tended to produce roots under these in vitro grafting and growth conditions.Micrografting efficiency varied dramatically among different species of a Figure . N. atteN. attenuata plants (chimeras are named in a shoot/root manner throughout this paper) were compared weekly to intact WT individuals and no significant differences were observed  and root  dry mass were similar in intact versus grafted plants   and seeds per capsule  (data not shown) were also not significantly different.Growth parameters of scions and rootstocks of grafted WT/WT d Figure . Rosetted Figure  as well s Figure . Compari) Figure , capsuleThe graft junction of three-week-old grafted plants was located just below the rosette  or overexpress (ov) a particular gene are the main approaches by which gene function can be studied in N. attenuata system. However, the value of micrografting is mainly limited by the transmission of signals through the graft site [TPI and PMT genes as innate reporters of the up- and down-ward spread of silencing since the endogenous expression of these target genes in N. attenuata are particularly useful for this purpose: TPI is mainly expressed in shoots and PMT in roots [ov) mRNA of a foreign gene could also be transmitted to WT scions or rootstocks. Importantly, these lines are not morphologically distinguishable from WT N. attenuata plants [The main reason for establishing a grafting method for aft site . Therefoin roots . We furta plants ,10,15, hTPI transcripts in leaves of irTPI plants . Likewise, PMT transcript levels in roots of irPMT plants , as previously characterized by Steppuhn et al. [TPI and PMT of intact plants were maintained when comparing grafted irTPI/irTPI and irPMT/irPMT plants to grafted WT/WT plants, respectively. irTPI/irTPI and irPMT/irPMT plants yield only 0.8% and 3% of TPI and PMT transcript levels of WT/WT plants, respectively . In addition, TPI transcript levels of scions of WT/irTPI plants . Roots of WT/irPMT grafts . However, the same low levels of PMT transcript .Leaves of s Figure  failed ts Figure  didn't ss Figure  expresset Figure  were fouTPS in shoot or roots of non-grafted ovTPS plants . In addition, levels of TPS transcripts in scions of WT/ovTPS and rootstocks of ovTPS/WT .Transcript levels of s Figure  didn't dT Figure  resembleN. benthamiana [N. attenuata with transgenic lines. Our data support the employment of N. attenuata grafts that combine WT genotype either to ov- or ir-transgenic lines, this latter only when used as rootstocks, given the altered transcript accumulation of WT roots promoted by silenced shoots. The motivation to adopt this well-known grafting method for N. attenuata is grounded in the several layers of inter-related plant defenses that have described in N. attenuata. Systemic signals produced upon herbivore attack enhance the indigestibility of N. attenuata leaves by augmented TPI activity [N. attenuata can change its flower opening time in order to recruit new pollinators that do not have larval stages that are herbivores of the plant and thereby reduces future herbivore loads [N. attenuata will allow us to examine the systemic signals and gene function in above- versus below-ground parts of a plant in an ecological context.As previously described for other species ,17,18, ithamiana ,20, hereactivity  and diteactivity . Systemiactivity ,22, as wactivity . In addire loads . Taken tN. attenuata plants resulted in low success rate may be due to poor callus formation in stem tissues. The hypocotyls of germinated seeds have proved to be the most reliable explant to induce meristematic activity for tissue culture and regeneration of N. attenuata [Meristematic activity in the graft junction is an important determinant of attaining high grafting success rates . The reattenuata  and thisN. attenuata is that its impact on a plant's adult life is expected to be minor because the grafting takes place in an early phase of plant development. Only five to six days after grafting, completely healed and healthy grafted seedlings are obtained  and tolduction, ) to herbirCOI1, ), synthe[irLOX3, ) or acti[irLOX3, )  of N. a irRdR3, -40) enabof genes . TherefoN. attenuata which has no available mutant libraries, represents a key tool to evaluate gene-function in the many developmental and physiological processes that are governed by long-distance signals. The ectopic expression of ov or ir constructs was restrained in WT/ov, ov/WT and WT/ir grafts. Micrografting thus represents an important advance towards organ-specific characterization of gene function and detection of currently unrecognized long-distance signals, particularly focusing on root physiology which determines the relationships between their below- and above-ground parts and the contribution of this root-shoot communication to whole-plant performance. Given the potential of this method in unraveling root and shoot interplay and the hitherto overlooked importance of roots, we envisage that this procedure will be commonly applied to the study of gene function in N. attenuata.Micrografting combined with a collection of available stably transformed lines, especially for a non-model plant such as N. attenuata seeds are derived from an inbred collection from the DI Ranch, Utah [Nicotiana sp. were kindly supplied by Dr. Verne A. Sisson  and originated from collections made by Dr. T. H. Goodspeed [TPI (Accession number: AY426751) and PMT (Accession number: AF280402) silencing in N. attenuata, inverted repeat constructs (IR) containing either a fragment of the TPI gene (irTPI) or a consensus fragment for the two N. attenuata's PMT genes (irPMT) in inverted orientation were used and characterized by Zavala et al. [TPI and PMT are found reduced to below 1% in irTPI and 18% in irPMT plants, respectively, of levels found in WT plants. Consistent with the transcript analysis, no TPI activity was detected in leaf tissues of irTPI plants while levels of nicotine were reduced to 15% in leaves of irPMT plants [TPS10-overexpressing N. attenuata plants (ovTPS) harbor a sense sequence based on the TPS10 gene of maize (Accession number: AY928078), driven by CaMV 35S promoter [TPS10 gene encodes a terpene synthase involved in the herbivory-induced production of (E)-α-bergamotene and (E)-β-farnesene which are the major components of the volatile blend emitted by attacked plants and function as attractants of herbivore's natural enemies [ir lines included in this study, no morphological or developmental differences between WT and ovTPS are observed [th until the 8th week of development and the total number of produced flowers was compared. For seed production, mature capsules were collected from plants (n = 5) over 5 days starting from the first mature capsule harvested.ch, Utah . Seeds ooodspeed . For TPIa et al.  and Stepa et al. , respectT plants . TPS10-o enemies . As for http://www.stratagene.com) and qPCR Core Kit for SYBR® Green I . Transcript levels were quantified relative to N. attenuata elongation factor 1A (NaeEF1A) and primers were designed according to Steppuhn et al. [RNA was extracted from shoots of twenty-day-old or from leaves of five-week-old plants using Tri Reagent . For roon et al.  and Zavan et al. . All reat- or normal distribution, Welch's t-test or Kruskall-Wallis test, followed by Fisher's PLSD as a post-hoc test, were performed using R-2.11.1 http://www.R-project.org or StatView5 softwares .After verifying data for The authors declare that they have no competing interests.irTPI and irPMT grafts, ITB and SK conceived the project and oversaw the research. All authors wrote, read and approved the final manuscript.VF carried out the lab work, HG helped with the Nicotiana attenuataTable 1: List of stably transformed lines of . Lines harbor sense (ov), antisense (as) or inverted repeats (ir) constructs and were created in two different accessions of natural population  [tah, Ut) -74.Click here for file


Pinus ponderosa Dougl. ex. Laws) and mixed-conifer forests of western North America. These extensive montane forests are considered to be adapted to a low/moderate-severity fire regime that maintained stands of relatively old trees. However, there is increasing recognition from landscape-scale assessments that, prior to any significant effects of fire exclusion, fires and forest structure were more variable in these forests. Biota in these forests are also dependent on the resources made available by higher-severity fire. A better understanding of historical fire regimes in the ponderosa pine and mixed-conifer forests of western North America is therefore needed to define reference conditions and help maintain characteristic ecological diversity of these systems. We compiled landscape-scale evidence of historical fire severity patterns in the ponderosa pine and mixed-conifer forests from published literature sources and stand ages available from the Forest Inventory and Analysis program in the USA. The consensus from this evidence is that the traditional reference conditions of low-severity fire regimes are inaccurate for most forests of western North America. Instead, most forests appear to have been characterized by mixed-severity fire that included ecologically significant amounts of weather-driven, high-severity fire. Diverse forests in different stages of succession, with a high proportion in relatively young stages, occurred prior to fire exclusion. Over the past century, successional diversity created by fire decreased. Our findings suggest that ecological management goals that incorporate successional diversity created by fire may support characteristic biodiversity, whereas current attempts to “restore” forests to open, low-severity fire conditions may not align with historical reference conditions in most ponderosa pine and mixed-conifer forests of western North America.There is widespread concern that fire exclusion has led to an unprecedented threat of uncharacteristically severe fires in ponderosa pine ( Pinus ponderosa Dougl. ex. Laws) and Jeffrey pine , or those mixed with ponderosa/Jeffrey-pine and other conifer species , an imperiled fire-dependent species, largely avoids previously thinned forest areas burned at high-severity Strix occidentalis caurina), a Threatened Species under the USA Endangered Species Act Pekania pennanti), a Candidate Species under the USA Endangered Species Act In recent decades, to address the widespread concerns about uncharacteristically severe fire in western North America, fuel reduction treatments have been implemented on millions of hectares of ponderosa pine and mixed-conifer forests at a cost of billions of dollars Most descriptions of the fire regimes that characterize the ponderosa pine and mixed-conifer forests of western North America e.g.,  emphasizIn contrast, mixed-severity fire regimes are characterized by more variable fire and forest structure across a wide range of spatial and temporal scales The concepts and nomenclature used to describe fire regimes in western North America can be ambiguous. Part of the problem with defining fire regimes for the drier forests of western North America is the classification of fire regimes into distinct categories of low-, mixed-, and high-severity In addition to unclear terminology, other factors create difficulties for identifying which historical (i.e. prior to fire exclusion) fire regime applies to a particular forest region. Where fire has been excluded from a mixed-severity landscape for 100 years, early- and mid-successional patches created by high-severity fire become late-successional patches, making it more likely that these patches, indicative of a mixed–severity regime, will be undetected. For example, high-severity fire patches may be detected in old but not recent aerial imagery The US Forest Service Inventory and Analysis (FIA) program provides an extensive dataset that is a probabilistic sample of forest structure in large landscapes. This dataset allows for landscape-scale inference and statistical analyses of forest age and structure parameters consistent with a low- or mixed-severity fire regime.Using the FIA data, and published sources of landscape-scale  data, our objectives were to address two broad questions: (1) How prevalent were mixed-severity fire regimes historically in ponderosa pine and mixed-conifer forests of western North America; and (2) How have mixed-severity fire patterns in these forests changed with fire exclusion? Consistent with common perceptions and restoration models applied to these forests, we hypothesized that: (1) forest age-class diversity was low, reflecting long-term effects of low/moderate-severity fire regimes ; and (2)http://www.epa.gov/wed/pages/ecoregions/level_iii_iv.htm) to define the Sierra Nevada, Klamath, and eastern Cascades regions. The Sierra Nevada was split along the distinct crest of the range into the east and west slopes. The portions of the northern Cascades east of the crest and the main Cascades within California were combined into the eastern Cascades. The Modoc Plateau and eastern Sierra Nevada was also combined with the eastern Cascades. The northern Rockies were in Idaho and Montana, and the central Rockies were in Colorado, Wyoming and South Dakota. The southwestern USA included Arizona and New Mexico.FIA and published sources of landscape-scale  data with inference to pre-settlement fire severity and forest structure were available from the following regions of western North America: Baja California, the Sierra Nevada, the Klamath Region, the eastern Cascades, the northern Rockies, the central Rockies, and the southwestern USA . We usedAbies concolor (Gord. and Glend.) Lindl.), and/or grand fir (A. grandis (Douglas ex D. Don) Lindl.). In the Sierra Nevada and Klamath regions, ponderosa pine is common and may be dominant, especially in low-elevation forests, and mixed-conifer forests generally include components of ponderosa pine, white fir, Douglas-fir, incense-cedar  Florin), sugar pine (P. lambertiana Dougl.), California black oak (Quercus kelloggii Newb.) and evergreen canyon live oak (Q. chysolepsis Liebm.). Mid-elevation forests of the Sierra Nevada and Cascades are often dominated by Jeffrey pine, ponderosa pine, white fir and sugar pine. Low- to mid-montane forests of the eastern Cascades are dominated by ponderosa pine and Douglas-fir, and can include components of white fir, grand fir (Abies grandis Dougl. ex D. Don.) Lindl.), and western hemlock (Tsuga heterophylla (Raf.) Sarg). Low- and mid-elevation forests of the Rocky Mountains are dominated by ponderosa pine and Douglas-fir. In the northern Rockies, these two dominants may co-occur with white fir and grand fir, and with western hemlock, western redcedar (Thuja plicata Donn. Ex D. Don.) and quaking aspen (Populus tremuloides). Forests of the southwestern U.S. are heavily dominated by ponderosa pine, with some white fir and Douglas-fir at middle elevations. Precipitation and temperature data for each region in this study are provided in The dominant conifer over most of the low- to mid-elevation, montane forests in these regions is ponderosa pine, often with lesser amounts of Douglas-fir, white fir  in ponderosa pine and mixed-conifer forest landscapes of western NA over a period of 70 or more years. The high-severity fire rotation is equal to the average interval between high-severity fire across the affected landscape . Additiohttp://www.fia.fs.fed.us/tools-data/) by region. These data capture the average age of the trees dominating the canopy layer in forest stands  stand ages  and crown position of each tree and the ring count from cores of the dominant and co-dominant trees  of each tree species are measured in each subplot We selected FIA data from low- to mid-elevation forest types in Wilderness, Inventoried Roadless Areas, and National Parks to ensure as best we could that stand initiation was not caused by commercial harvesting of trees or other land use . We had A small number of plots had different stand ages for different subplots due to disturbances that affected some, but not all, subplots. In FIA split-age plots where both plot ages were ≤100 years, plots were split into two stand ages by FIA if they differed by as little as 1 year. In split-age plots in which both ages were 100–199 years old, plots were split into two stand ages if they differed by as little as 2 years. In split-age plots where both ages were ≥200 years, plots were split into two stand ages if they differed by as little as 15 years. To assess the within plot variability in tree ages, we calculated the standard deviation of the trees used to age each plot. We standardized this across the range of stand ages by calculating the standard deviation of the proportional difference between stand age, and the individual trees used to determine stand age in each plot, over the range of stand ages.We reasoned that, prior to fire suppression, under a low/moderate-severity fire regime, successional, or age-class diversity, would be low, while it would be high under a mixed-severity fire regime. With fire exclusion and greater amounts of uncharacteristically severe fire the pattern should reverse in both cases (i.e. increased age-class diversity in low-severity systems and decreased diversity in mixed-severity systems). We used a Chi-square test of proportions We included only plots where there was one stand age for the full plot because we wanted to evaluate high-severity fire occurrence in patches at least 1 ha in size, rather than include smaller torching of groups of trees. Excluding the split-age plots  omits some additional evidence for local high-severity fire effects; thus our results may be conservative.We used FIA data drawn from 2001–2009, comprising 90% of available plots, in our classification of low/mid-elevation forests in the Sierra Nevada, Klamath, and eastern Cascades. In the other regions, FIA plots represented 100% of the data from low- to mid-elevation, montane forests. The number of plots in the 0–39 year age bins may be slight underestimates of the amount of high-severity fire in the last 40 years because severe fire could have occurred subsequent to the sample date . To estimate the number of plots that burned severely after the sample date, we increased the 0–39 year old bin by a factor of 40/36 in the Sierra Nevada, Klamath and eastern Cascades, 40/34 in the northern Rockies and 40/32.5 in the central/southern Rockies and southwestern USA region. The denominator in these weightings is based on 40 minus the mean amount of time in which plots in each region could have burned after being sampled.We used year of the recent fire disturbance, captured in the disturbance data field, to define the age of very young FIA plots not containing trees that could be aged in a non-destructive way . These “non-stocked” plots were relatively rare, as reported in the results, and ages, based on fire dates, all fell within the 0–39 age category. Some non-stocked stands had no disturbance coded. In California, Oregon, and Washington (Pacific states), disturbances were only coded if they were <6 years old. We placed all non-stocked plots where no disturbance was coded in the database into the 0–39 stand age bin.Next, we considered whether the age distributions as shaped by fire were consistent with mixed- or low/moderate-severity fire regimes. We reasoned that a wide range in the plot stand ages in a landscape would be consistent with age-class diversity created by mixed-severity fire, while stand ages that were evenly distributed in predominantly older age classes would be consistent with a low/moderate-severity fire regime. To test whether stand age distributions were consistent with mixed- or low/moderate-severity fire regimes, we again used a Chi-square comparison of proportions Third, we tested, again using a Chi-square comparison of proportions The studies that allow calculation of rotations of high-severity fire over large, ponderosa pine and mixed-conifer forest landscapes of western North America over time periods of at least 70 years include areas ranging from 40,700 to 1,193,200 ha . These lThe high-severity fire rotations in Numerous other studies that describe historical patterns of fire behavior also have documented or described evidence for mixed-severity fire effects in the ponderosa pine and mixed-conifer forests of western North American, including the occurrence of large high-severity fire patches , and higPrevious studies  have useA total of 2119 FIA plots representing a sample population of about 5.1 million ha of unmanaged low- to mid-elevation, montane forests in six regions , Table 5P<0.001, The comparison of actual stand ages from 1930–2009 and the rescaled (expected) stand ages from 1810–1889 assuming no effect of fire exclusion are shown in Chi-square comparisons between actual stand-age distributions at the onset of fire exclusion versus the expected stand-age distributions for a low/moderate-severity fire had exceptionally low probabilities in all regions . This was because plots were mostly dominated by young and intermediate aged trees prior to fire exclusion . The mea2 = 0.85, n = 676, P>0.6). Likewise, in the southwestern USA, 98% of ponderosa pine stands (n = 96 plots) and 92% of the non-ponderosa stands (n = 37 plots) that established in or before 1900 had stand-ages between 1800 and 1900 . However, when all plots were considered, significantly more stands established from 1800–1900 in ponderosa pine than non-ponderosa forests , indicating higher fire disturbance in pine forests.The hypothesis that mixed-severity fire prior to fire exclusion would be lower in the driest (ponderosa and Jeffrey pine) forests than other forests also was not supported. Based on stand-ages (not shown), there was as much as or more mixed-severity fire in the pine forests. In the Pacific states, we found almost identical stand-age distributions from 1800–1900 in ponderosa/Jeffrey pine stands (n = 20 plots) versus all non-ponderosa stands (n = 204 plots). Plots from the time period 1800–1900 accounted for 70% and 73%, respectively, of all plots with dominant trees that established in or before 1900. In the northern and central Rockies, 86% of ponderosa pine stands (n = 66 plots) and 81% of the non-ponderosa pine stands (n = 615 plots) that established in or before 1900 had stand-ages between 1800 and 1900  with the arrival of settlers FIA stand ages in the unmanaged forests in all regions reflect a pattern of high age-class diversity occurring prior to federal fire suppression policies and reductions in Native American burning , one in the Sierra Nevada , and one in the southwestern USA (307 ha)) that reported very little or no historical high-severity fire, and hence low-severity regimes . In contth century and early 20th century indicate much denser and more variable forest conditions  may subject virtually any conifer forest, regardless of fuel density, to crown fire The largest patch sizes of high-severity fire likely occurred during the most extreme conditions for fire behavior. While patch sizes of high-severity fire are difficult to document, it follows from commonly observed heavy-tailed distributions of patch sizes created by fire Ceanothus.” For mid-elevation forests, he reports (page 37): “Nearly all the type situated at altitudes below 7,000’ [2134 m] carries a vast amount of undergrowth. It consists mainly of manzanita [Arctostaphylos spp.], ceanothus, and scrub oak .” Similarly abundant shrub fuels were also documented historically in the westside of the central/southern Sierra Nevada There is abundant evidence that past forests may not have required extreme weather and climate for mixed-severity fire to have occurred. Younger, more flammable forests We also hypothesized, consistent with existing concerns about unprecedented fire severity in western North America e.g., , that fiThe importance of multiple lines of evidence has been stressed in determining whether mixed-severity fire regimes applied historically Our findings suggest a need to recognize mixed-severity fire regimes  as the pFor management, perhaps the most profound implication of this study is that the need for forest “restoration” designed to reduce variation in fire behavior may be much less extensive than implied by many current forest management plans or promoted by recent legislation. Incorporating mixed-severity fire into management goals, and adapting human communities to fire by focusing fire risk reduction activities adjacent to homes Table S1Evidence of historic fire severity in ponderosa pine and mixed-conifer forests of western North America. A summary of published studies and historical documents that provide evidence regarding mixed-severity fire in the ponderosa pine and mixed-conifer forests of western North America, but do not provide sufficient information to estimate high-severity fire rotations, or were conducted in smaller landscapes. Many fire scar studies have also been done in these forests, but fire scars alone are not sufficient to distinguish low- from mixed-severity regimes.(DOCX)Click here for additional data file.


Statins are increasingly prescribed to prevent cardiovascular disease (CVD) in asymptomatic individuals. Yet, it is unknown whether those at higher CVD risk – i.e. individuals in lower socio-economic position (SEP) – are adequately reached by this high-risk strategy. We aimed to examine whether the Danish implementation of the strategy to prevent cardiovascular disease (CVD) by initiating statin (HMG-CoA reductase inhibitor) therapy in high-risk individuals is equitable across socioeconomic groups.Design: Cohort study.Setting and participants: Applying individual-level nationwide register information on socio-demographics, dispensed prescription drugs and hospital discharges, all Danish citizens aged 20+ without previous register-markers of CVD, diabetes or statin therapy were followed during 2002–2006 for first occurrence of myocardial infarction (MI) and a dispensed statin prescription (N = 3.3 mill).Main outcome measures: Stratified by gender, 5-year age-groups and socioeconomic position (SEP), incidence of MI was applied as a proxy for statin need. Need-standardized statin incidence rates were calculated, applying MI incidence rate ratios (IRR) as need-weights to adjust for unequal needs across SEP.Horizontal equity in initiating statin therapy was tested by means of Poisson regression analysis. Applying the need-standardized statin parameters and the lowest SEP-group as reference, a need-standardized statin IRR > 1 translates into horizontal inequity favouring the higher SEP-groups.MI incidence decreased with increasing SEP without a parallel trend in incidence of statin therapy. According to the regression analyses, the need-standardized statin incidence increased in men aged 40–64 by 17%, IRR 1.17 (95% CI: 1.14-1.19) with each increase in income quintile. In women the proportion was 23%, IRR 1.23 (1.16-1.29). An analogous pattern was seen applying education as SEP indicator and among subjects aged 65–84.The high-risk strategy to prevent CVD by initiating statin therapy seems to be inequitable, reaching primarily high-risk subjects in lower risk SEP-groups. A steep inverse relationship between socioeconomic position (SEP) and incidence of cardiovascular disease (CVD) has consistently been shown across high-income Western countries -3. The sAs CVD is one of the leading causes of premature death in the Western world, preventive strategies are on political agendas, all focusing on the conventional risk-factors, either through their socio-cultural determinants  strategies) or through individual behaviour/risk-factors, such as the high-risk strategy to prevent CVD in general practice. In the high-risk strategy (often termed ‘primary prevention’), asymptomatic individuals are screened to determine the need for preventive interventions, such as antihypertensives or lipid lowering drugs . In the present study, we focus on statins (HMG-CoA reductase inhibitors), introduced in 1994  to, reduThe high-risk strategy has been implemented in Denmark as an opportunistic screening strategy i.e. clients who show up in the general practitioner’s (GP) office may be screened for high CVD risk for possible prescription of preventive drugs. In line with the European guidelines and the European Systematic Coronary Risk Evaluation , Danish The Danish health care system is mainly tax-financed and based on the egalitarian principle, with equal health care (access/treatment/outcome) for equal (legitimate) health care needs  – also cThere is an ongoing debate about the high-risk strategy ,27-29, eIn the present study, we focus on initiation of preventive statin therapy in the high-risk strategy as implemented in Denmark. Due to the social gradient in incidence of CVD we expect an increasing need for CVD preventive drugs with decreasing SEP i.e. unequal needs across socioeconomic groups. In line with other studies focusing on equity in health care delivery ,42, we aThe aim of this study was to examine whether the Danish implementation of the strategy to prevent CVD by initiating statin therapy in high-risk individuals is equitable across socioeconomic groups; hypothesising that this high-risk strategy will not adequately reach groups with a lower SEP, characterised by having a higher risk of CVD.From nationwide Danish registers maintained by the National Board of Health and Statistics Denmark, we retrieved individual-level information on dispensed prescription drugs, hospital discharges, dates of death or emigration, and socioeconomic indicators. Data were linked by means of a unique encrypted person-identifier, allowing authorised researchers to follow individuals in multiple individual-level registries hosted in Statistics Denmark. Register-based studies in Denmark do not require approval by an ethics board.A cohort corresponding to all Danish citizens aged 20+ without previous register markers of CVD, diabetes or statin treatment were followed, during 2002–2006, in the registries, for dispensed statin prescriptions and the first occurrence of MI (N = 3.3 mill). We applied two different SEP indicators: disposable family income and highest attained education (as of 2002). Table th revision until 1993, and 10th revision thereafter (9th revision has not been used in Denmark) along with codes for diagnostic and surgical procedures. We included main and secondary diagnoses for admitted patients and patients in ambulatory care. From the Registry of Causes of Death, we retrieved date and cause of death (primary and contributory).From the Danish National Patient Registry, we retrieved information on patient discharge from non-psychiatric hospitals since 1977. Records include the admission and discharge dates, discharge diagnoses according to the International Classification of Diseases (ICD), 8Information on dispensed prescription drugs was retrieved from the Danish National Prescription Registry (DNPR), containing full information since 1996 on all out-of-hospital purchases of prescription drugs at Danish pharmacies including those of nursing home residents . RecordsTo identify asymptomatic individuals, we applied historical register data on in/out-patient diagnoses and procedures along with dispensed prescription drugs as register-markers for a range of CVD conditions, including ischemic heart disease (IHD) with or without myocardial infarction (MI), stroke, a range of other atherosclerotic conditions, and diabetes. We define asymptomatic individuals as individuals without register-markers of CVD or diabetes, as defined in a recent publication .While measures such as the Gini-coefficient of inequality, concentration index and the slope index of inequalities provide means for quantifying the degree of for example income-related inequality in health or health care delivery , a measuDue to the high validity of the diagnosis of MI in the Danish registries , we applWe applied a fixed SEP level corresponding to the beginning of the observation period (primo 2002). In order to capture income fluctuations over time, we calculated the average annual income between 1996 and 2001 (inflated to 2000 purchasing power), divided into income quintiles within gender and age group. The highest attained educational levels as of 2002 were divided into four groups according to length of formal education, cf. Table To evaluate horizontal inequity in initiation of preventive statin therapy, we adjusted the observed incidence of statin therapy according to the different needs across SEP-groups, applying stratum specific MI-incidence as proxy for needs. By means of indirect standardisation, we calculated the expected incidence of statin therapy (‘need-standardized statin incidence’), assuming that incidence of statin therapy must increase proportionally to the need across SEP-groups - for equity to be met. The need-standardized statin incidence was calculated as the observed statin incidence divided by the stratum specific need weights corresponding to the incidence rate ratio of MI (MI-IRR), Table ‘horizontal inequity gradient’  reflecting the increase in need-standardized statin-IRR for each increase in SEP. Owing to a gender and age specific pattern of both MI-incidence (need) and incidence of preventive statin therapy, we stratified the analyses according to gender and ages +/−65, cf. Figure Based on the need-standardized statin incidence parameters (observed statin events (counts), need-standardized PYR (exposure)), Poisson regression analyses were applied to test the overall horizontal equity across SEP. With the lowest SEP-group (and age group) as reference, a need-standardized statin-IRR > 1 translates into horizontal inequity favouring the higher SEP-groups. The null hypothesis, horizontal equity, corresponds thus to statin-IRR = 1. We estimated a Nonparametric bootstrapping  was applAll analyses were performed using Stata Release 11.1 . Access to data was provided and secured through collaboration between the University of Copenhagen and Statistics Denmark. Register-based studies in Denmark do not require approval by an ethics board.Figure Table Table The HIE-gradient concerning educational level reveals a similar pattern, i.e., 19%  in men aged 30–64, and 24%  in women.Table Applying indirect standardisation and MI-incidence as a proxy for need, we developed a pharmacoepidemiological method to explore horizontal equity in initiation of preventive statin therapy across SEP-groups with unequal needs, adjusting the observed statin incidence according to relative needs across socio-demographic groups. Our study indicates that the high-risk strategy (as implemented in Denmark) to prevent CVD by initiating preventive statin therapy is inequitable, reaching primarily high-risk individuals in low-risk SEP-groups. The favouring of more advantaged groups holds for both genders, independently of applying income quintiles or educational level as SEP indicator. In men aged 30–64, the need-standardized statin incidence increased by 17% for each increase income quintile  – in women the increase was 21%.observed incidence statin therapy tended to increase with increasing SEP, but due to a steeper social gradient in MI-incidence among women, the horizontal inequity gradient of initiating preventive statin therapy was steepest in women.Only among men, the Given the inverse relationship between SEP and CVD, the challenge of this study examining equity in the medicamental high-risk strategy to prevent CVD was twofold: firstly, to operationalize need and equity in CVD preventive drug therapy across SEP-groups with unequal needs, and secondly, to develop appropriate pharmacoepidemiological methods for testing horizontal equity.We opted to apply nationwide register data on MI-incidence in the statin free and asymptomatic background population stratified by gender, age and SEP as need-proxy, instead of calculating individual-level CVD-risk based on survey information on CVD-risk factors and risk scoring,This is a strength for at least three reasons:1) The nationwide approach is without the well-known selection bias problems from cohort studies/surveys where people are invited to participate.2) The risk-score charts generally have low predictive value – both at the individual and the group level. Various cohort studies indicate that standard risk-score charts tend to underestimate CVD-risk in worse-off groups, overestimating the risk in better-off groups , and attempts have been made to modify risk-score charts according to the actual background populations [51] and/or socioeconomic groups . Yet, individual risk prediction is notoriously difficult , and as most CVD events occur in people with modest risk-factor values, overlapping with those seen in people without CVD, the appropriateness of applying individual risk-factor levels as a screening tool has been questioned . The risk-score charts applied in Denmark seems, in fact, to have a very poor predictive value [56]. In the present study we applied risk (MI-incidence) at the sub-group level  as need-proxy rather than risk at the individual level. Yet, individual risk estimates based on risk-scoring relies on risk calculated at the group level (age and gender) from historic survey data.3) Register-based information on MI-diagnoses is regarded as valid in-hospital diagnosis information [47]. Thus, the estimated MI-incidences should reveal the actual SEP gradient in MI-incidence. However, due to potential differential symptom awareness and communication barriers, a social gradient in admission for MI may exist, leading to an underestimated MI-incidence in less advantaged groups. The better-off, on the other hand, may be more likely to prevent MI by means of invasive coronary procedures, leading to a potential underestimated MI-incidence here. Yet, including acute invasive coronary procedures as marker for MI revealed the same results (data not shown). Thus, we regard this bias of minor importance. In a sensitivity analysis, stroke/MI as a combined endpoint and CVD-death were tested as two alternative need-proxies. Independently of need-measure, the sensitivity analysis demonstrated similar patterns of horizontal inequity. Yet, when applying the combined endpoint, the horizontal inequity gradient was – especially in women – attenuated, presumably due to a less pronounced social gradient in the incidence of stroke than in MI-incidence. Applying CVD-death as a need-proxy (corresponding to the risk measure applied in Danish risk-scoring charts), the horizontal inequity was enhanced. Yet the ‘validity’ of actual MI-incidence as the need-proxy rests upon the assumption that the need for statin therapy is a question of CVD-risk rather than a single risk-factor level – and partly on the finding that conventional risk-factors to a large extent mediate the social gradient in CVD-risk (in absolute terms). However, high cholesterol level has not consistently been found to increase with decreasing SEP , potentially explained by the rather imprecise cholesterol parameters applied: risk thresholds for cholesterol levels and measurement of total cholesterol without distinguishing between the various lipid-fractions. Conversely, other studies have demonstrated an association between the metabolic syndrome  and elevated low-density lipid cholesterol (LDL) [58], indicating that the inverse social gradient in LDL may follow the social stratification in physical inactivity and obesity.horizontal inequity index’  was estimated. In our pharmacoepidemiological approach, we calculated, instead, a need-standardized incidence rate of statin therapy. Applying need-standardized statin incidence parameters as a dependent variable and a SEP indicator as an ordinal explanatory variable , we estimated a horizontal inequity gradient. We consider this methodological analogy to be a strength. Yet, while it is intuitively reasonable to ‘adjust’ for differential health conditions when evaluating horizontal equity in ambulatory visits, it may be less obvious that incidence of preventive CVD-drug therapy should be proportional to the risk of disease for equity to be met.Analogous to studies within economic equity research ,22,42, winequality in prescription of CVD preventive drugs [While a range studies have demonstrated ve drugs ,59-61, nve drugs  showing ve drugs . With thve drugs , indicatve drugs . Psychosoutcome or beneficial effect of lifelong preventive statin therapy may be greater in less advantaged groups – provided adherence to therapy.In line with other studies ,65 our sVarious incentives have been proposed to enhance adherence, requiring often GPs to be more actively involved ,69. In aThe incidence of preventive statin treatment in this study was found to peak around the age of 65, and to decrease steeply hereafter. This pattern may reflect the widespread use of the risk-score charts, covering the age range of 40–65, potentially representing an issue of ageism. The finding that statin incidence is considerably higher in asymptomatic women than men although MI-incidence is higher in men  this ‘population’ strategy will be palliative and not radical as structural/population strategies tends to be [In contrasts to the opportunistic screening strategy applied in Denmark, a universal screening programme to prevent CVD is actually being implemented in the UK . Here, ads to be . Proposids to be  demonstrOur study indicates that the high-risk strategy to prevent CVD by means of preventive statin therapy – as practiced in Denmark – is inequitable, primarily reaching high-risk individuals in low-risk groups, i.e., individuals in higher SEP-groups. The inequity is likely to be the consequence of using a screening tool with low predictive value and a screening programme with differential socioeconomic uptake. Provided long-term adherence and a beneficial effect of preventive statin therapy independent of SEP, the strategy may contribute to accentuating the inverse relationship between SEP and CVD. Facing the challenges posed by an ageing population, one might question to what extent scarce GP resources should be allocated for better-off, asymptomatic individuals.SEP, Socioeconomic position; CVD, Cardiovascular disease; RCTs, Randomised clinical trials; GP, General practitioner; DNPR, Danish National Prescription Registry; ICD, International Classification of Diseases; ATC, Anatomical Therapeutic Chemical classification system; IHD, Ischemic heart disease; MI, Myocardial infarction; PYR, Person-years at risk; IRR, Incidence rate ratio; HIE gradient, Horizontal inequity gradient.The authors declare no conflicts of interest. Morten Andersen participates, however, in a research project on statins funded by AstraZeneca, Merck Sharp & Dohme and Pfizer.All authors read and approved the final manuscript.Finn Diderichsen, Allan Krasnik and Theis Lange contributed equally to the work.Helle Wallach-Kildemoes, first and corresponding author.Morten Andersen contributed as last author and principal co-author.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/610/prepub


Purpose. It is unclear whether sociocultural and socioeconomic factors are directly linked to type 2 diabetes risk in overweight/obese ethnic minority children and adolescents. This study examines the relationships between sociocultural orientation, household social position, and type 2 diabetes risk in overweight/obese African-American (n = 43) and Latino-American (n = 113) children and adolescents. Methods. Sociocultural orientation was assessed using the Acculturation, Habits, and Interests Multicultural Scale for Adolescents (AHIMSA) questionnaire. Household social position was calculated using the Hollingshead Two-Factor Index of Social Position. Insulin sensitivity (SI), acute insulin response (AIRG) and disposition index (DI) were derived from a frequently sampled intravenous glucose tolerance test (FSIGT). The relationships between AHIMSA subscales , household social position and FSIGT parameters were assessed using multiple linear regression. Results. For African-Americans, integration (integrating their family's culture with those of mainstream white-American culture) was positively associated with AIRG  and DI . For Latino-Americans, household social position was inversely associated with AIRG  and DI . Conclusions. Sociocultural orientation and household social position play distinct and opposing roles in shaping type 2 diabetes risk in African-American and Latino-American children and adolescents. Type 2 diabetes and prediabetes have emerged as significant health issues in overweight/obese African-American and Latino-American pediatric populations in the United States (US). Data from the SEARCH for Diabetes in Youth Study indicate that incidence rates for type 2 diabetes were three times higher in African-Americans and Latino-Americans aged 15–19 years compared to non-Latino whites .While several behavioral mechanisms have been proposed to explain the increased diabetes risk in African-American and Latino-American children and adolescents , 5, reseUnger et al.  have argThe limited number of studies assessing the influence of sociocultural orientation on type 2 diabetes risk suggests that, for African-Americans, integrating into mainstream American culture while retaining aspects of their own family's culture is inversely associated with diabetes risk through health-related behaviors . The litI), decreased acute insulin response (AIRG), and decreased disposition index (DI) derived from a frequently sampled intravenous glucose tolerance test. Independent of household social position, integration (combining aspects of one's family's culture with aspects of mainstream American culture) would be associated with lower diabetes risk in overweight/obese African-American children and adolescents, whereas this same sociocultural adaptive style would be associated with increased diabetes risk in Latino-Americans.Sociocultural attitudes, beliefs, and behaviors are heavily influenced by an individual's socioeconomic environment . Ethnic All participants met the following inclusion criteria: age- and gender-specific BMI ≥ 85th percentile, African-American or Latino-American ethnicity , and between the ages of 8–18 years. Prior to any testing, informed written consent and assent were obtained from the participants and parents. All studies were approved by the University of Southern California Institutional Review Board. Participants arrived at the General Clinical Research Center (GCRC) where a licensed pediatric health care provider conducted a medical/family history and physical examination which included an assessment of Tanner stage , 23. BodI, AIRG, and DI. SI was defined as the net capacity for insulin to promote the disposal of glucose and to inhibit the endogenous production of glucose. AIRG was defined as the area under the plasma insulin curve between 0 and 10 minutes. DI, an index of beta-cell function, was calculated as the product of AIRG and SI. Detailed descriptions of the FSIGT methods and protocols used in this study have been previously published [A frequently sampled insulin-modified intravenous glucose tolerance test (FSIGT) was used to assess type 2 diabetes risk . Fastingublished –28.During the GCRC visit, participants also completed a questionnaire regarding sociocultural orientation and parents of participants answered questions regarding parental educational attainment and occupational rank. Sociocultural orientation was assessed using the Acculturation, Habits, and Interests Multicultural Scale for Adolescents (AHIMSA) questionnaire . The eign = 42). The Hollingshead method has relatively good interrater agreement (67–96%) [r = 0.73–0.86) [Household social position was measured using the Hollingshead's Two-Factor Index of Social Position . The Hol(67–96%)  and corr73–0.86) , 31. t-tests, Chi-square, and ANCOVA. All analyses were performed using SPSS 18.0 .Data analysis included data summarization, Spearman correlations, and multivariate regression modeling. Data were evaluated for normality before analysis and natural log transformations were made when necessary. Our total sample size included 156 participants. Of the 156 participants, 20 participants were missing data for dietary intake, moderate-to-vigorous physical activity, and/or sedentary time and were not included in the multivariate regression analyses. Mean variable differences by ethnicity were analyzed by independent I, AIRG, and DI). Multivariate regression models were used to further explore the relationships between the independent variables  and dependent variables . Specifically, partial correlations and parameter estimates were used to describe the relationship between sociocultural and socioeconomic variables and FSIGT variables after controlling for a priori covariates. A priori covariates included sex, Tanner stage, fat mass, fat-free mass, energy intake, moderate-to-vigorous physical activity, sedentary time, household social position , and SI (for AIRG only). Statistical analyses that addressed the objectives of this study were stratified by ethnicity for the reason that both qualitative and quantitative variables were examined in this study with African-Americans using a modified version of the AHIMSA questionnaire  and Latino-Americans using the standard version. A priori significance level was set at P < 0.05. All assumptions for multiple linear regression were satisfied and FSIGT variables were log transformed in order to meet these assumptions. Data reported are means ± SE.Spearman correlations were used to explore the associations between sociocultural and socioeconomic variables  and FSIGT parameters ; however, dietary patterns were similar across ethnic groups. For biological factors, African-American adolescents were significantly taller and heavier than their Latino-American counterparts, with higher BMIs, volumes of fat mass and fat-free mass . After controlling for sex, Tanner stage, fat mass, fat-free mass, and SI (for AIRG only), fasting glucose and SI were significantly lower in African-American children and adolescents compared to Latino-Americans (both P's < 0.01). In addition, HOMA, AIRG, and DI were significantly higher in African-Americans compared to Latino-Americans . Ethnic differences were not observed in age, Tanner stage, BMI percentile, total dietary intake, and sedentary time. I  and marginalization was positively associated with log AIRG . However, these relationships were no longer significant in our multivariate regression analyses suggesting confounding of biological and behavioral covariates included in our model. For Latino-American children and adolescents, Spearman correlations revealed a negative association between integration and DI ; again this relationship was no longer significant after controlling for biological and behavioral covariates in our regression analyses. Household social position was negatively associated with DI  in Latino-Americans; this relationship remained significant in our regression analyses. Spearman correlations revealed that, for African-American children and adolescents, assimilation was positively associated with log SG and log DI. Also, log AIRG was positively associated with integration  after controlling for sex, Tanner stage, fat/fat-free mass, energy intake, moderate-to-vigorous physical activity, sedentary time, and SI. Additionally, log DI was also positively associated with integration , after controlling for covariates. From these results and with all confounding effects of covariates being equal, predicted mean AIRG was 92% higher for African-American children and adolescents at the 75th compared to 25th percentile of the AHIMSA integration subscale. Predicted mean DI was 93% higher for African-American children and adolescents at the 75th compared to 25th percentile of the AHIMSA integration subscale. Although the bivariate analysis did not reveal a significant correlation between integration, log AIRG, and log DI, once other variables were accounted for, these relationships became significant, suggesting negative confounding by covariates in our model. There were no significant relationships between household social position, other AHIMSA subscales , and FSIGT parameters. G, and DI. Moreover, log AIRG was inversely associated with household social position , after controlling for sex, Tanner stage, fat/fat-free mass, energy intake, moderate-to-vigorous physical activity, sedentary time, and SI. In addition, DI was inversely associated with household social position , after controlling for biological and behavioral covariates. To better understand which of the two socioeconomic indicators measured was driving the inverse relationship between household social position and diabetes risk, we also calculated parameter estimates and partial correlations for educational attainment, occupational rank, log AIRG, and DI. These analyses revealed that log AIRG and DI were significantly associated with parental educational attainment , whereas the associations between parental occupational rank, log AIRG, and DI were nonsignificant (data not shown). From these results and with all covariates being equal, predicted mean AIRG was 129% lower for Latino-American children and adolescents at the 75th compared to 25th percentile of parental education. The model for DI contained an intercept of 3600 × 10−4 min−1 and a parameter estimate of −189.6 × 10−4 min−1 for every one-unit increase in parental education. From these results and with all covariates being equal, DI was 31% lower for Latino-American children and adolescents at the 75th compared to 25th percentile of parental education. There were no significant relationships between AHIMSA subscales and FSIGT parameters.G) in response to insulin resistance. This nonlinear hyperbolic relationship between sensitivity and secretion is best described as DI [G and DI typically represent an ability to compensate for insulin resistance in order to maintain normal glucose tolerance . In contrast, lower AIRG and DI represent an inability of the pancreas to secrete enough insulin at a given level of insulin resistance where impaired glucose tolerance may arise . Indeed, our laboratory has shown both increased AIRG and DI as potential compensatory mechanisms for decreased SI in minority children and adolescents [G and DI). These relationships remained significant after adjusting for household social position and other behavioral and biological covariates. In contrast, household social position was positively associated with type 2 diabetes risk in Latino-American children and adolescents (via decreased AIRG and DI). Taken together, these findings suggest that sociocultural factors may be important predictors of type 2 diabetes risk in overweight/obese African-American children and adolescents whereas socioeconomic factors, rather than culture, may be more important for Latino-Americans.Pancreatic beta-cells have the ability to increase insulin secretion , independent of household social position, physical activity, sedentary time, dietary intake, sex, Tanner stage, and fat/fat-free mass.African-Americans are a heterogeneous ethnic group who vary in the extent to which they both retain their black-American culture and also adopt aspects of white-American culture . PreviouAn association between culture and type 2 diabetes risk, independent of physical activity and diet, is plausible, given what is known about the physiological mechanisms linking psychosocial stress to insulin resistance and subsequent type 2 diabetes risk via hypothalamic-pituitary-adrenal axis activation , 36. In Many more researchers have investigated the influence of sociocultural factors on diabetes risk in Latino-Americans . The infA protective effect of socioeconomic position and educational attainment in particular on type 2 diabetes risk has been well established among adults and non-Latino whites ; howeverG, and DI in African-Americans as well as significant associations between household social position, AIRG, and DI in Latino-Americans. Thus, our findings may be an underestimation of the true effect of sociocultural orientation and household social position on type 2 diabetes risk in overweight/obese African-American and Latino-American children and adolescents. Nevertheless, additional research examining these relationships in a larger, more homogenous sample may better elucidate the role of sociocultural and socioeconomic factors in shaping type 2 diabetes risk in overweight/obese ethnic minority pediatric populations.Several limitations of this study should be noted. First, data limitations precluded analysis of other factors known to influence diabetes risk in this analysis including genetic admixture , smokingG and DI). For Latino-Americans, increased diabetes risk was independently associated with increased household social position, higher parental education in particular, via decreased AIRG and DI. Future research should continue to examine these factors over time to better understand the relationships between the sociocultural orientation, household social position, and type 2 diabetes risk in overweight/obese African-American and Latino-American children and adolescents. Moreover, behavioral interventions and public policies are needed to better address sociocultural and socioeconomic factors associated with type 2 diabetes risk in ethnic minority pediatric populations.In summary, sociocultural orientation and household social position appear to play distinct and opposing roles in type 2 diabetes risk in overweight/obese African-American and Latino-American children and adolescents. For African-Americans, maintaining a sense of their own family's culture while integrating into mainstream white-American society was independently associated with decreased diabetes risk (as represented by increased AIR


Background. We obtained pilot data to examine the clinical and neuroendocrine effects of short-term mifepristone treatment in male veterans with PTSD. Methods. Eight male veterans with military-related PTSD completed a randomized, double-blind trial of one week of treatment with mifepristone (600 mg/day) or placebo. The primary clinical outcome measures were improvement in PTSD symptoms and dichotomously defined clinical responder status as measured by the CAPS at one-month follow-up. Additional outcome measures included self-reported measures of PTSD symptom severity, CAPS-2 symptom subscale scores, and morning plasma cortisol and ACTH levels. Results. Mifepristone was associated with significant improvements in total CAPS-2 score. At one-month follow-up, all four veterans in the mifepristone group and one of four veterans in the placebo group achieved clinical response; three of four veterans in the mifepristone group and one of four veterans in the mifepristone group remitted. Mifepristone treatment was associated with acute increases in cortisol and ACTH levels and decreases in cytosolic glucocorticoid receptor number in lymphocytes. Conclusions. Further controlled trials of the effects of mifepristone and their durability are indicated in PTSD. If effective, a short-term pharmacological treatment in PTSD could have myriad uses. Within the last decade, two selective serotonin reuptake inhibitors (SSRIs), sertraline and paroxetine, have been approved by the FDA for the treatment of posttraumatic stress disorder (PTSD). In studies of civilian trauma survivors, each has been superior to placebo in achieving clinical response and reducing core PTSD symptoms, which represents a significant advance. However, their use as PTSD monotherapy has significant limitations. Even in the most favorable studies, some key symptoms are resistant to treatment , remission is uncommon, and continuous treatment is often necessary to prevent relapse. Additionally, SSRIs have been shown to be largely ineffective in combat veterans with PTSD. There is a need to develop better pharmacological treatments that specifically target PTSD symptoms and/or pathophysiology. Given the abundant evidence for hypothalamic-pituitary adrenal (HPA) axis dysregulation in PTSD, we performed a preliminary study to examine whether mifepristone—which recalibrates the HPA axis through peripheral and central mechanisms—could be of therapeutic benefit.Mifepristone is a selective antagonist of glucocorticoid receptors (GRs) that induces increases in cortisol and adrenocorticotropin hormone (ACTH) levels through blockade of cortisol's feedback inhibition of the HPA axis. At high doses, mifepristone has central effects , 2. MifePreliminary data suggest that short-term administration of high doses of mifepristone can have beneficial effects in other neuropsychiatric conditions , 8–10, bMale veteran outpatients who met DSM-IV criteria for chronic military-related PTSD and who were without a history of adrenal insufficiency (or morning plasma cortisol <5 mcg/dL), diabetes mellitus or endocrinopathy, a history of moderate to severe traumatic brain injury, stroke, or other neurological illness, a history of schizophrenia, schizoaffective disorder, or bipolar disorder, current suicidal ideation, or a known allergy to mifepristone were eligible to participate. Veterans who were taking oral corticosteroids, receiving specialized trauma-focused psychotherapy, or who were unwilling to use effective contraceptive methods during the study were excluded. Veterans receiving psychotropic medications for PTSD were eligible to participate if they had been stabilized at a therapeutic dose for a minimum of six weeks prior to randomization. The protocol was approved by the Institutional Review Board of the James J. Peters VA Medical Center. Written informed consent and HIPAA authorization were obtained from all participants prior to the initiation of any study procedures. Participants underwent a complete diagnostic assessment, including a medical history, physical examination, laboratory testing, and administration of the Clinician Administered PTSD Scale (CAPS) and the Structured Clinical Interview for DSM-IV (SCID). Questionnaires were administered to assess combat exposure severity and characteristics of deployment (combat exposure questionnaire (CEQ)), childhood trauma (childhood trauma questionnaire (CTQ)), and symptoms of depression (beck depression inventory (BDI)) and posttraumatic stress (PTSD checklist (PCL)). Eligible veterans were randomized to receive mifepristone (600 mg/day) or matched placebo for one week (7 days), to be taken at bedtime. The randomization log was maintained by the VA pharmacy, and both the participant and research staff were blinded to the treatment assignment. Clinical assessments were performed at baseline, treatment endpoint, and four weeks after drug discontinuation. Vital signs, adverse events, and concomitant medications were obtained at every visit to monitor safety. The primary clinical outcome measures were severity of PTSD symptoms, as measured by the total severity score of the CAPS-2, and dichotomously defined clinical responder status. The liberal responder criterion, defined as a 12-point or greater reduction in total CAPS-2 score, reflects reliable clinical changes in veterans with PTSD. The more conservative criterion, a 30% or greater reduction in total CAPS-2 score, is a frequently used definition in PTSD clinical trials. Secondary clinical outcome measures included self-reported symptoms as measured by the PCL and the BDI, and the three PTSD symptom subscale scores from the CAPS-2 . Dichotomous remitter status, defined as no longer meeting diagnostic criteria for PTSD at four-week follow-up, was also explored.50-DEX was used to measure mifepristone's antagonism of peripheral GR sensitivity [Morning plasma cortisol and ACTH levels and GR binding per cell were measured as indicators of the magnitude of mifepristone's effects on negative feedback inhibition. The lysozyme ICt-tests for continuous variables and chi-square analysis for categorical variables. Changes within groups for the primary and secondary outcome measures were analyzed using a series of paired samples t-tests, pairing initial rating and endpoint or four-week follow-up. For between groups-analysis, changes in primary and secondary outcome measures were analyzed using independent samples t-tests. The Mann-Whitney U test, which compares the sum of the ranks and is less likely to be influenced by outliers, was used to assess whether findings from the independent samples t-tests remained significant when nonparametric methods were employed. The dichotomous clinical response and remitter status variables were analyzed using chi-square analysis. All tests were two sided with statistical significance set at P < 0.05. Measures of central tendency and variability (mean and SD) were calculated at treatment baseline, endpoint, and four-week follow-up for all continuous primary and secondary outcome measures. Baseline comparisons of group differences were conducted using independent samples n = 8), the mean (SD) age was 48.88 (12.68) years (range 26–63 years). With respect to race and ethnicity, 12.5% were Caucasian, 75% were African American, and 12.5% chose not to identify their race; 25% were Hispanic. The mean level of education was 13.5 (2.39) years. Four participants served in the Vietnam or post-Vietnam era, and four participants were Operation Iraqi Freedom/Operation Enduring Freedom (OIF/OEF) veterans. The mean score on the CEQ at screening was 55.88 (12.26), indicating moderate exposure to combat stress. As shown in  Thirteen veterans were enrolled in the clinical trial; three were lost to follow-up or found to be ineligible, one withdrew prior to randomization, nine were randomized, and eight completed study procedures. For completers (P = 0.465). At one-month follow-up, all four veterans in the mifepristone group and one of four veterans in the placebo group achieved clinical response (P = 0.028). Similar improvements were observed using the clinical response definition of a 30% change in total CAPS-2 score; three of four veterans in the mifepristone group achieved clinical response at treatment endpoint compared to one of four in the placebo group (P = 0.157). At one-month follow-up, two of four veterans in the mifepristone group and no veterans in the placebo group maintained clinical response (P = 0.102). With respect to remitter status, one of four veterans in the placebo group and three of four veterans in the mifepristone group no longer met diagnostic criteria for PTSD at four-week follow-up (P = 0.157). Based on the clinical response definition of a 12-point or greater decrease in total CAPS-2 score from initial evaluation, three of four veterans in the mifepristone group and two of four veterans in the placebo group achieved clinical response at treatment endpoint (P = 0.047) at one-month follow-up when compared to the placebo group. Using the Mann-Whitney U test, our findings were similar to those obtained using parametric methods, with significant improvement in total CAPS score (P = 0.021) across groups from screening to four-week follow-up. The mifepristone group also had a significantly greater improvement in CAPS-2 avoidance symptom scores at four-week follow-up when analyzed with both parametric (P = 0.027) and nonparametric (P = 0.028) methods. Within-groups analysis showed a significant decrease in total CAPS-2 score from initial evaluation to four-week follow-up (P = 0.039) in the mifepristone group; this included significant decreases in intrusive symptom score (P = 0.001) and avoidance symptom score (P = 0.008). There were no significant changes in total CAPS-2 score or CAPS-2 symptom subscale scores within the placebo group. The mifepristone group also had a significant decrease in PCL score at four-week follow-up (P = 0.036). There were no significant differences between groups for improvement in PCL or BDI scores.Clinical data are shown in P = 0.007), ACTH (P = 0.009), and GR binding (P = 0.041) from baseline to treatment endpoint were observed within the mifepristone group. There were no significant biological changes in the placebo group, and neither group showed significant changes in lysozyme IC50-DEX. Between-groups analysis revealed significant differences in changes in cortisol (P = 0.001), ACTH (P = 0.009), and GR binding (P = 0.003) from baseline to treatment endpoint. Nonparametric tests also found significant differences between groups for the change in cortisol (P = 0.021) and ACTH (P = 0.021) from baseline to treatment endpoint. There were no significant changes within groups or between groups for cortisol, ACTH, GR binding, or lysozyme IC50-DEX at one-month follow-up. Neuroendocrine outcomes are also presented in  No side effects, adverse events, or serious adverse events were reported by participants in either treatment condition.This is the first study to examine mifepristone in PTSD. In these preliminary data, mifepristone was significantly more effective than placebo based on the primary outcomes of clinical responder status and PTSD symptom severity. Rates of remission were also higher in the mifepristone group than the placebo group. Interestingly, these beneficial effects were more marked at four-week follow-up than at treatment endpoint, suggesting that the results do not merely reflect an acute change in neurohormonal activity. The precise central mechanism of action of mifepristone is not known.Indeed, mifepristone induced acute increases in cortisol and ACTH levels, as would be predicted by blockade of negative feedback inhibition. To our knowledge, this is the first study that also demonstrates changes in glucocorticoid receptor number in lymphocytes with mifepristone. Since cytosolic GR declined substantially following mifepristone administration, the data raise the possibility that the acute increase in cortisol levels may have increased binding to cytosolic GR, resulting in greater translocation to cell nuclei and enhanced glucocorticoid signaling. At four-week follow-up, the acute neurohormonal changes had resolved, but beneficial clinical effects were evident. These findings may reflect some form of recalibration of the HPA axis; mifepristone also impairs consolidation of fear memory  and may The results of this pilot study are limited by the small sample size and all the male veteran sample. Additionally, half of all study participants were veterans of the Vietnam or post-Vietnam era; these veterans may have had PTSD of a much longer duration when compared to OIF/OEF veterans, which may confound the results of the study. However, this clinical trial had a high retention rate and no adverse events or complaints about study procedures, suggesting that mifepristone treatment is safe, tolerable, and acceptable to veterans with PTSD, as has been found in other populations. The positive findings of this pilot clinical trial provide an important proof of the concept that treatment with mifepristone is a feasible strategy for improving clinical outcomes in veterans with PTSD; accordingly, further study is indicated. Should this neuroendocrine agent yield evidence of efficacy in PTSD, there are many potential uses which could be explored: as single or intermittent monotherapy in patients reluctant to take long-term medications, as an adjunct to pharmacotherapy, psychotherapy, or other standard treatments, or as prophylaxis in acutely traumatized persons.Additional funding is needed to conduct a larger, multisite trial of mifepristone in veterans with PTSD to determine if this medication is an effective psychopharmacological treatment option for PTSD.


Whole-cell models promise to accelerate biomedical science and engineering. However, discovering new biology from whole-cell models and other high-throughput technologies requires novel tools for exploring and analyzing complex, high-dimensional data.We developed WholeCellViz, a web-based software program for visually exploring and analyzing whole-cell simulations. WholeCellViz provides 14 animated visualizations, including metabolic and chromosome maps. These visualizations help researchers analyze model predictions by displaying predictions in their biological context. Furthermore, WholeCellViz enables researchers to compare predictions within and across simulations by allowing users to simultaneously display multiple visualizations.WholeCellViz was designed to facilitate exploration, analysis, and communication of whole-cell model data. Taken together, WholeCellViz helps researchers use whole-cell model simulations to drive advances in biology and bioengineering. Mycoplasma genitalium[Whole-cell computational models promise to predict how complex cellular behaviors such as growth and replication arise from individual molecules and their interactions. Recently, we developed the first whole-cell model of a single cell, the Gram-positive bacterium enitalium. The modenitalium.However, the large number of whole-cell model predictions – over 50 billion data points in a typical dataset – makes directly analyzing the predictions time consuming and cumbersome. Furthermore, directly analyzing the model’s predictions requires deep knowledge of mathematical modeling, computer programming, and the unique data structures used to represent the model’s predictions.Data visualization software is critically needed to help researchers realize the full potential of whole-cell models by enabling researchers to more quickly and efficiently analyze whole-cell model simulations. We developed WholeCellViz to enable researchers to easily visualize whole-cell model predictions. WholeCellViz provides researchers interactive animations as well as time series plots to easily explore whole-cell model predictions. Furthermore, WholeCellViz facilitates comparisons within and across simulations by enabling researchers to view grids of animations and plots.Halobacterium energy transduction by visualizing its protein interaction network [Drosophila development [Interactive data visualization is becoming increasingly important as biological data continues to grow in complexity and volume. Data visualization can help scientists identify subtle patterns in large data sets leading to important scientific findings. For example, Lum et al. used Iris to visualize genetic data from 272 breast cancer patients . Baliga  network . Pathway network . Chang e network ,7. Multeelopment ,9.Here we describe WholeCellViz’s implementation, features, and visualizations. We also provide two examples of how WholeCellViz can be used to analyze whole-cell model predictions.WholeCellViz is composed of a web-based front-end application and a back-end web server. The front-end displays visualizations to the user. The back-end server stores over 2 TB of simulation data using a combination of a MySQL relational database and JSON (JavaScript Object Notation) files, and sends this data to the front-end as requested by the user. WholeCellViz was developed as a web application in order to enable platform independence, simple installation, instant developer updates, and data streaming.Our whole-cell model software stores the predicted values of each biological variable at each time point using a set of MATLAB data files. We converted this data into the JSON format using custom Python scripts. We stored the metadata for each simulation, and the label and units for each data point in the database. The WholeCellViz front-end requests metadata and JSON file(s) from the back-end server as needed to display visualizations.http://jquery.com) to implement event handling, animations, and AJAX calls.The WholeCellViz front-end was implemented in HTML5 and JavaScript using the native canvas to maximize performance. We used JQuery  plotting library. We used the JQuery and JQuery UI (http://jqueryui.com) libraries to implement WholeCellViz’s grid layout and animation controls.We developed the time series plots using the Flot  storing their simulations on their own server using the hybrid MySQL/JSON format described here, and (3) editing the back-end server URL configuration option from the WholeCellViz front-end. Researchers can achieve this either by installing the whole-cell model and WholeCellViz software on their own machine or by using our Linux virtual machine which contains both the whole-cell model and WholeCellViz software (see below for more information about availability). In the future, we also plan to enable researchers to configure and run whole-cell simulations through a simple graphical interface within WholeCellViz. However, this will require the development of more computationally efficient whole-cell model simulations.To date only one whole-cell model has been developed. Consequently, we chose to focus WholeCellViz on the over 3,000 Overall, whole-cell modeling is an emerging field that has the potential to accelerate the pace of biological discovery and enable rational bioengineering and personalized medicine. Data visualization software such as WholeCellViz is critically needed to help researchers access, explore, and analyze complex, high-dimensional whole-cell model simulations, as well as to accelerate model-driven biological discovery. With the current influx of big data in research and industry, WholeCellViz also serves as an example of how to use animation for scientific communication. We anticipate that WholeCellViz will play a critical role in realizing the full potential of whole-cell models.Project name: WholeCellVizProject home page:http://wholecellviz.stanford.eduOperating system(s): Platform independentProgramming language: HTML, JavaScript, PHPOther requirements: Web browserLicense: MIT licenseAny restrictions to use by non-academics: Nonehttp://wholecellviz.stanford.edu. The hosted version visualizes the over 3,000 simulations described in Karr et al., 2012 [http://simtk.org/home/wholecell. A Linux virtual machine containing the whole-cell model and WholeCellViz server and client software is also available at SimTK.WholeCellViz is available under the MIT license at AJAX: Asynchronous javascript and XML; ATP: Adenosine triphosphate; dNTP: Deoxynucleotide triphosphate; HTML: Hypertext markup language; JSON: Javascript object notation; oriC: Origin of replication; PHP: PHP: hypertext preprocessor; SVG: Scalable vector graphics; TB: Terabyte; URL: Uniform resource locator; XML: Extensible markup language.The authors declare that they have no competing interests.RL and KR contributed equally to the conception and development of WholeCellViz. MC supervised the project. All authors wrote and approved the final manuscript.


In recent years, numerous new targets have been identified and new experimental therapeutics have been developed. Importantly, existing non-cancer drugs found novel use in cancer therapy. And even more importantly, new original therapeutic strategies to increase potency, selectivity and decrease detrimental side effects have been evaluated. Here we review some recent advances in targeting cancer. In 1977, Andrzej “Andrew” V. Schally won Nobel Prize in medicine for his research into peptide hormone production in the brain. He described the neurohormone GnRH and other releasing hormones (RH). As initially unexpected application, agonists and antagonists of these hormones have become investigational anti-cancer agents . As furtYet, this is only one of hundreds examples for new therapeutic targets and new types of drugs that have been developed recently in cellular and animal models. Searche for new targets has continued with many promising lead compounds identified -38.Among promising targets are cancer stem cells -42, micrTargeting the androgen receptor also has also shown significant progress -103. An Among numerous new experimental therapeutics, a small-molecule inducer of polyploidy, R1530, interferes with tubulin polymerization, leads to abortive mitosis, endoreduplication and polyploidy. In the presence of R1530, polyploid cancer cells underwent apoptosis or became senescent which translated into potent in vitro and in vivo efficacy. Normal proliferating cells were resistant to R1530-induced polyploidy thus supporting the rationale for cancer therapy by induced polyploidy. BubR1 plays a key role in polyploidy induction by R1530 and could be exploited as a target for designing more specific polyploidy inducers . VosaroxThere were several developments in targeting p53 and its cousins, p73 and p63 -121. NutSalmonella Typhimurium [hsp70 gene. A combination of non-toxic concentrations of quinacrine and proteotoxic stress inducers resulted in rapid induction of apoptosis in cancer cells. Therefore, quinacrine, a non-toxic drug long used for treatment of malaria, has significant clinical potential in cancer therapy [There was continued development of new ways of drug delivery including liposomes and nanoparticles , 127-130himurium -133 and himurium . Several therapy , 135-137 therapy . Also it therapy .Aerobic glycolysis, characterized by high glucose uptake, low oxygen consumption and elevated production of lactate, is associated with a survival advantage and is a hallmark of cancer. Targeting key metabolic enzymes involved in glycolysis may provide a novel therapeutic approach -145.There was also further development of the concept of synthetic lethality -149. SynIntriguingly, activation of mTOR converts arrest caused by p53 into senescence -156. AndThere was also further development of the concept of protection of normal cells . Pre-treAs a side effect, CPT-11 can cause severe diarrhea caused by symbiotic bacterial beta-glucuronidases that reactivate the drug in the gut. The strategy was suggested to target these enzymes without killing the bacteria essential for human health. Bacterial beta-glucuronidase inhibitors were identified, which have no effect on the orthologous mammalian enzyme. Inhibitors were effective against the enzyme target in living bacteria, but did not kill the bacteria or harm mammalian cells. Oral administration of an inhibitor protected mice from CPT-11-induced toxicity . In anotIn addition to quinacrine, other well known, non-toxic drugs are under re-development for cancer therapy. One of them is metformin, an anti-diabetic drug -186. SevThus there have been significant advances to the targeting of various cancers, both with selective inhibitors and with drugs such as rapamycin and metformin which have been used to treat organ transplant patients and diabetics respectively. Further studies will continue to evaluate the effectiveness of targeting the various pathways mentioned in this review with signal transduction inhibitors, natural products, chemotherapeutic drugs and drugs used for different medial purposes either by themselves or in various intelligent combinations based on the knowledge of the critical signal transduction pathways altered in the particular cancer cell.


Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) have emerged in high-HIV-prevalence settings, which generally lack laboratory infrastructure for diagnosing TB drug resistance. Even where available, inherent delays with current drug-susceptibility testing (DST) methods result in clinical deterioration and ongoing transmission of MDR and XDR-TB. Identifying clinical predictors of drug resistance may aid in risk stratification for earlier treatment and infection control.We performed a retrospective case-control study of patients with MDR (cases), XDR (cases) and drug-susceptible (controls) TB in a high-HIV-prevalence setting in South Africa to identify clinical and demographic risk factors for drug-resistant TB. Controls were selected in a 1∶1∶1 ratio and were not matched. We calculated odds ratios (OR) and performed multivariate logistic regression to identify independent predictors.We enrolled 116, 123 and 139 patients with drug-susceptible, MDR, and XDR-TB. More than 85% in all three patient groups were HIV-infected. In multivariate analysis, MDR and XDR-TB were each strongly associated with history of TB treatment failure  and hospitalization more than 14 days . Prior default from TB treatment was not a risk factor for MDR or XDR-TB. HIV was a risk factor for XDR , but not MDR-TB. Comparing XDR with MDR-TB patients, the only significant risk factor for XDR-TB was HIV infection .In this high-HIV-prevalence and drug-resistant TB setting, a history of prolonged hospitalization and previous TB treatment failure were strong risk factors for both MDR and XDR-TB. Given high mortality observed among patients with HIV and drug-resistant TB co-infection, previously treated and hospitalized patients should be considered for empiric second-line TB therapy while awaiting confirmatory DST results in settings with a high-burden of MDR/XDR-TB. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) continue to emerge in high HIV prevalence settings, and their mortality in HIV co-infected patients remains high Reliable assessment of a patient's risk of drug-resistant TB can enable targeting of DST where resources are limited. Further, it can aid clinicians in identifying patients for early initiation of second line anti-tuberculosis drugs while awaiting DST results. Indeed, an approach of early, aggressive management of drug-resistant TB has been shown to contribute to good patient outcomes and higher cure rates While several studies have shown previous TB treatment to be a risk factor for MDR TB We undertook a case-control study in a community with a high prevalence of HIV and drug-resistant tuberculosis in rural South Africa to assess clinical predictors of multi-drug resistant and extensively drug-resistant tuberculosis.The study protocol was approved by the institutional review boards of the Albert Einstein College of Medicine, Yale University, and University of KwaZulu-Natal, and by the KwaZulu-Natal Department of Health. The data used in this study were collected as part of routine medical care in the hospital clinical chart. As these were simply for clinical care, patients were not asked to give informed consent at the time of these clinical encounters. For the purposes of this retrospective study, the requirement for informed consent was waived by the ethics committees listed above, since all data used were previously collected during the course of routine medical care and did not pose any additional risks to the patients.This study was conducted from June 2005 to January 2007 in Tugela Ferry, South Africa, a rural community of approximately 200,000 Zulu people. The case notification rate of tuberculosis in this community is over 1,100 per 100,000 population; MDR and XDR TB incidence were 118 and 72 per 100,000, respectively, in 2007 During the study period, culture and DST were available for clinicians to order for any tuberculosis suspect, and clinicians were encouraged to obtain these tests for all tuberculosis suspects. This practice differed from other district hospitals in South Africa, and from that recommended by national policy, which recommended restriction of culture and drug-susceptibility testing to TB patients who were failing their current first-line regimen or who were re-treatment cases Mycobacterial culture was performed at the provincial TB reference laboratory using both liquid -960 system) and solid media (Middlebrook 7H10), as previously described We selected all patients with MDR and XDR TB diagnosed in outpatient clinics or inpatient wards at COSH for whom complete medical records were available for inclusion as cases in the study. A control group of patients with drug-susceptible (DS) TB was selected to achieve a 1∶1∶1 ratio of DS: MDR: XDR TB through review of consecutive patients listed in the TB DOTS office register diagnosed during the same period as cases. In order to fully examine potential risk factors for drug-resistant tuberculosis, patients were not matched by any demographic or clinical variables.We collected data from hospital medical records, TB DOTS clinic records, and HIV clinic records. Variables of interest included age, sex, HIV status, CD4 cell counts, antiretroviral therapy (ART) use, extrapulmonary TB, presenting vital signs and laboratory parameters, TB treatment history, and hospitalization at COSH (for any reason) history.We compared clinical and demographic variables of patients with DS, MDR and XDR TB by Fisher's exact test for categorical variables and the Student's t-test or Mann-Whitney U test for continuous variables as guided by normality of the data. All three groups were compared against each other (i.e. the MDR TB group and XDR TB group were separately compared against the DS TB group and compared against one another in two-way comparisons).We calculated odds ratios (OR) and adjusted odds ratios (aOR) using bivariate and multivariable logistic regression to assess independent predictors of MDR TB or XDR TB compared with DS TB controls, as well as XDR TB compared with MDR TB. A cutoff criteria of p-value <0.2 on bivariate analysis was used to select variables for inclusion in the multivariate models comparing MDR TB and XDR TB with DS TB (models 1 and 2 respectively). We performed an Allen-Cady modified, backward selection procedure with gender and age included by default in all models Medical records were reviewed for 116, 123 and 139 patients with DS, MDR and XDR TB, respectively, diagnosed between June 2005 and January 2007. MDR TB patients were resistant to a mean of 2.9 drugs (SD 0.6); XDR TB patients were resistant to a mean of 5.3 drugs (SD 0.8). The most common drug resistance pattern for MDR TB isolates was resistance to isoniazid, rifampicin, and streptomycin ; the majority of XDR TB isolates were resistant to all six drugs tested: isoniazid, rifampicin, streptomycin, ethambutol, ciprofloxacin and kanamycin . There were no differences in the median age of patients in all three groups . Female The majority of patients with MDR (75%) and XDR TB (69%) had been previously treated for drug-susceptible tuberculosis, whereas only 27% of patients with DS tuberculosis had been treated previously  than those with MDR TB  or DS TB . There were no significant differences in median CD4 cell count among the drug resistance groups, though there was a trend towards lower CD4 cell counts among patients with MDR or XDR TB. Among HIV-infected patients, those with MDR and XDR TB were more likely to be receiving antiretroviral therapy at the time of their drug-resistant TB diagnosis .In multivariable analysis comparing MDR or XDR TB cases with DS TB controls, MDR and XDR TB were strongly associated with a history of TB treatment failure  and hospitalization more than 14 days .Recent global data have shown rising rates of drug-resistant TB in sub-Saharan Africa, the region also suffering from the world's highest burden of HIV/AIDS Prior tuberculosis treatment is a well-established risk factor for drug-resistant tuberculosis Our study also identified hospitalization as an important risk factor for MDR and XDR tuberculosis, with prolonged hospitalization associated with a 3- to 6-fold increase in risk, respectively. This finding further supports nosocomial transmission as a likely driver of the epidemic in this setting, consistent with previous studies from our site There has been conflicting evidence about whether HIV is an independent risk factor for primary or acquired drug-resistant tuberculosis Though the numbers were small, an unexpected finding in our study was a significantly higher rate of antiretroviral therapy (ART) use among patients with MDR and XDR TB. This may be explained by the higher rate of previous TB treatment among MDR and XDR TB patients  as all TB patients are routinely tested for HIV in Tugela Ferry and initiated on ART within the first few months of treatment. Another possible explanation is that HIV patients receiving ART may have been exposed to other undiagnosed cases of MDR or XDR TB while attending clinic visits. A recent active TB case-finding study at our site identified high rates of undiagnosed MDR and XDR TB among patients attending the HIV clinic . Further molecular epidemiology studies are needed to better define transmission patterns in hospital, clinic, and community settings in Tugela Ferry. Nonetheless, improved efforts to actively screen for TB and strengthen infection control in HIV care and treatment facilities are needed to avoid undermining gains achieved by ART roll-out programs Few studies have compared patients with MDR and XDR TB to determine whether they differ in clinical or demographic characteristics. A report from Estonia, a country with low HIV prevalence, found that they shared common predictor variables in comparison to drug-susceptible TB, though direct comparisons were not made These data must be interpreted in the context of the study design and setting. First, this was a hospital-based study and may reflect severity of disease and risk factors for TB patients diagnosed at a hospital or at hospital-based clinics. Second, the prevalence of MDR and XDR TB is higher in Tugela Ferry than that reported from other districts in South Africa. The contribution of various risk factors – such as hospitalization – is likely closely related to the prevalence of drug resistance in the hospital and community. However, the more widespread use of culture and DST in Tugela Ferry provides a more representative sample of the true MDR and XDR TB epidemic. As other high HIV prevalence communities undertake similarly representative surveys, as recently performed in Khayelitsha, South Africa, high drug-resistant TB prevalence rates, similar to ours, may be uncovered Third, because DST results from the first TB episode were not available on most re-treatment patients in our study, it is not possible to know whether previous TB treatment was a risk factor due to acquisition of drug-resistance or exogenous infection (or re-infection) with drug-resistant strains. However, our prior studies have found exogenous re-infection with drug-resistant strains to be an important mechanism in this setting Lastly, we did not evaluate certain demographic and clinical variables identified in other studies as risk factors for MDR or XDR TB – such as homelessness, alcohol use, and imprisonment Although there has been renewed enthusiasm for the development and roll-out of low-cost, rapid drug-susceptibility tests for TB, the majority of patients in resource-limited settings do not have access to these new tests or even conventional drug-susceptibility testing, even in settings such as South Africa with a relatively advanced laboratory infrastructure