The gold standard to identify rejection is endomyocardial biopsy. This technique is complex, cumbersome and requires a lot of expertise in the correct interpretation of stained biopsy sections. Traditional histopathology cannot be used actively or quickly during cardiac interventions or surgery. Our objective was to develop a stain-less approach using an emerging technology, Fourier transform infrared (FT-IR) spectroscopic imaging to identify different components of cardiac tissue by their chemical and molecular basis aided by computer recognition, rather than by visual examination using optical microscopy. We studied this technique in assessment of cardiac transplant rejection to evaluate efficacy in an example of complex cardiovascular pathology. We recorded data from human cardiac transplant patients’ biopsies, used a Bayesian classification protocol and developed a visualization scheme to observe chemical differences without the need of stains or human supervision. Using receiver operating characteristic curves, we observed probabilities of detection greater than 95% for four out of five histological classes at 10% probability of false alarm at the cellular level while correctly identifying samples with the hallmarks of the immune response in all cases. The efficacy of manual examination can be significantly increased by observing the inherent biochemical changes in tissues, which enables us to achieve greater diagnostic confidence in an automated, label-free manner. We developed a computational pathology system that gives high contrast images and seems superior to traditional staining procedures. This study is a prelude to the development of real time  The success of cardiac transplantation depends foremost on the immune response to the new implant. The golIn routine cases of monitoring allograft reception, biopsy sections are stained and the inflammatory response is observed, which is predominantly lymphocytic. This apAs opposed to the standard practice of staining tissue with dyes or molecular imaging of specific epitopes, the emerging technology of chemical imaging can utilize the inherent molecular contrast within samples to provide histologic data. One approach in particular, infrared (IR) spectroscopic imaging, offers strong contrast, high sensitivity and rapid data recording. It has shown potential broadly in biomedical applications for understanding metabolomics and molecular diagnostics,14. CombWritten consent was obtained in all patients for study of their archived pathological specimens. The consents were recorded and maintained securely and separately. The consent process for this study was reviewed by the IRB at Rush University Medical Center and approved. All specimens were anonymized, de-identified and no clinical or demographic information was recorded. Thirty five anonymized human EMB sections from ten patients, formalin fixed and paraffin embedded were examined. The biopsies were taken using a bioptome, which is an instrument inserted through the internal jugular vein, and directed under fluoroscopy to be positioned in the right ventricle. The biopsies are then taken as small pieces of tissue, typically measuring 1 mm x 1 mm x1 mm. The section thickness was 5μm. Of the thirty five sections obtained; three sections had to be discarded due to damage to the sections. Out of the 10 patients analyzed, patient 1–5 had no rejection; thus counted as control. Patient 6, 7 and 8 had moderate rejection; and patient 9 and 10 had mild rejection. In current practice, it is very rare to find samples with grade 4 severe rejection, and hence such samples could not be included in the study.Samples were microtomed onto reflective low-emission (Low-E) glass slides for IR imaging. These slides provide a reflective substrate for the sample in IR light but are transparent to visible light. Although when using Low-E slides, the IR beams pass through the sample twice and suffers from distortions in the spectrum,41, we h-1  exceeding 500:1 in all cases. Data was collected over the mid-infrared region and truncated for storage (800cm-1 to 4000cm-1). The image resolutions were nominally 6.25 μm and 25 μm. Since the samples were large,  and irregularly shaped, each image was acquired by breaking it down to 5–10 smaller rectangular regions and using raster scanning of these parts. Each region was separately focused by the instrument to remove any error due to change in focus and the composite image was stitched back together using ENVI-IDL 4.8. Processing time for a square section of 1mm X 1mm at 6.25 μm, starting with imaging and obtaining computational stain was about 2 hours. Processing time for the same section at 25 μm was about 10 minutes. It has to be kept in mind that this imaging was performed by sweeping through all the wavenumber bands from 4000 cm-1 to 800 cm-1 at 4 cm-1 resolution. After building the classifier, one can realize that only a segment of this range is actually necessary for classification (discussed in results), and thus scanning at discrete frequencies for detection can enable reduction in imaging time by three folds or larger[De-paraffinized sections were imaged under mid-infrared light on the IR imaging system. FT-IR imaging was performed using a Spotlight 400 system from Perkin Elmer. Spectra were collected using a liquid nitrogen cooled mercury-cadmium-telluride (MCT) 16-element linear array detector. The background was collected on a clear area of low-E slide at 4cmor larger,43.Serial sections were preserved and stained with H&E for initial determination of rejection grade by the pathologist. In addition, after IR imaging was performed on sections, the sections on low-E slides were stained with Hematoxylin and Eosin stains for future comparisons and imaged using Zeiss visible microscope. All the data analysis done in this manuscript used H&E images from same section imaged by infrared spectroscopy and not serial section.Acquired data were imported in ENVI-IDL 4.8 software for analysis. A figure annotating important IR peak assignment is shown in With the aim of understanding spectral differences in various components of the section and in order to build a Bayesian classifier for automated classification of the sections, we first built a classification grid comprising of 16 sections by combining data from 16 images in a single file. The breakdown of 16 chosen sections for classification training set was as follows: 3 sections from patient 1, 2 sections each from patient 5, 6,9,10 and 1 section each from patient 2, 3, 4, 7, and 8. Sections were chosen to sufficiently represent each class, namely Myocardium, Endocardium, Fibrosis (Endocardium), Fibrosis (Myocardium) and Lymphocytes and to provide maximum inter-patient diversity to the training set. Remaining sections were used for validation set. The breakdown of validation set was as follows: 2 sections each from patient 3, 4, 5, 6, 7, 8, and 9 and 1 section each from patient 1 and 10.-1 to see contrast between lymphocytes and muscle. The 1236 cm-1 peak is associated with CH2 wagging vibrations associated with proteins [-1) to account for the variations in sample thickness[We then used the peak height of vibrational mode at 1236 cmproteins ,45; whicproteins . Next, eproteins ). This pthickness. We thenOur Bayesian classifier works by determining the likelihood that an unknown pixel belongs to a particular class by using biochemically significant features called metric parameters defined by the user. Each of these parameters can have different weights in the classification process depending on their ability to differentiate between classes. We used a protocol that has previously been established and validated ,48,49. UUse of ratios instead of absolute values also ensures that these metric definitions are independent of variability in instrumentation and sample preparation steps. We evaluated these metrics in terms of their ability to separate the classes by using minimum error in identification of class and the area under the curve (AUC) for the Receiver Operating Characteristic (ROC). We further tested the Bayesian classifier built using these parameters on an independent set of sections to evaluate its accuracy in identification of classes. The findings are described in the following section.Samples were imaged using IR microscopy and correlated to features in H&E images that were marked by the pathologist’s review as the ground truth. Computerized pattern recognition of IR imaging data from unstained EMB samples led to every tissue pixel being classified into a specific histological class. Compared to the ground truth, the resultant probability of detection at the pixel level for the training set was quite high for lymphocytes (0.991), fibrosis-endocardium (0.999), fibrosis-myocardium (0.997) and myocardium (0.952) and somewhat lower for endocardium (0.860) with approximately 0.10 probability of false alarm . There iWe performed validation using an independent set of 16 samples with approximately 300,000 pixels. Uniformly high probability of detection with low probability of false alarm (0.13) was found  associated with glycogen[According to the ISHLT criteria, for the collagen,45,49,54amide III , infrared spectroscopy can potentially detect changes undergone by the tissue which are indicative of transplant rejection even when lymphocytes are not picked in sampling, reducing the error rates, false negatives and avoiding significant trauma to the patient tissue. Apart from chemical information associated with tissues, tissues digitally stained with IR imaging approach are capable of providing a much better contrast and easy quantification of lymphocytes which can greatly reduce the time and effort spent per section by the pathologist.in vivo imaging can be attempted based on chemical molecular imaging. This can be made possible by touch probe based fiber optic technology on which work is currently under progress. Multiple studies show that spectral in-vivo analysis is promising using probe based instruments and have previously been applied to study atherosclerosis [This report stresses the capabilities of this approach in a complex condition such as cardiac transplant rejection, which traditionally needs careful tissue preparation, multiple stains and review by experienced cardiac pathologists to provide accurate diagnoses. Combined with the speed of the data acquisition and emerging technologies for high speed IR microscopy,43, we bclerosis –66 and tclerosis ,68. Usinclerosis .Although the present study has shown excellent promise in terms of on-site detection, we are currently limited by spatial resolution and speed of data acquisition and processing. Spatial resolution used in this study can identify single lymphocytes larger than 10 μm, and lymphocytes smaller than this can be identified in large enough cluster. While the current study can successfully identify grade 0 through grade 4, in order to accurately identify grade 2(mild rejection), one would require much better spatial resolution to identify single lymphocytes. This task can be accomplished in near future using high definition imaging systems which have spatial resolution of the order of single micron, reducing the problem of boundary pixels and enabling us to identify every cell more accurately in tissue. Another challenge faced by pathologists is to identify whether the rejection is cellular or antibody mediated. Good spatial resolution is necessary to identify individual cells and to classify cells that are present in low density in tissues, for example macrophages, basophils; or bacterial cells in case of pathogen infections. As the project expands, we hope to be able to identify many other cell populations in the region such as activated mononuclear cells and pathogens; making spectroscopic analysis of specific cells possible. This could in turn enable us to understand other pathological mechanisms of disease development. While we were limited by speed in terms of imaging and data processing in this study, progress is now being made to reduce data acquisition time by manifolds using discrete infrared spectroscopy ,43. The The chemical molecular imaging approach offers numerous advantages over traditional sample examination techniques, providing a new avenue for clinical diagnosis. Chemical information, along with morphologic and architectural tissue information provides for a comprehensive analysis of tissue. Computer algorithms allow us to dispense with staining and pathological recognition is aided by color coded images. In this study, we have shown an example of how chemical imaging can be applied in cardiac tissues to achieve automated pathology while providing a high probability of detection and low probability of false alarm. We identified specific spectral characteristics which related to the biochemical changes undergone by the tissue which could be used for chemical detection of rejection. In future, we can make this even more extensive by differentiating between acute cellular rejection and Quilty lesions. This is the first study to show that the chemical molecular imaging approach can be used to diagnose complex cardiac conditions, with results equivalent to and probably superior to conventional pathology.This technique would also be useful in identifying other cell populations that can be present in cardiovascular environment such as activated immune cells, antibody mediated rejection and bacterial infections to name a few. It is also possible to integrate this digital data with patient history to provide an even more nuanced scientific assessment of disease and prognosis. The idea here is to kick start the development of an approach which can give an all-encompassing rapid diagnosis at the site of collection of sample without stains and more importantly, assist during surgery for identification of diseased and problem areas in the heart & vasculature.S1 Fig(TIF)Click here for additional data file.S2 FigArrows show lymphocytic infiltration. Top panel: No rejection; Middle panel: Mild rejection; Bottom panel: Moderate rejection.(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.


To apply genetic analysis of genome-wide association data to study the extent and nature of a shared biological basis between migraine and coronary artery disease (CAD).Four separate methods for cross-phenotype genetic analysis were applied on data from 2 large-scale genome-wide association studies of migraine  and CAD . The first 2 methods quantified the extent of overlapping risk variants and assessed the load of CAD risk loci in migraineurs. Genomic regions of shared risk were then identified by analysis of covariance patterns between the 2 phenotypes and by querying known genome-wide significant loci.PHACTR1) and insulin homeostasis (GIP).We found a significant overlap of genetic risk loci for migraine and CAD. When stratified by migraine subtype, this was limited to migraine without aura, and the overlap was protective in that patients with migraine had a lower load of CAD risk alleles than controls. Genes indicated by 16 shared risk loci point to mechanisms with potential roles in migraine pathogenesis and CAD, including endothelial dysfunction (The results suggest that shared biological processes contribute to risk of migraine and CAD, but surprisingly this commonality is restricted to migraine without aura and the impact is in opposite directions. Understanding the mechanisms underlying these processes and their opposite relationship to migraine and CAD may improve our understanding of both disorders. If migraine and CAD have a shared biological basis, one might anticipate that they will also share genetic variants that affect their risk. In this study, we utilized data from 2 large-scale nonoverlapping GWAS meta-analyses of migraine p value and effect size) at single nucleotide polymorphism (SNP) level from 2 recently performed meta-analyses of genome-wide association data on migraine (IHGC)12 and CAD (CARDIoGRAM)13 were used in the present study. After excluding overlapping samples, the 2 studies consisted of 19,981 cases with migraine vs 56,667 controls, and 21,076 cases with CAD vs 63,014 controls. A proportion of the migraine cases were phenotyped in sufficient detail to allow subclassification into MO  and MA . In addition, individual-level genotype data were available for a proportion of the migraine cohorts . All data sets were imputed by using the HapMap release 21 or 22 as reference. An overview of the study design and the included cohorts is given in Neurology.org/ng.Summary statistics  r2 > 0.05 (based on HapMap CEU release 27) were removed. This process was repeated until a set of 92,654 SNPs in approximate linkage equilibrium remained. For each of 5 separate p value cutoffs , we counted the number of SNPs above and below the cutoff in each of the 2 studies, resulting in one 2 × 2 table for each p value cutoff. The Fisher exact test was used to estimate deviation from the expected distribution, and false discovery rate correction was performed on all 6 tests using the p.adjust function in R.15 A corrected Fisher p < 0.01 was taken to indicate an excess of overlapping signals. In order to obtain a more robust estimate of the significance of the observed overlap, this was also assessed through permutations. In each permutation cycle, the relation of p values to SNPs was randomized within each of the LD-pruned migraine and CAD data sets, and a Fisher p for overlap was calculated for each p value cutoff. We generated 100,000 permutations to produce an empirical null distribution of p values.To assess whether more association signals were shared between the migraine and CAD studies than would be expected by chance, we used a set of 2,342,101 overlapping SNPs that were directly typed or imputed in both studies. Following the same procedure as described in a previous study,In an equivalent manner, secondary analyses were performed for MO  and MA .16 We first generated 3 sets of CAD risk SNPs by selecting SNPs with strong , moderate , or weak  association to CAD among the 2,342,101 SNPs with information in both migraine and CAD studies. As suggested in the original description of the method,16 the analysis was based on non–LD-pruned SNP sets in order to optimize sensitivity. Using each set of CAD risk SNPs, we calculated a per-individual CAD polygenic risk score by summing the number of CAD risk alleles , each weighted by the log odds ratio from the CAD study. We subsequently assessed whether CAD polygenic risk score was associated with migraine status by applying a logistic regression model of the effect of CAD polygenic risk score (continuous) on migraine status , adjusted for sex and dummy-coded covariates representing the 6 individual migraine study cohorts.If shared genetic risk variants are in part or fully responsible for comorbidity between migraine and CAD, we would expect an accumulation of CAD risk alleles in migraineurs. To test this hypothesis, we used the 6 migraine cohorts in which individual-level genotype data were available for analysis  were examined using an existing expression quantitative trait locus database from peripheral blood17 (e-Methods).In order to identify shared risk loci between migraine and CAD, we applied a novel method, Cross-Phenotype Spatial Mapping . This method compares 2 sets of 13 The lead SNP at each locus was cross-analyzed for association to the other phenotype, Bonferroni correcting for the number of SNPs tested. All 13 of 13 reported migraine loci and 22 of 25 reported CAD loci were available in our data set and could be tested .Lastly, we analyzed loci with previously reported genome-wide significant association to migraine or CAD .p values ≤1 × 10−2, 1 × 10−4, and 1 × 10−6. This was supported by permutation testing, which indicated a sharing of association signals at p value cutoff 1 × 10−5 as well. For reference, the full list of SNPs with association p value ≤1 × 10−2 to both CAD and migraine is given in table e-3. Secondary analyses by migraine subtype revealed an overlap of association signals between MO and CAD at all p value cutoffs , while no overlap was seen between MA and CAD at any of the p value cutoffs. The direction of effect for overlapping association signals did not consistently agree between migraine and CAD, as evidenced by nonsignificant binomial p values for concordance . Secondary analyses revealed a similar, but more pronounced, association between CAD polygenic risk score and MO . No association was seen for MA. In the analysis of the weak CAD risk SNP set, there was no association to CAD genetic risk score for either migraine category, indicating that the observed associations were driven by a fairly limited number of loci that are at least moderately associated with CAD. These findings were consistent across men and women (figure e-2) and across individual independent cohorts within the same migraine subtype (figure e-3).To examine this further, the second analysis compared the load of genetic risk variants for CAD between migraineurs and controls, using individual-level data. The results indicated that a high CAD polygenic risk score was associated with a reduced risk of migraine . The second strongest overlapping signal was on 17q21 (locus no. 2), where the lead CAD SNP  was intragenic in UBE2Z, whereas the lead migraine SNP  was intergenic between SNF8 and GIP. It is interesting that both lead SNPs are in high LD (r2 > 0.9) with 2 functional variants in GIP: Ser103Gly (rs2291725) and a splice site variant (rs2291726) that is predicted to lead to a prematurely truncated transcript18 (table e-6). The locus was also found to have a potential effect on the expression level of UBE2Z (table e-7). Lead SNPs in 5 loci were in high LD (r2 > 0.8) with nonsynonymous or splice site variants in nearby genes (table e-6). Ten of the 16 loci showed opposite direction of effect for migraine and CAD. In the secondary analyses, 12 of the 16 lead migraine SNPs had a lower association p value in MO than in MA , and all 16 SNPs had the same effect direction in each of the 2 migraine subtypes. Local Manhattan plots and covariance plots for the identified loci are given in figure e-4.CPSM yielded 16 loci that overlapped between migraine and CAD , where bWhen considering previously reported risk loci for migraine and CAD, 3 CAD risk SNPs were associated to migraine at study-wide significance, and 2 migraine risk SNPs were associated to CAD . These c10 and an increased risk for CAD-related mortality,11 although one cross-sectional study failed to find an association between CAD and any migraine subtype.19 Studies not differentiating on migraine subtype have been less conclusive, with some21 but not others22 indicating an increased risk of CAD related to migraine overall.In this study, we used data from 2 recently performed large-scale nonoverlapping GWAS to examine shared genetic risk between migraine and CAD. We found that association signals overlapped in excess of what would be expected by chance. Stratifying by migraine subtype further revealed that MO and MA behaved differently. MO had a genetic overlap with CAD, whereas MA did not. These results are unexpected, given the epidemiologic evidence that comorbidity with CAD is more common in MA than MO. Patients with MA were found to have a 2-fold increased risk for CADp value cutoff was used to define signals. Intriguingly, the impact was in the opposite direction, in that patients with MO had a lower load of CAD risk alleles than migraine-free controls. This association seemed to be driven by a limited number of loci. Only a proportion of the included migraine patients were phenotyped in sufficient detail to allow subclassification into MA or MO. When using the considerably larger set of all migraine patients, a similar association was seen as for MO, likely driven by this migraine subtype. While the results suggest that there are shared common risk variants between migraine and CAD, they do not indicate that these variants explain comorbidity between the 2 disorders.For MO, we found a clear overlap of association signals with CAD, whichever PHACTR1) was associated with cervical artery dissection, with effect in the same direction as for migraine but opposite of CAD.23 Two further migraine SNPs showed evidence of association to cervical artery dissection with the same effect direction as for migraine  but opposite direction for CAD.The opposite direction of effect for some of the loci is consistent with a recent GWAS in which the migraine and CAD risk SNP rs9349379 .25 The strongest overlapping region (locus 1) is entirely intragenic in PHACTR1 (which encodes phosphatase and actin regulator 1 protein). This locus is associated with both migraine and CAD at genome-wide significant levels in the current and previous studies30 and has also been associated with coronary artery calcification and stroke.32PHACTR1 is highly expressed in the brain, and its transcript is an important regulator of synaptic activity and dendritic morphology through the control of protein phosphatase 1 and actin binding.33 More recently, PHACTR1 has been identified as a key regulator of endothelial function, including endothelial cell survival and angiogenesis,34 and it is associated with altered vasomotor tone.35 Both endothelial and vasomotor dysfunctions have been implicated in migraine,37 and this locus offers a potential focus for future studies. Alternatively, the pleiotropic effects of this gene on both synaptic and vascular functions may give rise to independent causal pathways for the 2 disorders.Six of the overlapping loci have previously been associated with CAD at genome-wide significant levels  and migraine variants (rs11079844) are in strong LD (r2 = 0.94), and both are in strong LD (r2 > 0.90) with 2 potentially functional variants in GIP (which encodes gastric inhibitory polypeptide). GIP regulates glucose-induced insulin release from pancreatic β-cells and helps resensitize the insulin response.38 It is also expressed in the brain, where it may be involved in proliferation of neuronal progenitor cells.39 Whether GIP is involved in the observed tendency for insulin resistance and metabolic syndrome in migraineurs should be investigated.40The second strongest overlapping region (locus 2) is a previously identified risk locus for CAD.Strengths of this study include the use of large-scale nonoverlapping GWAS of migraine and CAD, stringent quality control measures, and sufficiently rich phenotyping to allow secondary analyses of the 2 migraine subtypes. Nevertheless, some limitations should also be acknowledged. First, only summary statistics and not individual-level genotype data were available for the majority of the samples included in this study. Second, in each included cohort, phenotype information was available on only migraine or CAD, not both. This prevented us from performing more in-depth analyses, including analysis for potential gene-gene interactions or identification of CAD risk loci specific to migraineurs. Third, considerable effort was devoted to the careful avoidance of shared controls between studies, and stringent quality control measures within each data set were enforced to reduce the risk of spurious effects resulting from biases within the data sets. Nevertheless, we cannot rule out subtle biases that could affect the current results. Two such concerns are the effects of migraine on survival and the possibility that migraineurs may be more likely to seek medical treatment and therefore be under closer surveillance with regards to other disorders. Future efforts should aim to replicate these findings in sufficiently large prospective data sets where both phenotypes are measured in the same individuals.Our study provides novel insights into the relationship between migraine and CAD. Intriguingly, and unexpectedly, there was no genetic overlap between MA and CAD, for which epidemiologic studies suggest comorbidity, but there was compelling evidence for a genetic overlap between MO and CAD, where the impact of risk variants overall was in opposite direction for the 2 disorders. The results do not demonstrate that shared common genetic risk factors drive comorbidity between the 2 disorders. However, dissecting the mechanisms underlying the shared risk loci may improve our understanding of both disorders.


To describe patient preferences in selecting specific biologics and compare clinical response using patient reported outcomes (PROs) among patients with rheumatoid arthritis (RA) started on different anti-tumor necrosis factor (TNF) therapies.Participants were enrollees in Kaiser Permanente Northern California. Patients with RA who had at least two provider visits and started a new anti-TNF therapy from 10/2010–8/2011, were eligible for participation in this longitudinal study. Using a telephone survey, patient preferences in biologic selection and RAPID3, MDHAQ, and SF-12 scores were collected at baseline and at 6 months. Patient scores rating injection/infusion-site burning and stinging (ISBS) were collected at 6 months.In all, 267 patients with RA responded to the baseline survey, of whom 57% preferred an injectable biologic, 22% preferred an infused biologic, and 21% had no preference. Motivation for injectable biologics was convenience (92%) and for infusion therapy was dislike or lack of self-efficacy for self-injection (16%). After 6 months of treatment with anti-TNF, 70% of the 177 patients who answered the ISBS question reported ISBS with the last dose; on a scale of 1 (none) to 10 (worst), 41% of these reported a score of 2–5; and 29% reported a score of 6–10. Adalimumab users experienced 3.2 times  the level of ISBS that etanercept users experienced. There were no significant differences in RAPID3, MDHAQ, or SF-12 scores between etanercept or adalimumab initiators.Convenience and fear of self-injection were important considerations to patients selecting a biologic drug. Although more convenient, adalimumab associated with more ISBS than did etanercept, and this rate was higher than reported in clinical trials. At 6 months, PROs did not differ between etanercept and adalimumab users. In general, when medical treatments are being selected, most patients want to be offered choices and the opportunity to give their opinion, even though a sizable proportion may want their physician to make the final decision . For thiFurthermore, self-efficacy to administer injections is thought to be important to patients with RA when selecting a biologic agent . It is iPatients eligible for this study included English-speaking enrollees in Kaiser Permanente Northern California, a large, well-characterized, integrated care organization. Patients with RA were considered preliminarily eligible for the survey if they had at least two provider visits for RA and started a new anti-TNF therapy while enrolled in Kaiser Permanente during the period of October 2010 through August 2011. These individuals were identified within 1 week of initiation using computerized pharmacy information from the electronic medical record and were invited to complete a baseline telephone survey and 6-month follow-up survey. Patients either could have been biologic naïve or could have switched from a previous biologic agent. Permission from the treating rheumatologist and confirmation that the patient had RA were also required. The baseline survey was conducted at the time of initiation of anti-TNF therapy and another survey was conducted 6 months later.Patients participated in a phone survey. Participants provided a verbal informed consent; a written consent was not feasible given that the survey was conducted over the phone. The assent was recorded in the study database, mainly by the mere fact that we quit the call if they said no. Kaiser Northern California IRB approved this consent procedure and the study. Patient demographics and descriptive information on preferences and factors in selecting an injectable or infused biologic were collected on the baseline survey “Descriptive statistics were used to summarize factors related to preferences for route of administration and for specific biologics, and to summarize the magnitude of ISBS and correlations between ISBS and PROs. This analysis focused on the two biologics used most commonly in the Kaiser Permanente population: adalimumab and etanercept. We used nonparametric statistics to compare the median ISBS scores of adalimumab and etanercept users. Polynomial logistic regression analysis was used to assess factors associated with ISBS after dividing patients into three categories based on their ISBS score: 1–5 (reference group), 6–8, and 9–10. The polynomial logistic regression compared adalimumab with etanercept after adjusting for potential confounders including age, sex, RA disease duration, prior biologic use, and fibromyalgia. The latter two variables were forced into the model based upon clinical interest.To evaluate the comparative effectiveness of biologics with regard to PROs, we used a multivariate ordinary least square regression to evaluate change in each PRO at 6 months, comparing adalimumab initiators with etanercept initiators and controlling for multiple potential confounders including age, sex, RA disease duration, and prior biologic use. The analysis included all patients who contributed both a baseline and a 6-month survey , irrespective of whether or not they remained on the therapy that they initiated. We also conducted a sensitivity analysis using an intent-to-treat analysis in which the baseline survey PRO was carried forward to the 6-month survey for patients who were lost to follow-up. Patients provided consent to participate, and approval for the study was granted by the Kaiser Foundation Research Institute Institutional Review Board.The number of preliminarily eligible patients with RA was 428 , of whicMean age of the 242 patients included in the study was 54.4 (SD ± 12.1) years and 73% were females. Further details about patient characteristics are listed in In all, 177 responders rated ISBS with the last dose of their injectable biologic. The incidence of ISBS for these responders was 70%; 30% of responders reported no ISBS (score = 1 out of a maximum of 10), 41% reported levels between 2 and 5, and 29% reported levels between 6 and 10. Forty-five percent of the patients on injectable biologics listed the level of ISBS or difficulty with needles as a primary inconvenience of using injectable biologics. After 6 months of treatment, 2% of patients initially on an injectable biologic reported that ISBS was the reason they switched to another injectable biologic and 1% reported ISBS as the reason they switched to an infusion biologic. This suggests that despite the fact the ISBS was highly prevalent (70%) it was not a major reason for patients to switch biologics.Of the 187 adalimumab and etanercept initiators who completed the 6-month follow-up survey, 154 (82%) answered the question regarding ISBS. The relationships of infused biologic with improvement in the RAPID3, MDHAQ, and SF-12 are shown in To better characterize preferences for biologics and their tolerability and effect on PROs, we obtained and analyzed longitudinal data for a community-based population of patients with RA starting new biologic agents. The study revealed that 56% of patients with RA shared the decision about biologic selection with their physician and that the route of administration (injectable vs. infusion) was important in choosing one biologic over another. The decision about whether to use an infused or injected biologic was largely influenced by the patient’s preference to have the medication administered by a clinician at an infusion clinic or to administer the medication by themselves at home. In addition, the study found no differences in PROs between patients treated with adalimumab and etanercept.These results were very similar to those observed in a separate community-based study of RA, in which 50% of patients with RA  chose inThe findings we report here also are similar to those of a previous study in which the prevalence of ISBS was 59%, compared with our estimate of 70% . AlthougPrevious studies demonstrated no difference in ISBS between adalimumab and etanercept, except when the delivery mechanism was taken into account  , 24. TheSeveral studies have suggested differences in treatment response with different anti-TNFs depending on immunogenicity –30; howeOur study has several strengths. Comprehensive outcomes included patient preferences, ISBS, and PROs. Compared with data in past reports; our data has greater generalizability to community-based populations treated by numerous physicians across geographic locations. Furthermore, the sample size N = 267) was larger than in past studies (N<100) , 24; the7 was larThe study also has limitations, including its setting in a single region of the U.S. within a single healthcare system and the inclusion of too few golimumab and certolizumab users for analysis. A minority of patients did not complete the follow-up survey, and we were unable to assess the reason for lack of follow-up, be it an adequate response to treatment or other, although the intent-to-treat analysis indicated that this weakness was unlikely to have a major effect on the study. Another limitation is the short follow-up period between the baseline and follow up survey (6 months). Finally, it is possible that our survey did not include items that could be representative of how the patients felt regarding the use of injectable biologics .In conclusion, the selection of biologics for the treatment of RA appears to be a shared decision between patient and physician. Although the prevalence of ISBS was high, especially in adalimumab patients, this did not lead to discontinuation or changes in medication. Etanercept and adalimumab were similar in effectiveness with respect to PROs. Further investigation with longer follow-up data to evaluate patient expectations about effectiveness, safety, and tolerability with regard to medication adherence would be useful for improving long-term outcomes on biologic therapies and to confirm the results of the present study.S1 Document(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Dear Editor,Inside the previous issue of "Research in Cardiovascular Medicine", Arya et-al presented a nice review of novel anticoagulation strategies in patients with non-valvular atrial fibrillation . ThroughLower risk AF patients may be defined as those with a CHADS2 score < 2 and they comprise the majority of the AF population  agents in the recent years has been associated with dramatic changes in treatment planning of non-valvular AF. However, the issue of optimal anticoagulation in lower risk AF remained unsolved. For instance, In Re-LY study the risk of major bleeding in the low to intermediate-risk subgroup on Dabigatran was approximately 3% per year while the rate of stroke was less than 1%. Also, in ARISTOTLE, nearly the same results have been reported for the lower risk subgroup on Apixaban. The ROCKET AF study for evaluation of Rivaroxaban enrolled a higher risk population with a mean CHADS2 score of 3.5, but the trial did not show a net clinical benefit over warfarin either for efficacy or safety. In a recent meta-analysis of all the three new OACs, there was no clear benefit over warfarin regarding bleeding risk reduction  occlusion devices have been tested with acceptable procedural safety . This ap


Data herein reported and discussed refer to vaccination with the recombinant fatty acid binding protein (FABP) family member of the schistosomes, called Sm14. This antigen was discovered and developed under a Brazilian platform led by the Oswaldo Cruz Foundation, from the Health Ministry in Brazil, and was assessed for safety and immunogenicity in healthy volunteers. This paper reviews past and recent outcomes of developmental phases of the Sm14-based anti schistosomiasis vaccine addressed to, ultimately, impact transmission of the second most prevalent parasitic endemic disease worldwide. Schistosoma mansoni in the Americas, of whom 95% are in Brazil  for one 50 μg dose.The Sm14 project has been mostly funded by public funds at Oswaldo Cruz Institute/FIOCRUZ, belonging to the Brazilian Ministry of Health. Since the early stage of the development, there was a critical concern to reduce the cost of production at its lowest level and to ensure the use of non-proprietary components in the production process in order to get a low final price for the vaccine. The strategy adopted to reduce the cost of production of the human Sm14 vaccine involved the following steps:Mycobacterium bovis BCG , were undertaken to gradually convert the original laboratory-bench protein into a clinical product. First, it was demonstrated that rSm14 could be produced in a non-fused form while retaining its protective immunogenicity . A genomovis BCG , 24, andovis BCG . Last, aovis BCG .Pichia pastoris based expression system for the protein  (Number NCT01154049). These trials demonstrated that the vaccine is safe and immunogenic. The vaccine was administered intramuscularly in three 0.5 mL doses, each containing 50 μg of both rSm14 and GLA-SE. The second dose was administered 8 weeks after the first one, while the third dose was given 1–2 months later. There were no serious adverse events reported with the only side effects being mild local pain at the site of vaccination in some individuals. With the support of Infectious Disease Research  clinical trial team, cells and sera from Brazilian volunteers were shipped to Seattle and extensively screened for the immune response generated by vaccination with Sm14 + GLA-SE toward the identification of the immunological signature of human immunization. Vaccination stimulated anti-Sm14 IgG antibodies as well as a Th1 T-cell response, resulting in gamma-interferon production in the vaccinated individuals. (manuscript submitted to Vaccine).Following approval by the local Ethics Committee and the Brazilian Regulatory Agency, ANVISA, two separate Phase I clinical trials of the rSm14/GLA-SE vaccine have been undertaken with healthy volunteers. The first trial involved 20 males and the second involved 10 females  approved the results of phase 1 clinical studies, and Fiocruz has licensed Sm14 technology to a Brazilian company, Ourofino, for final development and commercialization of Sm14 vaccine for use in cattle herds against Field based immunogenicity and safety phase 2 trials of the Brazilian Sm14 + GLA-SE anti schistosomiasis vaccine are planned to start in 2015 in endemic areas (Brazil and Africa).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cognitive theories of depression suggest that beliefs of low self-worth and the tendency to attribute negative events to causes that are global (widespread rather than specific) and stable (will persist rather than change in the future) are associated with the development of depressed mood. Such theories are supported by evidence from prospective studies and have guided the development of successful treatment and prevention strategies such as CBT. However, the relative importance of different psychological constructs within cognitive theories is unknown. This is important to refine cognitive theories and develop more efficient prevention strategies.We used prospective data from over 3500 young adults from the Avon Longitudinal Study for Parents and Children (ALSPAC) cohort in the UK to investigate the association between cognitive style, measured by short forms of the Dysfunctional Attitudes Scale (DAS) and Cognitive Styles Questionnaire-Short Form (CSQ-SF) at age 18, and future depressed mood at age 19. Structural equation modelling techniques were used to separate cognitive style constructs.Cognitive styles were associated with future depressed mood, independently of baseline mood, both as measured by the DAS-SF and the CSQ-SF. Of the different CSQ-SF constructs, only global attributions were associated with both baseline and future mood independently of other constructs.The study was subject to attrition and the follow-up was relatively short (10 months).The findings suggest that the tendency to attribute negative events specifically to global causes could be particularly important for depression. Reducing global attributions is potentially important in the prevention and treatment of depression. It is associated with substantial disruption to social, educational and occupational functioning . PreventCognitive Behavioural Therapy (CBT), which is based on cognitive theories of depression, can be effective in treating and preventing depression . Howeverself-worth׳ (means we are flawed in some may) and that are internal (our fault), global  and stable (will impact our future)  . In thei future) . Cogniti future) .There are commonalities between the negative belief systems measured by the DAS and attributional styles. For example, we draw upon our belief systems to explain the world around us: if we believe we are flawed, when something bad happens to us we attribute it to such flaws . Therefoprospective association between the CSQ and/or the DAS and later depression. Several of these studies report evidence for an association with future depression (N<400). In larger low-risk samples results are less consistent: some report little evidence for a main association between cognitive style and later depression  any ‘true’ differences between constructs may be masked. For example, if all observed global attribution item scores are summed up, scores represent: 1) true differences in the individual׳s global attribution construct, 2) measurement error or 3) differences in the salience of the scenarios to individuals. By deriving construct specific latent traits, SEM separates the construct variance from the other two ‘nuisance’ sources see .1.Is there an association between cognitive styles and later depressed mood , having adjusted for baseline depressed mood?2.Which specific cognitive style constructs (separated using latent traits) are independently associated with later depression? This may enable refinement of the cognitive theory of depression using empirical data.In the present study we used SEM techniques to model prospective data on cognitive styles measured by short forms of the DAS and CSQ at age 18 and depressed mood at both age 18 and 19 from over 3500 young adults from the ALSPAC cohort in the UK. We have previously reported a positive cross-sectional association between CSQ scores and depression in this sample ; however22.1http://www.alspac.bris.ac.uk. Detailed information has been collected on the cohort, including regular self-reported information from mothers and children and face-to-face assessments in research clinics. The present study reports data from the sample of ALSPAC offspring who attended the most recent research clinics for the children at age 18, where CSQ and DAS measures were taken.The sample comprised participants from the Avon Longitudinal Study of Parents and Children (ALSPAC). All pregnant women resident in the former Avon Health Authority in south-west England, having an estimated date of delivery between 1 April 1991 and 31 December 1992, were invited to take part. The children of 15,247 pregnancies were recruited . EthicalN=3845). A sample of 1698 also had data for depressed mood (outcome) at both 18 and 19. However, missing depression data was imputed and all analyses described below were repeated using the same starting sample size (N=3845) in sensitivity analyses.Our starting sample was those with complete primary exposure (cognitive style measures) data  developed from the original Cognitive Style Questionnaire was administered to the ALSPAC children at the fourth Teen Focus clinic (age 18). The CSQ is a frequently employed measure of negative cognitive style in relation to depression. The reliability and validity of the short version (CSQ-SF) have been established in young adults . The sho studies . Interna2.2.2α=0.80.The DAS-SF was also administered at the focus clinic. The DAS-SF is a self-report questionnaire containing nine items taken from the original Dysfunctional Attitude Scale , using i2.2.32.2.3.1The SMFQ was also administered at the clinic at age 18 and again approximately 10 months later in a postal questionnaire (age 19). The SMFQ is a brief measure of depressive symptoms designed for children and adolescents aged 6–18 years . The 13-2.2.3.2Depression in the offspring at 18 was also measured using the computerised version of the Clinical Interview Schedule-Revised (CIS-R). The CIS-R is a computerised interview which derives a diagnosis of depression according to ICD-10 criteria. The CIS-R is designed for, and has been widely used within, community samples including in the National Surveys of Psychiatric Morbidity and the 1958 birth cohort . A binar2.2.4Maternal age at the index child׳s birth (in years), social class (1–5) and child sex were recorded at the beginning of the ALSPAC data collection and these variables were included as covariates. Adjusting for baseline depressed mood should also provide a further proxy for shared risk variables.2.32.3.1In the first stage of the analysis we investigated the cross-sectional and prospective associations between CSQ-SF total and sub-scale scores and DAS-SF total scores with SMFQ binary outcomes (depressed/not depressed) at age 19, using logistic regression analyses in Stata version 12. Baseline depression was controlled for in two ways: 1) to account for clinical depression, those with a diagnosis of depression at 18 using the CIS-R were removed and 2) to account for sub-threshold symptoms we adjusted for SMFQ total scores at age 18.auxiliary measures that can be employed for this purpose. For example, given that there is substantial information on socio-demographic variables in ALSPAC which predict missingness, missing information can be assumed dependent on observed data (missing at random assumption). We employed a fully conditional specification as implemented in the MICE algorithm in STATA 12 using all variables described in the analyses and additional socio-demographic indicators of missingness (list available on request) to predict missing data across 100 imputed datasets. Monte Carlo errors were less than 10% of the standard error and FMI values were no larger than 0.8. Earlier measures of child depression and neuroticism were used to predict later depression allowing imputation to those with complete exposure data (n=3845)  . The impN=8446). Analyses were conducted post-imputation by combining estimates across imputed data sets using Rubin׳s rules  were available we extended sensitivity analysis to a larger sample which was more representative of the original ALSPAC cohort .In the next stage of analysis we investigated the association between separate constructs of the CSQ-SF and depressed mood at both 18 and 19, using structural equation modelling techniques in order to separate ‘of interest’ variance from ‘nuisance’ variance. As described in plus version 7. Construct-specific items (sum of 2 items relating to each construct from each scenario) were loaded on to ‘construct’ factors. There were 3 construct factors: global (f_glob), stable (f_stab) and self-worth (f_self) each derived from 8 items, one for each scenario. Scenario-specific variance was accounted for by deriving ‘scenario’ factors; items also loaded on to a factor representing the scenario within which they appeared. Each item thus cross-loaded on to one ‘construct’ and one ‘scenario’ factor (see construct-specific variance across the 8 scenarios. Loadings for construct items on to construct factors ranged from 0.2 to 0.6 (note these are cross-loadings). Separate latent factors for SMFQ scores at both time points were also derived from CFA analyses loading all 13 items onto a single factor.Latent factors for each construct of the CSQ-SF were derived using confirmatory factor analysis (CFA) in Mctor see . As scenIn assessing the fit of the CFA model, three model fit statistics were used: 1) root-mean-square error of approximations RMSEA; ; 2) compFor each CSQ-SF item, factor loadings, factor variances and residual variances were used to calculate the proportion of variance which was explained by the construct factor (‘of interest’), the scenario factor and residual variance (in combination: ‘nuisance variance’). These are presented in http://www.statmodel.com/download/Plausible.pdf) for each construct factor were imputed using a Bayesian estimator across 50 datasets. Estimated values for each construct factor across these 50 models were then used to investigate the association between each construct factor and SMFQ in separate models.We then used these latent factors in a series of linear regression models to investigate the association between each construct factor and SMFQ factors at a) age 18, b) age 19 and c) age 19 adjusted for SMFQ scores at age 18. These associations were investigated initially in a combined model  using a maximum likelihood estimator. In order to look at the association between SMFQ and each construct factor in isolation, plausible values .As can be seen in 3.2.1.2n=3845). In contrast, once this was extended to a larger sample (n=8446) many of the independent associations were diminished, potentially indicating that these associations were over-estimated in the complete case sample. However, there was remaining evidence that the global attribution sub-scale was independently associated with future depressed mood.As can be seen in 3.2.2global attributions and later depressed mood, independently of baseline depressed mood and other CSQ-SF constructs. Therefore, the conclusion of this analysis is that global attributions are the only construct to be independently associated with depression; other constructs did not appear to explain any unique variance over and above that explained by global attributions.As can be seen in 4latent constructs, global attributions were the only construct to be associated with both baseline and future mood independently of other constructs.The findings provide evidence that cognitive style is associated with future depressed mood independently of baseline mood, both as measured by the DAS-SF and the CSQ-SF. However, there was little evidence that dysfunctional attitude scores (DAS-SF) were associated with later depression after adjusting for CSQ-SF scores. In contrast, the association between the CSQ-SF and depression was unaffected by including the DAS-SF. This is consistent with theoretical models . Observational studies alone do not provide causal evidence. However, the findings suggest that attributing negative events to such global causes could be important to the development of later depression, irrespective of whether the cause is also perceived as stable and reflecting low self-worth. In contrast, in the absence of a global attribution to the cause, attribution of negative life events to causes which are stable and reflect low self-worth may not be sufficient to lead to future depression.4.1There are several strengths of the current study. To our knowledge this is the largest sample to date in which prospective associations between measures of cognitive styles and later depressed mood have been investigated in early adulthood. We accounted for potential reverse causality in the association between depression and cognitive styles by adjusting for baseline symptoms of depression. That said, however, it remains possible that there were some individuals in the analyses who had past depression prior to 18 and who may have retained negative cognitive styles from this time, i.e., the ‘scarring hypothesis’.Finally, the study took full account of different aspects of cognitive styles by including both the CSQ-SF and DAS-SF and by using structural equation modelling techniques to separate constructs of the CSQ-SF. The fact that the unique contribution of global attributions was only identified using latent traits further highlights the importance of this approach. Indeed, as shown in However some limitations should also be acknowledged. Firstly the sub-sample of ALSPAC who completed the CSQ-SF at age 18 was a relatively advantaged sub-group of young adults, meaning that the results may be less representative of higher risk populations. Indeed it is hypothesised in cognitive models of depression, that the association between cognitive styles and depression would be greater in high risk samples undergoing stress (diathesis stress). It is therefore, important to investigate the potentially moderating effect of stressful circumstances on the associations between cognitive styles and depression in future studies. It is also important to note that associations were attenuated in sensitivity analyses which extended to a larger and less advantaged sample. One possible explanation for this is that the association between depressed mood at age 18 and 19 was under-estimated in the complete case sample because those with more chronic depression were most likely to have missing data. Thus, once this association was more accurately estimated it had a greater effect on the associations between cognitive styles and later depressed mood. Nonetheless, the association between global attributions and later depression remained in this sensitivity analysis.In addition, the follow-up was relatively short (10 months) and was within a specific time period (between 18 and 19 years of age). This period reflects the transition to adulthood. At this time changes in home, social, educational and career environments are inevitable and cognitive styles may be particularly relevant to depression . It is a4.2global attributions may be important in preventing depression. The importance of global attributions also has implications for prevention strategies for depression where challenging beliefs of low self-worth is a core focus. The present findings suggest that concentrating on making such beliefs less global could be just as effective as attempting to remove such low self-worth beliefs entirely. This approach could provide a more focused and thus achievable target for prevention. It may be acceptable  to accept that we are not good at something, as long as this does not extend to other domains. For example, we could accept that a failed job application may say something about our skills relating to that job specification, while also focusing on the fact that this bears no implications for our aptitude for other jobs, or our skills as a friend/partner. Encouragement of more specific  attributions to negative events by parents and teachers could thus potentially provide an effective prevention and treatment strategy (see case example). Finally, the findings that the scenarios accounted for a large proportion of variance on the CSQ also highlight the possible importance of the scenario in both the measurement and understanding of cognitive styles.XXX was an 18 year old student who recently started a summer internship in a medical lab. She experienced a lot of anxiety about doing things well and reported difficulty in coping. During a course of Cognitive Behavioural Therapy a conditional belief was identified that could be summarised as “If I don׳t do things perfectly then I am a failure as a person”. She often felt stressed when she was slightly late for school and her new internship, or had forgotten things. In one incident, she had forgotten to take some extra work into school. She then had thoughts “I am hopeless” and “I can never do anything right” and her mood dropped. The therapist worked with the patient to reduce the global way she interpreted these minor things going wrong. Rather than thinking that one minor event means that everything else will go wrong, she was encouraged to treat it as an isolated incident with little influence over other events in her life or her own view of herself.The findings suggest that understanding the developmental origins specifically of 4.3global could be particularly important for future depression. Reducing global attributions in response to events may thus be key in the prevention and treatment of depression, through CBT or other psychological therapies.Consistent with cognitive theories of depression, the present findings provide evidence for the association between ‘depressogenic’ cognitive style and baseline and future depressed mood in a large community sample. The findings suggest that the tendency to attribute negative events to causes that are The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.UK Medical Research Council (Grant ref: 74882) the Wellcome Trust (Grant ref: 076467) and the University of Bristol provide core support for ALSPAC. The current project was supported by a Wellcome grant held by GL (084268/Z/07/Z) and an Elizabeth Blackwell Institute for Health Research Institutional Wellcome Strategic Award supporting an early career fellowship held by Dr. Pearson. This article is the work of the authors, and Dr Pearson will serve as guarantor for the contents of this article and the analyses of data.The None.