Registering CTs for patients receiving external beam radiotherapy (EBRT) with a boost dose from high-dose-rate brachytherapy (HDR) can be challenging due to considerable image discrepancies . This study is the first to comparatively evaluate image processing and registration methods used to register the rectums in EBRT and HDR CTs of prostate cancer patients. The focus is on the rectum due to planned future analysis of rectal dose-volume response.For 64 patients, the EBRT CT was retrospectively registered to the HDR CT with rigid registration and non-rigid registration methods in VelocityAI. Image processing was undertaken on the HDR CT and the rigidly-registered EBRT CT to reduce the impact of discriminating features on alternative non-rigid registration methods applied in the software suite for Deformable Image Registration and Adaptive Radiotherapy Research (DIRART) using the Horn-Schunck optical flow and Demons algorithms. The propagated EBRT-rectum structures were compared with the HDR structure using the Dice similarity coefficient (DSC), Hausdorff distance (HD) and average surface distance (ASD). The image similarity was compared using mutual information (MI) and root mean squared error (MSE). The displacement vector field was assessed via the Jacobian determinant (JAC). The post-registration alignments of rectums for 21 patients were visually assessed.The greatest improvement in the median DSC relative to the rigid registration result was 35 % for the Horn-Schunck algorithm with image processing. This algorithm also provided the best ASD results. The VelocityAI algorithms provided superior HD, MI, MSE and JAC results. The visual assessment indicated that the rigid plus deformable multi-pass method within VelocityAI resulted in the best rectum alignment.The DSC, ASD and HD improved significantly relative to the rigid registration result if image processing was applied prior to DIRART non-rigid registrations, whereas VelocityAI without image processing provided significant improvements. Reliance on a single rectum structure-correspondence metric would have been misleading as the metrics were inconsistent with one another and visual assessments. It was important to calculate metrics for a restricted region covering the organ of interest. Overall, VelocityAI generated the best registrations for the rectum according to the visual assessment, HD, MI, MSE and JAC results.The online version of this article (doi:10.1186/s13014-015-0563-9) contains supplementary material, which is available to authorized users. Radiotherapy dose-volume parameters for specific organs have been associated with normal tissue toxicity . Howeverα/β) [α/β doses [Therapies with different fractionation can be adjusted for fractionation effects by converting to equieffective dose given in 2 Gy fractions (EQD 2α/β) , 6. Howeα/β) . A rigidα/β) . A total/β doses , 8. Comb/β doses  and is sThe accuracies of DIR algorithms have been examined experimentally using deformed phantoms or image modification to include deformations , 11. ThePublications are lacking in the context of registering an EBRT pelvic CT to a high-dose-rate brachytherapy (HDR) pelvic CT. Image-intensity based DIR algorithms applied to such CTs are susceptible to errors when there are major image differences . This apThis study examines the performance of image processing and non-rigid registration tasks available in commercial software and customizations to an open-source package when applied to register the rectums in prostate EBRT and HDR data. Specifically, how did they perform in terms of the Dice similarity coefficient , HausdorThis study used treatment plans for 64 prostate cancer patients who were treated with EBRT followed by a boost dose from Iridium-192 HDR via after-loading hollow metal needles at Sir Charles Gairdner Hospital in the period 2004–2008. Patient criteria and treatment methodology were as specified for the Trans-Tasman Radiation Oncology Group (TROG) 03.04 Randomized Androgen Deprivation and Radiotherapy (RADAR) trial . A plannThe external wall of the rectum was manually delineated by treating clinicians in the EBRT CTs using the Elekta Focal treatment planning software  and in the HDR CTs using the Brachyvision planning software . Rectum outlines were reviewed (by author MK) for consistency between patients. The superior border of the rectum structures in the EBRT CTs were defined by the level that the rectum turns horizontally into the sigmoid colon and the inferior border defined on the most inferior axial image slice on which the ischial tuberosities were visible. Any further references to rectum ‘structure’ refer to the 3D manual outline of the external rectum wall while ‘contour’ refers to the 2D section of this outline on a particular image slice.The TROG 03.04 RADAR Trial is registered with the National Institutes of Health Clinical Trials Registry (number NCT00193856). This trial has approval from the Hunter New England Human Research Ethics Committee , the Sir Charles Gairdner Group Human Research Ethics Committee (2003-050) and the University of Western Australia Human Research Ethics Office (RA/4/1/5601). Patients participating in the trial signed consent forms.The signed patient consent forms for the trial informed patients that their medical information may be used to publish the results of the study. In accordance with the signed patient consent forms, this publication includes only anonymized information and does not include information identifying any patient.Figure A manual rigid registration  was performed in Velocity Advanced Imaging 2.8.1  to align the bony anatomy in the EBRT and HDR planning CTs. An automatic rigid registration was then performed to optimize the registration.TM R2010a , CERR (version 4.1) [Copies of the HDR CT, the re-sampled rigidly-registered EBRT CT and the rectum structures from the HDR and rigidly-registered EBRT CTs were exported from Velocity Advanced Imaging (VelocityAI) in DICOM format for further image preprocessing in MATLABion 4.1)  and DIRAion 4.1) . At the The HDR needles, HDR rectum packing material and HDR rectum low CT number artefacts were replaced with the average CT number of neighboring tissue pixels.A Gaussian smoothing and blurring process was applied to avoid features in the HDR image caused by the previous pixel adjustments.Rectum painting  with a uPrior to DIR in DIRART the image processing detailed below was applied as the image processing led to a considerably improved post-registration rectum alignment. In Additional file Image processing was not applied prior to DIR in VelocityAI as the post-registration alignment in VelocityAI was reasonable relative to registrations obtained in DIRART without image processing. The multi-pass DIRs in VelocityAI (version 2.8.1) were based on the B-spline algorithm with the Mattes mutual information metric . AdditioThe DIR in DIRART was applied to the EBRT rigidly-registered CT and the HDR CT after image processing as this led to a considerably-improved post-registration rectum alignment and made it more comparable with the VelocityAI alignments. The original Demons and original Horn-Schunck optical flow (HSOF) algorithms were used. These DIRs use the root of the mean of the squared-intensity differences as the image-similarity metric . The defThe anatomical alignment for 64 patients was initially inspected by the researcher running each registration (author CRM). The post-DIR anatomical alignments for 21 of the 64 patients were inspected by a combination of in-training (author VL) and experienced (author CIT) radiation oncologists. The alignment between the rectums in the HDR CT and the registered EBRT CT was graded slice-by-slice using the spyglass tool in VelocityAI. The grades were ‘approved’, ‘indifferent’ or ‘unapproved’. The grading was based on whether the misalignment was clinically relevant and was similar to the situation where an observer has to decide if a contour is sufficiently inconsistent with anatomy to warrant re-contouring. The results were assessed by calculating the proportion of slices with grades of the ‘approved’ type.The Dice similarity coefficient (DSC) was calculated as the volume of overlap of the two structures and normalized by the average volume of the structures. The DSC range is zero (no overlap) to one (perfect overlap) . The HauImage similarity was examined via the percentage increase (decrease) in the image-similarity (dissimilarity) metric relative to that before the registration. The mutual information (MI) was used for similarity and the root of the mean squared error (MSE) for dissimilarity , 19. UsiPhysically unachievable organ deformations are indicated by negative Jacobian determinants (JAC) of the DVF . ConsequP-values were considered significant if less than 0.05.Paired percentage differences between the absolute DSC/ASD/HD results for different registration comparisons were tested for significance via exact Wilcoxon signed-rank tests against a zero median. The percentage JAC metric and the proportion of approved rectum-alignments for different registration comparisons were expressed in absolute difference and subject to the same test. Quantile-quantile plots showed that differences were not normally distributed. The tests were performed in R (version 2.15.2)  using thThe major misalignments after DIR were observed around the pubic symphysis, ischium near the inferior extent of the obturator foramen, superior ramus of pubis near the obturator canal, coccyx, medial aspect of the acetabulum and anterior side of the rectum  in the median MSE relative to the rigid registration value Fig. , whereasTo determine orderings, Wilcoxon signed-rank tests of the pairwise differences in the percentage of voxels with a negative JAC between the HS, D, V1 and V2 methods were performed for the 64 patients. When the V2, V1, HS and D methods were compared for the DVF across the whole image, the ordering of methods according to increasing percentage of voxels with a negative JAC was D, V2 and V1/HS. However, the medians of the percentages of voxels with a negative JAC were zero for the VelocityAI methods when calculations were restricted to the region contained by the volume of the rigidly-registered EBRT rectum structure. For this region, the ordering of registrations in terms of increasing percentages of voxels with a negative JAC was V1/V2, D and HS. Alternatively, Additional file The V1 method was superior to the V2, HS and D methods in terms of significant differences in the proportions of slices with the rectum-alignment approved according to the visual assessments. Additionally, the VelocityAI methods (V1 and V2) resulted in superior rectum alignment approval-proportions compared to the DIRART methods. This was inconsistent with the structure-correspondence metric results, where the HS and D methods achieved better DSCs with worse HDs. The inconsistency between the results of metrics and visual assessments has been identified before . AdditioThe visual assessment results can be confounded by intra-observer and inter-observer assessment variations ; howeverDIR was useful as, for example, the DSC, ASD and HD results were improved by applying DIR methods after rigid registration. The improvement in the median DSC was 35 % for the HS algorithm with image processing as compared to rigid registration. This compares well with the 31 % improvement in the mean DSC obtained by a study using the same algorithm with similar image processing tasks in the context of registering daily mega-voltage CT images to treatment planning kilo-voltage CT images . The resThe results for the structure-correspondence metrics indicate that the selection of structure-correspondence metrics should be made carefully. The HS method was superior to the D, V1 and V2 methods in terms of a better structure-volume match (DSC) and less overall shape discrepancy (ASD). The V1 and V2 methods were superior to the HS and D methods in terms of extreme shape discrepancy (HD). The inconsistency of these metrics contrasts with another study where they were useful for evaluations . In thisIt is important to calculate metrics over a restricted region that covers the area of concern or the organ at risk rather than the whole image when assessing whether the registration is acceptable in that area or for the organ at risk. The reason is that the registration is optimized over a region of interest and the performance can vary locally. For example, the V1/V2 methods provided optimal rectum results in terms of MI and MSE calculated in the region defined by the volume of the rigidly-registered EBRT rectum structure, whereas the HS method provided the best MSE result when calculated over the whole image. Additionally, unlike the DIRART algorithms the VelocityAI algorithms led to improvements in the image-similarity metrics calculated across the rectum relative to the rigid registration result. The choice of metrics can be important as elsewhere the MSE was found to not be useful for evaluation .The results for the rectum were sufficiently different to distinguish the best-performing VelocityAI registration from the best-performing DIRART registration. Relative to the DIRART algorithms, the VelocityAI algorithms did achieve better image similarity and visual alignment over the region contained by the volume of the rectum structure. Additionally, the VelocityAI algorithms appeared to do so with less physically-unrealistic displacements  and less extreme shape discrepancy between the fixed and propagated rectum structures . As there was no image processing prior to the VelocityAI algorithms, the VelocityAI algorithms achieved these superior results whilst exposed to rectum discrepancies. As such, this study demonstrates the VelocityAI DIRs (B-splines based) appeared to result in the best rectum alignment and achieve DVFs with the least physically-unrealistic displacements.This evaluation is based on the algorithms in the form they were released. Also, the user cannot change the registration parameters in VelocityAI. If the parameters in both packages were adjustable it would be a useful and difficult task to find optimal performance .The comparative evaluations of rectum registrations from different registration systems is important for adequately accumulating dose for combined EBRT/HDR prostate cancer treatment and correlating it with observed gastrointestinal toxicities. The assessment of impact of image registration on dose-outcomes correlation will provide additional validation of the alternative approaches, and this is the subject of ongoing investigation.Registrations may benefit from images immediately prior to the HDR insertion of needles as this may allow changes over the preceding months to be separated from changes due to HDR needles and treatment positioning.Given the image discrepancies, it would be useful to evaluate registrations including a recently-developed penalty term minimizing the volume of missing information , methodsEvaluation of registrations customized for the urethra, bladder, prostate and seminal vesicles would be useful as they require work on considerable image issues .Registration evaluation for patients can be difficult and involve a variety of methods as there is no direct measure of registration error due to no known ground truth. Information obtained from other evaluation methods such as landmarks, phantoms and deformed dose uncertainty , 33–36 wThis study demonstrated that structure correspondence, image similarity and visual assessments are useful for assessing registrations applied to EBRT and HDR CTs of prostate cancer patients. We found that using non-rigid registrations in VelocityAI or image processing plus non-rigid registrations in DIRART improved the alignment of the rectum according to visual assessment and various metrics. It would have been misleading to use a structure-correspondence metric as a sole indicator of rectum alignment given that such metrics were inconsistent with other metrics and visual assessments. It is recommended that image-similarity and displacement-vector-field metrics be calculated for a restricted region covering the organ of interest instead of using global values. Applying the DIR methods in VelocityAI provided the most optimal registration result for the rectum as assessed by the greatest rectum alignment approval-proportion, the least extreme shape discrepancy between rectum structures and the most optimal rectum image similarity. We encourage the development of registrations for the prostate and urethra in EBRT and HDR CTs as doses to the prostate and urethra are key clinical concerns in the RADAR trial.Additional file 1Online supplementary material providing additional method details, patient images, metric values and statistical analysis. (PDF 0.46 kb)Additional file 2Online supplementary material providing metric results for the 21 patient subsample. (PDF 796 kb)


Antibody response to carbohydrate antigens is often independent of T cells and the process of affinity/specificity improvement is considered strictly dependent on the germinal centers. Antibodies induced during a T cell-independent type 2 (TI-2) response are less variable and less functionally versatile than those induced with T cell help. The antigen specificity consequences of accumulation of somatic mutations in antibodies during TI-2 responses of Marginal Zone (MZ) B cells is a fact that still needs explanation. Germline genes that define carbohydrate-reactive antibodies are known to sculpt antibody-combining sites containing innate, key side-chain contacts that define the antigen recognition step. However, substitutions associated with MZ B cell derived antibodies might affect the mobility and polyspecificity of the antibody. To examine this hypothesis, we analyzed antibodies reactive with the neolactoseries antigen Lewis Y (LeY) to define the residue subset required for the reactive repertoire for the LeY antigen. Our molecular simulation studies of crystallographically determined and modeled antibody-LeY complexes suggests that the heavy-chain germline gene VH7183.a13.20 and the light-chain Vκ cr1 germline gene are sufficient to account for the recognition of the trisaccharide-H determinant Types 1–4, while the specificity for LeY is driven by the CDR3 backbone conformation of the heavy chain and not the side chain interactions. These results confirm that these monoclonals use germline-encoded amino acids to recognize simple carbohydrate determinants like trisaccharide-H but relies on somatic mutations in the periphery of the combining site to modify affinity for LeY through electrostatic interactions that leads to their optimized binding. These observations bring further attention to the role of mutations in T-cell independent antibodies to distinguish self from non-self carbohydrate antigens. Antigen binding by an antibody is mediated by atomic interactions between the paratope (an antibody-combining site) and the epitope (antigen/determinant). An emerging concept, based on analysis of high-resolution crystal structures of carbohydrate-reactive antibodies and high throughput screening, is that germline antibodies often have polyspecific carbohydrate-binding paratopes and that Complementarity-Determining Regions (CDRs) may, contrarily to current paradigm, play only a secondary role in modifying the fine specificity of determinant recognition The evolutionary conservation of preformed paratopes has been observed earlier for instance in studies on the anti-phosphoryl choline T15 idiotype To further the understanding of carbohydrate recognition by antibodies in these terms we reconsidered the restricted V gene usage and somatic adaptation, of antibodies to the neolactoseries carbohydrate antigen Lewis Y (LeY). The histo-blood-group Lewis antigens are cell-surface carbohydrates associated with a large number of human cancers, including lung, breast, colon and ovarian carcinomas. The Lewis antigens play an important role in tumor growth, progression and metastases cr1 germline gene, encodes the key structural features defining the recognition of the footprint of the trisaccharide-H determinant Types 1–4. Most notably, the LeY-reactive antibodies present an example in which the somatic mutation does not involve residues in direct contact with the LeY antigen but the substitutions lend to both a restriction and increase in antibody flexibility relative to the germline precursor. This suggests that these antibodies are most likely derived from low intensity mutation and selection process outside germinal centers that yields with high probability the desired specificity distinguishing between self and non-self carbohydrate epitopes by modulating structural flexibility and relying on electrostatic interactions with the LeY antigen.The LeY antigen is closely related to the murine embryonic antigen SSEA-1 (LeX) in its carbohydrate structure. LeX is a self-antigen in mice . The Variable regions of the four antibodies were structurally aligned using the Align Structures Module of the Protein Modeling Protocol in Discovery Studio (DS) 3.1 . The murine form of hu3S193 was constructed starting from the humanized structure, using the ‘Build Mutant’ protocol in DS to perform the step-by-step mutation of the humanized form in reverse order while calculating the Root Mean Square Deviation (RMSD) of the new structure at each step against the preceding structure. This was done to ensure that the conformation of the antibody does not change drastically compared to a single-step all-mutation process.The structure of the binding site was addressed using computer-simulated molecular models generated by homology modeling http://www.ncbi.nlm.nih.gov/igblast/) search with V region of the L and H chains of each murine protein separately as the query sequence. The VH and VL sequences ranked highest by E-value were chosen as the candidate sequence for the germline VH- and VL-model building. Single amino acid substitutions were made on the available mature murine structures  using DS’s ‘Build Mutant’ protocol, which resulted in the final germline antibody structures. At each mutation step, an all-atom-based RMSD calculation was performed against the initial structure to check conformational stability. RMSDs were calculated and compared among the germline structures differing in their starting structure to look for robustness of the modeling software The germline antibody model was built by identifying a suitable template structure based on sequence homology. The putative germline sequences were obtained by IgBLAST  simulation in vacuo, the structures were energy-minimized with 2000 cycles of Steepest Descent followed by another 1000 cycles of minimization using the Conjugate Gradient method to eliminate close contacts between atoms Calculations were performed under The resulting individual structures of the ensemble were evaluated for their total energy and how close each was to the initial starting structure based on an all-atom RMSD calculation. The lowest-energy structure with the lowest RMSD was picked for further analysis. The RMSD fluctuations were calculated using the ‘Analyze trajectory’ protocol in DS. The Van der Waals contacts for the final structures were determined using a cutoff of 4 A°. The potential intermolecular hydrogen bonds were listed out by ‘Intermolecular Hydrogen Bond’ monitor in DS and also visually confirmed. The interaction energy between the antibodies and the LeY was calculated using the ‘Interaction Energy’ protocol in DS. In some cases, individual interaction energies with respect to the amino-acid residues were also calculated. Individual H- and L-chain contributions towards antigenic interaction were also determined for some specific cases.in vacuo by setting the dielectric equal to one. While minimization with charged residues in the absence of a solvent  is very unrealistic and is sure to move a structure far from its X-ray-determined position, we compensated for this effect by restraining interaction sites corresponding to an X-ray-determined position The monoclonal antibodies were also subjected to restrained dynamics where residue-specific harmonic and distance restraints were used. MD calculations were performed first by restraining those germline residues using a distance restraint that were observed in the human anti-LeY antibody crystal structure to participate in intermolecular hydrogen bond interactions with LeY (pdb code: 1S3K). This was repeated next by restraining those residues that the germline model of the human anti-LeY antibody picked up for intermolecular hydrogen bonding with LeY at the binding pocket. All calculations were conducted H or VL domain, and (2) by mutation type as R (replacement) versus S (silent) according to their effects on the antibody’s amino-acid residues. The resulting data for all four antibodies were then analyzed for whether R mutations occurred significantly more often in one region compared to the other, using a stratified-analysis approach with each antibody as a stratum, in order to prevent bias due to Simpson’s Paradox H and VL domains.In each murine antibody considered, the nucleotide changes from the germline sequences were individually classified (1) by region as FR (framework region) versus CDR (see above) according to their locations in the VL and the VH domains . Moreover, this result was accompanied by a non-significant Breslow-Day test result , which indicates that the individual OR of each antibody does not differ significantly from the common OR of 6.26.The murine V domains  of the f domains  and Vκ c domains  gene fam domains  and also domains , followevy chain , BR96 haL domains (data not shown) was also supportive of increased odds favoring R mutations in CDRs (common OR of 2.42), but the increase was not significant for VL . Overall these results suggest that transversion type mutations, associated with affinity maturation, are observed in these antibodies despite their evident TI origin.For light chains , more trH and VL domains is the same in the bound conformation for all four antibodies being compared. RMSD values observed to be within 0.5Å were calculated taking into account the backbone Cα atoms and a pairwise comparison of the bound conformations were also made. The CDRs were also separately structurally aligned following a pairwise superimposition. The LeY antigens from the four antibodies were also individually superimposed to further ensure that they are all structurally similar.The sequence comparison of the germline sequence with the crystallographically determined anti-LeY antibodies provides a template for the variable region of the germline sequence. Super-positioning of the heavy and light chains of the respective antibodies suggest good structural similarity between the four antibodies, and the relative orientation of the VThe germline model antibody was constructed in accordance with the homology-based modeling approach and the resultant model structure of the germline sequence was subjected to energy minimization and MD simulations. To determine the relative importance of individual amino acid replacements that might affect antibody structure, a BLAST search of the VH7183.a13.20 gene on structurally known antibodies was performed. High levels of homology were observed with the several crystallographically resolved antibodies. The highest homology level  was with an anti-canine lymphoma monoclonal antibody (Mab231) Based upon the LeY-crystal structures and sequence homologies, the germline polar contacts involve hydrogen-bonding residues that include the side-chain interactions of L-His 27D and H-Tyr 35 contacting Gal, side-chain interactions of L-Asn 28 and L-Ser 27E contacting Fuc1, the side chain of H-Tyr33 and its backbone atoms contacting NAG, the backbone interactions of H-Tyr 96, H-Asp 97, H-Gly 99 and H-Ala 100, contacting Fuc4 . The intAnother site of substantial change is the CDR3-H region, where some of the germline antibody’s contact residues have undergone somatic mutation. This is in keeping with arguments that the VH CDR3 loop exerts the greatest influence on antigen-binding specificity D) for the interaction   . As noteD trend. The most predominant change in amino acid residues occur at L-Ser 27E, which is conserved in all antibodies except for BR96. The emphasis on electrostatics in the interactions would suggest that the transition-type substitution of Asn for Ser at position L-27E, as found in BR96, would increase the binding energy for LeY. Indeed, this substitution in the germline antibody model results in an interaction energy of −232 Kcal/mol and no significant change in the Van der Waals energy component   .rd highest, even though they are tied for having the fewest number of mutations (four) in this hypervariable region, whereas B3 has the 2nd highest CDR-2 contribution even though it has the most number of mutations (six) in this region. These results suggest that there is no direct correlation between the number of mutations and the electrostatics –an argument in favor of the existence of multiple short pathways for the somatic evolution of these antibodies.Since the majority of the mutations were found to occur in the CDR2-H region, and since the Van der Waals component of the total interaction energy is already optimized from the germline to the mature antibodies, it becomes important to analyze the electrostatic contribution to the total interaction energy of the antibody-antigen complex. The interaction-energy calculations between our antibody-LeY complexes and their respective CDR2-H regions  show thaTo determine if the few somatic mutation optimized the binding-site conformation for LeY recognition, we compared the degree of conformational diversity between the various states of the antibodies generated during the MD simulation. Analyzing the backbone RMSD from the average structure over the course of the trajectory illustrates an antibody’s stability and plasticity. The RMS fluctuations for the mature antibody-LeY complexes were compared individually with respect to the RMS fluctuation curve of the germline antibody-LeY complex over time. As shown in LeY-reactive antibodies are associated with the VH7183 family, suggesting that they arise from the mouse natural autoantibody repertoire Greater affinity is accomplished through the optimization of electrostatic interactions in the binding site to fine-tune the nuances of the antigen’s fine structure (e.g. stereo-specificity in LeY). The observed R/S ratios for CDRs in the monoclonals indicate that these regions are more prone to R-type mutations than the framework regions despite their evolving from T-cell independent events as typically thought of for T cell-dependent (TD) antigens. When compared against the inherent R/S ratio We also observe that small structural changes in a binding site can completely change the balance between the enthalpy contributions of the scaffold and directional interactions that enable a variety of different structural solutions to be explored to achieve the same functional outcome. Changes in the electrostatic contribution in interaction with a ligand need not involve contact or recognition site residues, nor is one substitution type more important than another.For antibodies against T-cell-dependent antigens, the acquisition of high affinity to antigens is known to proceed via antigen-driven evolution based on somatic hypermutation In some species somatic diversification exists also outside of the germinal center microevolution and instead of adapting the BCR to the selecting antigen it rather ensures globally the completeness of the repertoire prior to the antigen encounter The hypothesis of an alternative way for adaptation of TI-2 antibodies is complementary rather than antidogmatic. The current paradigm considers only two alternatives: TD responses with SHM and TI-2 responses without a SHM adaptation. Anything in between is considered a misclassified case of one of the above. Repertoire generation and affinity maturation are different outcomes of somatic mutation process. Yet, in the LeY antibodies studied there are signs of adaptation of the paratope to the epitope through somatic mutations. Apart from the possibility of TD GC process, unlikely in view of the IgG3 isotype of the studied antibodies and the low level of mutations, is it possible that the “tick over” mutation process leads to an affinity selection happening over several cycles of reactivation of the respective MZ B cell clones? This process may be orders of magnitude slower than the GC reaction but in line with the dynamics of the formation of the MZ compartment.LeY and LeX are very similar. Antibodies raised against LeY expressing cells show a typical TI-2 profile and rarely cross-react with LeX while those raised against synthetic LeY coupled glycoproteins are TD and cross-react with LeX The VH7183 germline antibody family, to which the LeY-reactive antibodies belong, is likely to possess a polyspecific-binding site. It is possible that evolution of the germline immunoglobulin genes favored preformed flexible paratopes like that of VH7183, which are only a couple of mutations away from several different biologically relevant specificities. Similar maturation can be easily and consistently achieved during the slow (non-SHM) somatic diversification due to the presence of low level of AID in the early stages of the development of the TI B cell population Our studies show that germline LeY reactive B cells yield higher affinity clones as a result of a somatic diversification. The germline antibody is preformed to recognize salient glycan fragments of LeY. Accumulating discrete mutations as part of a process of diversification that has only recently been addressed yields with high probability clones that accommodate better the fine structure of the antigen fragments. This reflects a strategy different from germinal center related somatic hypermutation for exploration of the paratope space close to the limit of tolerance, as target and self-carbohydrate antigens are naturally highly cross-reactive. While this study highlighted the occurrence of anti-LeY thymus independent antibody related to a single germline V gene family, it might be true to some extent for other types of TI- 2 antigens as well. Thus, as our knowledge about the structural details of antibody-antigen interaction broadens, we may be able to design better antibodies, as well as design improved antibody based vaccines through more refined reverse-engineering concepts based on the knowledge of antibody structural plasticity and specificity, paving the path towards improved immunotherapy for cancer patients.


Despite the detailed characterization of the inflammatory and endothelial changes observed in Sickle Cell Disease (SCD), the hierarchical relationship between elements involved in the pathogenesis of this complex disease is yet to be described. Meta-analyses of gene expression studies from public repositories represent a novel strategy, capable to identify key mediators in complex diseases. We performed several meta-analyses of gene expression studies involving SCD, including studies with patient samples, as well as in-vitro models of the disease. Meta-analyses were performed with the Inmex bioinformatics tool, based on the RankProd package, using raw gene expression data. Functional gene set analysis was performed using more than 60 gene-set libraries. Our results demonstrate that the well-characterized association between innate immunity, hemostasis, angiogenesis and heme metabolism with SCD is also consistently observed at the transcriptomic level, across independent studies. The enrichment of genes and pathways associated with innate immunity and damage repair-associated pathways supports the model of erythroid danger-associated molecular patterns (DAMPs) as key mediators of the pathogenesis of SCD. Our study also generated a novel database of candidate genes, pathways and transcription factors not previously associated with the pathogenesis of SCD that warrant further investigation in models and patients of SCD. Sickle cell disease (SCD) is a genetic disorder that affects approximately 300,000 newborns worldwide each year, mostly in developing countriesAlthough SCD is caused by a single amino acid substitution in the β chain of hemoglobin, the disease is characterized by multisystem and progressive organ damage affecting almost every system of the body4High-throughput genomic technologies such as microarrays have contributed to our understanding of complex interactions in multisystem diseases such as diabetes and cancer6812In order to refine our understanding and generate new hypothesis about the different biological systems involved in the pathogenesis of SCD we performed a meta-analysis of two recent gene expression studies involving patients with SCD. In addition, to explore the role of heme in the inflammatory response observed in these patients, we also performed meta-analyses comparing the gene expression pattern of heme-stimulated EC, with that observed in patients with SCD.Four studies fulfilled the inclusion criteria and were selected for our meta-analysis. All of them provided high-quality metadata that allowed the meta-analysis. We first identified relevant biological terms  enriched in each individual study that used samples from SCD patients . The terWe performed a meta-analysis of the two studies that evaluated gene expression signatures in patients with SCD (GSE53441 and GSE35007). Although the GSE35007 database includes 250 samples from children with SCD, we selected only SS homozygous patients with a severity scoreP-value) biological pathways, gene ontology terms, protein-protein interaction (PPI) hub proteins and kinases predicted from the meta-analysis are shown in To gain further insights into the biological processes associated with the expression signature identified in the meta-analysis, a comprehensive gene set analysis was performed, using EnrichR and the list of DE genes. The top significant , which was up-regulated in the meta-analysis involving SCD patients, was again up-regulated in both meta-analyses involving heme-stimulated EC. In addition, the gene coding for Bone Marrow Stromal Cell Antigen-1 (BST2), which is involved in B cell growth and redox reactions was also identified in more than one meta-analysis , and 3 different gene expression studies, respectively: (i) EC exposed to plasma from SCD patients during acute chest syndrome (GSE1849), (ii) SCD adult patients in steady-state (GSE53441), and (iii) SCD children in acute crisis (GSE35007). The list of top 10 DE genes is presented for each meta-analysis . The genanalysis .A gene set functional analysis was then performed to identify biological pathways and gene ontology terms overrepresented in each of the three meta-analyses using data from heme-stimulated EC. Again, pathways and gene ontology terms associated with coagulation and platelet activation, innate immune response, response to oxidative stress and angiogenesis were consistently overrepresented in these meta-analyses .p-value, as well as the top 10 predicted kinases that might control the formation of these TF complexes are shown in To gain insight into the regulatory system upstream of the DE genes identified in the meta-analysis, we used a bioinformatics tools (Expression2Kinase) to identify: (i) the predicted transcription factors (TFs) that likely drove the identified expression pattern; (ii) the intermediate proteins that could be forming a regulatory complex with these TFs; and (iii) the kinases most likely involved in the formation and activation of the regulatory complexes identified. With the candidate regulatory proteins identified, we built a subnetwork/protein complex that connects the TFs to each other and to their activation systems . The topet al. proposed a probable hierarchy of sub-biological process involved in the pathogenesis of SCD, in which reperfusion injury was regarded as the most likely proximate mechanism of inflammationSickle cell disease is a monogenic, yet multisystem chronic progressive disease characterized by tissue damage and complications in nearly every body system18525Here, we used a complementary strategy to further explore the relationship between the biological processes involved in the pathogenesis of SCD consisting of meta-analyses of public databases of gene expression studies. The past 15 years have witnessed an explosive increase in the number of studies using microarray technology in a variety of diseases and phenotypes28311214BMPR2 have been associated with an increased risk of this complication in SCDRobust experimental data demonstrate the prominent role of pathways associated with the innate immune system3418RUNDC3A (RUN Domain Containing 3A) and RAP1GAP (RAP1 GTPase Activating Protein), which are involved in the regulation of GTPase activity. RAP1GAP was also upregulated in monocytes from patients with chronic lymphocytic leukemiain vitroTMCC2 (Transmembrane And Coiled-Coil Domain Family 2), a transmembrane protein whose functions are not well characterized, but that was recently associated with mean platelet volume in a large GWAS studyOSBP2, which codes for Oxysterol binding protein 2 was also upregulated. Oxysterols are cytotoxic components that increase oxidative stress and have been shown to be increased in red cells from SCD patientsNRF2, a major anti-oxidant transcription factor, in astrocyte cellsIFI27  was upregulated in both meta-analyses, of clinical samples and heme-stimulated cells. In two recent publications, this protein was identified as a mediator of vascular injury in inflammatory animal models48PARK7, a gene that encodes a redox-sensitive hypoxia-induced protein associated with cell death from acute ischemia-reperfusion injury50Despite the inherent limitation of analyzing lists of individual DE genes, the list generated by our meta-analysis  also supcAMP responsive element binding protein 1) was identified as the most significant transcription factor. CREB1 regulates cell proliferation, survival and differentiation by mediating the transcription of genes containing a cAMP-responsive element. These include several genes from the immune system such as IL-2, IL-6, IL-10 and TNF-α. It has been proposed that phosphorylated CREB1 directly inhibits NF-κB activation, limiting pro-inflammatory responsesWe also used the gene list generated in our meta-analysis to predict gene regulatory network associated with SCD, and generate new hypothesis about the regulatory complexes governing these gene expression patterns. CREB1 (in vitro experiment using EC)8in vivo human models, we believe that our strategy could add relevant information about the role of heme in the pathogenesis of SCD. Interestingly, pathways and gene ontology terms identified in these 3 meta-analyses were similar to those identified in the meta-analysis between clinical samples, in that innate immunity, hemostasis, angiogenesis, response to oxidative stress and porphyrin metabolism dominated the top enriched terms , and Array-express (http://www.ebi.ac.uk/arrayexpress/). Search was conducted with the terms (“sickle cell disease” and “homo sapiens”). Only studies that offered sufficient metadata for the analysis were included. GEO accession number, sample type, platform used for gene expression analysis, number of samples and gene expression data were extracted from the databases for each study.Microarray datasets that examined potentially DE genes in SCD, and that were publicly available by May 2015 were searched in two public repositories: NCBI Gene Expression Omnibus (GEO)  associated with each genep-value < 0.05 was considered statistically significant. Heatmap visualization of a subset of genes from different studies was performed using the “Pattern extractor” tool from INMEX.Meta-analysis were performed using a web-based tool named INMEX , designed to support and facilitate meta-analysis of multiple gene-expression data sets59p-value significance level of ≤0.05), followed by the Bonferroni adjustment for the terms and the groups created by ClueGO. Fusion criteria to reduce the redundancy of the terms that have similar associated proteins was also applied allowing the maintenance of the most representative “parent” or “child” terms in the generated networks. The Kappa-statistics score threshold was set to 0.3, and leading term groups were selected based on the highest significanceIn order to obtain additional biological information from the list of DE genes, a comprehensive functional gene set analysis was performed using the EnrichR platformp-value was uploaded on Genes2Networks (G2N) module of X2K to identify transcriptional complexes associated with these gene signatures. These complexes were used to identify the protein kinases that are most likely responsible for TF complex formation and functional regulation. Finally, the regulatory network was visualized using yEd Graph EditorTo further refine our exploratory study, we analyzed the regulatory networks of transcription factors and kinases predicted from the list of up-regulated genes derived from the meta-analysis, and from each individual gene expression study. Each gene list was uploaded into the transcription factor (TF) inference module of Expression2Kinases (X2K) software, and the TFs that were most likely involved with identified genes were extracted using the Position-Weight-Matrices (PWMs) databaseHow to cite this article: Hounkpe, B. W. et al. Role of innate immunity-triggered pathways in the pathogenesis of Sickle Cell Disease: a meta-analysis of gene expression studies. Sci. Rep.5, 17822; doi: 10.1038/srep17822 (2015).


Engineering T cells with chimeric antigen receptors (CARs) is proving to be an effective method for directing T cells to attack tumors in an MHC-independent manner. Current generation CARs aim to recapitulate T cell signaling by incorporating modular functional components of the TCR and costimulatory molecules. Development of next generation CARs has relied upon trial and error evaluation of signaling domains. We sought to develop an alternate method to re-direct the T cell receptor which does not rely upon the incorporation of signaling domains into the chimeric receptor. To this end, we developed a tri-functional molecule which is membrane-anchored and redirects the TCR in the presence of tumor antigen. We also included components of the CD4 co-receptor to provide requisite Lck signaling upon ligation of the tumor antigen. Our prototype receptor was directed against the HER-2 proto-oncogene. We have determined that engineering peripheral blood T cells with this novel receptor (termed a Tri-TAC) engenders tumor-antigen specific activation of numerous T cell functions, including cytokine production, degranulation and cytolysis - equivalent to, if not greater than, a 2nd generation CAR bearing the CD28 and CD3zeta signaling domains. Future iterations of the engineered T cells will include chimeric costimulatory receptors to enhance T cell functionality and reduce off target toxicity. This research was supported by the Canadian Institutes of Health Research and the Terry Fox Foundation.


Escherichia coli and plant plastids, a third component – TatB – is involved that resembles TatA but has a higher affinity to TatC. It is not known why most TatA dissociates from TatBC complexes in vivo and distributes more evenly in the membrane. Here we show a TatBC-independent substrate-binding to TatA from Escherichia coli, and we provide evidence that this binding enhances Tat transport. First hints came from in vivo cross-linking data, which could be confirmed by affinity co-purification of TatA with the natural Tat substrates HiPIP and NrfC. Two positions on the surface of HiPIP could be identified that are important for the TatA interaction and transport efficiency, indicating physiological relevance of the interaction. Distributed TatA thus may serve to accompany membrane-interacting Tat substrates to the few TatBC spots in the cells.The Tat system can transport folded, signal peptide-containing proteins (Tat substrates) across energized membranes of prokaryotes and plant plastids. A twin-arginine motif in the signal peptide of Tat substrates is recognized by TatC-containing complexes, and TatA permits the membrane passage. Often, as in the model Tat systems of  E. coli as well as in plant plastids, TatBC-containing complexes recognize the twin-arginine motif and thereby bind the Tat substrates -cofactor absorption that indicates complete cofactor insertion and thus stable folding of HiPIP.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


In humans, search for evidence linking total GABA concentration to gamma oscillations has led to promising -but also to partly diverging- observations. Here, we provide the first evidence of a direct relationship between the density of GABAA receptors and gamma oscillatory gamma responses in human primary visual cortex (V1). By combining Flumazenil-PET (to measure resting-levels of GABAA receptor density) and MEG , we found that GABAA receptor densities correlated positively with the frequency and negatively with amplitude of visually-induced gamma oscillations in V1. Our findings demonstrate that gamma-band response profiles of primary visual cortex across healthy individuals are shaped by GABAA-receptor-mediated inhibitory neurotransmission. These results bridge the gap with in-vitro and animal studies and may have future clinical implications given that altered GABAergic function, including dysregulation of GABAA receptors, has been related to psychiatric disorders including schizophrenia and depression.High-frequency oscillations in the gamma-band reflect rhythmic synchronization of spike timing in active neural networks. The modulation of gamma oscillations is a widely established mechanism in a variety of neurobiological processes, yet its neurochemical basis is not fully understood. Modeling,  Neuronal oscillations across multiple frequencies are a prominent feature of brain function. In particular, high-frequency neural oscillations and broadband activity in the gamma-band ca. 30–200 Hz) have been demonstrated to be involved in a wide range of primary and high-level cognitive processes100 Hz hav34567891013Yet, despite being the subject of a thriving field of research, the neurochemical mechanisms underlying the emergence of gamma-band oscillations in the human brain remain poorly understood. Evidence from animal studies suggests that they are generated by a neuronal network containing interconnected pyramidal cells and GABA (gamma-aminobutyric acid)-ergic inhibitory interneurons15172122A receptor density, which is more closely related to inhibitory neurotransmission than total GABA concentration, could provide a more direct assessment of the relationship between GABAergic signaling and gamma oscillations in humans. Recent findings in rodents have shown that high-frequency oscillations in the ripple range (80–250) are abolished following focal GABAA receptor blockadeA receptor subunit clustering has been associated with a reduction of gamma power in the ca. 40–70 Hz rangein-vitro studies point to the key role of GABAA receptor mediated inhibition in the generation of gamma oscillations81627We hypothesized that GABArange 80–0 are aboA receptor distribution, estimated during rest, on rhythmic activity recorded during visual perception. To this end, we investigated the relationships between the timing, amplitude and frequency of alpha-, beta- and gamma-band oscillatory activity (determined with MEG) and GABAA receptor density  in primary visual cortex (V1). We hypothesized that the PET-based GABAA receptor measure would form an informative proxy for the strength of inhibition in the V1 neural network, and, thus, predict the properties of visual gamma-band oscillations across individuals. Our results demonstrate that the density of GABAA receptors correlates positively with gamma peak frequency and negatively with gamma amplitude in human primary visual cortex.We set out to evaluate the effects of variations in GABAA receptor density using FMZ-PET and gamma oscillation dynamics with MEG in the same population of healthy individuals . The FMZ-PET data used to determine the GABAA receptor density were collected while the subjects were resting in the scanner, and the characteristics of oscillatory neuromagnetic signals in each individual were estimated from their visual responses in a standard n-back working memory task.To investigate the relationship between GABAergic inhibition and high-frequency oscillations in V1, we measured GABAA receptors across the cortex was evident in the bilateral medial occipital cortex  prominent gamma-band response within a window of 0–600 ms in bilateral occipital and occipito-temporal cortices . In paral cortex . The obsl cortex . In addil cortex . The alpl cortex .A receptor density and gamma peak frequency  nor for frequency .The correlation analysis between FMZ-PET data and MEG neural response properties in V1 revealed a significantly positive correlation between GABAA receptor density and V1 gamma oscillation properties, we conducted extensive additional analyses exploring multiple potential confounds. First, to make sure that the observed correlations were specifically linked to the GABAergic properties of the V1 region, we recomputed the correlations replacing V1 receptor density with the overall brain-wide GABAA-density. The results . We also tested whether correlations could arise between the gamma-activity properties and anatomical properties of V1: No significant correlation was detected between the gamma properties and volume  or surface area  of V1 , whereas the correlation between the receptor density and gamma-band amplitude only showed a trend . The correlations results summarized in To assess the robustness and specificity of the correlation observed between GABA results  demonstr1) of V1 . FurtherA receptor density in randomly selected sets of voxels across the brain . The magnitude of the correlations between the V1 MEG estimates and PET GABAA receptor data from any of the 200 random sets of PET-voxels was systematically lower than the ones obtained with the corresponding V1-PET values. We also explored whether our findings depend on the way the gamma-band modulations were evaluated. By replacing absolute with percent signal change, a significant correlation was still present between the GABAA receptor density and the peak gamma frequency ; however, the correlation between GABAA receptor density and peak amplitude was no longer significant . Finally, we found that neither peak frequency nor amplitude of V1 gamma correlated with the subjects’ age .To further probe the robustness of our findings, we also computed correlations between V1 gamma oscillation characteristics (peak frequency and amplitude) and the PET-based GABAA receptors correlates positively with gamma peak frequency and negatively with gamma amplitude in primary visual cortex. These results support the view that gamma-range neuronal response profiles across individuals are shaped by inhibitory neurotransmission, assessed by GABAA receptor density.By combining MEG and Flumazenil-PET measurements the current study reveals that the density of GABAA receptors and gamma peak frequency is in-line with modeling workThe prominent correlation between GABAA receptor density and gamma amplitude was not observed in these studies. Interestingly, previous results have, in fact, shown that the administration of both alcoholA but not the gamma-amplitude- GABAA correlation  predicts saccade distractibilityA receptor density rather than total GABA, neither FMZ-PET nor MRS-derived measures provide a direct quantification of inhibition strength. Flumazenil cannot be used to isolate a specific synaptic GABA-benzodiazepine receptor signal: The [11C]Flumazenil radioligand binds with similar affinity to synaptic (α1-α3) and to extrasynaptic (α5) subunits of GABA-benzodiazepine receptors. In contrast, recent findings indicate that the inverse agonist GABA-benzodiazepine receptor PET tracer [11C]Ro15-4513 can be used to quantify specific synaptic  signals[11C]Ro15-4513 PET with MEG measurements could help fine-tune the relationship between GABAergic inhibition and gamma oscillations. Moreover, it should be noted that at present it is difficult to associate our findings with the extensive modeling, animal and imaging work that shows that increased GABAergic efficacy (as measured by the inhibitory postsynaptic current decay times) leads decreased gamma frequencies32334748A receptor density measurement can serve as a proxy for the total inhibition strength that has been suggested to also affect the gamma frequencyA receptor density links to GABAergic efficacy and the various network kinematics properties that have been shown to influence the frequency of gamma oscillationsFinally, it is noteworthy that although FMZ-PET provides an assessment of GABAA receptor density and MEG based recordings of gamma-band oscillations in primary visual cortex. These results provide the first evidence in humans for a direct link between gamma oscillation properties and inhibitory neurotransmission indexed by GABAA receptor density. Moreover, our findings also suggest that Flumazenil based PET-GABAA estimates provide a spatially specific measure of GABAergic inhibition that, in combination with MEG measurements, could help elucidate the mechanisms that determine the properties of gamma-band oscillations and their variability across individuals in health and disease.In conclusion, the present results show a prominent correlation between PET based measurements of GABAA receptor density were collected while the subjects were resting eyes-closed in the scanner.Thirteen healthy, native French-speaking subjects participated in the study. For one subject, the MEG recording failed due to technical reasons, and a second subject did not participate in the PET recording. In addition, the MEG data of a third subject had to be discarded due to excessive blinking. Both PET and MEG data of sufficient quality were thus recorded from 10 subjects . Informed consent was obtained from all subjects, in agreement with the prior approval of the Institutional Review Board and by the National French Science Ethical Committee (CPPRB). All methods were conducted in accordance with the CPPRB guidelines. The MEG experiment consisted of a classical working memory task of visually presented letters, with 3 s intervals between consecutive letters. Each letter was shown for 300 ms. The paradigm comprised three different memory load conditions , and the subjects indicated with a button press, after each letter, whether the letter matched or did not match the appropriate preceding letter. Here, we focus on the initial average visual response across the three tasks for the non-target trials; we excluded the target-trials where the subjects’ behavior would have been markedly different with respect to the non-target trials. These trials and the early time-window contain the neural responses that reflect the perception and encoding of the presented letters to memory. The PET data used to determine the GABA[11C]flumazenil (FMZ), an agent that binds to benzodiazepine (BZD) receptor, was used. FMZ (RO15-1788) was labeled with 11C, using the methylation process3 FWHM . A 68Ge transmission scan was used to measure the attenuation correction. A partial saturation protocol consisting of a single intravenous injection of a mixture of 5 mCi of [11C]FMZ and 0.01 mg/kg of unlabeled FMZ was used, followed by acquisition of the emission data for 55 minutes. This single injection allowed the calculation of B′max parametric images, i.e., estimates of receptor density, without arterial blood samplingAll data  were collected from the same participants at the CERMEP imaging centre . The MEG data were recorded in a magnetically shielded room using a 275-channel CTF whole-head system. The signals were band-pass filtered at 0.016-150 Hz and sampled at 600 Hz. The PET data were recorded with a Siemens HR + camera, and Neural activity estimates were obtained from the MEG signals with event-related Dynamic Imaging of Coherent Sources (erDICS)Static PET images were obtained by summing frames 8–12 from the dynamic imaging (corresponding to the acquisition period from 20 to 55 min post-injection). These static images of the PET volumes were realigned to the anatomical MRIs. Ten millimeter circular ROIs were placed over the midportion of the pons, a suitable reference region for the calculation of the non-specific FMZ bindingThe MEG estimates were computed separately in each voxel of a grid that covered the gray matter surface of the brain. An equivalent spatial sampling across subjects was achieved by first forming a volumetric grid with 6-mm sampling in the template brain. A grid point was included in the analysis when it was within 1.5 cm of the brain surface. The template brain grid, consisting of 3922 points, was transformed, via an elastic transformation , to each individual’s anatomy. The MEG beamforming estimates were calculated directly at these individual-level grid points. The PET-data were coregistered with each individual’s anatomical MRI in SPM8.A estimate we used the total GABAA receptor density in the unique set of V1-PET voxels. The correlation between the PET-GABAA estimates and the MEG estimates were computed using Spearman’s rho.The MEG-PET correlation analysis was conducted in the bilateral primary visual cortex (V1). The anatomical definition of V1 see c, 2 was A estimates in random voxel sets outside the visual cortex . Here, we selected 100 sets of fully random voxels and 100 sets of random contiguous voxels (clusters); for both set-types, an equal number of voxels to voxels in each subjects’ V1 were selected. We also investigated whether it would matter if one quantifies the modulations as an absolute or percent signal change between the post-stimulus and baseline time-windows. In addition to the MEG-PET correlation analysis, we also evaluated the correlation between the size of V1 (volume and surface area) and the MEG responses. Moreover, because FMZ-PET based measures of GABAA receptor density have been used as a surrogate marker for neuronal densityA density estimates and rhythmic activity could be due to inter-subject differences in neuronal volume of V1. Moreover, previous reports have suggested that the gamma-band frequency is dependent on the surface area of V1A receptor density on one hand and oscillatory amplitude or frequency on the other, across the population, we used uncorrected p-values.We also evaluated the possibility of finding correlations between the V1-specific MEG estimates and PET-GABAFinally, in addition to Spearman’s rank correlation analysis, we also computed Pearson’s correlation coefficient and a Bayes factor (BF) based on the approach described by Wetzels and WagenmakersAs with low number of subjects the correlation estimates may be influenced by outliers in the data, we computed the 95% confidence limits for the correlation estimates using bootstrapping. In the approach we left out data pairs for 2 random subjects 200 times, and computed the correlation and p-values for the re-sampled data. From the obtained distribution of 200 correlation-values we calculated the 95% upper and lower confidence limits. This procedure was applied to all main correlation analyses.How to cite this article: Kujala, J. et al. Gamma oscillations in V1 are correlated with GABAA receptor density: A multi-modal MEG and Flumazenil-PET study. Sci. Rep.5, 16347; doi: 10.1038/srep16347 (2015).


Diagnosis of coeliac disease (CD) relies on a combination of clinical, genetic, serological and duodenal morphological findings. The ESPGHAN suggested that biopsy may not be necessary in all cases. New guidelines include omission of biopsy if the concentration of CD-specific antibodies exceeds 10 times the upper limit of normal (10 ULN) and other criteria are met. We analysed the 10 ULN criterion and investigated multiple antibody-assays. Serum was collected from 1071 children with duodenal biopsy . IgA-antibodies to tissue transglutaminase (IgA-aTTG), IgG-antibodies to deamidated gliadin peptides (IgG-aDGL) and IgA-endomysium antibodies (IgA-EMA) were measured centrally. We considered 3 outcomes for antibody test procedures utilizing IgA-aTTG and/or IgG-aDGL: positive , negative  or unclear (perform biopsy). Positive (PPV) and negative (NPV) predictive values were based on clear test results. We required that they and their lower confidence bounds (LCB) be simultaneously very high (LCB >90% and PPV/NPV >95%). These stringent conditions were met for appropriate antibody-procedures over a prevalence range of 9–57%. By combining IgG-aDGL with IgA-aTTG, one could do without assaying total IgA. The PPV of IgG-aDGL was estimated to be extremely high, although more studies are necessary to narrow down the LCB. The proportion of patients requiring a biopsy was <11%. The procedures were either equivalent or even better in children <2 years compared to older children. All 310 of the IgA-aTTG positive children were also IgA-EMA positive. Antibody-assays could render biopsies unnecessary in most children, if experienced paediatric gastroenterologists evaluate the case. This suggestion only applies to the kits used here and should be verified for other available assays. Confirming IgA-aTTG positivity (≥10 ULN) by EMA-testing is unnecessary if performed on the same blood sample. Prospective studies are needed. Coeliac disease (CD) is an autoimmune mediated enteropathy with tissue transglutaminase (TTG) as autoantigen and is triggered by an abnormal immune response to wheat gluten and related cereal peptides in genetically predisposed persons. The clinical presentation ranges from typical malabsorption signs to rather atypical symptoms and conditions that can affect any organ system.Until recently, the diagnosis of CD was based on the assessment of the highly variable clinical status, assays of different specific antibodies, the histological evaluation of intestinal biopsies, and the response to gluten-free diet The 10 ULN suggestion was derived from 3 studies, all applying the same antibody test. The first study found only CD patients among 91 biopsied adults with IgA-aTTG ≥10 ULN but none of the 7 control patients The performance of antibody tests can be assessed by estimation of their positive predictive values  and their negative predictive values .PPV and NPV depend strongly on prevalence (pre-test probability). The studies cited above were based on very high prevalence of up to 100%. However, pre-test probability in symptomatic patients in clinical practice may be as low as 3 to 10% Here, we report on antibody data from 1071 children, who underwent endoscopy due to gastrointestinal complaints. We investigate diagnostic procedures based on IgA-aTTG measurements alone and in combination with IgG-aDGL. We chose this second antibody because it is directed against an antigen different from TTG and since it may detect antibodies in the case of IgA deficiency. In particular we look at diagnostic procedures with three possible test outcomes: clearly negative, clearly positive (≥10 ULN) and indeterminate. The idea is that clear cases can be spared biopsy while indeterminate cases form a grey zone and require additional diagnostic information (e.g. biopsy). We determine ranges of prevalence for which these antibody based diagnostic procedures are reliable. Our predictions are to be prospectively confirmed in an ongoing antibody trial, AbCD The study was approved by the ethical committees of the University of Leipzig, the Medical Faculty of the Technical University Carl Gustav Carus Dresden, the Medical University Graz, the Medical Faculty of the Eberhard Karls University and the University Hospital Tübingen, the Medical Faculty of the Ludwig Maximilians University of Munich, the Medical Faculty of the Justus Liebig University Hospital Giessen, the Universitaire Ziekenhuizen Kuleuven, and the Azienda Ospedaliera di Padova. Informed consent was not given by all participants and/or next of kin/caregiver. If there was no informed consent, patient records/information was anonymized and de-identified prior to analysis.We analysed serum from 1071 children consisting of 376 CD patients and 695 control patients  in whom CD was excluded by means of duodenal biopsy. A flowchart describing how we arrived at these 1071 children can be found in Selective IgA deficiency (sIgAD) was found in three control patients and 24 CD patients, one of whom had common variable immunodeficiency (CVID). There were 23 children with type 1 diabetes mellitus (20 CD patients and three control patients). The control group also comprised 81 patients with inflammatory bowel disease.Patients were diagnosed and antibodies were tested as described IgA-aTTG and IgG-aDGL were measured  in all sera with test kits from EUROIMMUN, Lübeck, Germany . IgA-EMA) were assessed in all except three sera by indirect immunofluorescence analysis using a combination of primate oesophagus, primate small intestine, and primate liver (EUROIMMUN).We will argue in the results section below that a diagnostic procedure cannot simultaneously reach a PPV >90% and a NPV >90% for a broad range of prevalence without introducing a “grey zone”. We therefore define three possible outcomes for a diagnostic procedure: clearly negative, clearly positive and a grey zone containing the unclear cases. For a single antibody test this requires two cut-values.Using the results of the individual antibody tests, we define two test procedures . The first makes use of the IgA-aTTG test alone and the second combines it with the IgG-aDGL test.We use the manufacturer's cut-off to define clearly negative cases and - following the suggestion of ESPGHAN - define a positive result using tenfold cut-off values. Values between these cut-values comprise the grey-zone.One-test-procedure: CD if IgA-aTTG ≥10 ULN;no CD if IgA-aTTG <1 ULN;otherwise: unclear.Two-test-procedure: CD if IgA-aTTG ≥10 ULN OR IgG-aDGL ≥10 ULN;no CD if IgA-aTTG <1 ULN AND IgG-aDGL <1 ULN;otherwise: unclear.The diagnostic properties of the IgG-aDGL test alone will be discussed separately.The three possible outcomes of the diagnostic procedure and two possible states of disease diagnosis lead to a 2 by 3 contingency table, Assuming that sensitivity and specificity do not depend on prevalence, we can use Bayes' formula to determine the PPV and NPV and the proportion of indeterminate results, all as functions of prevalence. A detailed calculation is provided in the supplement.We use maximum likelihood estimates for sensitivity, specificity, etc. Confidence bounds for PPV and NPV are determined based on a normal approximation for the logit-transformed quantity to avoid being overly optimistic Calculations and graphics were produced with R version 2.14.0 We consider a diagnostic procedure sufficiently reliable over a range of prevalence if two conditions are met: Firstly, that the PPV and NPV estimates both lie above 95%. Secondly, that the 95% lower confidence bound (LCB) for both predictive values be simultaneously above 90% over the whole range of prevalence. The second condition guaranties sufficient statistical precision for the first and can only be fulfilled with large enough data sets.The results of the antibody tests are presented in Many commercially available antibody tests only consider the outcome positive or negative. Applying company cut-offs for children without known sIgAD to our data , specifiThe three-valued procedures defined in the statistics section above do perform very well over a broad range of prevalence, because PPV and NPV are only calculated in cases in which the test procedures make a definite prediction.It is necessary to check the performance of the test procedures at much lower prevalence such as encountered in clinical settings: The dependence of predictive values on prevalence are shown in As we see in Considering the IgG-aDGL test alone for all patients , one finds 106 true positives, no false positives, 675 true negatives, 47 false negatives and 243 unclear cases. Although this yields a PPV of 100%, the low number of true positives compared to the other procedures means that this estimate cannot be provided with a comparable certainty, i.e. the LCB is lower and one meets the stringent criteria only over a prevalence range of 19–29%.Some literature suggests that very young children show a delayed antibody response and thus antibody tests might have a compromised sensitivity Thus, there is no evidence supporting an age restriction for the application of the diagnostic procedures discussed here. Since the conditional distributions of the antibody titres are super-imposable further subgroup analyses in age groups appear pointless and would lack power due to limited sample sizes.ESPGHAN guidelines From 81 control patients with inflammatory bowel disease, 79 (one-test procedure) or 77 (two-test procedure) are correctly identified as negative and the remaining two/four are in the grey zone and would require a biopsy.Until recently, the diagnosis of CD mainly relied on intestinal biopsies. New guidelines propose diagnosing CD in symptomatic patients without biopsy if the concentration of CD-specific antibodies exceeds 10 ULN and further confirmatory tests are positive In the future, the diagnostic strategy to exclude or confirm CD will mainly rely on CD-specific antibodies with intestinal biopsies relegated to difficult border-line cases. However, such diagnostic procedures must be shown to be reliable and in concordance with the earlier definition. This requires that PPV and NPV be simultaneously high over a broad range of prevalence.For such a validation, large sample sizes for both CD and control patients are necessary. To the best of our knowledge our study including 376 CD patients and 695 controls is the largest study investigating antibody diagnostics in paediatric CD.Simultaneously high PPV and NPV are very difficult to achieve unless one introduces a third diagnostic result “indeterminate” in addition to the conventional “positive” and “negative” test results. Calculating PPV and NPV only for those cases in which the diagnostic procedure has a definite result, i.e. excluding the grey zone cases, increases the predictive values.Inspired by the ESPGHAN proposal We showed that if sIgAD is excluded both test procedures are reliable for a prevalence between 9 and 57%. Presumably, the range of prevalence is even broader, but more data would be needed to show this. The PPV for IgG-aDGL test alone with the ≥10 ULN is also very promising, but more studies are needed to verify these results Specialised gastroenterological clinics report very high prevalence of CD (39 to 92%) Both the one and two-test procedure performed comparably well. Nevertheless we argue that the two-test procedure may be safer and advantageous in special situations. When total IgA measurements are not available  measuring IgG-aDGL picks up IgA-deficient CD cases. Indeed, not excluding sIgAD patients from our data, the NPV of the one test procedure is compromised.In addition, there are reports that the percentage of IgA-aTTG negative CD (without sIgAD) is as high as 8% Current guidelines The diagnostic procedures also appear safe in children under two years of age. The point estimates of PPV and NPV for both diagnostic procedures are not markedly different from those in the entire population. We found no false positives among 139 control patients and three false negatives among 42 CD cases. Although some research suggests that one should be wary of negative antibody test results for children under the age of two, we see no indication in our data suggesting that the tests perform worse.A few remarks on the limitations and properties of our data are in order. They were collected over a long period (15 years). Although the CD specific antibodies were measured centrally, we did not re-measure total IgA and thus had to rely on local results assessing sIgAD. We were not able to perform confirmatory assays on a second blood sample. Patients with sIgAD are enriched in our sample inflating differences between the one and the two-test-procedures when they are not excluded. Moreover, the method of acquiring data may well have led to an underrepresentation of unclear cases, both in terms of contradictory antibody results and uncertain diagnoses. When selecting our patients, we only had to exclude six cases with unclear diagnoses To conclude, antibody assays could render biopsies unnecessary in the majority of children if experienced paediatric gastroenterologists evaluate the case, as recommended Figure S1Selection of patients for current data analysis starting from the 1502 data sets with informed consent and antibody data. Only six patients were excluded due to unclear diagnosis.(DOCX)Click here for additional data file.Figure S2Calculation of PPV, NPV and proportion of patients without reliable diagnosis for given prevalence. D- disease controls, D+ CD patients, T- test negative, T+ test positive, T? test result in grey zone, FN-false negative, FP-false negative, TN- true negative TP- true positive.(DOCX)Click here for additional data file.Table S1Double positive (IgA-aTTG and IgG-aDGL) control patients.(DOCX)Click here for additional data file.Table S2IgA-aTTG negative CD patients.(DOCX)Click here for additional data file.


Human respiratory syncytial virus (HRSV) is an important respiratory pathogens amongchildren between zero-five years old. Host immunity and viral genetic variability areimportant factors that can make vaccine production difficult. In this work,differences between biological clones of HRSV were detected in clinical samples inthe absence and presence of serum collected from children in the convalescent phaseof the illness and from their biological mothers. Viral clones were selected byplaque assay in the absence and presence of serum and nucleotide sequences of the G2and F2 genes of HRSV biological clones were compared. One non-synonymous mutation wasfound in the F gene (Ile5Asn) in one clone of an HRSV-B sample and one non-synonymousmutation was found in the G gene (Ser291Pro) in four clones of the same HRSV-Bsample. Only one of these clones was obtained after treatment with the child's serum.In addition, some synonymous mutations were determined in two clones of the HRSV-Asamples. In conclusion, it is possible that minor sequences could be selected by hostantibodies contributing to the HRSV evolutionary process, hampering the developmentof an effective vaccine, since we verify the same codon alteration in absence andpresence of human sera in individual clones of BR-85 sample. Human respiratory syncytial virus (HRSV) is one of the most important respiratory pathogen since thNasopharyngeal aspirates were obtained from children hospitalised in University Hospital ofSão Paulo University (HU-USP), in the city of São Paulo, Brazil, during 1998, as previouslydescribed . Informe230 was maintained  was sequenced in50 clones and the F2 region (nt 1-595) in 77 clones . Clones intained . These mintained .N-glycosylation .Four biological clones from Br98-85 exhibited nucleotide alterations in the absence ofserum , to Pro. The samealteration was found in clone 85CS3 in the presence of child's serum  showed one non-synonymous modification atposition 886 leading to a change at residue 291 from Ser, a potential site for's serum . Ser is  pattern . In siliA limitation in this study was the amount of sera that was only enough to carry out onepassage. This limitation involving works using human sera has also been reported . MoreoveThe nucleotide differences between some clones and the master sequence either in presenceor absence of sera observed in this work could inThe minority sequences in a viral population can be selected by neutralising antibodies and theyBesides, they can be selected by human serum, even if just one amino acid has been altered,showing the adaptability of this virus.In summary, we detected mutants in the same viral population and determined if human seracollected from children in the convalescent phase and from their biological mothers couldselect them. Two synonymous mutations were found in the G protein in the absence of serum,one of them in different HRSV-A samples. On the other hand, two non-synonymous alterations(Ser291Pro in G protein and Ile→5Asn in F protein) were found in three clones of HRVS-B inthe absence of serum and in one clone in the presence of child's serum. This findingsuggests that minority HRSV sequences can be selected by the host immune system over time.Information on intrapopulation HRSV variation could be important in understanding thegenetic diversity of this virus. However, more studies are necessary to understand HRSVquasispecies dynamics and the relation of viral mutation with human antibody pressure,since immunotherapy and vaccine success also depend on these factors.


Children with the recessive genetic disorder Xeroderma Pigmentosum (XP) have extreme sensitivity to UV-light, a 10,000-fold increase in skin cancers from age 2 and rarely live beyond 30 years. There are seven genetic subgroups of XP, which are all resultant of pathogenic mutations in genes in the nucleotide excision repair (NER) pathway and a XP variant resultant of a mutation in translesion synthesis, POLH. The clinical symptoms and severity of the disease is varied across the subgroups, which does not correlate with the functional position of the affected protein in the NER pathway. The aim of this study was to further understand the biology of XP subgroups, particularly those that manifest with neurological symptoms. Whole genome gene expression profiling of fibroblasts from each XP complementation group was assessed before and after UV-light exposure. The biological pathways with altered gene expression after UV-light exposure were distinct for each subtype and contained oncogenic related functions such as perturbation of cell cycle, apoptosis, proliferation and differentiation. Patients from the subgroups XP-B and XP-F were the only subgroups to have transcripts associated with neuronal activity altered after UV-light exposure. This study will assist in furthering our understanding of the different subtypes of XP which will lead to better diagnosis, treatment and management of the disease. The childhood skin cancer disorder Xeroderma Pigmentosum (XP), and the childhood neurological disorders Cockayne’s syndrome (CS) and trichothyodystrophy (TTD) all occur as a result of autosomal recessive mutations in 1 of 10 genes involved in the nucleotide excision repair (NER) pathway ,3,4. ChiUV-light induced DNA damage both distorts and covalently modifies DNA, requiring the precise NER DNA repair process to correct the damage. In XP, absence of functional NER results in up to 2000 times greater susceptibility to uniformly distributed melanomas and 10,000-fold increase in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) . The firXP can be further sub-divided into seven distinct subgroups, known as complementation groups, XP-A through to XP-G as well as a milder variant form known as XP-variant (XP-V). Each complementation group refers to the presence of a causative mutation in one of the seven XP genes involved in NER ,13 or PODNA damage recognition occurs via global genome repair (GGR) or transcription coupled repair (TCR). After damage recognition occurs, the remainder of the NER pathway removes and repairs the DNA lesion. As the name suggests, TCR detect DNA damage during transcription when RNA polymerase II stalls at a site of damage and recruits CSA and CSB. GGR is responsible for damage detection across the remainder of the genome and involves XPC and the DDB1/DDB2 complex. After the DNA damage is detected via GGR or TCR the remainder of the XP proteins are involved in DNA unwinding  and excision of the damage . The location of the affected gene in the NER pathway does not confer severity of DNA repair deficiency and subsequent clinical manifestation of the disease. Therefore, accurate diagnosis of XP, CS and TTD currently relies on a combination of clinical assessment and identification of mutations in the NER genes.A potential mechanism to understand the biological differences seen in each XP subtype is whole genome gene expression analysis. This approach has been used successfully for understanding subtypes of common cancers, particularly breast cancer . The aim2 UVC-light exposure for one minute. To ensure the pattern of altered transcript expression was robust, two separate cell lines were used for control fibroblasts and each XP complementation group with the exception of XP-E and XP-F where only one cell line was available . Similarity of transcript expression profiles is measured by distance; the vertical height of the branches of the dendrogram , the 4-fold increase in transcripts and the increase to two cell lines for the majority of complementation groups. Nevertheless, the same pattern of distinct changes in the complementation groups with neurological symptoms was present in both studies.Gene expression profiling has successfully been used to find subtypes of disease, with the most reproducible results seen for breast cancer subtypes . To reprThe study reported herein confirms fibroblasts are ideal to study whole genome transcript expression profiles in disorders with reduced NER activity. A high number of transcripts were altered in the control fibroblasts after UV-light exposure and an even higher number of transcripts were altered in most of the NER deficient XP fibroblasts providing a large dataset to use as the basis for determining if each XP complementation group displayed a distinct set of altered transcripts. The control fibroblasts had 2576 transcripts with altered expression after UV-light, which was significantly less than the majority of XP complementation groups  and the median age at death (29 years) in XP patients with neurodegeneration was significantly younger than those without neurodegeneration (37 years) (p = 0.02). The underlying biology of the neurodegeneration associated with XP remains to be elucidated but it appears to have a negative impact on the survival of XP patients. In this study, we found that the XP-B and XP-F complementation groups with confirmed neurological symptoms and lack of UV-sensitivity, clustered separately and had very distinct gene expression profiles. Although skin fibroblasts were used, the results indicated the function of neuronal related pathways was significantly affected by UV-light exposure. In the context of XP patients with neurological manifestations, it is likely that non-UV forms of DNA damage such as oxidative damage are accumulating, resulting in neurodegeneration [A recent study reported the long-term follow-up of 106 XP patients admitted to the National Institutes of Health (NIH) from 1971 to 2009 [neration . However2).Cell lines: Control and XP fibroblast cell lines were obtained from the NIGMS Human Genetic Cell Repository, Coriell Institute for Medical Research, the details of the cell lines are summarized in 2 for 1 min) and left to recover  before RNA was extracted for transcript expression analysis.UVC-treatments: Each cell line was irradiated with UVC-light  from treated and non-treated cell lines as per manufacturer’s instructions. The RNA was cleaned  and quantified using a fluorometer and RiboGreen reagent (Invitrogen). 500 ng of RNA was amplified and biotinylated using the Ambion Illumina TotalPrep kit . After quantification of the amplified RNA, 1–2 µg of biotinylated cRNA was hybridised with a Whole Genome Gene Expression BeadChip (Illumina) containing approximately 24,000 transcripts.p < 0.05) observed (O) number of transcripts compared to expected (E). The O/E ratio was used to indicate the over-representation of specific biological processes rather than absolute number of transcripts. To investigate the relatedness of the XP complementation groups to each other, cluster analysis was performed using Pearson’s correlation and average linkage algorithm for clustering.Data analysis: The transcript expression results were initially cubic spline normalised and the expression profiles and each individual transcript were normalized to the median resulting in two-way normalisation. Volcano plots were generated using Welch’s ANOVA and Benjamini and Hochberg false discovery rate (5%) and fold change of greater than 2. PANTHER ,25 was uThe expression profiles within XP complementation groups appeared to be highly specific and after UV-irradiation, also reflects the clinical symptoms seen in XP. The biological pathways with altered gene expression after UV-light exposure were distinct for each subtype and contained oncogenic related functions such as perturbation of cell cycle, apoptosis, proliferation and differentiation. Patients from the subgroups XP-B and XP-F were the only subgroups to have transcripts associated with neuronal activity altered after UV-light exposure. The results of this study confirm that cluster analysis can identify distinct gene expression patterns for each XP complementation group. The identification of distinct gene expression profiles for XP complementation groups will further our understanding of the biology of XP and may assist in diagnosis of the disease, which currently relies on complementation studies . In addi


The highlands and mountains of Iceland are one of the largest remaining wilderness areas in Europe. This study aimed to provide comprehensive and up-to-date data on non-native plant species in these areas and to answer the following questions: (1) How many non-native vascular plant species inhabit highland and mountainous environments in Iceland? (2) Do temporal trends in the immigration of alien species to Iceland differ between highland and lowland areas? (3) Does the incidence of alien species in the disturbed and undisturbed areas within Icelandic highlands differ? (4) Does the spread of non-native species in Iceland proceed from lowlands to highlands? and (5) Can we detect hot-spots in the distribution of non-native taxa within the highlands? Overall, 16 non-native vascular plant species were detected, including 11 casuals and 5 naturalized taxa (1 invasive). Results showed that temporal trends in alien species immigration to highland and lowland areas are similar, but it is clear that the process of colonization of highland areas is still in its initial phase. Non-native plants tended to occur close to man-made infrastructure and buildings including huts, shelters, roads etc. Analysis of spatio-temporal patterns showed that the spread within highland areas is a second step in non-native plant colonization in Iceland. Several statically significant hot spots of alien plant occurrences were identified using the Getis-Ord Gi* statistic and these were linked to human disturbance. This research suggests that human-mediated dispersal is the main driving force increasing the risk of invasion in Iceland’s highlands and mountain areas. While it is well known that the average proportion of non-native species in polar regions is very low , the totThe beginning of human impact on the vascular flora of Iceland dates back to the ninth century, when the island was first reached by Norse settlers migrating across the North Atlantic. The vegetation of Iceland has changed significantly since that time of settlement. Growing anthropogenic pressure and climatic changes during the “Little Ice Age” (1600–1900 AD) led to significant alteration of the vegetation cover, mainly due to erosion and desertification . HoweverThe latest research on the non-native flora of Iceland has shown that there are significant differences in the composition of the local floras in Iceland . The resThere are two main factors responsible for the low rate of colonization of these areas by non-native species: the low frequency of human-mediated plant dispersal and the harsh climate see , which iHow many non-native vascular plant species inhabit highland and mountainous environments in Iceland?Do temporal trends in the immigration of alien species to Iceland differ between highland and lowland areas?Does the incidence of alien species in disturbed and undisturbed areas within the Icelandic highlands differ?Does the spread of non-native species in Iceland proceed from lowlands to highlands?Can we detect hot-spots in the distribution of non-native taxa within the Icelandic highlands?Future changes in the flora of highland areas of Iceland will be best understood only if measured against a credible baseline. Given the fact that no attempts have been made to summarize existing data concerning non-native plant species in the Icelandic highlands, the present study aimed to answer the following questions:This study is focused on non-native plant species as defined by Data were obtained from the Icelandic Institute of Natural History. The Institute has the largest repository of biodiversity data in Iceland, with over 500,000 georeferenced records of plant species distributions. Only records of non-native taxa  were conhttp://gatt.lmi.is). Elevation (in meters) was then assigned to each data point using the QGIS point sampling tool. A database was developed that contained georeferenced species occurrences linked with elevation data. This database was queried to identify records with an altitude of at least 400 m above sea level  using locally weighted regression - LOESS , using axi is attribute value for feature j, iw, j is the spatial weight between feature i and j, n is equal to the total number of features andHot Spot Analysis  was emplThe A 10 × 10 kilometer grid used in Iceland to record and map plant occurrences  was usedpost-hoc test (http://gatt.lmi.is).The presence or absence of non-native plant species in each grid cell (10 × 10 km) was determined using QGIS software  as well hoc test . Statisthoc test . GIS layLupinus nootkatensis can be classified as an invasive plant. Comparison of the geographic origin of non-native plant taxa showed that most have a European origin, constituting 66% of all non-native flora in the highlands and 49.2% of all non-native plants in Iceland  is the most widespread and invasive non-native plant in the Icelandic highlands. Due to the presence of nitrogen-fixing bacteria in the roots, the species can increase the availability of this nutrient in the soil, which otherwise is low, as in most of the Arctic environments . All investigated non-native species showed clear tendency to occur within approximately 5 km from man-made objects  and roads  and undisturbed areas (10 × 10 km polygons) differ in terms of the occurrence of non-native species  were analyzed to test the hypothesis that the colonization of highland habitats in Iceland can be considered as a “second step” in the process of colonization of the island by non-native taxa. Results showed that species naturalized within highland areas were first well-established in lowland areas, and only then started to spread within the highlands  theFairly accessible highland areas close to Mývatn Lake (north-eastern Iceland) have been strongly influenced by naturalized alien plant species. The climatic conditions in the northeastern part of the country allowed human settlement and farming activities at greater than 400 meters above sea level, facilitating the introduction of non-native plants. In contrast, areas located at greater than 400 meters above sea level in other parts of Iceland (e.g. the Western Fjords and Eastern Fjords) appear to be almost free of non-native plant species. This pattern of spatial occurrence can be explained by human-mediated dispersal. A closer examination of places with very high numbers of non-native species records shows that they are mostly found in areas with tourist attractions such as hot springs  and areas with geothermal activity , as well as near hiking huts and shelters along the highlands (e.g. Jökulheimar).D.caespitosa susbp. beringensis).The study seems to confirm that human-mediated dispersal along a road network is one of the most important factors contributing to plant dispersal . It is cAlpoecurus pratensis and Phleum pratense - and possibly suggests that penetration of agricultural species into the highlands within this area might be more dynamic than in other regions.Only one spatial cluster identified during the present study (areas around southern part of Skagafjörður) seems to depart from what was said above. This cluster contains mostly occurrences of grass species commonly used in Iceland as fodder - Digitaria ischaemum (Poaceae), which seems to have spread to thermal areas in southern Iceland via visitors’ hiking shoes. Future actions to facilitate travel through the central highlands (e.g. construction of new road tracks or improvement of existing routes), will inevitably increase the number of visitors, leading to a greater inflow of seeds and other plant propagules of non-native taxa and therefore a higher risk of invasion. Increased colonization due to increased propagule transfer, may be further facilitated by climatic changes. It has been shown that climatic niche of many non-native species in Iceland will increase dramatically in forthcoming decades (In recent years, Arctic wilderness environments have become a major tourist attraction, while the Central Highlands in Iceland are considered one of the largest remaining wilderness areas in Europe . The inc decades .1.Sixteen non-native vascular plant taxa were recorded in the Icelandic highlands and mountains.2.Temporal trends in alien species immigration to lowland and highland areas of Iceland are similar, but the process of colonization of highland areas is still in its initial phase.3.Non-native plants tend to occur close to all types of man-made infrastructure and buildings including huts, shelters, and road networks.4.The spread of non-native species in Iceland proceeds from lowlands to highlands.5.Several statically significant hot-spots of alien plant occurrences can be identified and are linked to human disturbance.