We derive the Iterative Confidence Enhancement of Tractography (ICE-T) framework to address the problem of path-length dependency (PLD), the streamline dispersivity confound inherent to probabilistic tractography methods. We show that PLD can arise as a non-linear effect, compounded by tissue complexity, and therefore cannot be handled using linear correction methods. ICE-T is an easy-to-implement framework that acts as a wrapper around most probabilistic streamline tractography methods, iteratively growing the tractography seed regions. Tract networks segmented with ICE-T can subsequently be delineated with a global threshold, even from a single-voxel seed. We investigated ICE-T performance using ex vivo pig-brain datasets where true positives were known via in vivo tracers, and applied the derived ICE-T parameters to a human in vivo dataset. We examined the parameter space of ICE-T: the number of streamlines emitted per voxel, and a threshold applied at each iteration. As few as 20 streamlines per seed-voxel, and a robust range of ICE-T thresholds, were shown to sufficiently segment the desired tract network. Outside this range, the tract network either approximated the complete white-matter compartment (too low threshold) or failed to propagate through complex regions (too high threshold). The parameters were shown to be generalizable across seed regions. With ICE-T, the degree of both near-seed flare due to false positives, and of distal false negatives, are decreased when compared with thresholded probabilistic tractography without ICE-T. Since ICE-T only addresses PLD, the degree of remaining false-positives and false-negatives will consequently be mainly attributable to the particular tractography method employed. Given the benefits offered by ICE-T, we would suggest that future studies consider this or a similar approach when using tractography to provide tract segmentations for tract based analysis, or for brain network analysis. Diffusion weighted imaging (DWI) provides a novel and unique method with which to study white-matter microstructure within the brain  and the Partial Volume Effect (PVE), modelling limitations in regions of crossing fibres, and data limitations due to an image resolution that is far coarser than the axons themselves. These issues hamper the inference of any robust form of true anatomical connectivity  from these connection-confidence maps .Furthermore, the application of tract-specific statistics, and therefore also many forms of network analysis e.g. , relies The origin of the PLD effect lies in the mechanics of the probabilistic streamline tractography method, and is simply due to the step-wise dispersion of the propagating streamlines along the length of a tract. Hence image resolution is also a contributory factor. PLD is an inherent side-effect of the probabilistic approach and is manifested as a monotonic, non-linear down-modulation of the calculated probability as a function of the propagation distance from the seed point . Hence tThe PLD effect is a well-known phenomenon and has been previously reported in early tractographic human studies, Whilst few studies have looked into moderating PLD itself, some attempts have been made to compensate for its effects. Normalisation by a distance-correction factor has been suggested , but dueTo be able to delineate specific longer tracts, many studies use constrained tractography (using a priori information such as waypoints) to enable a global threshold. Although this does not solve the PLD issue, the extra a priori info can help to delineate specific tracts. This approach was used together with additional heuristics by Sherbondy et al. thresholdICE-T and streamsICE-T) and their generalizability, and investigates the method's parametric behaviour. We confirm both the applicability of our ICE-T method against the results derived by Here, we visualise the PLD effect and its non-linear behaviour. We propose a heuristic method, based upon probabilistic tractography, to segment out a given tract system emanating from a seed region, with minimal influence from PLD. Based upon a region-growing approach, Iterative Confidence Enhancement for Tractography (ICE-T) is an easy-to-implement framework that is applicable as a wrapper function to most probabilistic streamline tractography methods. This work introduces ICE-T, its modifiable parameters , and the threshold applied to the end-of-iteration connection-confidence map (ICE-Tthreshold).A tractography pipeline generally includes the following steps: voxelwise fibre-reconstruction and generation of uODFs, followed by the streamlining tracking process. ICE-T is a simple modification of this, introducing a feedback loop around the streamlining tracking process, and is described as pseudo-code in i, (where ‘i’ indicates the iteration count) along the tract branches it comes across, as outlined in threshold to produce ICE-T ROIi+1. This ROI, if it has increased in size, is then fed back to the tracking step where it is used as the updated seed region for the next iteration. By employing the connection-confidence value of a voxel as the predicate, a connectivity constraint is automatically imposed upon ICE-T ROIi and all its voxels are thereby classified as being highly connected to one another and hence also to the original seed region. In addition, the connectivity constraint is enhanced at each iteration by the application of a streamline waypoint region through which all streamlines are required to pass. The waypoint region for iteration ‘i’ is defined as ICE-T ROIi-2 . This guarantees that the original seed region will be included in the final segmented tract. For efficiency, the streamline computations are stored between iterations meaning that at each iteration only streamlines from the newly-included voxels need to be generated. The final grown seed region ICE-T ROII , representing the segmented tract system from the original seed, can then be used for tract-based analysis. The resultant ICE-T ROII is not a PICo map, but instead each voxel's value represents an index of connection confidence with every other voxel within the tract, and is hereby defined as the Intra-Tract Confidence.The ICE-T framework consists of iteratively growing the seed region-of-interest (ROI), ICE-T ROIAll procedures followed “Guidelines for the Care and Use of Experimental Animals” and were approved by the Danish Animal Experiments Inspectorate.The participant signed an informed consent following the guidelines of the declaration of Helsinki. The study protocol (KF 01 – 131/03) was approved by the local ethics committee (“De Videnskabsetiske Komiteer for Københavns og Frederiksberg Kommuner”).The data, including ground-truth seed regions defined by tracer injection, from three young and normal Göttingen mini pig brains , as used in 2. Diffusion sensitisation gradient duration δ = 27 ms, time between gradient-pulse onsets Δ = 33.5 ms, gradient strength 56 mT/m. A slice thickness of 0.5 mm, gap 0.5 mm and two sets of 35 interleaved slices ensured whole brain coverage. NEX = 2. The pig brain datasets consisted of 3×b = 0 s/mm2 and one b-value of 4009 s/mm2 (chosen as specified in MRI data were acquired ex vivo on a 4.7T Varian MR scanner using a pulse gradient spin echo (PGSE) sequence with single line read-out using the following parameters: TR = 6500 ms, TE = 67.1 ms; matrix  = 128×128, in-plane resolution  = 0.51×0.51 mmThe application of a spin-echo diffusion sequence, and the absence of both physiological and subject-generated motion, minimised the distortions in the ex vivo data. Visual inspection confirmed that no additional processing of the ex vivo data was required prior to tractography http://dig.drcmr.dk).For tractography, we used the same hand-drawn seed regions as defined in A single healthy volunteer , with no history of neurological or psychiatric disorders, or a family history thereof, nor hypertension, was recruited.2 and one b-value of 1200 s/mm2 were acquired along the 61 non-collinear directions available from Camino 3 isotropic voxels, FOV 256 mm, matrix  = 192×256×256, TR = 1540 ms, TE = 3.93 ms, TI = 800 ms, flip-angle  = 9°) was acquired for the generation of tissue segmentations and thereafter a brainmask.An in-vivo diffusion MRI dataset was acquired on a 3T Trio Siemens MR scanner with an eight-channel head coil  using a twice-refocused diffusion-weighted sequence to minimise eddy current distortion www.fil.ion.ucl.ac.uk/spm) was then used to estimate the voxel displacement map. The DWI images were then re-sliced into the space of the first b = 0 image via cubic b-spline interpolation within SPM8, with voxel intensities per volume scaled by the Jacobian determinant of the calculated transformation matrix. Finally, the rotational part of the affine model was applied to reorient the gradient directions Intra-volume subject motion and the undesired stretching or shearing caused by eddy-current build-up were simultaneously corrected for by estimating a 12 parameter affine model A brain mask was generated in the diffusion image space using the grey- and white-matter (GM & WM) segmentations generated by SPM8 from the MPRAGE T1 scan.For tractography, a cubic seed VOI was specified (by ML) using Matlab, defined so as to approximate the left subcortical motor area. The location and size of the VOI was defined following tractography seeded in the brainstem.For this work we chose, for comparative purposes, to use the same probabilistic tractography method as that was employed in Dyrby et al For comparison, results from tractography were subjected to a linear correction for PLD as described in streams parameter, and with the addition of the ICE-Tthreshold parameter, as described in For the ICE-T Framework, we employ the same setup and parameters for probabilistic tractography as described above, with the exceptions that the number of streamlines was defined by the ICE-TThe ICE-T Framework was implemented in Matlab (Mathworks Inc.) as a data-flow wrapper, handling file management and with calls to the required tractography functions from the Camino package threshold and ICE-Tstreams, using a single pig-brain dataset (P1) and sampling the following values:An initial experiment was performed to empirically investigate the combined impact of both ICE-T parameters, ICE-Tthreshold: ICE-Tstreams: .ICE-Tstreams parameter, one which could then be employed for all three pig-brain datasets.To reduce processing time and storage demands when investigating the ICE-T parameter space, a file repository of 500 projection streamlines per voxel for dataset P1 was generated a priori, from which streamline samples could be randomly drawn as required. This experiment permitted the derivation of a single value for the ICE-Tthreshold parameter was investigated using this fixed value of ICE-Tstreams on all three pig-brain datasets, with the following values:Subsequently, the impact of the ICE-Tthreshold: ICE-Tcanonical, from a collection propagated from the seed region to a waypoint. The canonical streamline, Scanonical, was estimated as that which had the greatest number of points of agreement with all other streamlines. For this we used the first 100 successful streamlines. For analysis and subsequent comparison of connection-confidence values produced with and without ICE-T, Scanonical was then employed as the LOI to extract the tract midline data.To assess the degree of PLD along a selected tract, we select a line-of-interest (LOI) along its midline (see below). To delineate the LOI, we determined the most-likely pathway by selecting the canonical streamline, Scanonical was derived from validated pathways described in In the pig-brain datasets, Sstreams parameter has no global effect upon the size of ICE-T ROII, however the latter does decrease with increasing ICE-Tthreshold. A slight variation in the size of the ICE-T ROII can be observed in the PFC and MC between 5 and 10 streamlines. An outlier is noted at a single point for the PFC at ICE-Tstreams 100 and ICE-Tthreshold 0.25. For the remaining experiments, we fixed the ICE-Tstreams at 20 streamlines, as a compromise between having a sufficient number of streamlines whilst minimising computational resources.The first experiment investigated the combined impact of both ICE-T parameters. threshold parameter significantly impacts the size of the ICE-T ROII as shown in I increases linearly as the ICE-Tthreshold is reduced, down to around ICE-Tthreshold = 0.025. Below this threshold, the increase is greater and non-linear, most likely due to the incorporation of adjacent tract networks into the segmented network along with a consequential increase in the number of false positives. For very low ICE-Tthreshold (approx. <0.001), ICE-T ROIi always grows at each iteration, and hence never generates a specific segmented tract network (results not shown). However, as shown in threshold above 0.005 results in a stable ICE-T ROII. threshold is increased. Further iterations provide no additional change, and the ICE-T halts at this point. The resultant ICE-T ROII therefore represents a segmented tract system emanating from the seed. Similar results are obtained for SC and PFC (results not shown). In the following experiments the number of ICE-T iterations is automatically limited, determined by the point at which the ICE-T ROIi and ROIi-1 show no size differences, and hence the number of iterations is not a parameter in itself.For any seed region, the ICE-TI from the SC seed region along the canonical streamline of the corticonigral tract that projects through a complex crossing fibre region  is illustrated in threshold>0.015 causes the region growing to halt at this complex region. However, ICE-Tthreshold values below this level permit the ICE-T ROII to grow along the entire canonical streamline. Similar ICE-Tthreshold values were also found for this seed region in the P2 & P3 datasets (results not shown).The spatial growth of the ICE-T ROII, thresholded at different levels. In order to extract the entire tract without using ICE-T i.e. using seed ROI (left column), thresholding of the obtained results would require application of a very low global threshold (<0.010). Using the seed ROI, the effect of low thresholding produces a near-seed flare, reflecting the high proportion of false positive connections found close to the seed, where the sampling is still sufficient . Exceptions can be seen in, for example, the projections from the PFC seeds towards substantia nigra in P1 vs those from P2 and P3  with those derived from a seed defined by the segmented tracts provided by ICE-T with an ICE-TThe tractography PICo values in threshold = 0.01 and ICE-Tstreams = 20). Tractography without ICE-T employed the seed ROI, whereas that with ICE-T used the ICE-T ROII as seed. The ICE-T tracts show, as in the ex vivo data, a uniform cross-sectional size along their length as a function of threshold. In contrast, such features are not seen in the tracts generated from probabilistic tractography without ICE-T due to the effects of PLD, as was also observed in the ex vivo pig brain. A possible false positive can be observed in the ICE-T results, seen as a descending tract in the region of the contra lateral capsula interna , and reduced distal propagation , confounds which have been speculated to bias structural connectivity analysis . We haveAt present, the PLD confound is rarely addressed. Indeed, it is common for the threshold-level of probabilistic streamline tractography experiments to either be left extremely low , but especially the tractography method as well as the topology of the particular tract network being analysed.ICE-T has a further benefit of generalizing the parameter choice. Conventional tractography, besides the definition of a seed region, requires the specification of the number of streamlines and usually the global threshold, applied to the probabilistic results in order to delineate the desired tracts. Aside from the seed region, the parameters of the ICE-T Framework are ICE-Tthreshold may be needed to permit successful penetration into the distal portion of the tracts. This underlines that external factors such as tractography method and dataset parameters  influence the selection of ICE-T threshold. However, we have shown how the parameters are generally transferrable to similar, ex vivo datasets , and have also successfully applied it to an in vivo clinical dataset. The ICE-T parameters are not expected to be generalizable across tractography methods or acquisition parameters, however it is expected that they will also exhibit a stable range. In future work we will investigate the effect of various tractography methods upon the ICE-T parameters.The tracking results obtained with ICE-T show wide agreement with those obtained in A major difference between tractography with and without ICE-T is that while the latter outputs a PICo map based upon tracking from a given seed region, ICE-T generates an Intra-Tract Confidence map of all connections within the segmented tract. Interpretation of the Intra-Tract Confidence map is therefore different from that of a PICo map. The direct interpretation of the values has not been considered herein. However, since a PICo map is a metric of streamline propagation from a seed region, it is affected by tract integrity, but is not a direct measure of it. In contrast, the Intra-Tract Confidence map from ICE-T is a metric that reflects the sum of connections from every member voxel, most of which will, by construction, lie within the ICE-T_ROI, i.e the segmented tract. However, this in turn means that it cannot be used directly for network analysis as it does not reflect a probability of being connected to the seed, but instead it can be used as a binarized version of the tract system emanating from the seed. The latter is often used for creation of structural connectivity matrices.i region. This infers the possibility that the segmented tract network might reflect bi-directional pathways along the entire delineated tract. If such behaviour is undesirable then a simple forwards-only directionality constraint could be applied at the end of each ICE-T iteration.In tractography, streamlines are propagated in both directions from the seed region. It should be noted, however, that the ICE-T method we have implemented here is based upon the Camino toolbox and does not include directionality constraints applied to each ICE-T_ROIWhen specifying the initial seed region, we expect that any subset of voxels within the region of interest could be employed. Due to the region-growing feature of the seed region when using ICE-T, it is expected that the iterative region-growing will expand the seed to approximate the entire tract network. This same argument would also imply that care must be taken to ensure that over-inclusive regions are not employed as seeds. For example, we found false-positive lateral branching only in the results using the overly-large ROI when ICE-T is not applied . This suThe impact of PLD on the results of probabilistic streamline tractography is a confound which should be considered. We have shown the non-linear spatial variation of PLD along any given pathway, challenging the application of a global threshold and introducing both false positives (near-seed flare) and false negatives (premature tract termination). We have shown how a novel re-appraisal of the probabilistic streamline tractography pipeline, termed ICE-T Framework (ICE-T), offers the possibility to segment tract systems without the problems imposed by PLD. With ICE-T, PLD issues are substantially reduced to the point where tract networks can be delineated using a global threshold, leading to a reduction in the PLD-related confounds. Importantly, ICE-T only addresses the PLD issue, and preserves all the characteristics of the individual tractography methods. It is recommended that future work should consider handling PLD in order to minimise the risk of bias in tract statistics and structural network analysis.


Activation of eIF4B correlates with Abl-mediated cellular transformation, but the precise mechanisms are largely unknown. Here we show that eIF4B is a convergent substrate of JAK/STAT/Pim and PI3K/Akt/mTOR pathways in Abl transformants. Both pathways phosphorylated eIF4B in Abl-transformed cells, and such redundant regulation was responsible for the limited effect of single inhibitor on Abl oncogenicity. Persistent inhibition of one signaling pathway induced the activation of the other pathway and thereby restored the phosphorylation levels of eIF4B. Simultaneous inhibition of the two pathways impaired eIF4B phosphorylation more effectively, and synergistically induced apoptosis in Abl transformed cells and inhibited the growth of engrafted tumors in nude mice. Similarly, the survival of Abl transformants exhibited a higher sensitivity to the pharmacological inhibition, when combined with the shRNA-based silence of the other pathway. Interestingly, such synergy was dependent on the phosphorylation status of eIF4B on Ser422, as overexpression of eIF4B phosphomimetic mutant S422E in the transformants greatly attenuated the synergistic effects of these inhibitors on Abl oncogenicity. In contrast, eIF4B knockdown sensitized Abl transformants to undergo apoptosis induced by the combined blockage. Collectively, the results indicate that eIF4B integrates the signals from Pim and PI3K/Akt/mTOR pathways in Abl-expressing leukemic cells, and is a promising therapeutic target for such cancers. Abl oncoproteins, including v-Abl, Bcr-Abl and Tel-Abl, are activated non-receptor tyrosine kinases that are associated with various malignant transformations of hematopoietic cells –3. v-AblGrowing evidence suggests that alterations in protein expression occur in various cancers , 9. HoweFor eukaryotic translation initiation, an essential event is the assembly of the translation pre-initiation complex (PIC) , 14, whieIF4B is involved in translation of numerous proliferative or anti-apoptotic mRNAs with highly structured 5′UTR and subsequently affect cell growth and survival , 18. AbnThe activity of eIF4B relies on its phosphorylation status , 23. It In this study, we found that eIF4B was a common substrate of oncogenic Pim and PI3K/Akt/mTOR pathways in Abl transformants, and such redundant activation of eIF4B is responsible for the compromised effect of pharmacological inhibition of single pathway on Abl tumorigenesis. By contrast, combined treatment to block both pathways caused a deeper inhibition of eIF4B phosphorylation, induced more apoptosis in Abl transformed cells, and synergistically inhibited K562 xenograft growth in nude mice. Overexpression of eIF4B phosphomimetic mutant S422E remarkably attenuated the synergistic effects of these inhibitors on suppression of Abl oncogenicity. These results establish a key role of eIF4B in Abl tumorigenesis by integrating the functions of oncogenic Pim and Akt signaling pathways, and highlight eIF4B as a novel therapeutic target for Abl positive cancers.Recently, we observed that phosphorylation of eIF4B was not completely abolished by disrupting the expression of Pim-1 and Pim-2, implying that there exists other Ser/Thr kinase(s) responsible for eIF4B phosphorylation in Abl-expressing leukemic cells . To veriFurthermore, we observed that the phosphorylation level of eIF4B Ser422 was obviously reduced by treatment with Akti-1/2, an isozyme-selective inhibitor of Akt Figure . Consistin vitro kinase assay showed that Akt can directly phosphorylate eIF4B on Ser422 [In addition, rapamycin was employed to determine the functional relevance of mTOR, a downstream effector of PI3K/Akt pathway, in eIF4B phosphorylation. Interestingly, treatment of cells with rapamycin also significantly impaired the eIF4B phosphorylation on Ser422 but had no obvious effect on Ser406 phosphorylation . Becausen Ser422 , we askeOur previous studies have shown that Akt is upregulated in v-Abl-transformed cells derived from Pim triple knockout cells as compared to Pim wild-type counterparts . In addiNext, we asked whether STAT/Pim signaling could be activated by pharmacological inhibition of PI3K/Akt/mTOR pathway. Indeed, persistent blockage of PI3K by LY294002 resulted in activation of STAT5 and increased expression of Pim-1 Figure . SimilarTo corroborate whether the restoration of eIF4B phosphorylation resulted from enhanced activation of the other pathway due to persistent inhibition of one signaling pathway, we performed similar experiments as described in Figure Our results presented above indicate that eIF4B is a convergent target of oncogenic Pim and PI3K/Akt/mTOR signaling in Abl transformants. Hence, we reasoned that combined inhibition of the two pathways would suppress eIF4B phosphorylation more effectively than inhibition of a signaling pathway. To test this concept, K562 cells were treated with SMI-4a and LY294002 simultaneously and eIF4B phosphorylation was examined at indicated time points Figure . As expePhosphorylated eIF4B was previously shown to play a crucial role in regulating cell survival , we founTo rule out the possibility that the higher concentrations of combined compounds or vehicle DMSO induced such synergy, we tested the effect of high concentrations of each inhibitor on apoptosis in Abl transformants . The resNext, the synergistic effect of the combined treatment on inducing apoptosis was tested in v-Abl-transformed cells. Strikingly, treatment with SMI-4a/LY294002 or SMI-4a/Akti-1/2 greatly sensitized NS2 cells to undergo apoptosis Figure . ApproxiTo further define the relationship between STAT/Pim and PI3K/Akt/mTOR pathways and their role in regulating eIF4B phosphorylation, genetic manipulation of individual signaling molecules in the two pathways was taken to examine the effects of suppressing these pathways on eIF4B phosphorylation and Abl transformant survival. For this, we generated Abl-transformed cells stably expressing shRNAs to either PDK1, Akt1, S6K1, or Pim-1. These cells were then treated with either Pim inhibitor or PI3K/Akt/mTOR inhibitors, followed by assessing eIF4B S422 phosphorylation and cell survival, as described above. We found that knockdown of PDK1 strengthened the inhibitory effect of Pim inhibitor SMI-4a on eIF4B S422 phosphorylation in K562 cells Figure . Using AIn addition, we determined whether forced expression of Akt1 wild-type (WT) or the active form Akt1-E17K affects eIF4B phosphorylation and cell survival in response to SMI-4a treatment. Interestingly, overexpression of Akt1, especially Akt1-E17K, conferred high resistance to SMI-4a treatment, as evidenced by attenuated inhibition of eIF4B Ser422 phosphorylation and cell survival, compared to control .On the other hand, Pim-1 knockdown cells were treated with PI3K/Akt/mTOR inhibitors, followed by the same analyses as described above. As shown in Figure th day after inoculation, tumors were removed from mice and their volumes were measured as previously described [In an attempt to explore the feasibility of this combined therapeutic strategy for treatment of Abl-mediated tumorigenesis, we tested the efficacy of inhibiting both Pim and PI3K/Akt/mTOR pathways in a mouse xenograft model. Each mouse was inoculated subcutaneously with the same amount of K562 cells, and administration of either single agent (SMI-4a or Akti-1/2), combination (SMI-4a and Akti-1/2), or mock was then conducted on day 11 after inoculation of the cells. On the 29escribed  and in MIn addition, similar experiments using SMI-4a and rapamycin were performed. As shown in Figure Having revealed that eIF4B is a common substrate of Pim kinase and PI3K/Akt/mTOR pathways, we hypothesized that eIF4B phosphorylation on Ser422 might be critical for synergic apoptosis of Abl transformants induced by the combinational treatment. To test this hypothesis, firstly we generated K562 cells stably expressing shRNAs targeting eIF4B Figure . We founTo confirm the importance of eIF4B Ser422 phosphorylation in Abl-mediated tumorigenesis, we further performed xenograft experiments using K562 cells stably expressing either eIF4B-WT, eIF4B-S422E, or empty vector. Mice were inoculated with equal amount of each cell type and treated with the combination of SMI-4a and rapamycin as described above. Indeed, the synergic inhibition of tumor growth by the combined treatment was greatly compromised by the expression of eIF4B-S422E in K562 cells Figure . The ectAbl-mediated tumorigenesis of hematopoietic cells involves various complex mechanisms including dysregulation of gene transcription and alterations in protein synthesis important for cell growth and apoptosis , 10. In It has been shown that components of the translation initiation machinery are often deregulated in various cancer cells , 30, 37.Accumulating evidence suggests that redundant activation of multiple signaling pathways may converge on common targets that integrate their functions in cancers. For example, BCL2-associated agonist of cell death (BAD) functions as a shared target of Akt and ERK signaling in phosphatase and tensin homolog (PTEN) deficient tumor cells . Phospho+/CD19+ pre-B cells. Abl transformants stably expressing short hairpin RNA (shRNA) targeting human or mouse PDK1, Akt1, S6K1, Pim-1, eIF4B were generated by infection with lentiviruses as described previously [Cell lines K562 and 293T were purchased from American Type Culture Collection (ATCC) and cultured in RPMI-1640 or Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) as described previously [eviously , 47. PaiThe following antibodies were used in this study: anti-phospho-eIF4B (Ser422) , anti-eIF4B , anti-phospho-STAT5 (Tyr694) , anti-STAT5 , and ani-PDK1 . The inhibitors were purchased as follows: Pim kinase inhibitor SMI-4a , PI3K inhibitor LY294002 , Akt inhibitor Akti-1/2 , and mTOR inhibitor rapamycin . All other antibodies and reagents were obtained as previously described .Apoptosis assay was performed as previously described , 5. Brie6 cells per mouse) were transplanted subcutaneously into Balb/c nude mice (5-6 weeks old) . Starting from the 11th day after inoculation, mice were treated with inhibitors or mock every other day and tumor volume was monitored simultaneously. SMI-4a (50 mg/kg) and Akti-1/2 (80 mg/kg) were administered orally every other day. Rapamycin (10 mg/kg) was injected intraperitoneally every two days. At 8 h after the 10th treatment, mice were sacrificed and tumors were removed for further analysis. Tumor sizes were analyzed for synergism according to the formula AB/C < A/C × B/C, where A was the tumor volume treated with inhibitor 1, B was the tumor volume treated with inhibitor 2, AB was the tumor volume treated with inhibitors 1 and 2, and C was the tumor volume of control group [K562 cells (5 × 10ol group . Biolumiol group .Short hairpin RNA (shRNA) expressing constructs were generated as previously described . The shRhuman Akt1: 5′-CGCGTGACCATGAACGAGTTT-3′ and 5′-CGAGTTTGAGTACCTGAAGCT-3′; mouse Akt1: 5′-CGTGTGACCATGAACGAGTTT-3′ and 5′-CGAGTT TGAGTACCTGAAGCT-3′); human PDK1: 5′-CAAAGT TCTGAAAGGTGAAAT-3′ and 5′-GCAGCAACATAGA GCAGTACA-3′; mouse PDK1: 5′-GAATTTGCACCAG CAGACA-3′ and 5′-GGCTAGAGATCTTGTGGAA-3′.The mouse experimental design and protocols used in this study were approved by “the Regulation of the Institute of Microbiology, Chinese Academy of Sciences of Research Ethics Committee” (Permit Number: PZIMCAS2013008). All mouse experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People's Republic of China.Data were displayed as mean values ± standard error (mean ± SE) of at least three independent experiments. Statistical significance was determined by Student's t-test and the P values < 0.05 was considered to be significant.


Kinetic measurements of pancreatic lipase revealed that the pinhão coat tannin is an effective inhibitor. Inhibition was of the parabolic non-competitive type. The inhibition constants, 50). Consistently, the pinhão coat extract was also effective at diminishing the plasma triglyceride levels in mice after an olive oil load; 50% diminution of the area under the plasma concentration versus the time curve occurred at a dose of 250 mg/kg. This observation is most probably the consequence of an indirect inhibition of triglyceride absorption via inhibition of pancreatic lipase. For the pinhão coat tannin, this is the second report of a biological activity, the first one being a similar inhibition of the absorption of glucose derived from starch as a consequence of an inhibitory action on α-amylases. Taken together, these effects represent a potential anti-obesity action, as suggested for other polyphenol or tannin-rich preparations.The purpose of the present work was to characterize the possible inhibition of pancreatic lipase by a tannin-rich extract obtained from the  Pinhão is the name generally given to the seed of the coniferous tree Araucaria angustifolia ) and inhibitor ([I]) concentrations /Ki1 term in Equation (2) non-significant. The same applies to the Ki2 value, which greatly reduces the significance of the [I]/Ki1 term in Equation (2) and also suggests that the concentrations of the ESI and ES2I complexes are low. An alternative to Equation (2) would be Equation (3) in which binding of at least two molecules of the inhibitor is considered to occur practically at a single step, so that only binary complexes of the inhibitor with the various enzyme forms are considered, namely EI2, ESI2 and ES2I2 50 ≈versus inhibitor concentration curve in In Equation (3),ive type ,14. In tp-nitrophenyl palmitate was not very pronounced, as revealed by comparison of the KM and KiS values, the latter exceeding the former by a factor of 24.4  were administered to mice in the same manner as the pinhão coat extract. It is apparent that both orlistat  and the A. mearnsii tannin were quite effective at diminishing triglyceride absorption, the area under the corresponding curves representing less than 5% of the area under the control curve. This is not surprising for orlistat, which is a covalent inhibitor of pancreatic lipase [A. mearnsii tannin, which is a much weaker lipase inhibitor than the pinhão coat tannin . One cannot exclude that, under physiologic conditions, with the natural substrates (long-chain triglycerides)) and in the presence of bile acids and colipase, the A. mearnsii tannin reveals itself to be a better inhibitor than the pinhão coat extract. It should also be considered that the enzyme used in the in vitro assays was obtained from pig pancreas, and the in vivo experiments were done with mice. The actions of both the A. mearnsii tannin and the pinhão coat extract on both enzymes can be different. Finally, it is possible that the mouse responds differently to both preparations. This includes different gastric movements, but also the possibility that the A. mearnsii tannin site of action is not restricted to pancreatic lipase. Irrespective of the real reason for the discrepancy, a definitive explanation depends on additional conclusive evidence.c lipase , but is nnin see . It shou studies , which dA. mearnsii polyphenol preparation has been proposed as consisting of a combination of lipase inhibition and maltase and sucrase inhibition [pinhão coat tannin based on the results obtained in the present work and on the inhibitory actions of the same preparation on both salivary and pancreatic α-amylases and on starch absorption that have been demonstrated in a previous work [pinhão coat extract are able to alter the expression of obesity and diabetes-related genes in the adipose tissue, muscle and liver, as demonstrated to occur when mice are treated with an A. mearnsii polyphenol preparation [pinhão coat extract are similar not only to those proposed for the A. mearnsii polyphenols [A mechanism for the anti-obesity actions of an hibition . The firous work . It remaparation . On the yphenols ,19, but yphenols  and seveyphenols ,21.pinhão coat tannin requires binding to these enzymes, a phenomenon that was clearly demonstrated to exist for several combinations of tannins and proteins, including enzymes [pinhão coat condensed tannin, where procyanidins predominate [Inhibition of pancreatic lipase, as well as of α-amylases by the  enzymes ,23,24. B enzymes . In the dominate , bindingpinhão coat tannin is an effective inhibitor of pancreatic lipase. Consistently, it was also effective at diminishing the plasma triglyceride levels in mice after a load of olive oil. This is most probably the consequence of an indirect inhibition of triglyceride absorption via inhibition of pancreatic lipase [pinhão coat tannin, this is the second report of a biological activity, the first one being a similar inhibition of the absorption of glucose derived from starch as a consequence of an inhibitory action on α-amylase [The results obtained in the present study revealed that the c lipase . For the-amylase . All of -amylase ,6,20,21.


For the molecular clock rate, we found that at least 3–4 months of temporal divergence from the start of sampling was required to make precise estimates that also agreed with long-term values. For whole-gene dN/dS, we found that at least 2 months of data were required to generate precise estimates, but 6–9 months were required for estimates to approach their long term values. For site-wise dN/dS estimates, we found that at least 6 months of sampling divergence was required before the majority of sites had at least one mutation and were thus evolutionarily informative. Furthermore, 8 months of sampling divergence was required before the site-wise estimates appropriately reflected the distribution of values expected from known protein-structure-based evolutionary pressure in influenza. In summary, we found that evolutionary metrics calculated from gene sequence data in early outbreaks should be expected to deviate from their long-term estimates for at least several months after the initial emergence and sequencing of the virus.With the expansion of DNA sequencing technology, quantifying evolution during emerging viral outbreaks has become an important tool for scientists and public health officials. Although it is known that the degree of sequence divergence significantly affects the calculation of evolutionary metrics in viral outbreaks, the extent and duration of this effect during an actual outbreak remains unclear. We have analyzed how limited divergence time during an early viral outbreak affects the accuracy of molecular evolutionary metrics. Using sequence data from the first 25 months of the 2009 pandemic H1N1 (pH1N1) outbreak, we calculated each of three different standard evolutionary metrics—molecular clock rate , whole-gene  While modern medicine has been able to control the prevalence of traditional human pathogens through vaccines and medications, emerging infectious diseases remain a major threat to public health. Many emerging infectious diseases are caused by zoonotic viruses, which are normally endemic to a reservoir animal and are transmitted to humans upon exposure to the respective reservoir. Some viruses that have successfully undergone zoonoses into humans during the last century include HIV, Zaire ebolavirus, Hantavirus, Machupo virus, Marburg virus, and Chickungya virus, as well as most major influenza virus subtypes, including pandemic 1918 H1N1, H2N2, H3N2, pH1N1, H5N1, and the recent H7N9.dN/dS  and neuraminidase (pN1) sequences from the 2009 pandemic H1N1 (pH1N1) to systematically examine how divergence time influences clock rate and dN/dS estimates both across the entire protein and at each site individually. Previously, 0, an analysis of the time dependence in the molecular clock rate, and the identification of the most recent common ancestor with whole-genome data. We have performed a more detailed analysis of the two genes that dominate influenza antigenicity and have included calculations of the gene-wise molecular clock rate, the whole-gene dN/dS, and the site-wise dN/dS  . We specFor molecular-clock rate estimation, we grouped sequences for each gene into time-aggregated datasets, as follows. We established a dataset for April 2009, a dataset for April–May 2009, a dataset for April–June 2009, and so on, until all months were included. Because datasets containing more than 2 months of data were too large for the estimation procedure to be computationally tractable, we down-sampled the datasets, as follows: For estimates in the third month, we aggregated twenty-five sequences from the first month, twenty-five sequences from the second month, and twenty-five sequences from the third month. Thus, estimates from June 2009 contained seventy-five sequences sampled at random from each month. For any month that did not have twenty-five sequences, we simply added all of the sequences from that month. For each subsequent time window, we added an additional twenty-five sequences from the next month , such that the 25-month time window contained 470 sequences. For all subsampling, we used completely random sampling within each month’s sequences.After compiling all datasets for all time windows, we converted each set of nucleotide sequences to amino acids and aligned them with MAFFT , specifyFor each alignment, we used BEAST  to inferSupplementary Fig. S5). In addition, to ensure that sample divergence was increasing during the sampling period, we plotted the root height of the tree against the sampling time. We found a progressively taller phylogenetic tree as the sample time increased .To be sure that codon site heterogeneity was not affecting the molecular clock rate calculations, we repeated the MCMC calculation adding the gamma site heterogeneity model with four rate categories. The results were statistically identical to those we found previously  and a set of 490 distances in each column . To be clear, for hemagglutinin, the first column in the matrix (a 1 × 490 column) represents the distances from the first amino acid to all amino acids in the protein; the second column represents to distances from the second amino acid to all amino acids in the protein, etc. Then, for each column in the matrix, we calculated the correlation of site-wise ng PyMOL . The samdN/dS estimates were calculated with the aggregate of data for all previous months. Thus, at each time point, we were left with a distribution of correlations between distance and rates calculated with data up to that time point. The distribution of correlations was plotted as a violin plot for each time point  emerged in Mexico during March and April of 2009 and spread across the globe within weeks . AlthougWe calculated the molecular clock rate for pH1 and pN1 for each month in the outbreak from April 2009 to 2011, aggregating all available data up to each respective month . We founImportantly, the 95 per cent highest probability density (HPD) intervals estimated 1–2 months after the start of the pandemic, although wide, did not include the long-term average . Thus, tdN/dS rate ratio. Traditionally, a value of dN/dS rate ratio .We inferred whole-gene  of data . For botdN/dS estimates for pH1 and pN1. In dN/dS estimates for pH1 . Thus, the site-wise dN/dS distribution had after 25 months largely, but not completely, reached its final shape.We next analyzed how sample divergence time influenced site-specific  for pH1 A and pN1 for pH1 B. Both gdN and dS are undefined. In this case, the maximum-likelihood inference algorithm outputs the arbitrary starting value for dN/dS of 1. More generally, for site-wise dN/dS estimates to be reliable, the sequence alignment needs to be sufficiently diverged at each codon site. We estimated overall divergence by counting the fraction of codon sites in each alignment that had 1, 2, 3, etc. distinct codons, and plotted these fractions over time . The fraction of sites with one distinct codon corresponds to the sites that have not experienced any mutations, while the fractions of sites with two, three, etc. distinct codons correspond to the sites that have undergone at least one, two, etc. mutations. We found that the mode at dN/dS declined in proportion to the number of completely conserved sites. At 5–6 months, approximately 50 per cent of all sites were still completely conserved in both proteins , and even after 25 months, 16 out of 503 sites in hemagglutinin and 14 out of 387 sites in neuraminidase still did not show any evidence of evolutionary divergence.We investigated the origin of the mode at dN/dS  of site-wise dN/dS . More spy colder .dN/dS, we expected to find a similar trend in both pH1 hemagglutinin and pN1 neuraminidase. In contrast to the receptor binding protein hemagglutinin, however, neuraminidase is an enzyme with a functionally constrained chemical active site. Therefore, we expected neuraminidase to display the opposite trend from that of hemagglutinin: sites closer to the active site of neuraminidase should appear colder, that is, more evolutionarily conserved.Assuming sufficient divergence for accurate site-wise estimates of Supplementary Videos S1 and S2.We found for both proteins that these expected trends emerged with sufficient evolutionary divergence. For pH1, just as for H3, sites closer to the sialic acid-binding site appeared hotter on our maps . SimilardN/dS evolutionary rate ratio, are widely used to infer the evolutionary dynamics of viruses. With the uptake of rapid sequencing technologies, such evolutionary estimates have found widespread use in tracking emerging epidemics. However, the underlying models used to infer these metrics make a key assumption that genetic variation arises solely from fixed differences among sequences, and the influence of segregating mutations is ignored. Theoretical work has demonstrated that this assumption produces biased or inaccurate clock rate and dN/dS estimates at small divergence times  and the ce times . ImportadN/dS. Moreover, we found that site-specific dN/dS values were largely uninformative during the first 6 months of the outbreak but started to become reliable for longer time windows. Our results provide empirical evidence of the theoretical predictions that short-time scales produce biased and inaccurate estimates for both the molecular clock and dN/dS. Since we obtained these results for influenza virus, which is one of the most rapidly evolving viruses, it is possible that more slowly evolving viruses will require substantially more time before estimates converge to their long-term values.We found that reasonably accurate measurements for each metric could be obtained with 4–6 months of data for clock rate and 8 months of data for dN/dS . For pH1, that pattern started to emerge clearly in November of 2009 with 8 months of accumulated mutations. Moreover, this date could be inferred from the distribution of site-wise dN/dS estimates; it is the first month when substantially more sites are informative than are uninformative . Likewise, for pN1, we showed that a similar proximity metric can be used for the enzyme active site. However, in the case of neuraminidase, the active site was more constrained rather than less constrained, as was the case in hemagglutinin. This finding makes sense intuitively, since enzymes must retain a functional active site at all costs. Further, we found that the expected pattern began to emerge after the same 8-month interval, and again, this interval could have been inferred based on the site-wise dN/dS distribution . Therefore, we have found that by inspecting the change in the distribution of site-wise dN/dS estimates over time, we could identify the time point at which site-wise estimates became reliable.We showed here that, after 25 months of divergence, the number of uninformative sites dropped to a sufficiently low number, and the resultant site-wise estimates were sufficiently accurate, to display the same proximity-based pattern as seen in H3 hemagglutinin (Supplementary data is available at VEVOLU Journal online.


As a consequence, venom researchers and medical practitioners are in deliberate quest of potent molecules alongside ASV to tackle the brutal local manifestations induced by aforesaid venom toxins. Based on these facts, we have demonstrated the protective efficacy of inhibitor cocktail containing equal ratios of N,N,N’,N’-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and silymarin (SLN) against progressive local toxicity induced by Echis carinatus venom (ECV). In our previous study we have shown the inhibitory potentials of TPEN towards Zn2+MPs of ECV (IC50: 6.7 μM). In this study we have evaluated in vitro inhibitory potentials of SLN towards PLA2s (IC50: 12.5 μM) and HYs (IC50: 8 μM) of ECV in addition to docking studies. Further, we have demonstrated the protection of ECV induced local toxicity with 10 mM inhibitor cocktail following 15, 30 min (for hemorrhage and myotoxicity); 60 min  of ECV injection in murine model. The histological examination of skin and thigh muscle sections taken out from the site of ECV injection substantiated the overall protection offered by inhibitor cocktail. In conclusion, the protective efficacy of inhibitor cocktail is of high interest and can be administered locally alongside ASV to treat severe local toxicity.Viperbite is often associated with severe local toxicity, including progressive hemorrhage and myotoxicity, persistent even after the administration of anti-snake venom (ASV). In the recent past, investigations have revealed the orchestrated actions of Zn Over the past decade, systemic approach of drug discovery has identified combinatorial therapy as a new approach . As idea2+ metalloproteases, also called as snake venom metalloproteases (SVMPs) were mainly blamed for such complications, as they degrade proteins of extra cellular matrix (ECM), basement membrane and blood coagulation cascade, resulting in a wide range of hemostatic alterations and local tissue damage , fatty acids, n-acetyl glucosamine (NAG), p-dimethylaminobenzaldehyde (p-DMAB), pentobarbital sodium salt, silymarin (SLN), and N,N,N',N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) were purchased from Sigma Aldrich . Hyaluronic acid was purchased from Across Organics . BSA, dimethyl sulfoxide (DMSO), ethanol (HPLC grade), and water (HPLC grade), were purchased from Sisco Research Laboratories . All other chemicals and reagents used in this study were of analytical grade and were procured from local firms .Adult Swiss Albino mice weighing 25–30 g were used for pharmacological studies. Animals were collected from University Central Animal Facility and housed under a controlled environment. All experimental protocols were approved by the Institutional Animal Ethical Committee (No: UOM/IAEC/25/2011), University of Mysore, Mysuru, and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Control, venom injected and treatment groups were monitored regularly  until the end of incubation period and there were no unintended death of animals. After incubation, animals were euthanized by administering pentobarbital sodium salt  and dissection was performed.2s; cromolyn sodium salt (CSS), sodium aurothiomalate hydrate (SAH), and silymarin (SLN) against ECVHYs induced fit docking (IFD) [2 (PDB ID: 1OZ6) [E. ocellatus venom hyaluronidase (UniProt ID: A3QVN2) [2 and predicted homology model of ECVHY were prepared using "protein preparation wizard" of the Schrodinger software as it facilitates assignment of proper bond order, addition of charges, and protonation state prior to minimization. In addition, hydrogen bond network optimization and identification of proper ionization states (His tautomers) were also performed. Further, terminal angle of Asn, Gln, and His residues were allowed for 180° rotations and impref module was invoked for final constrained energy minimization with root mean square deviation cut off of 0.30 Å. The amino acid residues of ECVPLA2 (His48 and Asp49) and ECVHY  were used to locate the active site for docking compounds of interest.To assess the inhibitory potentials of aristolochic acid (AA), 6-O-palmitoyl-L-ascorbic acid (AP), oleanolic acid (OA), and ursolic acid (UA) against ECVPLAng (IFD)  analysisD: 1OZ6)  was retrD: 1OZ6) –19. Wher A3QVN2)  retrieve A3QVN2) . The tem A3QVN2)  and huma A3QVN2)  were use A3QVN2) –25 with  A3QVN2)  were useLigands were downloaded from the PubChem structure database in 3D SDF format and prepared using the ligprep module ; pH 7±2 2 (PLA2) activity was measured using 14C-oleate labeled autoclaved E. coli as substrate as described in [Phospholipase Aribed in . For inhn-acetyl glucosamine (NAG) released. For inhibition studies, hyaluronidase activity was determined after pre-incubating 100 μg ECV with various concentrations of AP and SLN (0.1 nM-10 mM) for 15 min at 37°C.Hyaluronidase activity was carried out according to the method in  using hyTPEN and SLN stock solutions (200 mM each) were separately prepared in ethanol and DMSO respectively. Required concentration of inhibitor cocktail  was formulated by mixing 1:1 molar concentrations of TPEN and SLN and final volume was adjusted using 50% ethanol. Aliquots were kept at 4°C until further use. After several repetitions and standardization experiments 1, 3, and 10 mM concentrations were used for inhibition studies.Inhibition studies were carried out by ‘independent injection’ method (treatment mode) where, AP and SLN were separately used for edema inhibition. Whereas, different doses of inhibitor cocktail containing TPEN and SLN were used for the inhibition of hemorrhage, and myotoxicity at different time points following the administration of ECV.Edema-inducing activity of ECV was determined as described in . For inhExperimental animals were randomly divided into different groups (n = 3) and hemorrhagic activity was determined as described in . InhibitMyotoxicity studies were performed according to the method in . A groupt-test. The comparison between the groups were considered significant if p ≤ 0.05. In vitro and animal experimental data were analyzed using the statistical package Graph Pad Prism version 5.03  and docking analysis was performed using schrodinger software package .The results of experiments were expressed as mean ± SD of three independent experiments performed in triplicate. Statistical analyses were carried out using Student’s 2, and 3D structures of inhibitor of interest were prepared as described in materials and methods and inhibitors were initially screened using IFD method. AA, AP, OA, and UA , OA (PubChem ID: CID 10494), and UA (PubChem ID: CID 64945) exhibited different interactions on repeated trials. The steady hydrogen bond  and hydrophobic interactions  of AP with ECVPLA2 was evidenced by its low glide score  and glide energy  compared to AA, OA and UA  is proved to be one of the most potent inhibitors of hyaluronidases, particularly against streptococcal and bovine testicular hyaluronidase [2. Similarly SLN is known for effective antioxidant and free radical scavenger properties [2s and ECVHYs. Up on docking studies both AP and SLN showed different yet stable hydrogen and hydrophobic interactions with active site residues of ECVPLA2 and ECVHY. However, the glide score of SLN  was 1.2 and 1.7 times better than that of AP  against ECVPLA2 and ECVHY respectively. The low glide score of SLN compared to AP on priority suggested SLN as a potent inhibitor towards tested enzymes  and recruitment of inflammatory cells at the bitten site [2 was further evaluated by ECV induced edema inhibition studies. Paw edema was induced in mice by intra-plantar injection of ECV. Edema induction was concentration dependent and the minimum edematic dose (MED) was found to be 1 μg. For inhibition studies, percentage increase of 3 μg ECV injected (170 ± 5%) and saline injected (120 ± 3%) limbs compared to their respective un-injected limbs were used as positive and negative controls as per the mathematical calculations indicated in materials and methods. SLN and AP, upon independent injections following 15 min of ECV administration, dose dependently inhibited the edema-inducing effect of ECV. At 10 mM dose both AP and SLN significantly decreased the edema ratio . However SLN was quite effective compared to AP in preventing ECV induced edema  was found to be 1 μg. Three MHD was used for inhibition studies as it could progressively increase the area of hemorrhagic spot time dependently. ECV induced the hemorrhagic lesion of 40 ± 4% at 15 min, 50 ± 5% at 30 min, and 63 ± 5% at 60 min respectively compared to hemorrhagic spot formed at the end of 180 min (100%). Initially different molar concentrations  of inhibitor cocktail (containing 1:1 molar concentrations of TPEN and SLN respectively) was tested following 15 min of ECV administration. Among the tested doses 3 mM  and 10 mM  inhibitor cocktails significantly prevented the progression of hemorrhage. On the basis of results obtained 3 mM and 10 mM inhibitor cocktails were further used for hemorrhage inhibition studies following 30 and 60 min of ECV administration. At tested doses and time points inhibitor cocktail considerably protected the progression of hemorrhage. The results were statistically significant with 61 ± 13%; p = 0.0005 inhibition for 3 mM and 82.5 ± 30%; p < 0.0001 inhibition for 10 mM at 30 min compared to 50 ± 8%; p < 0.001 inhibition for 3 mM and 68 ± 20%; p = 0.0005 inhibition for 10 mM at 60 min respectively . However, in this study we documented such hemorrhage protective effect with 3 mM inhibitor cocktail . Further, we have shown the protection of ECV induced progressive hemorrhage for succeeding time intervals (30 and 60 min following ECV administration). This clearly substantiates the efficacy of mixture of inhibitors which are specific and can work in a combinatorial fashion against progressive hemorrhage induced by mixture of toxins in crude venom.The above results were supported by histological examination of skin sections. The skin section of ECV injected hemorrhagic spot showed extensive damage of dermis, basement membrane surrounding blood vessels and massive infiltration of inflammatory cells. Saline and inhibitor cocktail alone injected skin sections showed intact dermis and basement membrane. Further no infiltration of inflammatory cells was observed. Treatment groups showed reduction in tissue damage and infiltration of inflammatory cells . In our 2+SVMPs, myotoxic SVPLA2s and SVHYs further leads to severe tissue damage—dermo and myo-necrosis; which in turn depends on the concentration of these hydrolytic enzymes in crude venom [2s (enzymatic/non-enzymatic) exert direct action of on the plasma membrane of muscle cells and tissues; SVHYs rapidly degrade hyaluronic acid. Overall, the combinatorial action of these enzymes causes decreased viscosity at the envenomed site and lead to progressive myotoxicity [p = 0.0007 and 1, 425 ± 400 U/L; p = 0.0005 for CK and LDH respectively). The variations in CK and LDH values in control, ECV and inhibitor cocktail treated groups were supported by microscopic observations of thigh muscle tissue sections. ECV treated groups showed muscle necrosis and accumulation of inflammatory cells at the site of ECV injections in comparison with intact, elongated and striated appearance of control and 10 mM inhibitor cocktail injected groups  was comparable to that of 20 mM TPEN administered following 15 min of ECV injection . However, the inhibitor cocktail was twice more potent than TPEN in terms of molar concentration and it extended the inhibitor administration time to 30 min supporting the hypothesis of combinatorial approach.The progressive hemorrhage induced by orchestrated actions of Znde venom , 43. SVMtoxicity , 10, 44.d groups . The myoE. carinatus is one among the “BIG FOUR” venomous snakes of India and accounts for large number of mortality in Indian subcontinent [Snakebite is a serious health concern in many regions of the world, particularly in tropical and subtropical countries including India as it accounts for large number of mortality and morbidity among the rural population in these areas . In viewontinent . In addiontinent , 47.2+MPs, PLA2s and HYs) present in ECV [E. carinatus bite. In addition, they are also known to trigger remarkable amounts of reactive oxygen/nitrogen species—the key players of oxidative stress and vital organ damage [Orchestrated action of locally acting, cell and/or matrix degrading principle toxins , they often fail to tackle the progressive tissue damage induced by mixture of toxins in crude venom upon independent injection experiments [in vitro and in vivo findings [2s and ECVHYs among the screened inhibitors using IFD has been detailed in this study. Further, we have demonstrated the efficacy of inhibitor cocktail containing TPEN and SLN against the progressive tissue damage induced by ECV in independent injection type experiments.Although, mono specific inhibitors  are highly effective against crude venom on pre incubation experiments  in the progression and aggravation of tissue damage induced by Zn2+ECVMPs. In this direction, potent inhibitors against these hydrolytic enzymes were successfully screened using in silico, in vitro and in vivo methods, and SLN was chosen as common ECVPLA2s and ECVHYs inhibitor for the formulation of inhibitor cocktail along with TPEN. As expected, upon independent injection studies following 15 min of ECV administration, 3 mM inhibitor cocktail was equally efficient to that of 20 mM TPEN alone in protecting progressive hemorrhage . Further, 10 mM cocktail administration following 30 and 60 min of ECV injection efficiently protected progressive hemorrhage. Similarly, myotoxicity inhibitory potentials of 10 mM inhibitor cocktail administered following 30 min of ECV injection was comparable to that of 20 mM TPEN alone administered following 15 min of ECV injection (cocktail increased the efficacy of TPEN ~2 times and doubled the inhibitor administration time). This clearly substantiates the effectiveness of combinatorial approach against progressive tissue damage induced by mixture of toxins in crude venom in treatment mode. Besides, upon evaluating stability, inhibitor cocktail was stable for more than six months and successfully exhibited its inhibitory potentials against ECV induced tissue damage with ~92 ± 2% accuracy compared to freshly prepared inhibitor cocktail. Further, it demonstrated negligible signs of toxicity at tested concentrations as evidenced by histopathology of skin and thigh muscle sections.In summary, TPEN—a potent ZnOverall, the formulated inhibitor cocktail was highly efficient than TPEN alone in protecting progressive tissue damage induced by ECV with negligible adverse effects. With this it opens up a new avenue of combinatorial drug approach to treat viper venom-induced severe local toxicity alongside ASV. Furthermore, as the experimental results accounts for time elapsed from venom injection to inhibitor cocktail administration, topical application of inhibitor cocktail of TPEN and SLN can be a part of first aid in the management of venomous snake bites, particularly viperid and crotalid bites alongside ASV.


Despite the key role of IgE as the allergen recognizer at the initiation of allergic responses our knowledge on the molecular interactions between human IgE and allergen is limited. To date, most studies aiming at identification of IgE-reactive epitopes have had to rely on polyclonal IgE-preparations, non-human antibodies or antibodies of isotypes other than IgE. However, due to their intrinsic ability to recognize highly relevant, in the context of allergy, epitopes on allergens, allergen-specific human monoclonal IgE, represented by recombinant human antibody fragments, offer an opportunity to decipher the IgE-epitome of major allergens with precision and at high resolution. Such information may guide us in development of novel therapeutic candidates and better diagnostic tools.We have isolated a large pool of antibody variable domain fragments, specific for several major allergens, from the IgE repertoires of allergic donors. Three sets of such binders, targeting the major birch pollen allergen Bet v 1 (n = 4) and the major timothy grass pollen allergens Phl p 1 (n = 5) and Phl p 5 (n = 10), and their interactions with respective allergen have been studied by immunochemical methodologies.We have, using our own technology and through collaborations, been able to define multiple non-overlapping IgE-reactive epitopes on all three investigated allergens. Two novel epitopes on Bet v 1 were identified, which allowed for identification of single amino acid residues with crucial importance for the interaction between human IgE and allergens in the PR-10 protein family. Further, it was shown that the relatively small allergen Phl p 5 carries a complex composition of IgE-reactive epitopes, providing an explanation to its high allergenicity. The less complex epitope composition on Phl p 1, on the other hand, with an IgE-binding hot-spot located on the surface of its immunodominant C-terminal domain, allowed us to, in a rational manner, design a hypoallergenic group 1 grass pollen allergen fragment, with the potential to be used as a safer alternative to the today used extracts in specific immunotherapy.We here summarize data that show the great usefulness of human monoclonal IgE as a tool in studies of the molecular interactions taking place at the initiation of allergic responses, and how information gained from such studies can aid us in, among other things, the design of novel hypoallergenic allergen variants.


Natural Killer (NK) cells are critical in the defense against viruses in general and against influenza in particular. We previously demonstrated that the activating NK cell receptor NKp46 is involved in the killing of influenza-virus infected cells through its interaction with viral hemagglutinin (HA). Furthermore, the recognition by NKp46 and consequent elimination of influenza infected cells were determined to be sialic-acid dependent. Here, we show that the human co-activating receptors 2B4 and NTB-A directly recognize the viral HA protein and co-stimulate killing by NK cells. We demonstrate that the 2B4/NTB-A-HA interactions require the sialylation of these receptors, and we identified the binding sites mediating these interactions. We also show that the virus counters these interactions through its neuraminidase (NA) protein. These results emphasize the critical role played by NK cells in eliminating influenza, a significant cause of worldwide morbidity and mortality. NK cells are bone marrow derived lymphocytes that are part of the innate immune system . They arOther receptors such as 2B4 and NTB-A function as co-receptors  . The 2B4Influenza virus infections annually result in worldwide health and economic burdens that affect the lives of millions . InfluenThe mouse orthologous protein of NKp46 is named NCR1 . Using mIn the present study, we identified two NK cell receptors, 2B4 and NTB-A, that recognize HA in a sialic acid dependent manner. We show that the recognition of HA by 2B4 and NTB-A co-stimulates killing by NK cells. Furthermore, we were able to reveal the HA binding site on 2B4 and NTB-A.We have previously shown that NK cells kill influenza infected cells through the interaction of NKp46 with the viral HA , 26, 28.To test whether 2B4 and NTB-A recognize influenza virus directly, we generated fusion proteins composed of the extracellular portions of human 2B4 and NTB-A fused to the Fc portion of human IgG1 (named 2B4-Ig and NTB-A-Ig). Next, we incubated JEG-3 cells with influenza PR8 and BRI viruses and stained them with an anti-influenza antibody which recognizes components of both viruses Figure . We usedThe enhanced binding of 2B4 and NTB-A was specific, as no additional NK cell receptor fusion proteins bound the PR8 infected cells Figure . SimilarWe previously demonstrated that NKp46 interacts directly with viral HAs and that this interaction is sialic acid-dependent . To inveTo further demonstrate that 2B4, NTB-A and NKp46 bind directly to HA we performed ELISA assays in which we coated ELISA plates with the different fusion proteins and tested the binding to biotinylated HA-Ig. A significant binding of HA to NKp46-Ig was detected and a moderate binding to 2B4-Ig and NTB-A-Ig, Figure .To test whether sialylation of 2B4 and NTB-A is required for their binding to HA, as described for NKp46 , we treaThe NA protein is expressed on influenza-infected cells and on the virus itself Figure . A substTo test whether 2B4 and NTB-A can be directly activated by influenza virus we used a cell based reporter system. In this system, we expressed chimeric proteins composed of the extracellular portions of NKp46, 2B4 and NTB-A fused to the mouse ζ-chain in mouse BW cells. The BW cells secrete IL-2 upon engagement of the chimeric protein with an appropriate ligand , thus reTo identify the NTB-A and 2B4 binding site that mediates their interaction with HA, we focused on the glycosylated residues of these two receptors, because we demonstrated that glycosylation is essential for their binding. The 2B4 receptor contains three N- glycosylated residues . We further demonstrated (as previously reported) that these glycosylated residues are also involved in the binding of 2B4 and NTB-A to their corresponding cellular ligands: CD48 and NTB-A, respectively. Interestingly, in NKp46 an O-glycosylated residue (Threonine 225) mediates the activating interaction with HA, while in 2B4 and NTB-A N-glycosylated residues mediate co-stimulatory interactions with HA.We previously showed that the mouse NKp46 orthologous protein named NCR1 directly interacts with HA, and that increased mortality of mice infected with influenza virus is observed in the absence of NCR1 . ConversThe identification of two additional NK cell receptors that directly interact with viral HA and the understanding that NA counters the interactions of these two co-stimulating receptors with HA might lead to the development of new therapeutic approaches against this dangerous virus that are based on either blocking the NA activity or on augmenting the sialylations of NKp46, NKp44, 2B4 and NTB-A receptors.The cell lines used in this study were the human choriocarcinoma cell line JEG-3, the EBV transformed B cell line 721.221, and the murine thymoma BW cell line. The human influenza viruses A/Puerto Rico/8/34 H1N1 and A/Brisbane/59/2007 H1N1 used in this study were generated as previously described . NK cellMonoclonal antibodies (mAbs) used in the present study included anti-influenza type A mAb (anti Flu) (Center for Disease Control Atlanta Georgia) that recognizes components of Influenza A viruses, anti-HA1 mAb (H17-L2) that recognizes the HA protein of A/Puerto Rico/8/34 H1N1, anti-NA1 (NA21C1) and the blocking antibody anti-HA1 (H28E23) . The anti-2B4 (c1.7), anti-NTB-A (NT-7) and anti-NKp46 (9E2) mAbs were purchased from Biolegend. Anti-CD48 (eBio156-4H9) was purchased from eBioscience. The polyclonal anti-2B4, anti-NTB-A and anti-NKp46 were generated by immunization of male C57BL mice with 2B4-Ig, NTB-A-Ig and NKp46-Ig, respectively. The specificity of these polyclonal antibodies was tested against transfectants expressing the appropriate receptors. PE-conjugated AffiniPure donkey anti-human IgG and Alexa Fluor 647-conjugated AffiniPure goat anti-mouse IgG were purchased from Jackson ImmunoResearch. The generation of the following proteins: NKp46-Ig, LIR1-Ig, NKp30-Ig, CEACAM1-Ig, DNAM1-Ig, TIGIT-Ig, NKG2D-Ig, HA-Ig was previously described . All proFor the generation of the WT 2B4-Ig fusion protein, the sequence encoding the extracellular part of 2B4 was amplified by PCR using the 5′ primer CCCACCGGT GCCGCCACC ATG CTG GGG CAA GTG GTC ACC (including AgeI restriction site) and the 3′ primer GGATCCGG CCA AAA TCT GAA TTC CTG (including BamHI restriction site). Single point mutations in 2B4 were generated by PCR- based site directed mutagenesis using the 5′ primer AAG TGG GAG GCT GGC TCT TTG and the 3′ primer CAA AGA GCC AGC CTC CCA CTT for N71A mutation, the 5′ primer TTG CCT TCC GCT ACT TCC AAT and the 3′ primer ATT GGA AGT AGC GGA AGG CAA for the N77A mutation, the 5′ primer ATA GTC AAG GCC TTG AGT CTTC and the 3′ primer GAA GAC TCA AGG CCT TGA CTAT for the N89A mutation. For the generation of the WT NTB-A-Ig the 5′ primer CCC ACCGGT GCCGCCACC ATG TTG TGG CTG TTC CAA TCG (Including AgeI restriction site) and the 3′ primer GGG ACTCAT TTT GGT ATC TGT ATA TTG (Including BamHI restriction site) were used. The single point mutations of NTB-A were generated by PCR based site directed mutagenesis using the 5′ primer GCC TTG GGA GCC ACA CTT TCA and the 3′ primer TGA AAG TGT GGC TCC CAA GGC for the N171A mutation, the 5′ primer AGT CAG CCA GCC CTC ACT GT and the 3′ primer AC AGT GAG GGC TGG CTG ACT for the N178A mutation. The N171A PCR fragment was used with the N178A primers to generate a double mutated PCR fragment. These PCR fragments were cloned into an expression vector containing a mutated Fc portion of human IgG1 (CSI-Ig Puro plasmid). For the generation of the mouse 2B4-Ig fusion protein, the sequence encoding the extracellular part of mouse 2B4 was amplified by PCR using the 5′ primer CCC ACCGCT GCCGCCACC ATG TTG GGG CAA GCT GTC CTG (including AgeI restriction site) and the 3′ primer GGG ATCCGG CAG AAA TCT GAA ATT CGAA (including BamHI restriction site). For the generation of the mouse NTB-A-Ig fusion protein, the sequence encoding the extracellular part of mouse NTB-A was amplified by PCR using the 5′ primer CCCACCGGT GCCGCCACC ATG GCT GTC TCA AGG GCT CAA (including AgeI restriction site) and the 3′ primer GGGATCCCA GGG TGG ATT AGT TAG AAC (including BamHI restriction site).Cells were coated overnight with 50μl of the A/Puerto Rico/8/34 H1N1 virus strain at 37°C. The cells were then washed, incubated with the appropriate antibody (0.2μg/well) or fusion protein (1μg to 5μg/well), stained with the appropriate secondary antibody and then analyzed by fluorescence-activated cell sorting (FACS). For NA inhibition or HA blocking, the cells were incubated for 1h on ice with Tamiflu (1:1000) or anti-HA (H28E23 0.5μg/well)) and then stained with the appropriate fusion protein. All results were analyzed with the CellQuest software.35S release as described previously [NK cells were incubated for 5 hours at 37°C with various targets and the cytotoxic activity was measured by eviously . For bloThe cloning of chimeric 2B4- ζ chain constructs was performed by PCR amplification. The primers used for the extracellular portion of 2B4 were: The 5′ primer CCC AAGCTT GCCGCCACC ATG CTG GGG CAA GTG GTC AC (Including HindIII restriction site) and the 3′ primer TCC ATC TAG CAA GTA GCA GAG CGG CCA AAA TCT GAA TTC CTG A (including the seven first amino acids of the mouse ζ-chain transmembrane portion). The mouse ζ-chain was amplified by using the 5′ primer CAG GAA TTC AGA TTT TGG CCG CTC TGC TAC TTG CTA GAT GGA A (including the last seven amino acids of the human 2B4 extracellular portion) and the 3′ primer GGC TCG AGT TAG CGA GGC C AGGGTC (Including XhoI restriction site). The two amplified fragments were mixed and PCR was performed for the generation of 2B4- ζ-chain construct. The cloning of chimeric NTB-A- ζ chain constructs was performed by PCR amplification. The primers used for the extracellular portion of NTB-A were: The 5′ primer C GAATTC GCCGCCACC ATG TTG TTG CTG TTC CAA TCG (Including EcoRI restriction site) and the 3′ primer TCC ATC TAG CAA GTA GCA GAG CAT TTT GGT ATC TGT ATA TTG AATT (including the seven first amino acids of the mouse ζ-chain transmembrane portion). The mouse ζ-chain was amplified by using the 5′ primer CAA TAT ACA GAT ACC AAA ATG CTC TGC TAC TTG CTA GAT GGA A (including the last seven amino acids of the human 2B4 extracellular portion) and the 3′ primer GGC TCG AGT TAG CGA GGC C AGGGTC (Including XhoI restriction site). The two amplified fragments were mixed and PCR was performed for the generation of NTB-A- ζ-chain construct. The constructs (2B4- ζ-chain and NTB-A- ζ-chain) were cloned into a pcDNA3 expression vector and transfected into BW cells. BW-NKp46 cells were previously generated in our lab .BW assays were performed as previously described . ParentaVarious fusion proteins were coated to an ELISA plate (0.5ug/well). Blocking was performed with BSA 1%. 0.1ug/well of biotinylated HA-Ig was added followed by incubation with Streptavidin HRP (Jackson ImmunoResearch). Subsequently TMB solution is added and binding is measured by a spectrophotometer at an OD of 650nm.


The cyclic AMP phosphodiesterases type 4 (PDE4s) are expressed in a cell specific manner, with intracellular targeting directed by unique N-terminal anchor domains. All long form PDE4s are phosphorylated and activated by PKA phosphorylation within their upstream conserved region 1 (UCR1). Here, we identify and characterise a novel PKA site (serine 42) within the N-terminal region of PDE4D7, an isoform whose activity is known to be important in prostate cancer progression and ischemic stroke. In contrast to the UCR1 site, PKA phosphorylation of the PDE4D7 N-terminus appears to occur constitutively and inhibits PDE4 activity to allow cAMP signalling under basal conditions. Specificity of receptor action is often underpinned by the compartmentalisation of intermediates within the cAMP-signalling cascade. Discrete positioning of enzymes that synthesise cAMP (adenylate cyclase), are activated by cAMP  or degrade the second messenger (phosphodiesterases) allow the cell to tailor cellular responses following signals generated by a number of receptors coupled to Gαs Of particular interest is the PDE4 family of enzymes, which is made up of over 25 different isoforms, many of which have important, non-redundant functions In addition to phosphorylation of UCR1, the long isoform PDE4D3 undergoes PKA phosphorylation within its unique N terminus 22.134EPYLVRRL(p)SCRN45. Total PDE4D7 antibody was custom made by Altabioscience (UK) against a GST-fusion of the whole unique N terminal region of PDE4D7.Forskolin (Sigma) and KT5720 (Enzo) were dissolved in dimethyl sulfoxide. Anti-PKA phospho substrate (RXXpS) antibody was supplied from Cell Signalling, USA: Cat. No. 9621. Anti-phospho PDE4D7-serine42 antibody was custom made by AMSBIO (Europe) in rabbits against a phosphorylated peptide corresponding to residues 2.22, 0.5 mM CaCl2, 1 mM DTT, 0.2 mg/ml BSA, 1 mM ATP) and incubated with arrays at 30 °C for 30 min with shaking.Peptide libraries were produced by automatic SPOT synthesis and synthesized on continuous cellulose membrane supports on Whatman 50 cellulose membranes using Fmoc-chemistry with the AutoSpot-Robot ASS 222  as previously described by us 2.3Site-directed mutagenesis was performed using the Quickchange kit (Stratagene) according to manufacturer’s instructions. The following primers were used to create the required full length and N terminal mutants. PDE4D7 S42A mutant, forward primer: AGACTTGCATGTCGCAATATTCAGCTTCCCCCTCTC, reverse primer: ATTGCGACATGCAAGTCTCCGGACAAGATAGGGTTCCATTCC. S42D mutant, forward primer: CGGAGACTTGACTGTCGCAATATTCAGCTTCCCCCTCTC, reverse primer: GGGATAGAACAGGCCTCTGAACTGACAGCGTTATAAGTCG.2.4d-thiogalactopyranoside (IPTG). Following protein induction, cells were lysed by sonication and lysate was incubated with Glutathione Sepharose beads (Amersham). Following an incubation period, the beads were washed in elution buffer (50 mM Tris pH 8.0) + glutathione (6.8 mg of reduced glutathione (Sigma) per 1 ml of elution buffer). The beads were pelleted by brief centrifugation and the eluate was collected. Overnight dialysis of the collected eluate was carried out in dialysis buffer  in slide-a-lyzer cassettes (Piercenet) at 4 °C to remove any detergents or glutathione. Following recovery of the eluates from the cassettes, the protein concentration was determined by a Bradford assay.Briefly; BL21 cells were transformed with the fusion protein and induction was brought about by addition of 1 mM of isopropyl-β-2.52, 0.5 mM CaCl2, 1 mM DTT, 0.2 mg/ml BSA, 1 mM ATP) for 1 h at 30 °C with gentle agitation. The samples were run on an SDS–PAGE gel and immunoblotted with the PKA phospho-substrate and phospho-specific antibodies.Purified (2 μg) wt GST-PDE4D7 unique N terminal region and S42A wt GST-PDE4D7 unique N terminal region E/F or VS2.6VSV tagged wild type PDE4D7, S42A PDE4D7, S42D PDE4D7 and dnPDE4D7 were transfected into HEK293 cells using Polyfect transfection reagent (Qiagen). Cells were treated with KT5720 (4 μM) for 20 min prior to forskolin (100 μM) treatment for the time points indicated, or forskolin alone (100 μM). Control cells were treated with DMSO or left untreated (NT). Cell lysates were harvested in KHEM buffer  (for PDE activity assays) or 3T3 lysis buffer  containing Complete, EDTA-free Protease Inhibitor Cocktail Tablets (Roche) and PhosStop Phosphatase Inhibitor Cocktail Tablets (Roche).2.73H] adenosine cyclic-3′,5′-mono-phosphate was sourced from Amersham Biosciences  and cyclic-3′,5′-mono-phosphate from Sigma. The substrate concentration used for PDE assays was 150 nM, and the specific PDE activity was determined as pmol cAMP hydrolysed/min/mg protein. PDE activities were then normalised for expression of construct, and the data were normalised to untreated (NT) wild type PDE4D7 activity.Phosphodiesterase activity was measured using a radioactive cAMP hydrolysis assay that has been described previously 337LVRRLSCR44) in addition to the already known site in UCR1 (125QRRESFL131) (125QRRESF130) and the new site in the unique N-terminal (38VRRLSC43)  A. To detESFL131) B. Only tRRLSC43) B. No phoWild type N-terminal GST-PDE4D7 (WT-NT) and mutant N-terminal GST-PDE4D7-S42A (S42A-NT) were purified E and incTo determine whether PKA phosphorylation of PDE4D7 at serine 42 could occur in a cellular context, HEK293 cells were transfected with VSV-tagged constructs of wild type PDE4D7 and the mutants S42A and S42D. Cells were left untreated or pre-treated with the PKA inhibitor KT5720, before endogenous cAMP levels were elevated using the adenylate cyclase activator forskolin. Cell lysates were then probed with our phospho-specific S42 antibody and an antibody against VSV to evaluate protein loading and construct expression A. Basal p = 0.03, ANOVA) and forskolin challenge did not further increase its activity , no basal UCR1 phosphorylation could be detected, with a sustained phosphorylation being triggered by forskolin.4Fine control of compartmentalised cAMP signalling is underpinned by the discrete positioning of phosphodiesterase enzymes which act to both maintain basal cAMP concentrations and shape cAMP gradients following activation of cell surface receptors In this paper we provide evidence of a novel PKA phosphorylation site on PDE4D7 that alters its activity. Blockade of the phosphorylation of PDE4D7 on serine 42 dramatically increases its activity suggesting that phosphorylation at this site serves to inhibit the enzyme. Interestingly, the phosphorylation of PDE4D7 on serine 42 can be detected in the prostate cancer cell lines DuCaP and VCaP opening up the possibility that this post-translational modification may further promote the proliferative signalling observed following a reduction in PDE4D7 activity. Presumably, the increases in cAMP caused by this event could contribute to the AI phenotype by over exciting the autocrine and paracrine signalling systems that support androgen receptor transactivation


The allergen-induced IgE-mediated effector cell activation is the key mechanism in acute allergic inflammation. The aim of this study was to produce human monoclonal IgE antibodies specific for major allergens and the corresponding allergens to study basophil activation.Monoclonal human IgE antibodies specific for the major birch pollen allergen Bet v 1 and for the major grass pollen allergen Phl p 5 and the corresponding allergens as well as a hybrid protein consisting of the Bet v 1 and Phl p 5 allergens were expressed. Using circular dichroism spectroscopy we compared the fold of the hybrid protein with that of the mixture of the two allergens. Rat basophil leukemia cells transfected with the human FcεRI receptor were then loaded with the monoclonal IgE antibodies or patients serum containing polyclonal allergen-specific IgE and then exposed to the allergens and the hybrid protein to study basophil activation.We found that basophil activation was obtained with a mixture of two monoclonal IgE antibodies and the hybrid protein which increased in magnitude with rising concentrations of allergen-specific IgE antibodies. Thus increases in allergen-specific IgE lead to the release of larger amounts of mediators. When polyclonal IgE from allergic patients was used, basophil activation was obtained already with lower concentrations of the hybrid protein. An augmentation of the number of IgE epitopes thus increased the allergenic activity and thus the "potency" of the allergen. Preliminary titration experiments of the two monoclonal IgE antibodies also suggested that equimolar concentrations of the IgE antibodies with different specificity were most efficient in causing basophil activation, most likely due to the formation of large immune complexes.Results obtained in our molecular model for human basophil activation show that the levels of allergen-specific IgE dictate the extent of basophil degranulation and that the number of IgE epitopes determines the potency of an allergen to induce degranulation. The model should be useful to explore the factors determining allergen-induced basophil activation and to study therapeutic strategies controlling this event. This study was supported by grant P23318-B11 and in part by grants P23350-B11 and F4605 of the Austrian Science Fund (FWF).


Here we investigated whether main metabolites of metamizole (dipyrone) in human could be involved in the immune response using the basophil activation test (BAT). We studied subjects with confirmed selective immediate hypersensitivity to metamizole and performed BAT with metamizole and its metabolites: 4-methylamino-antipyrine (MAA), 4-aminoantipyrine (AA), 4-acetylamino-antipyrine (AAA) and 4-formylamino-antipyrine (FAA). BAT results showed an increase of positive results from 37.5% to 62.5% using metamizole plus metabolites as compared with the BAT carried out only with the parent drug, demonstrating that metamizole metabolites have a role in the reaction and can induce specific basophil activation in patients with immediate hypersensitivity to this drug. Our findings indicate that pyrazolone metabolites are useful for improving the in vitro diagnosis of allergic reactions to metamizole.Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common cause of hypersensitivity reactions, with pyrazolones the most frequent drugs inducing selective reactions. Immediate selective hypersensitivity to pyrazolones is thought to be mediated by specific-IgE. Sensitivity of  Adverse drug reactions (ADRs) constitute an important public health issue, causing 3 to 6% of all hospital admissions and occur in 10 to 15% of hospitalized patientsin vitro diagnostic tests for evaluating these patients.The mechanism involved in non-immunological reactions is believed to be based on the inhibition of cyclooxygenase (COX) enzymes in NSAID-sensitive patientsThe mechanism involved in immunological reactions, named single NSAIDs-induced reactions or selective reactions713141516in vitro method which safely substitutes to the direct application of the drug and additionally provides “proof of mechanism” in these reactionsThe diagnosis of selective allergic reactions to pyrazolones is mainly based on the clinical history, skin testing and/or drug provocation test (DPT)142122The aim of this study was to analyze whether metamizole metabolites could be recognised by IgE bound to basophil surface and induce their activation in subjects with confirmed immediate hypersensitivity to these drugs.in vivo method that confirms an IgE mechanism when positive.In order to perform this pilot study, a group of patients with a confirmed selective immediate allergic reaction to metamizole, and a group of tolerant controls were selected. The diagnosis of allergy was based on an immediate skin test, since it is the only With the aim of evaluating which structures derived from metamizole were recognized by IgE from allergic patients to this drug, BAT was performed using metamizole and four metabolites purified from human urine samples  at different concentrations. The optimal cut-off point that corresponds to the best sensitivity and specificity for the native metamizole and its metabolites was calculated by receiver operating characteristic (ROC) curve analysis. With this method we needed to make a balanced decision between sensitivity and specificity. In the case of drug allergy, and especially in severe reactions, the decision must be chosen to increase the sensitivity in spite of a loss of specificity. Finally, in order to confirm an IgE-mediated basophil activation, the phosphatidylinositol 3-kinase (PI3-K) inhibitor, wortmannin (WTM), was used in BAT for those metabolites that showed to induce relevant specific basophil activation.The study included 16 patients, 13 women and 3 men, the median age was 57.5 years (interquartile range (IR): 46.5–63.5 years), 8 had developed anaphylaxis and 8 urticaria accompanied or not by angioedema, and the median interval between the reaction and the study was 109 days (IR: 59.25–365 days). All patients were skin test positive in immediate reading to metamizole  and demonstrated good tolerance to ASA, therefore they were classified as selective reactors to metamizole . The stuDose-response curves studies (0.125 mg/mL–2.5 mg/mL) for the metabolites analyzed showed that the lowest concentration tested (0.125 mg/mL) was not relevant for all the metabolites and the highest concentration (2.5 mg/mL) produced non-specific basophil activation only for the metabolite MAA . BAT resWe tried to confirm the involvement of specific IgE by using a PI3-K inhibitor, WTM. BAT inhibition with WTM was performed with samples from four positive patients to MAA and found a mean percentage of inhibition of basophil activation of 73.96% for MAA and of 85.16%, for the positive control anti-IgE. As expected, no significant inhibition was observed with the positive control N-formyl-Met-Leu-Phe (fMLP), which is a positive control of non-mediated IgE basophil activation .in vitro tests such as BAT are also used for diagnosis with variable results in different studies142027et al.et al.vs. 74.5%).NSAIDs are the drugs most frequently involved in hypersensitivity drug reactions14These tests used the parent drug only, limiting their power to detect the relevant specific IgE antibodies. In the present work we verified if, in addition to metamizole, metabolites of this drug could induce basophil activation. Experimental evidence indicates that several pyrazolone structures can be recognised by a given antibody, indicating cross-reactivity3031Considering the response to the native molecule, metamizole, the sensitivity achieved here was lower than in previous studies14in vivo metabolite of metamizole. Although none of the metabolites are bound to more than 60% of plasma proteinsIt is thought that the molecules that induce basophil activation require covalent binding to proteins. In fact it has been suggested that pyrazolones become antigenic after binding to biomacromolecules following metabolic activationThere is some controversy as to whether selective reactions to NSAIDs are IgE-mediated because specific IgE antibodies had not been identified directly. However, in the case of immediate reactions to metamizole, the results of skin testing and BAT indirectly support that these reactions are IgE-mediated. Further support is given by the tendency for BAT results to decrease over time in these patientsin vitro for propyphenazoneGiven these findings, the next step would be to put these metabolites with the appropriate carriers in different solid phases and test for the presence of specific IgE antibodies using immunoassays. To our knowledge, specific IgE antibodies have only been detected in vivo events that occur after drug administration, including drug biotransformation, in order to improve the sensitivity of the test and to make it clinically viable.In summary, we have shown that metamizole metabolites can be specifically recognised by IgE antibodies bound to the surface of basophils, inducing their activation in subjects with anaphylactic reactions to metamizole. These results deserve further study with an increased number of patients in order to further explore whether MAA is a relevant structure recognized by IgE antibodies. Moreover, more studies are needed to identify the full range of metamizole metabolites and to assess their utility in testing for metamizole allergy. This is the first study demonstrating the role of metamizole metabolites in immediate reactions and the findings obtained here support the hypothesis that drug metabolites may underlie allergic reactions. This may be a major cause of the lack of sensitivity that has been observed when BAT is performed using the parent drug only. It is highly recommended to design BAT to mimic as much as possible the We selected patients with confirmed immediate allergic reactions to pyrazolones. The diagnosis was established by the presence of clinical symptoms of anaphylaxis or immediate urticaria after a pyrazolone derivative intake  and a positive skin test . To ruleThe study was conducted according to the Declaration of Helsinki principles and was approved by the Malaga Research Ethics Committee. All subjects were informed orally about the study and signed an informed consent form.Skin testing was carried out as previously described14To exclude a cross-intolerant response we carried out a single-blind procedure in all patients, as previously describedg. The organic phase was transferred to fresh tubes and dried by evaporation under a nitrogen stream. Then, the tubes containing the evaporated metabolites were closed to maintain the nitrogen environment, and kept frozen at −80 °C until use. The concentration and purity of the metabolites was checked by HLPC analysis as described elsewhere30Metamizole metabolites were purified from human urine samples, obtained from healthy individuals who received an oral dose of 575 mg of metamizole , with the exception of 4-aminoantipyrine (AA) which was purchased from SIGMA chemical Co. . Because the vast majority of metabolites are recovered in urine in the first 24 h302 (w/v); 0.033% MgCl2 (w/v); 0.1% HSA (w/v); 1 M HEPES and 10 μL/mL IL-3) for 10 min at 37 °C. Then, metamizole  and their metabolites  obtained as described above, were added at different concentrations chosen from dose-response curves and cytotoxicity studies .


To the Editor: Rhabdoviruses are enveloped, single-stranded, negative-sense RNA viruses that comprise a large and diverse family in the order Mononegavirales and infect arthropods, plants, fish, and mammals. There are 9 genera of rhabdoviruses: Cytorhabdovirus, Ephemerovirus, Lyssavirus, Novirhabdovirus, Nucleorhabdovirus, Perhabdovirus, Sigmavirus, Tibrovirus, and Vesiculovirus. In addition, a substantial number of plant, vertebrate, and invertebrate rhabdoviruses have not been classified  comprise members that infect fresh water and marine fish  that had beached along the coast of the Netherlands  with DRV-infected Vero cell supernatants as described  consists of 11,141 nt and has a typical rhabdovirus gene arrangement of 5 major open reading frames (ORFs) in the order 3′-nucleoprotein (N), phosphoprotein (P), matrix (M) protein, glycoprotein (G), and large (L) protein-5′  , and Siniperca chautsi rhabdovirus (SCRV); P (18%–20%) with eel virus European X (EVEX), HSHV, MARV, and SCRV; M (27%–33%) with lake trout rhabdovirus, Swedish sea trout rhabdovirus, and EVEX; G (30%–32%) with perch rhabdovirus, lake trout rhabdovirus, Swedish sea trout rhabdovirus, HSHV, MARV, SCRV, and EVEX; and L (54%–56%) with perch rhabdovirus, HSHV, and EVEX. This close relationship with fish rhabdoviruses is surprising because DRV was isolated from tissues of a mammal and propagated in mammalian cell lines at 37°C, which does not occur with related viruses isolated from fish.Deduced amino acid sequences of the 5 major genes had the highest, although weak, homology with those of various fish rhabdoviruses by pairwise identity analyses: N (48%) with hybrid snakehead virus (HSHV), Lagenorhynchus acutus), 79 harbor porpoises (Phocoena phocoena), 9 striped dolphins (Stenella coeruleoabla), and 6 white-beaked dolphins (Lagenorhynchus aIbirostris). Serum samples from 145 bottlenose dolphins (Tursiops truncates) from the collection of the Dolphinarium Harderwijk  were also tested. DRV-neutralizing antibodies were detected in serum samples from 1 bottlenose dolphin (7%), 5 striped dolphins (55%), 1 white-beaked dolphin (17%), and 3 harbor porpoises (4%). These results suggested that DRV or closely related viruses continue to infect members of cetacean species (To evaluate whether DRV or related viruses circulate among species of cetaceans, we performed serologic screening by using a virus neutralization assay as described (Although rhabdovirus evolutionary pathways are complicated (