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Neurosurgery is characterized by the delicate balance between surgical success and potential for devastating side effects . Thanks to multiple technological improvements, the morbidity of neurosurgical interventions has substantially decreased over the last decades, allowing for the resection of previously inoperable lesions . In particular, image - guided neurosurgery (igns) devices allow the use of coregistered and fused multimodality 3d images to guide the surgeon's hand and help define preoperatively the boundaries of pathological and predefined functional structures . Meanwhile, new modes of medical imaging have also improved the localization of pathological lesions and their characterization . Medical imaging nowadays includes a wealth of different techniques, such as computed tomography (ct), structural and functional magnetic resonance imaging (smri and fmri), diffusion tensor imaging (dti), and positron emission tomography (pet). Although the overall accuracy of igns is estimated to be 12 mm, current neuronavigation systems cannot, however, adapt to changing conditions over time . Skull - opening brain shift, brain retraction, cerebrospinal fluid suction, lesion resection, perfusion, and pharmacological manipulation during surgery indeed all alter the 3d morphology of the structures [25]. These changes can lead to localization errors that are one order of magnitude larger that igns accuracy [1, 2, 6] and may result in incomplete resections or unexpected damage to normal brain . Such inaccuracies could be reduced if one could acquire, throughout surgery, fresh images of the same modalities and quality as the preoperative ones intraoperative images such as intraoperative mr (imr) images are with the exception of a handful surgical facilities usually acquired using low - field mri scanners that provide lower resolution and contrast than their preoperative counterparts, and, to this date, several useful imaging modalities, such as pet and possibly meg, cannot be acquired intraoperatively . One solution is to bring over the high - quality preoperative multimodality images into the intraoperative configuration of the brain using a nonrigid registration technique [710]. One category of nonrigid registration techniques uses physics - based models, where landmarks are tracked in successive reduced - quality intraoperative images, and their displacement fields drive the deformation of a biomechanical model . So far, most of the mechanical conditions of the brain cannot be estimated in the operating room, such as the volume of cerebrospinal fluid flowing out of the skull cavity, intercellular fluid volume changes that result from mannitol injection, or changes in blood volume and vessel permeability . The fact that an intraoperative image can provide the knowledge of the current state of the brain after some deformation partly eliminates the need for a complete evaluation of these mechanical conditions . The nonrigid registration technique replaces them with the landmark displacements evaluated from successive intraoperative images . Using a nonrigid registration technique based on a biomechanical model, three types of brain deformations have been identified that require specific modeling, although they depend on common parameters, such as csf suction, perfusion, or pharmacological manipulation . The first deformation is the brain shift, which appears at the beginning of surgery with the opening of the skull and dura . The suction or leakage of csf, as well as the release of intracranial pressure caused by tumor growth, generally cause such shift of the brain (note that in this work, we name brain shift the specific shift of the brain that occurs after the opening of the skull and dura, before any other surgical act has happened). However, for these deformations, we will consider that the shift is a part of these two deformations . The second deformation is the retraction; when target tissues are located deep inside the brain, the surgeon incises brain tissues and inserts a retractor to spread out the tissues, and to create a path towards the target . The third deformation is the resection, that is, the removal, of lesion tissues . Three deformations can thus be defined in terms of the two elemental actions that change the topology of the brain: the introduction of a discontinuity and the removal of some tissues . Most studies of brain deformation based on biomechanical models have focused on shifts (the topology of the brain is not modified), that occurs just after the opening of the skull and dura [1125]. A good review of these different studies can be found in [24, 2628]. Resection and retraction are more complex to model than (brain) shift . Until recently, their modeling for the specific application of preoperative image update has received much less attention . One of the difficulty for modeling resection and retraction is that both induce a topological change of the brain because some tissue are cut . A method of mesh adaptation [2931] or remeshing [3235] must be used in conjunction with fem if an accurate representation of the location of the cut, for example, the resection cavity or retraction path, is needed to deform the model . Indeed, fem cannot directly handle discontinuities that go through the fes, and requires to realign the discontinuity with fe boundaries . In the field of fracture mechanics, which studies the growth and propagation of cracks in mechanical parts, some methods were developed to avoid using fem in conjunction with mesh adaptation or remeshing . The extended finite element method (xfem or x - fem) appeared in 1999 and has been the object of considerable research since then . Xfem works by allowing the displacement field to be discontinuous within some fes of the mesh . The mesh does not have to conform to the discontinuities, so that these can be arbitrarily located with respect to the underlying fe mesh . Because xfem allows an accurate representation of the discontinuities while avoiding mesh adaption or remeshing, and because of the similarity between cracks in mechanical parts and cuts in tissue, we proposed the use of xfem for handling cut, resection, and retraction in the updating of preoperative images . This paper presents a complete 3d framework for updating multimodal preoperative images with respect to surgical brain deformations, due to brain shift and successive resections, followed and quantified using imr images . Our approach is modular, and is applied iteratively each time a new intraoperative image is acquired . We take into account successive deformations based on a linear elastic biomechanical model which is deformed using fem or xfem, depending on the type of deformation occurring between the pair of imr images under consideration, namely, brain shift or resection . While some 3d results have already been presented for brain shift, and initial 3d results for resection modelings, this paper is the first complete and detailed account of the generalization to 3d of our 2d previous work . We present the state - of - the - art of resection modeling for preoperative image update . In section 3, we describe our basic strategy for updating preoperative images based on successive intraoperative images . In section 4, we give detail about our methods and algorithms . In section 5, we consider two patient cases that illustrate our approach for handling brain shift followed by successive resections . Among studies that take into account resection for preoperative image update, one should distinguish two categories . The first category of studies models brain deformation using two time - point images, the first image being acquired before surgery has started, the second image being acquired after resection . In this category, the methods that existed for a second image showing some brain shift are adapted for a second image showing some resection . The second category of studies models brain deformation using more than two time - point images, and models at least two successive resections . Among the first category of studies, hagemann et al . Developed a 2d method for modeling brain deformation between a preoperative mr image and a postoperative mr image, the postoperative image showing a complete resection . The 2d mesh of the biomechanical model corresponded to the underlying pixel grid of the 2d image . The biomechanical model included four different linear elastic laws for the skull / skin region, the whole - brain region, the csf region, and the image background . They computed the correspondence of the skull boundary, the whole - brain region boundary in the neighborhood of the tumor, and the posterior midline between the two images . They also computed the correspondence between the internal tumor region boundary visible in the preoperative image, and the resection cavity boundary visible in the postoperative image, both boundaries corresponding under the assumption that the resection is complete . The displacements fields of these landmarks drove the deformation of the biomechanical model . As a result, the biomechanical model presented high deformation in the tumor region, which is not physically plausible . However, the resection was complete, and, thus, they were not interested by the displacement field of the biomechanical model in the tumor region itself . Clatz et al . Developed a 3d method for modeling the brain deformation between a preoperative mr image and an imr image, the latter showing partial or complete resection . The biomechanical model was deformed based on a sparse volume displacement field evaluated from the two images, using a block matching algorithm . In their algorithm, blocks of voxels that presented discriminant structures were selected in the preoperative image . The blocks were then matched to blocks in the imr image using a similarity criterion, for example, a coefficient of correlation . The value of the similarity criterion was used as a value of confidence in the displacement measured by the block matching algorithm . The biomechanical model was then deformed iteratively, driven by the sparse displacement field of the matched blocks, where a block rejection step was included for measured block displacements initially selected but considered as outliers . In the imr image, a part, or the totality, of the tumor tissues the blocks were thus selected and matched in only the healthy - brain region of the two images . They tested their algorithm on six patient cases, and used for validation nine landmarks picked up manually in each image . They found a mean and maximum error on displacements of 0.75 mm and 2.5 mm, respectively . They explained this phenomenon by the fact that a substantial number of block matchings were rejected in the tumor neighborhood . The deformation of the biomechanical model in the tumor neighborhood was thus essentially governed by the linear elastic law, and the result might show the limitation of this model . Archip et al . Also tested the nonrigid registration method presented in on eleven patient cases, and used the 95% hausdorff distance for evaluating the alignment of the nonrigidly registered images . As a result, they obtained a mean error of 1.82 mm . Among the second category of studies, miga et al . They built a linear poroelastic biomechanical model and preoperatively tagged the tetrahedron fes that were going to be removed to simulate the brain deformation due to successive resections . Second, a boundary condition was applied to the new boundary of the resection cavity, in order to model the relaxation of strain energy, induced by preoperative tumor growth or surgery acts, stored in the resected tissues, and released after their removal . In this approach, the tissue discontinuity was represented as best as possible with a jagged topology defined by the fe facets defining the boundary of the resection cavity . [45, 46] also modeled the removal of tetrahedra in order to model the action of an ultrasonic aspirator in the context of real - time surgery simulation . [13, 47, 48] modeled successive resections based on several time - point imr images . Between two successive images, they deformed the biomechanical model, in its current state of update, to take into account the (partial) resections(s) that took place between these two images . First, the biomechanical model, in its current state of update, was deformed in accordance with the displacement field of the healthy - brain boundary between the pair of images under consideration . Second, the fes that fell into the resection cavity in the second image of the pair were removed, while the fes that laid across the resection - cavity boundary were cut . To ensure the link between the successive deformed configuration of the biomechanical model, their algorithm kept track of the topology modification between fes and nodes of the mesh before and after the removal of fes . They tested their algorithm on one patient case including five imr images (the first two imr images being used for brain shift modeling), and used for validation thirty - two landmarks picked up manually in each image . They found a mean and maximum error on the displacements of 0.9 0.7 mm (mean standard deviation) and 3.7 mm, respectively . They explained this phenomenon by the limited accuracy in the process of picking landmarks in that region, and because the retraction occurring between the second and third images was modeled as a resection, that is, a removal of tissues, even though the tissues were not removed but simply spread out . The methods described above have been all developed using an fem - based biomechanical model for intraoperative image registration . The objective of a surgical simulator is to provide an interactive manipulation with force feedback of the anatomical part to be operated using various surgical instruments . In order to model a large range surgical procedures, jebkov and kuhlen have applied nonlinear xfem for simulating cut, and have shown that xfem is successfully efficient for such purpose . The block diagram of figure 1 shows our global approach for updating preoperative images using successive imr images acquired at different critical points during surgery . Although the principles of the approach are quite general, they are tailored for use based on images acquired with a 0.5 tesla intraoperative ge signa scanner, which guarantees that the full volume of brain tissues is included in the image field of view . In our present strategy, the preoperative images are updated incrementally . At the end of each update, the preoperative images should be in the best possible alignment with the last imr image acquired . Prior to surgery, a patient - specific biomechanical model is built from the set of preoperative images . Because the patient does not necessarily lie in the same position during the acquisition of each of the preoperative images, one may need to perform a rigid registration (involving translations, rotations, and scales) to bring these images into correspondence, assuming, in first approximation, there is no local, that is, nonuniform, brain deformation between preoperative images . Once the 1st imr image has been acquired prior to the opening of the skull, the set of registered preoperative images and the biomechanical model are registered to the 1st imr image via a rigid transformation . In the present situation, it is assumed that the patient's brain imaged in the 1st imr image has the same physical shape as the brain imaged in the preoperative images (note that in the following, when an imr image is defined by a number, this number is the index of the imr image in the series for a specific patient case . The 1st imr image thus corresponds to the very first imr image of the series). As each imr image is acquired, this new image and the preceding imr image are used to estimate the deformation of the brain . The update of the preoperative images is done incrementally with each new pair of successive imr images . For each pair, we proceed as follows . A set of common anatomical landmarks the use of surface structures rather than volume structures seems more appropriate given the reduced - quality of typical intraoperative images, and would be more easily adapted to intraoperative modalities other than imr, such as ius . The landmark surface displacement fields resulting from the matching are then applied to the biomechanical model, which is deformed using fem or xfem, depending on the type of deformation occurring between the acquisition times of the imr images in the pair under consideration, namely, brain shift, or resection . The resulting displacement field of the biomechanical model is finally used to warp the set of preoperative images in their current state of updating . Note that, for each deformation modeling, the biomechanical model is deformed in accordance with the landmark displacements tracked between the pair of successive imr images under consideration . Because intraoperative deformation can follow a reverse direction, it is important to track the landmarks between the next - to - last and the last acquired imr images, rather than track the landmarks between the first and the last acquired imr images . For the patient cases treated in section 5, we assume that the brain undergoes relatively small deformations (small strains and small displacements), and we use a linear finite - element formulation in the biomechanical model . A consequence of using this linear formulation (linear elasticity) is that the equations of solid mechanics can be solved based on the initial configuration of the solid . Actually, knowing the displacement field increment un = u u at the anatomical landmarks between configuration n and increment n + 1, one can apply this constrained displacement field increment un to the initial configuration, and the finite element analysis will lead to the deformation tensor increment n between the configuration n and n + 1 . The final deformation tensor or the body is thus simply obtained from = k=0k . Remark that rigorously, the increment of constrained displacement field at the landmark should be applied to the balanced solution of the solid reached after increment n, but as we are using a linear elasticity model, this step can be skipped owing to the superposition principle: if = c, then = k=0k = ck=0k = c, where c is the hooke tensor . As a summary, with this approach, the process of deformations is modeled as a succession of deformations k, for example, brain deformation composed of shift followed by successive resections and the current configuration of the brain biomechanical model, after a specific deformation can then be recovered by adding the computed volume displacements for all successive incremental deformations . Remark that this is not a limitation of the method as we could easily extend it to nonlinear model by simply keeping in memory the previous deformed configuration n and adding the constrained displacement field increment un to compute the new deformed configuration at increment n + 1, simply this would be less computationally efficient . Because we use a linear formulation (and, thus, the incremental volume displacement fields can be added to recover the current configuration of the biomechanical model), we could theoretically obtain an identical deformed configuration of the biomechanical model using the two following approaches . The first one would consist of computing and adding the successive incremental deformations of the biomechanical model based on the landmarks tracked between the next - to - last and the last acquired imr images . The second approach would consist in computing directly the deformed configuration of the biomechanical model based on the landmarks tracked between the first and the last acquired imr images . However, the landmarks selected to drive the deformation of the biomechanical model vary depending on the type of deformation, namely, brain shift or resection . In addition, part of the biomechanical model is cut, using xfem, to model resection . Consequently, we would not get an identical deformed configuration of the biomechanical model by these two approaches . In order to use a maximum of information from the imr images, we track, as explained for the first approach, the landmarks between the next - to - last and the last acquired imr images . The problem of updating preoperative images between more than two critical points during surgery, that is, based on more than two imr images, is addressed in only a small number of studies . In our previous work [39, 41], and in, the biomechanical model was successively deformed, and this was done using a linear formulation . The framework proposed here, where the initial biomechanical model is always used, instead of using it in its successive states of deformation, has the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively deteriorates with each successive deformation modeling, and which would require remeshing or mesh adaptation for getting back good fe quality . To summarize, for each deformation, the landmarks are tracked between the two successive imr images under consideration . Because we use a linear formulation, the displacement fields of these landmarks are applied to the initial, rather than current, configuration of the biomechanical model . The resulting volume displacement field corresponds to the deformation that the brain undergoes between the two imr images . This volume displacement field is used to deform the preoperative images in their current state of update, that is, registered (at the previous step, if any) to the first imr image of the pair . After the deformation, the preoperative images are thus in as good as possible registration to the second imr image of the pair . In all the rest of this work, we make a simplification of the approach just presented, by using the 1st imr image as a substitute for the preoperative images . The biomechanical model is thus built based on structures visible in the 1st imr image, instead of in the preoperative images, and the structures used in the model are limited to the ones visible in the intraoperative image . Except for the rigid registration between the preoperative images, the biomechanical model, and the 1st imr image, this simplified approach allows us to discuss, illustrate, and test all key aspects of the system . The above strategy allows us to focus on the main issue of this paper, that is, the estimation and handling of 3d deformations . Even though the issues involved in the update of preoperative images will need to be addressed in a operational image update system, the present strategy of deforming the imr images remains useful for calibration purpose, even in the operating room . This section details the different methods that are commonly used for updating preoperative images in presence of brain shift and resection . More specifically, the block diagram of figure 2(a) shows the building of the biomechanical model from the preoperative images . Specific regions from the preoperative images are segmented, meshed, and assigned appropriate constitutive laws . The block diagram of figure 2(b) shows, for any pair of successive imr images, a detailed view of the calculation of the volume displacement field of the initial biomechanical model that corresponds to the deformation that has occurred between the acquisition time of these images . All along surgery, the patient is lying inside the 0.5 tesla intraoperative ge signa scanner . Although the patient's head is fixed, one cannot totally rule out the possibility of slight head motion . Imr images thus have to be rigidly coregistered to take into account this potential rigid motion . The rigid registration that we use is the point - based landmark transform available in vtk (http://www.vtk.org/). The segmentation of imr images into specific regions, for example, healthy - brain and tumor regions, is first performed manually using 3d slicer (http://www.slicer.org/) and then smoothed to minimize the dependance of the results on segmentation roughness . It is clear that performing a manual segmentation in the operating room is not acceptable, and that this process needs to be automated as completely as possible to test the feasibility of our framework online . However, while there exist sophisticated segmentation algorithms that could be used [5052], in particular for extracting the whole - brain region (skull and external cerebrospinal fluid masked out), the segmentation of the tumor region is still challenging . As mentioned above, the biomechanical model is built, in the present context, from the 1st imr image rather than from the preoperative images . Thanks to the use of xfem instead of fem for modeling discontinuities, this biomechanical model can be built offline before the operation starts and does not need to be repeated (through remeshing) during the surgery . With respect to fem - based approaches, the execution time thus ceases to be a limiting parameter, which is a remarkable advantage of our approach . It thus requires specific techniques, and we use the meshing software tool isosurf (http://svr-www.eng.cam.ac.uk/~gmt11/software/isosurf/isosurf.html). Our goal is to model the boundaries of healthy - brain and tumor regions as two connected surfaces meshes . However, isosurf can only mesh the boundaries of one or several separate regions, and, thus, does not allow one to mesh connected region boundaries with common nodes at their intersections . We thus start by building two separate surfaces meshes that we connect using our own routines based on vtk . The two connected triangle surfaces are then jointly meshed into a single volume mesh of tetrahedra that conform to the two surface meshes using gmsh (http://www.geuz.org/gmsh/). Further details on the building of the biomechanical model, in particular the building of the connected surface meshes, can be found in . A linear elastic law is assigned to the biomechanical model, with young modulus e = 3000 pa and poisson ratio = 0.45 . Because displacements, rather than forces, are applied to the model using a linear formulation, the fem or xfem solution is independent of young modulus e . We choose as surface landmarks the whole - brain and internal tumor region boundaries . To evaluate the surface deformations of these region boundaries between two imr images, we use an active surface algorithm [55, 56]. Because these region boundaries to match must be closed surfaces, we thus use as surface landmarks the whole - brain and healthy - brain region boundaries . The surface deformation of the internal tumor region boundary will be derived from the active surface algorithm of the healthy - brain region boundary . In our active surface algorithm coming from [13, 47, 48], the external forces f(x) are computed using a gradient descent on a distance map of the region boundary . With such external forces, the active surface algorithm is not able to take correctly into account local rigid motion due, as an example, to lateral or tangential movement depending on the head orientation . For the whole - brain region, any rigid transformation that could have occurred has already been taken into account by the rigid registration of the imr images (section 4.1). However, for the healthy - brain region, it can happen that the internal tumor region boundary moves partly in a rigid way . Therefore, the active surface, initialized from the healthy - brain region boundary in the first imr image, is first locally transformed in a rigid way along the internal tumor region boundary using the iterative closest point transform available in vtk . Then, this resulting surface is deformed using the active surface algorithm as explained above . Further details on the local rigid registration of the healthy - brain region boundary can be found in . Before applying the displacements whole - brain and internal tumor region boundaries to the biomechanical model nodes, the two surface displacement fields are smoothed based on a weighted - distance average, that is, the displacement of each node is averaged with the displacements of its n closest neighbor nodes . This smoothing will make them consistent with each other, and compatible with the volume mesh in order to avoid element flipping, in particular at the intersections between whole - brain and internal tumor region boundaries . The displacement fields of the surface landmarks are applied to the biomechanical model, which deforms according to the laws of solid mechanics . The equations of solid mechanics are solved using fem or xfem, depending upon the type of circumstances, namely, brain shift or resection . We use the fem - software tool metafor (http://metafor.ltas.ulg.ac.be/) developed in our mechanical - engineering department, to which we have added an xfem module . The initial stress state of the brain is unknown and is thus set to zero for each fem or xfem computation, as in [10, 13]. Fem discretizes the solid of interest into a mesh, that is, into a set of fes interconnected by nodes, and approximates the displacement field u(x) by the fem displacement field u(x) defined as (1)ufem(x)=iii(x)ui, where i is the set of nodes, the i(x)'s are the nodal shape functions (nsfs), and the ui's are the nodal degrees of freedom (dofs). Each i(x) is defined as being continuous on its compact support i, which corresponds to the union of the domains of the fes connected to node i . In our approach, we use linear nsfs . In contrast, xfem handles a discontinuity by allowing the displacement field to be discontinuous within fes [37, 5860]. The xfem displacement field generalises the fem displacement field (1) with (2)uxfem(x)=iii(x)ui+iji(x)j=1neigj(x)aji . The first term corresponds to the fem displacement field (1), where i is the set of nodes, the i(x)'s are the fem nsfs, and the ui's are the nodal fem dofs . The heart of xfem is the enrichment that adds a number, n, of dofs aji to each node i of the set j, which is the subset of nodes of i whose support is intersected by the discontinuity of interest . These dofs are multiplied by the nsfs i(x) and the discontinuous functions gj(x). The use of specific xfem enrichment functions gj(x) for a node i j depends on the type of discontinuity, for example, crack, hole, material interface, and so forth, to be modeled . Suppose that our goal is to model a crack, characterized by a discontinuity in the displacement field (as opposed to a material interface for instance, characterized by a discontinuity in the derivative of the displacement field). When the crack fully intersects the support of the node, a simple choice is a piecewise - constant unit function that changes sign at the boundary across the crack, that is, the heaviside function (3)h(x)={1for (xx)en>0,1for (xx)en<0, where x is again the position of a point of the solid, x * is the position of the point on the crack that is the closest to x, and en is the outward normal to the crack at x * . In case of resection deformation, the goal is to model a discontinuity such that the part of tissues corresponding to tissue removed by the resection has no influence on the deformation of the remaining part of the tissues . One is actually interested in the deformation of the remaining part of the tissues only . In that sense, the hole function as the following equation: (4)v(x)={1for (xx)en>0,0for (xx)en<0, could be used as xfem enrichment function, instead of the heaviside function, and would be totally sufficient . The results that we would obtain on the remaining part of the tissues would be identical . However, because the heaviside function is necessary for retraction modeling, we have used the same function for the resection modeling even if it was not strictly necessary . When minimizing the total deformation energy, the resulting xfem equations remain sparse and symmetric as for fem . Whereas fem requires a remeshing and the duplication of the nodes along the crack to take into account any discontinuity, xfem requires the identification of the nodes whose support is intersected by the crack and the addition of dofs: (1) any node whose support is not intersected by the discontinuity remains unaffected and thus possesses three dofs; (2) any node whose support is fully intersected by the discontinuity is enriched with three heaviside dofs and thus possesses six dofs . To qualitatively estimate the similarity between two images, we compare the edges extracted from these images using the canny edge detector available in itk (http://www.itk.org/). Indeed, although potentially useful for the sake of comparing methods on a mathematical basis and defining unique correspondences, landmark - based target analysis presents several relevant limitations in the present setting . Having experts picking landmarks introduces significant intra- and interobserver variability . Picking landmark points, as ferrant et al . Did, is rather difficult when it comes to define enough visible landmarks especially in the tumor region on the 5 different images (and not 2 images only, as majority of studies focusing on brain shift are using). Rather than point targets, linear tumor contours, and limits between structures and potential eloquent structures matter most in the practical case of tumor ablation neurosurgery . These are the reason why we chose to use the canny edges in order to evaluate the registration . Besides, while it is true that these edges do not necessarily physically correspond between the successive imr images, these images have been acquired with the same image protocol (mr sequence, voxel size, grayscale value range), which should limit this problem . To quantitatively estimate the similarity of the two edge maps, we compute the modified hausdorff distance between the sets of edge points, that is, voxels representing the edges, in these two images . The modified hausdorff distance (a, b) between two sets of points a and b is defined as (5)(a, b)=max(h(a, b),h(b, a)) with h(a, b)=1naaad(a, b), where the directed hausdorff distance h(a, b) is a measure of the distance of the point set a to the point set b, na is the number of points in set a, and d(a, b) is the distance of point a a to the closest point in b, that is, d(a, b) = minbb||a b||, where ||a b|| is the euclidean distance . The directed hausdorff distance h(a, b) thus computes the average distance of points of a to points of b. the averaging minimizes the effects of outlier points, for example, due to image noise . The value of the modified hausdorff distance (a, b) increases with the amount of difference between the two sets of edges points . In the following, we denote by (ia, ib) the modified hausdorff distance of the edges extracted from the whole - brain region of the images ia and ib, that is, with the skull and external cerebrospinal fluid masked out from them . In this section, we apply our methods, respectively, of brain shift and resection (imr images are acquired with the 0.5 tesla intraoperative ge signa scanner of the brigham and women's hospital, boston, usa . Imr image size is 256 256 60 voxels, and voxel size is 0.9375 0.9375 2.5 mm). Two patient cases, each including five imr images, are treated to illustrate our modeling and brain shift followed by successive resections . In both cases, the 1st imr image was acquired prior to the opening of the skull; the 2nd imr image was acquired after the opening of the skull and dura, and shows some brain shift; the 3rd, 4th, and 5th imr images were acquired after successive resections . The modelings of brain shift, 1st, 2nd, and 3rd resection are performed using different techniques, as detailed below . Except where otherwise noted, the following discussion applies to both patient cases (the result of each deformation modeling is shown for the two patient cases at the end of section 5.2.3). To model brain shift based on the 1st and 2nd imr images, we estimate the surface displacement fields of the whole - brain region boundary and the internal tumor region boundary from the two imr images . No tissue discontinuity is involved in the brain shift deformation, so the biomechanical model is deformed using fem . This results in the volume displacement field of the biomechanical model, which is illustrated in figure 3 for the first patient case . This volume displacement field is used to warp the part of the 1st imr image corresponding to the whole - brain region . In the following sections, the three successive resections are modeled separately, because they require different types of processing . Matching two region boundaries to get a displacement field makes sense only if they correspond to the same physical entity . Once the resection has started, we can no longer rely on the entirety of the whole - brain region boundary, since a part of it is now missing . For modeling the successive resections, we thus evaluate the displacement field for the boundary of the healthy - brain region only . The 1st resection occurs between the times the 2nd and 3rd imr images are acquired . However, since the corresponding removal of tissues is most likely accompanied by deformation, one cannot exactly determine what tissue is removed based just on the two imr images . We thus decided to model the 1st resection by still relying on the displacement fields of key surfaces, here the healthy - brain region boundary, to deform the biomechanical model . This indeed appears to be the only reliable information concerning the deformation due to resection that we can extract from the 2nd and 3rd imr images . Consequently, we do not model explicitly the removal of tissue, but we model directly the deformation resulting from it, without introducing any tissue discontinuity . Using the surface displacement field of the healthy - brain region boundary, we compute the deformation of the biomechanical model via fem . Then, using the resulting volume displacement field, we warp the part of the 2nd imr image corresponding to the whole - brain region, in the same way as we did in the case of for brain shift . The image resulting from the 1st resection modeling is now registered to the 3rd imr image, except outside of the healthy - brain region boundary, that is, for the tumor region . Finally, we alter the resulting image to reflect the effect of resection . For this, we assign the background color to the voxels corresponding to the resected tissue volume the significant feature of the 2nd resection is that some tissue has already been removed by the 1st resection, which means that this tissue cannot have any physical influence on subsequent brain deformations because it does not recall that the biomechanical model has been deformed to model the brain shift and the 1st resection and is thus registered to the 3rd imr image . So, using the 3rd imr image, we can define the boundary of the 1st resection, that is, the tissue discontinuity to include in the deformed biomechanical model (figures 4(a) and 4(b)). We then enrich the nodes whose supports are intersected by the discontinuity with heaviside dofs . Consequently, when the xfem - based biomechanical model deforms, the part corresponding to tissue removed by the 1st resection has no influence on the deformation of the remaining part of the brain . For the first patient case illustrated in figure 4, the tetrahedron mesh consists of 3,317 nodes, which corresponds to 9,951 fem dofs . The biomechanical model is deformed in accordance with the displacement field of the healthy - brain region boundary evaluated from the 3rd and 4th imr images . The bottom part of the mesh, representing the tissue remaining after the 1st resection, has been deformed according to the displacement field of the healthy - brain region boundary, while the top part, representing the tissue removed by the 1st resection, has been subjected to a translation, but only for visualization purposes . Even though the mesh is displayed as two separate parts, it is, in fact, a single entity . Indeed, a main feature of xfem is its ability to handle the effect of a discontinuity without modifying the underlying mesh, that is, without remeshing . For modeling the 2nd resection, the edges of fes straddling the discontinuity have been made discontinuous and their nodes moved apart . Using the xfem volume displacement field, we warp the part of the 3rd imr image corresponding to the whole - brain region . The resulting image is then masked out with the whole - brain region segmented out from the 4th imr image . One significant feature of the procedure described for modeling the 2nd resection is that it can be applied repetitively for each subsequent resection visible on successive imr images, no matter how many there are . The modeling of the 3rd resection is thus identical to the modeling of the 2nd resection . The tissue discontinuity due to the 2nd resection is defined from the 4th imr image, and used to appropriately enrich the nodes of the biomechanical model . Then, this biomechanical model is deformed using xfem, in accordance with the displacement field of the healthy - brain region boundary evaluated from the 4th and 5th imr images . For the first patient case, a simplification for the modeling of the 3rd resection can be made because, by the time the 5th imr image is acquired, the resection is complete . This means that we only need to compute the volume displacement field of the healthy - brain region . Since we apply displacements exactly to the boundary of the healthy - brain region, the results obtained with fem and xfem will be identical . Using the fem (for the first patient case) or xfem (for the second patient case) volume displacement field, we warp the part of the 4th imr image corresponding to the whole - brain region . The resulting image is then masked out with the whole - brain region segmented out from the 5th imr image . Figures 5 and 6 show the results of warping the imr images, as well as the edges extracted from them, after brain shift and each successive resection modeling for the two patient cases . As explained in section 5.2.3, since we apply displacements exactly to the boundary of the healthy - brain region, the results obtained with fem and xfem are identical in the healthy - brain region . One can deduce that using xfem for modeling resection is interesting when the neurosurgeon needs to have an accurate displacement field of the remaining tumor tissues . In this case, it is interesting to evaluate the impact of using fem, instead of xfem, to model the resection as if no resection was performed before . Using fem for modeling resection is equivalent to ignoring the presence of resection on intraoperative images . To illustrate the comparison between fem and xfem results, we choose the 3rd resection modeling of the second patient case indeed, it is the deformation with remaining tumor tissues that shows the largest magnitude, and, thus, that is likely to give a maximum difference between the two computations . The healthy - brain and tumor regions segmented out from the 4th and 5th imr images are respectively shown in figures 7(a) and 7(b). The volume displacement fields of the biomechanical model using xfem and fem are respectively shown in figures 7(c) and 7(d). The part of the 4th imr image corresponding to the whole - brain region is warped, first with the volume displacement field obtained via fem, and then with that obtained via xfem . The difference between the two warped images is shown in figure 7(e). As expected however, the difference between the two volume displacement fields is smaller than the image resolution (although the difference between the two volume displacement fields is smaller than the image resolution, the difference between the images resulting of the warping using these two volume displacement fields is nonzero . This is explained by the fact that the (gray) value of each voxel of the warped image is defined as a weighted - value of voxels of the original image . The weights are defined based on the overlapping ratio of the voxel of the warped image, with voxels (determined using the volume displacement field) of the original image). In addition, the deformed 4th imr images, using the xfem- and the fem - based deformations of the biomechanical model, show the same similarity, computed based on the modified hausdorff distance, with the 5th imr . Two reasons explain that the differences between the fem and xfem results are so small . First, the brain deformation itself due to the 3rd resection is small, and, thus, it is expected to obtain small differences between the two resulting brain deformations . Second, in the case the remaining tumor tissues are close to the healthy - brain region boundary, it implies that they are close to the boundary where surface displacement fields are applied to drive the deformation of the biomechanical model . Although this comparison between fem and xfem should be done on more patient cases, we suggest that, in first approximation, fem could be used for modeling resection cases with small brain deformations . Nevertheless, the presentation of the successive resections using xfem shows the generality of our framework, and details how xfem is implemented . Note that in section 6 devoted to validation, the warped images are the ones deformed with xfem . For each deformation modeling based on a pair (ik, ik+1) of two successive imr images that are already rigidly registered, we compare the similarity between these ik and ik+1 images, as well as the similarity between the ik and ik+1 images, where ik is the result of warping ik . This gives us an estimate of how well we are able to capture, and compensate for, the local deformations between ik and ik+1 . The goal of the nonrigid registration is, however, to deform the preoperative images . By warping ik for each deformation modeling, we do not take into account the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . To evaluate the effect of this error amplification on the results, we also perform the required succession of warpings on i1, and we denote the resulting image by i1,k . We then compare, for each deformation modeling, the similarity between i1 and ik+1, together with the similarity between i1,k and ik+1 . This allows one to evaluate the propagation, that is, the amplification, of alignment error on the results . The modified hausdorff distance computed for each pair of imr images are given in tables 1 and 2 . Table 1 shows, for each deformation modeling based on a pair (ik, ik+1) of two successive imr images, the values of the modified hausdorff distances (ik, ik+1) and (ik, ik+1). These values are computed using the canny edges extracted from the pair of images (ik, ik+1) (figures 5 (d) and 6 (d)) and (ik, ik+1) (figures 5 (e) and 6 (e)). We observe that the values for the images nonrigidly registered are relatively constant, that is, 1 mm, for each deformation modeling . Six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are (rigidly and subsequently) nonrigidly registered . However, the modified hausdorff distance increases for the 3rd resection modeling of the first patient case, as well as for the brain shift modeling of the second patient case . To understand if the nonrigid registration is responsible for the increase of the misalignment of the two imr images everywhere in the whole - brain region, or if this effect is localized, we compute the modified hausdorff distance in the region and neighborhood of the tumor only (volume region that extents by 25 mm the tumor region segmented in i1 for both patient cases). The modified hausdorff distance decreases from (i4, i5) = 1.70 mm to (i4, i5) = 1.37 mm for the first patient case, while it decreases from (i1, i2) = 1.36 mm to (i1, i2) = 1.28 mm for the second patient case . This indicates that the nonrigid registration enhances the alignment of the two imr images within the tumor region and its neighborhood, which is in fact the location requiring the best modeling accuracy . This behavior could be explained by the fact that a maximum of information from the imr images is used in this region, that is, one or two (in case of brain shift modeling) surface displacement fields are applied around it . The increase of misalignment elsewhere in the brain volume could be explained by two reasons . First, the landmarks tracked from the imr images are surfaces . As a consequence, the nonrigid registration is expected to give better results near the tracked surfaces than far from them in the volume . The volume misalignment could point out the need for better parameters values and/or other constitutive laws . Table 2 shows, for each deformation modeling based on a pair (ik, ik+1) of two successive imr images, the values of the modified hausdorff distances (i1, ik+1) and (i1,k, ik+1). So far, igns systems allow one to rigidly register preoperative and successive imr images . (i1, ik+1) thus represents the navigation accuracy that we can obtain with an igns system at the present time . The comparison of (i1, ik+1) with (i1,k, ik+1) gives the improvement that could be practically achieved in the alignment with our approach . As expected, table 2 shows that the igns accuracy decreases through the successive deformations . Indeed, the modified hausdorff distance increases from (i1, i2) = 1.24 mm to (i1, i5) = 1.78 mm for the first patient case, and from (i1, i2) = 1.01 mm to (i1, i5) = 1.68 mm for the second patient case . Six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are nonrigidly registered . To understand if the modified hausdorff distance increases everywhere in the whole - brain region for the brain shift and 1st resection modeling of the second patient case, we compute the modified hausdorff distance in the neighborhood of the tumor region (in the same way as explained for table 1), and observe the improvement of the alignment within the tumor region and its neighborhood . As opposed to the values of the modified hausdorff distances in table 1, the values for the images nonrigidly registered in table 2 increase through the successive resection modeling . This amplification error is due to the fact that, after having modeled brain deformation between a pair of imr images, the deformed biomechanical model is not in perfect alignment with the second image of the pair . Since, for the subsequent deformation modeling, the surface landmarks are initialized based on the deformed biomechanical model, this can thus ampliy a misregistration error . We developed a complete 3d framework for serial preoperative image update in the presence of brain shift followed by successive resections . The nonrigid registration technique used an homogeneous linear elastic biomechanical model, driven by the deformations of whole - brain and internal tumor region boundaries for brain shift modeling, and healthy - brain region boundary for resection modelings, tracked between successive imr images . The biomechanical model was deformed using fem for brain shift modeling, and fem or xfem for resection modeling, depending upon whether some brain tissues were previously resected or not . We showed that our approach was modular, and could be applied each time a new imr image is acquired . We used a linear formulation to characterize the deformation of the brains of both patients because the brains underwent relatively small deformations and displacements . While nonlinear biomechanical models have proven effective to decrease yet do not abolish the inaccuracies of fem - based modeling methods of large brain deformations, the deformations observed in our patients during surgery remained moderate (47 mm), thus reducing the theoretical benefit of using nonlinear models . This allowed us to use simpler linear models and focus on the added value of xfem to simultaneously account for surgical deformations, namely, shift and resection . Using a linear formulation implied that, for each new deformation modeling, one could use the initial configuration rather than the last - deformed configuration of the biomechanical model . This had the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively degraded with each successive deformation modeling . This also had the advantage that we did no longer need to reconnect the deformed mesh for each new xfem calculation, which was one drawback of our previous method, presented in [39, 41], where the biomechanical model was successively deformed . We also showed how xfem could handle a discontinuity for modeling resection without any remeshing or mesh adaptation while the representation of the discontinuity remained accurate, that is, the representation of the discontinuity was not based on a jagged topology using fe facets . Xfem thus also avoided making the mesh resolution richer in the neighborhood of the resection - cavity boundary for improving the accuracy of the representation of the discontinuity for that purpose only . We showed that our nonrigid registration technique improved the alignment of the successive imr images for most of the deformation modeling of both patient cases . When our nonrigid registration failed, it still improved the alignment locally, that is, within the tumor region and its neighborhood . We tested the explicit modeling of the lateral ventricles' region with a soft, compressible law in addition to the whole - brain region law used in the homogeneous biomechanical model . In addition to the validation that is usually performed for successive deformation modelings, that is, validation between pairs of successive intraoperative images, shown in table 1 of section 6 or in the work of ferrant et al . [13, 47, 48], we also evaluated the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . As a result, shown in table 2 of section 6, we showed that our approach suffered from the propagation of misregistration through the successive deformation modelings . We expected that this was due, at least partly, to the algorithms used to evaluate intraoperative surface displacements fields from the whole - brain and healthy - brain region boundaries . The surface displacement fields were computed using active surface algorithms, and smoothed to make them compatible with the biomechanical model . Because of these two smoothings, the deformed biomechanical model was likely to not be in a perfect alignment with the imr image to which it was registered . Because the surface displacement fields evaluated for the next deformation modeling were initialized based on the deformed biomechanical model, we expected to observe an amplification of the misregistration, which was confirmed by our quantitative evaluation . At the present time though, commercial igns systems allow one to register preoperative images and successive imr images, but in a rigid way only . Consequently, although the effect of error amplification exists, our technique still enhances the current capabilities of commercial igns systems . Future work on modeling of brain shift followed by successive resection is required in five main areas . First, the effect of error amplification through the successive brain deformation modelings calls for further research . Consequently, the segmentation, and the subsequent smoothing, as well as the evaluation of surface displacement fields, should be improved to minimize the effect of error amplification . Second, further research is required to include additional structures in the biomechanical model in general, and to study the best way to include the lateral ventricles in particular . The use of a poroelastic model in order to model the cerebrospinal fluid filling the ventricles could be considered [17, 18]. Indeed, these images provide volume information (rather than surface information only), are of good quality in comparison to other intraoperative modalities, and possess a field of view that includes the full volume of brain tissues (for the 0.5 tesla ge signa scanner). These images thus allow one to evaluate what, and how, new structures of the brain could be used, to enhance the modeling of brain shift . Some regions, for example, the lateral ventricles' region, could be extracted from the two imr images, and used as surface landmarks to drive the deformation of the biomechanical model [13, 62]. Indeed, the workflow presented in this paper has the advantage of being easily adaptable . In case the tumor region would not be visible (enough) on the imr images, these new structures, easier to segment, could also adequately replace the tumor for driving the deformation . Fourth, our global approach should no longer be based on the 1st imr image used as a substitute for preoperative images, but on the preoperative images themselves . Fifth, we should implement, for the surgery cases involving large deformations of the brain, a nonlinear formulation of fem [63, 64], and, particularly, a nonlinear formulation of xfem, which is the subject of recent research [65, 66].
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The contribution to human health of the specific fatty acid (fa) composition of the diet has received considerable attention in the literature . Fatty acids are key nutrients that affect early growth and development as well as the prevention of chronic disease in later life.1 among the fas, omega-3 polyunsaturated fatty acids (n-3 pufa) and omega-6 polyunsaturated fatty acids (n-6 pufa) have been suggested to decrease and increase several human diseases, respectively . Pufa that contains more than one carbon double bond consists of two major classes such as n-6 and n-3 (fig . Linoleic acid (la) is a representative n-6 pufa, serving as a substrate to be converted into an arachidonic acid (aa) affecting the prevalence and severity of inflammation . N-6 pufa can induce cardiovascular disease, diabetes, cancer, and age - related disease.1 -linolenic acid (ala), eicosapentaenoic acid (epa 20:5) and docosahexaenoic acid (dha 22:6) are important n-3 pufa involved in human . They are essential fatty acids that cannot be synthesized by mammals, by which must be obtained from dietary sources such as cold - water fish, certain seeds (flax) and nuts (walnuts). A lot of studies suggest that n-3 pufa, as diet - dependent factors, may be critical to preventing disease backed up with authentic antioxidative and anti - inflammatory actions . Especially, n-3 pufa has been shown to exert beneficial effects on some chronic degenerative diseases such as cardiovascular disease,2,3 rheumatoid arthritis,4 diabetes,5 other autoimmune diseases,6,7 and cancer.8,9 increased fat consumption by western diet has been associated with the development of cancer such as breast, colon, pancreatic, and prostate cancers with the notable exception of n-3 pufa, which show to have multiple beneficial anti - tumor actions that affect the essential alterations that dictate malignant growth in a number of studies.10 a diet rich in n-3 pufa may protect from cancer, at least at certain sites . Studies on the fatty acid status of patients with several cancer types including bladder, pancreatic, lung and esophageal cancer show low concentrations of plasma phospholipid n-3 pufa, ranging from 55 to 88% of amounts in healthy individuals.1113 recent studies have found a positive association between n-6 pufa and cancer risk, whereas in the same model, n-3 pufa were shown to reduce the development of cancer . Epidemiological studies suggest that a high n-3 pufa to n-6 pufa ratio may be the optimal strategy to decrease breast cancer risk.14 solid epidemiological study shows that consumption of n-3 pufa appears to protect against the development of hepatocellular carcinoma, even among patients with hepatitis b virus (hbv) and/or hepatitis c virus (hcv) infection.15 recently, zhennan et al . Has reviewed prospective studies investigating the possible protective effects of the dietary intake of n-3 pufa on prostate cancer development.16 fasano et al also has reviewed a lot of in vivo and in vitro experimental studies providing strong indications of the anti - tumor action of n-3 pufa against lung cancer.17 the purpose of this review is to discuss the potential role of n-3 pufa in gastrointestinal (gi) cancer development . We believe that increased consumption of n-3 pufa may lower the risk of gi cancer development via various chemopreventive activities . Future studies should include combination treatment of n-3 pufa and nutrients with different and complementary mechanisms of chemopreventive action . Among various cancers, most of the gi cancers including esophageal cancer, stomach cancer, and colon cancer, have a natural history of multi - step transition from precursor lesions to malignant lesions, inflammation, adenoma formation, dysplastic changes.18 therefore, gi cancers usually have premalignant lesions before developing invasive cancers, for instances, barrett s esophagus for esophageal cancer, chronic atrophic gastritis accompanied with intestinal metaplasia for gastric cancer, and adenoma or dysplasia originating from chronic ulcerative colitis for colon cancer . Because the western diet contains disproportionally high amounts of n-6 pufa and low amounts of n-3 pufa, denoted as a high n-6 to n-3 pufa ratio, n-3 pufa may feasibly play a role in several stages of gi cancers management . Esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . According to the increasing incidence of gastroesophageal reflux disease (gerd), esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades . Suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [cases vs. population controls; or=0.46, 95% ci=0.220.97, 4th vs. 1st quartiles of intake] was associated with a lower risk of barrett s esophagus . In contrast, higher trans - fat intakes were associated with increased risk (or=1.11; 95% ci=1.031.21 per g / day). Moreover, it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy (crt) by reducing crt toxicity.21 in contrast to other gi cancer, little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa . Gastric cancer (gc) is the fourth most common cancer worldwide, and almost two thirds of affected individuals will die of their disease . Some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer . Recently, correa et al . Suggests that docosahexanoic acid (dha) inhibits helicobacter pylori (h. pylori) growth in vitro and mice gastric mucosa colonization.23 h. pylori are recognized as a major etiological factor in chronic active gastritis, gastric duodenal ulcers and gastric cancer . It has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde (mda), gastrin, and nitric oxide (no) and normalization of mucosal glutathione.25 especially, it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer, of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27 in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase (mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover, n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand, dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach . Colorectal cancer (crc) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . Among the gi tract cancer, crc cancer has raised the most attention over the past decades, as they share a long precancerous stage (the adenoma in crc) which provides a window of opportunity to intervene and prevent development of cancer . Recently there is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer . Although epidemiological studies of the association between fish intake, n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk . Dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity, compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality, the pooled relative risks for colorectal cancer incidence were 0.960.97 (95% confidence interval: 0.92, 1.00) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer, there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer, but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer . Kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake (or=0.61 for the highest vs lowest quartile), as well as for epa and dha intake individually in a case - control study of 1,872 patients (929 cases of distal crc and 943 controls). Pot measured and compared serum n-3 pufa levels in 861 patients (363 cases of colorectal adenoma and 498 controls). There was a significant reduction in colorectal adenoma risk (or=0.67) between low level of n-3 pufa (<1.8%) and high level of n-3 pufa (> 2.3%).37 recently, sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer . The preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine (dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours . Studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence, together with a 3070% reduction in tumor multiplicity, in both carcinogen and apc mouse studies . Similar findings have been reported for studies of growth of human crc cell lines such as hct26, ht-29, hct116, and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc . The excellent tolerability and safety profile of n-3 pufas combined with other health benefits, particularly cardiovascular, make n-3 pufas an attractive candidate for prevention and treatment of crc.9 esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . According to the increasing incidence of gastroesophageal reflux disease (gerd), esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades . Suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [cases vs. population controls; or=0.46, 95% ci=0.220.97, 4th vs. 1st quartiles of intake] was associated with a lower risk of barrett s esophagus . In contrast, higher trans - fat intakes were associated with increased risk (or=1.11; 95% ci=1.031.21 per g / day). Moreover, it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy (crt) by reducing crt toxicity.21 in contrast to other gi cancer, little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa . Gastric cancer (gc) is the fourth most common cancer worldwide, and almost two thirds of affected individuals will die of their disease . Some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer . Recently, correa et al . Suggests that docosahexanoic acid (dha) inhibits helicobacter pylori (h. pylori) growth in vitro and mice gastric mucosa colonization.23 h. pylori are recognized as a major etiological factor in chronic active gastritis, gastric duodenal ulcers and gastric cancer . It has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde (mda), gastrin, and nitric oxide (no) and normalization of mucosal glutathione.25 especially, it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer, of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27 in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase (mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover, n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand, dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach . Colorectal cancer (crc) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . Among the gi tract cancer, crc cancer has raised the most attention over the past decades, as they share a long precancerous stage (the adenoma in crc) which provides a window of opportunity to intervene and prevent development of cancer . There is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer . Although epidemiological studies of the association between fish intake, n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk . Dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity, compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality, the pooled relative risks for colorectal cancer incidence were 0.960.97 (95% confidence interval: 0.92, 1.00) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer, there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer, but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer . Kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake (or=0.61 for the highest vs lowest quartile), as well as for epa and dha intake individually in a case - control study of 1,872 patients (929 cases of distal crc and 943 controls). Pot measured and compared serum n-3 pufa levels in 861 patients (363 cases of colorectal adenoma and 498 controls). There was a significant reduction in colorectal adenoma risk (or=0.67) between low level of n-3 pufa (<1.8%) and high level of n-3 pufa (> 2.3%).37 recently, sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer . The preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine (dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours . Studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence, together with a 3070% reduction in tumor multiplicity, in both carcinogen and apc mouse studies . Similar findings have been reported for studies of growth of human crc cell lines such as hct26, ht-29, hct116, and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc . The excellent tolerability and safety profile of n-3 pufas combined with other health benefits, particularly cardiovascular, make n-3 pufas an attractive candidate for prevention and treatment of crc.9 n-3 pufa, especially, epa and dha, have been shown to have multiple anti - tumor actions . Current knowledge of the anti - tumor activity of n-3 pufa has been comprehensively reviewed elsewhere.9,16,39 recently, stephenson et al.10 has reviewed more recent underlying mechanisms providing strong indications of the anti - tumor actions of n-3 pufa on the hallmarks of cancer . N-3 pufa appear to down - regulate epidermal growth factor receptor (egfr), protein kinase c (pkc), ras, and nf-b, insulin like growth factor (igf), which are important cell signaling mediators often found to be elevated in carcinogenesis . Secondly, n-3 pufa induces cancer cell apoptosis via modulation of peroxisome proliferator - activated receptors (ppars), the bcl-2 family, and nf-b cell signaling . Thirdly, n-3 pufa decreases sprouting angiogenesis by suppressing vascular endothelial growth factor (vegf)- and platelet derived growth factor (pdgf)-stimulated endothelial cell proliferation, migration, and tube formation and by inhibition of mmps via no production and nf-b and -catenin cell signaling . Fourthly, n-3 pufa also decreases cell - cell adhesion via down regulation of rho - gtpase, which inhibits cytoskeleton reorganisation, and reduction in intercellular adhesion molecule (icam)-1 and vascular cell adhesion molecule (vcam)-1 expression . Cockbain et al.9 has also proposed the four main anti - tumor actions of n-3 pufa (i) modulation of cox activity; (ii) alteration of membrane dynamics and cell surface receptor function; (iii) increased cellular oxidative stress and (iiii) derived anti - inflammatory lipid mediators . Firstly, n-3 pufa can act as an alternative substrate for cox-2, instead of aa, leading to a reduction in formation of pro - tumorigenic secondly, incorporation of n-3 pufa into cell membranes alters the fluidity, structure and/or function of lipid rafts or calveolae . Especially, the localization of cell surface receptors, such as g protein - coupled receptors (gpcrs), toll - like receptors (tlrs), and epidermal growth factor receptor (egfr), in lipid rafts is believed to be crucial for downstream receptor signaling, controlling proliferation and apoptosis . Thirdly, n-3 pufa may have an anti - tumor effect through alteration in the cellular redox state . N-3 pufa can increase reactive oxygen species (ros) because it is highly peroxidisable . Therefore, n-3 pufa can induce cancer cell apoptosis via elevation of intracellular ros levels . Fourthly, n-3 pufa can be metabolized novel anti - inflammatory lipid mediators including resolvins, protectins and maresins . It is known that resolvins exhibit antineoplastic activity via anti - inflammatory and inflammation resolution activity in animal models of acute inflammation . Besides these multiple anti - tumor actions, it is also reported recently that n-3 pufa can activate nrf2 and induced nrf2-directed gene expression40,41 and can suppress lipopolysaccharide - induced inflammation through induction of nrf2 expression.42 nuclear factor erythroid 2-related factor 2 (nrf2) is a redox - sensitive master regulatory transcriptional factor that plays an important protective role in cells by regulating cellular redox balance.43 moreover, n-3 pufa significantly reduces oxidative stress - induced endothelial cell ca influx . This effect might be associated, at least in part, with altered lipid composition in membrane lipid rafts.44 recently, the engineered n-3 pufa desaturase transgenic mice (fat-1 mice), which can endogenously synthesize n-3 pufa in their tissues, allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase, which utilizes n-6 pufa as substrate . This allows production of high n-3/n-6 ratios in the animals, thus eliminating the potential diet variations . Hence, the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis . Since fat-1 mice were generated, xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice . Nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss (dextrane sodium sulfate) plus aom (azoxymethane) in the fat-1 transgenic mice via its anti - inflammatory properties . Song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells, panc02 cells, were inoculated into the fat-1 transgenic mice . Recently, mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice . They generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma (84%; p<0.02) in fat-1-kras mice compared to kras mice . Besides of gi cancers, the growth of hepatocellular carcinoma (hcc) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1, which were bred with mouse mammary tumor virus (mmtv)-neu(ndl)-yd5 mice (an aggressive breast cancer model), displayed significant (p<0.05) reductions in tumor volume (30%) and multiplicity (33%).54 recently, in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development (unpublished data). Fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice . A dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown . Moreover, in the case of stomach cancer development, h. pylori initiated-, salt diet - promoted - gastric tumor was also reduced in fat-1 mice . In conclusion, several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development . The fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets, which can create confounding factors that limit studies of this nature.55 recently, the engineered n-3 pufa desaturase transgenic mice (fat-1 mice), which can endogenously synthesize n-3 pufa in their tissues, allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase, which utilizes n-6 pufa as substrate . This allows production of high n-3/n-6 ratios in the animals, thus eliminating the potential diet variations . Hence, the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis . Since fat-1 mice were generated, xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice . Nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss (dextrane sodium sulfate) plus aom (azoxymethane) in the fat-1 transgenic mice via its anti - inflammatory properties . Song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells, panc02 cells, were inoculated into the fat-1 transgenic mice . Recently, mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice . They generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma (84%; p<0.02) in fat-1-kras mice compared to kras mice . Besides of gi cancers, the growth of hepatocellular carcinoma (hcc) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1, which were bred with mouse mammary tumor virus (mmtv)-neu(ndl)-yd5 mice (an aggressive breast cancer model), displayed significant (p<0.05) reductions in tumor volume (30%) and multiplicity (33%).54 recently, in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development (unpublished data). Fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice . A dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown . Moreover, in the case of stomach cancer development, h. pylori initiated-, salt diet - promoted - gastric tumor was also reduced in fat-1 mice . In conclusion, several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development . The fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets, which can create confounding factors that limit studies of this nature.55 gi cancer incidence and mortality are increasing in the eastern world and a high n-6 to n-3 pufa ratio in the western style diet may be a contributing factor . There is much evidence to suggest that higher consumption of dietary n-3 pufa is associated with a lower risk of gi cancer in animal models and humans . Especially, recent studies suggest that endogenous n-3 pufa delay the progression of colon and stomach cancer and elevating n-3 pufa may be an important strategy to delay / prevent gastrointestinal cancer in high - risk patients via various mechanisms mediating cancer prevention by n-3 pufa (fig . 2). In addition, using n-3 pufa in combination with other agents with complementary antitumor action may improve their efficacy in gi cancer prevention . Recently, manson et al . Has started the vitamin d and omega-3 trial (vital), a large randomized, double - blind, placebo - controlled, 22 factorial trial of vitamin d and n-3 pufa supplements in the primary prevention of cancer among a multi - ethnic population of 20,000 u.s . Men aged 50 and women aged 55.56 we expect that new findings in combination consumption of n-3 pufa with other nutrients will provide new approaches to public health implications with regard to prevention of gi cancer through dietary and lifestyle interventions.
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Sexual development in males is a multistep process, implying a delicate network of molecular events that directs the bipotential gonad to differentiate into testis (sex determination) and consequent differentiation of internal and external genitalia in the presence of testicular hormones (sex differentiation). Alteration of any genetic or endocrine factors involved in testicular development may lead to 46 xy disorders of sexual development (dsd). Genital ambiguity in patients with 46 xy dsd may either occur due to the disorders in gonadal development or due to the disruption in androgen synthesis or action . Patients with disorders of gonadal development may present with either complete or partial forms of gonadal dysgenesis . Swyer's syndrome or complete gonadal dysgenesis (cgd) is characterized by unambiguous female genitalia, bilateral streak gonads, and elevated gonadotropins . Partial gonadal dysgenesis (pgd) may have a wide spectrum of phenotypes associated with ambiguous genitalia, varying degree of hypospadias and testis of variable size and echotexture present along the path of decent . Ovotesticular dsd (ot - dsd) is a rare condition marked by the presence of both ovarian and testicular tissue in an individual while congenital bilateral anorchia (cba) is characterized by the complete absence of testicular tissue in genotypic males . Disorders of androgen synthesis may occur due to deficiency of various enzymes involved in the steroidogenesis . Congenital adrenal hyperplasia (cah) due to 17-hydroxysteroid dehydrogenase 3 (17-hsd3) is caused by the mutations in hsd17b3 gene which is required for the conversion of androstenedione to testosterone . Deficiency of this enzyme results in feminized genitalia in newborns due to the low testosterone levels . Mutations in srd5a2 gene impairs the activity of enzyme 5-reductase and hence the conversion of testosterone to its more potent form dihydrotestosterone (dht). Individuals with 5-reductase deficiency (5-rd) are often raised as females due to undermasculinized genitalia but the gender role changes soon after attaining puberty . Mutations in androgen receptor gene may lead to the androgen insensitivity syndrome (ais) which can be partial androgen insensitivity syndrome (pais) or complete androgen insensitivity syndrome (cais) and is the most common cause of 46xy dsd . The spectrum of phenotypes associated with ais may range from completely female through mixed male / female to completely male type . In 1995, charmian quigley and frank french proposed new grading system for the phenotypic features in ais based on prader classification for cah . Individuals diagnosed with 46xy dsd show an overlap in clinical and biochemical parameters which emphasizes the need for comprehensive evaluation with a multidisciplinary approach . This is an ambispective study where all new and old follow - up patients diagnosed with 46xy dsd, being managed at endocrine opd in a period of 16 months (june 2014october 2015) were included . External masculinization score (ems) were calculated based on the external genitalia of patients . Abdominopelvic ultrasound was done for the localization of gonads (magnetic resonance imaging (mri) also, when required). For patients on follow - up, previous records were reviewed to establish a diagnosis as they were on medication while in new patients, samples were collected and hormonal measurements included serum luteinizing hormone, follicle - stimulating hormone, testosterone, androstenedione (a), cortisol, dehydroepiandrostenedione, and plasma adrenocorticotropic hormone levels by electrochemiluminescence immunoassay using commercial kits (roche, germany). Radioimmunoassay kit - based method was used for evaluating 17-hydroxyprogesterone (17-ohp) levels (diagnostic systems laboratories, inc ., webster, tx, usa). 5dht was measured using radioimmunoassay (immunotech; prague, czech republic) after extraction from other hormones with diethyl ether and celite chromatography . The short - term human chorionic gonadotropin (hcg) stimulation test was carried out to check the functioning of testicular tissue . Testosterone levels were measured before and 2448 h after the last injection of a series of three intramuscular injections of hcg on alternate days (<1-year - old, 500 units; 110 years, 1000 units;> 10 years, 2000 units). Testosterone: androstenedione (t: a) and testosterone: 5 dht (t:5dht) ratios were calculated as and when required to rule out 17 beta hsd deficiency and 5 alpha reductase deficiency respectively . On first evaluation, 11 patients had presented with the primary complaint of ambiguous genitalia, three (who were being reared as female) with primary amenorrhea, three with undescended gonads and one with fever and vomiting . The presenting complaints, clinical, hormonal, and radiological findings are summarized in table 1 . Five (26%) of them were reared females and 14 (74%) as males . Ten patients (s.5, s.7, s.8, s.9, s.10, s.14, s.15, s.16, s.17, and s.18) had undergone corrective surgeries for ambiguous genitalia . Clinical profile of patients patient s.1 reared as female presented with primary amenorrhea, delayed development of secondary sexual characteristics and unambiguous female genitalia . Patients s.2, s.3, and s.5 had ambiguous genitalia, undescended gonads, raised fsh and low testosterone levels while patient s.4 had unilateral undescended testis with raised fsh and low testosterone levels, all three were suspected to be cases of pgd . Abdominopelvic ultrasonography (usg) and mri could not locate gonads in s.7 while in patient s.6, a nubbin was seen in the scrotal area . Inguinal exploration was done to locate testis but it revealed the absence of any testicular tissue . Fsh was raised in both [table 2] and there was no testosterone response to hcg stimulation . Hormonal profile of patients patient s.8 being reared as female had presented with ambiguous genitalia and raised gonadotropins . She had undergone bilateral gonadectomy and on histological evaluation ot tissue was found, she was diagnosed with ot - dsd . Patient s.9 being reared as female presented with primary amenorrhea, genital ambiguity and delayed development of secondary sexual characteristics . She underwent bilateral gonadectomy, histopathological examination revealed testicular tissue and was diagnosed with cais . Hormonal profile showed normal testosterone levels with t: dht ratio of 9.7 and t: a ratio of 5.8 . Patient s.11 being reared as female came with a complaint of hirsutism and delayed development of secondary sexual characteristics . Bilateral gonadectomy was done and histological studies showed the presence of testicular tissue which confirmed the diagnosis of pais as t: dht and t: a ratios were also normal . Testosterone levels were undetectable, but showed a good response to hcg stimulation with t: dht ratio of 7.25, t: a ratio of 5.8 and was diagnosed with pais . Patients s.13, s.14, s.15, and s.16 came with ambiguous genitalia and low baseline testosterone levels . Testosterone to dht ratio in s.13, s.14, and s.15 were 31.4, 18, and 13.9, respectively . Due to high t: dht ratio post - hcg stimulation, all three were diagnosed with 5rd while in s.16, t: dht ratio was not calculated as b / l gonadectomy was done at 2 years of age . Molecular analysis of srd5a2 gene in this patient revealed r246q mutation commonly seen in 5-rd . Records show that his post hcg serum t: a ratio was 0.06, he was thought to be a case of 17hsd deficiency . At 2 years of age he had fever, vomiting and loose stools with hyponatremia, hyperkalemia and hypoglycemia . Patient s.18 was admitted in pediatrics with a complaint of poor feeding and poor activity since day one of life . On evaluation, he was found to have hypernatremia, hypokalemia, and genital ambiguity with bifid scrotum and hypospadias . In view of electrolyte imbalance, incomplete masculinization, low cortisol, and elevated 17-ohp, he was suspected to be a case of cah and treatment was started . An accurate diagnosis could not be established when he came to us as he was already on steroids and had a history of salt wasting, so steroids could not be stopped for evaluation of the cause of cah . He had undergone three genital surgeries for hypospadias correction but still had difficulty in urination . The patient is kept on antihypertensives, but no definitive diagnosis could be made as the baseline hormonal reports are missing . With the help of clinical, biochemical and radiological evaluation, accurate diagnosis was made in 16 patients while rest 2 are on follow - up and managed in our clinic . Discordance between genetic (xx or xy), gonadal (testis or ovaries), external genital (vulva or penis), and internal sex (wolffian or mullerian ducts) results in dsd . In humans, the bipotential gonad differentiates into testis in the presence of sry gene present on y - chromosome . Testicular differentiation is followed by the development of internal sex ducts and external genitalia in the presence of fetal hormones produced by the testis . Sertoli cells secrete anti - mullerian hormone, which causes irreversible regression of paramesonephric or mullerian ducts around the sixth week of gestational age . Testosterone produced by leydig cells of fetal testis promotes the development of mesonephric or wolffian ducts and hence male internal genitalia; epididymis, vas deferens, and vesicular seminalis . The development of external male genitalia depends on the conversion of testosterone to a more potent steroid dht in the presence of 5-reductase enzyme . In 2006, the term intersex or hermaphrodite was replaced by dsd and 46xy dsd was subcategorized as disorders of testicular development and disorders of androgen synthesis or action . First - line testing in newborns includes karyotyping, imaging (usg / mri pelvis), serum electrolytes, and serum gonadotropins . Molecular diagnosis has been made in approximately 20% of cases with 46xy dsd, although confirmatory, but is limited by cost and accessibility . The inability of bipotential gonad to differentiate into testis results in testicular dysgenesis or agenesis which occurs due to the disruption of various genetic factors involved in the process of sex determination . In our study, four patients were diagnosed with pgd, one with cgd, one with ot - dsd, and two with testicular agenesis or cba . In the absence of gonadal development, patients with cgd usually present with unambiguous female genitalia due to the absence of gonadal steroid production while in pgd, the external phenotype depends upon the degree of testicular tissue present . In our study, out of four patients with pgd, three (s.2, s.3, and s.5) presented with ambiguous genitalia and one with undescended gonad (s.4). In such cases, corrective surgeries are done if required, and testosterone supplementations are given at pubertal age . Patient s.5 being reared as female had presented with ambiguous genitalia at 4 years of age . With parents consent clitorovaginoplasty and bilateral orchidectomy orchidopexy and biopsy are usually recommended for undescended gonads as the risk of malignancy ranges from 8.3% to 54% in patients with xy pgd . In 46xy cba is a rare condition and is marked by the absence of testis since birth . Previous studies have shown the occurrence of pure or partial forms of 46xy gonadal dysgenesis in families of patients with cba, but the genetic cause is still not known . In our study, two patients (s.6 and s.7) with cba were born out of nonconsanguineous marriage and had no family history of genital ambiguity but the father of patient s.7 gave history of oligospermia and assisted reproduction . 46xy ot - dsd is a very rare condition marked by the presence of ot tissue and is confirmed by histopathological examination . Parents of patient s.8 had sought medical attention for ambiguous genitalia at the age of 13 years . Bilateral gonadectomy was done, and the corrective surgeries for hypospadias and phallus are awaited . The complete or partial resistance to the action of androgens may result in cais or pais in xy individuals . Patients with cais may present with primary amenorrhea in adolescence or inguinal swellings in infants . Patient s.9 was being reared as female, parents sought medical attention at the age of 18 years for primary amenorrhea and delayed development of secondary sexual characteristics . Bilateral gonadectomy was recommended after confirming the gender identity of the patient to be female . A recent study done on 102 phenotypic women with y - chromosome showed an incidence of 17.6% malignancy . Of these 9 out of 30 patients (30%) of cais with a mean age of 20.7 years were found to have gonadoblastoma on histological analysis . Therefore, adult cais patients who receive late intervention are recommended to undergo immediate gonadectomy in order to avoid the risk of malignancy . The phenotype of patients presenting with pais depends on the responsiveness of genital tissues to androgens . Infants with pais present with the genital ambiguity of varying degrees while adults may present with gynecomastia . However, the risk of malignancy in individuals with pais is 15% if the testis is not scrotal in position . Hormonal profile showed normal testosterone levels with t: dht ratio of 9.7 and t: a ratio of 5.8 . Patient s.11 being reared as female sought medical attention for hirsutism at the age of 19 years . Biochemical examination showed normal t: dht and t: a ratios with testosterone levels in male range . Gonadectomy was done and histopathological examination showed the presence of testicular tissue, she was then diagnosed with pais . She had female gender orientation and is now on estrogen replacement therapy for breast development . His t: dht ratio was 7.25 and t: a ratio was 5.8 post - hcg stimulation, which excluded the diagnosis of 5rd and 17hsd . Though the presumptive diagnosis of patients s.10, s.11, and s.12 was pais, as t: dht ratio and t: a ratio were normal, [figure 1] diagnosis with needs further confirmation by molecular studies . Flow chart for diagnosis of 46xy disorders of sexual development the interrupted conversion of testosterone to dht due to the mutations in gene encoding 5-reductase enzyme may lead to 5rd . The differentiation of internal and external male genitalia occurs in the presence of testosterone (t) and dht . The t: dht ratio is used to diagnose this condition but interpreting these results is not always straightforward . The diagnosis of 5rd is suspected in newborns with undermasculinized genitalia (depending upon the activity of enzyme 5-reductase), low serum testosterone levels, good response to hcg stimulation and high testosterone to dht ratio (> 1012). A good response to hcg stimulation (rise by more than twice the baseline value) is observed in these patients . The patients may present with microphallus, inguinal gonads or gonads in labioscrotal folds and varying degree of hypospadias . Treatment with percutaneous dht increases the size of the phallus in infants and children with 5-reductase 2 deficiency . Most of them are reared as males and gender reassignment is required in those who are reared as females due to the spontaneous virilization at puberty . The decisions regarding management of masculinizing puberty need to be taken before puberty with a multidisciplinary approach . Female gender assignment in these patients by surgery should be undertaken postpuberty with full caution and counseling with parents . In our study, patient s.16 had severely undervirilized genitalia, bilateral gonadectomy had been done at 2 years of age and female gender had been assigned . At the age of 17 years, he sought medical attention for gender dysphoria . At this time, the presence of common r246q mutation on molecular analysis of srd5a2 gene confirmed the diagnosis of 5rd . 46xy patients with deficiency of 17-hsd enzyme usually present with female - like genitalia at birth with clitoromegaly and blind - ending vagina or male type genitalia with micropenis and hypospadias . At puberty, these patients experience significant virilization due to the abnormally elevated levels of androstenedione or extragonadal synthesis of testosterone, the exact cause of which is unknown . The diagnosis of 17-hsd3 can be suspected if testosterone to androstenedione ratio is <0.8 . The overlapping phenotype of individuals with 17-hsd and pais can be differentiated by molecular analysis or on the basis of hcg stimulated t: a ratio, which is <0.8 in cases of 17-hsd . Patient s.17 initially predicted to have 17 hsd deficiency, but later presented with features of mineralocorticoid and glucocorticoid deficiency at 2 years of age . Patient s.19 is presently 49 years of age and gave a history of ambiguous genitalia . He was on dexamethasone, sustanon and had undergone three corrective surgeries for hypospadias but still has a problem in urination . Routine examination revealed raised blood pressure for which he is on hypertensives . Limitations of this study are that, being an ambispective study, all tests were not done in all patients and baseline records / tests to make a diagnosis could not be obtained in two patients . Nonetheless, this gives an insight into how to diagnose and manage 46xy dsd, who may present at different ages, with different etiologies and different gender of rearing in our set up . There is overlap in the clinical, hormonal and anatomic profile of different subsets of 46xy dsd . The diagnosis of 46xy dsd needs to be confirmed by cytogenetic, hormonal, radiological, and genetic tests . Phenotype varies depending on the degree of genetic, hormonal and anatomic defect . At times, in spite of all the tests a definitive diagnosis may not be possible . The pediatrician, endocrinologist, surgeon, psychologist, and clinical geneticist need to work as a team for multifaceted management of these patients . This study was supported by department of biotechnology, ministry of science and technology (bt / pr7681/med/12/597/2013) and indian council of medical research (dhr / gia/1/2014), new delhi, india . This study was supported by department of biotechnology, ministry of science and technology (bt / pr7681/med/12/597/2013) and indian council of medical research (dhr / gia/1/2014), new delhi, india.
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Urinary tract infection (uti) is a frequent cause of morbidity both in the community and in the hospital setting . The causative pathogen can vary greatly geographically, and so it is prudent to identify those with resistant strains and have current data on the appropriate empirical therapy within a region . The most frequently encountered organisms associated with utis include enteric gram - negative bacteria (with escherichia coli being the most predominant), coagulase negative staphylococcus saprophyticus along with proteus mirabilis, klebsiella, and enterococcus, which account for less than 5% . However, recent studies have reported the increasing prevalence of staphylococcus aureus (sa) in utis . Sa is an opportunistic pathogen affecting both immune competent and immunocompromised individuals, frequently resulting in significant morbidity . Many strains of sa carry a wide variety of multidrug - resistant genes on plasmids, which aid the spread of resistance among species . Methicillin - resistant sa (mrsa) is widespread in many irish hospitals and is increasingly seen in community health care units such as nursing homes . Globally, it is considered that there has been an epidemic of mrsa within health care institutions . Bacteriuria with sa is hypothesized to occur through a number of mechanisms that includes catheterization, urologic procedures, or seeding of the genitourinary tract including nephrologically excreted bacteria in overt bacteremia . Bacteremia itself is associated with bacteriuria in patients infected with sa, which suggests that bacteremia is an important precursor for bacteriuria in some patient groups . Herein, we chronologically assessed the source, patient demographics, and antimicrobial susceptibilities of mrsa - positive isolates from the hospital and community setting over a 5-year period in university hospital waterford a tertiary referral hospital with the primary microbiology laboratory for 4 acute hospitals, serving a total catchment area of almost 500,000 people . The microbiology laboratory in university hospital waterford processes all inpatient urines in the catchment area of almost 500,000, excluding 2 small private elective inpatient institutions . It also processes all urine samples sent from the community (i.e., all samples from general practice and nursing homes) and all specimens from the catchments emergency departments . Laboratory diagnosis of mrsa bacteriuria was performed using accredited microbiological microscopy analysis with culture identification criteria and standardized susceptibility testing protocols . Our laboratory utilized clinical and laboratory standards institute (clsi; 20102014) and european committee on antimicrobial susceptibility testing (eucast; 2014) methodologies . A retrospective data extraction was performed, from the hospital's electronic microbiological system cognos and de - duplication was executed . We considered a separate episode as a urine specimen culturing mrsa at least 6 months following a previous positive culture . Further urine specimens sent within a 6-month period following an initial laboratory diagnosis mrsa bacteriuria were excluded . Resistance rates were then calculated for the pathogen's susceptibility to 5 commonly used antimicrobial agents ciprofloxacin, coamoxiclav, flucloxacillin, nitrofurantoin, and trimethoprim . For unwell patients, that is, those in whom there was clinical suspicion of sepsis or invasive infection, teicoplanin and vancomycin were also tested for sensitivity profiling . In order to obtain further clinical information from our patient group, we also assessed those patients who had an mrsa - positive swab (usually from groin, nose, or perineum), those who had documented mrsa bacteremia, and we analyzed their demographic details . Ethical approval was waived for this retrospective observational study in light of its non - interventional nature . Over a consecutive 5-year period, the laboratory cultured 425,013 urine samples from all sources, with a mean number of 85,003 specimens per year received (table 1). A total of 542 unique urine isolates cultured sa and 151 of species (27.9%) were methicillin resistant, meaning that 0.04% of all urine samples tested cultured mrsa . Number of urine cultures by year . Of these 151 samples that tested positive for mrsa, 92 (61%) were from mid - stream urine (msu) samples, 50 (33%) from catheter specimen urine (csu) samples, and 9 (6%) were from an unspecified source (fig . Csu = catheter specimen urine, mrsa = methicillin resistant staphylococcus aureus, msu = mid - stream urine . Mrsa was slightly more common in msu samples; however, the findings were not statistically significant (18.2% vs 13.9%, p = 0.388). Forty - nine (32.5%) specimens were from an inpatient cohort, 9 (6%) were from the emergency department, 79 (52.3%) from general practitioners, 12 (7.9%) were from nursing homes, with 2 (1.3%) from an unrecorded source (fig . Mrsa was seen in a similar proportion of inpatient and outpatient samples indicating that this is not solely a hospital - acquired phenomenon (29.1% vs 26.9%, p = 0.587). Mrsa = methicillin resistant staphylococcus aureus . The mean age of our patients who cultured mrsa within their urine was 72.7 years (range 10.9790.2) and most patients were aged between 70 and 90 years (table 2). Patients who cultured an mrsa bacterium were older than patients with mssa, and this was statistically significant (53 vs 73 years, p 0.001). The number of sa isolates grown over the 5-year study period doubled from 77 in 2010 to 161 in 2014 . The number of mrsa isolates had also increased; however, the percentage of sa isolates that were methicillin resistant has decreased (p = 0.145). This is true for both inpatient and outpatient samples (p = 0.313, p = 0.254, figs . Mrsa = methicillin resistant staphylococcus aureus, mssa = methicillin sensitive staphylococcus aureus, sa = staphylococcus aureus . Inpatient trends in sa mrsa = methicillin resistant staphylococcus aureus, mssa = methicillin sensitive staphylococcus aureus, sa = staphylococcus aureus . Mrsa = methicillin resistant staphylococcus aureus, mssa = methicillin sensitive staphylococcus aureus, sa = staphylococcus aureus . Regarding the clinical stance of patients within the cohort, 9 patients (7%) with mrsa - positive urine cultures had a documented history of mrsa bacteremia at that time . Each of these patients cultured mrsa isolates that were sensitive to nitrofurantoin, and 8 of the 9 patients (88.9%) were sensitive to trimethoprim . It was noted that all 9 patients in this cohort were aged 70 years or above (p = 0.079), although this did not reach statistical significance . Further antimicrobials agents were introduced to the testing panel in 59 (39%) samples, where there was a clinical concern regarding invasive infection or sepsis including the 9 patients with mrsa bacteremia . All 59 samples (100%) were sensitive to both vancomycin and teicoplanin . Eighty - eight (66.8%) of our 128 patients cultured mrsa - positive isolates on routine swabs from either mucosal or skin sites . All 3 patients with resistance to nitrofurantoin in their urine had a positive swab from either groin or nose . Eight of the 10 patients (80%) who had trimethoprim - resistant mrsa bacteriuria and 87 of the 125 (69.6%) of the ciprofloxacin - resistant mrsa bacteriuria also had mrsa - positive mucosal or skin swabs . There was no association between age and having an mrsa - positive swab in our cohort . Throughout the study period, there was 100% resistance of all mrsa isolates to flucloxacillin and coamoxiclav, as expected . Ninety - eight percent of isolates were resistant to ciprofloxacin, but of these samples, there was 97.2% sensitivity for nitrofurantoin and 92.4% sensitivity for trimethoprim (fig . Overall, there was only 2.7% resistance for nitrofurantoin, but all of these samples were sensitive to trimethoprim . Trimethoprim itself had a 7.4% resistance level, but all of these samples were sensitive to nitrofurantoin, meaning that no sample was resistant to both oral agents, that is, nitrofurantoin and trimethoprim . There was a statistically significant association between nitrofurantoin resistance and younger patients, in that 75% of patients with nitrofurantoin resistance were younger than 70 years (p = 0.025). There was no statistically significant association between age and resistance profile in any other of the antimicrobial panel . The impact of mrsa is considerable; in ireland, approximately 40% to 50% of isolates sa recovered from bloodstream infections are methicillin resistant . Despite available publications and guidelines on the management of mrsa skin - colonized patients, little has been published on the colonization of urine from patients with indwelling urinary catheters . The identification of an mrsa isolate in a urine culture has important ramifications for patients, both in the community and in the hospital setting . A recent study demonstrated that 22% of patients with mrsa bacteriuria went on to develop invasive mrsa infection within 12 months . Mrsa in urine clearly warrants treatment in symptomatic patients, but even in asymptomatic patients, it may require eradication before certain elective procedures such as endourological surgery . It also has consequences for patients and health care providers when it comes to providing isolation and other barrier precautions that should be administered in a nosocomial setting . The european association of urology guidelines outlines that colonization with microorganisms is a special risk factor for urological procedures (http://uroweb.org/guideline/urological-infections/), and furthermore, that an indwelling catheter is one of the most important risk factors for complications . Patients with asymptomatic bacteriuria who undergo traumatic genitourinary procedures associated with mucosal bleeding have a high rate of post - procedure bacteremia and sepsis . Bacteremia occurs in up to 60% of bacteriuric patients who undergo transurethral prostatic resection, and sepsis is clinically confirmed in 6% to 10% of these patients . Retrospective analysis and prospective, randomized clinical trials support the effectiveness of antimicrobial treatment in preventing these complications in bacteriuric men undergoing transurethral resection of the prostate . There is little information relevant to other genitourinary procedures, but any intervention with a high probability of mucosal bleeding should be considered as a risk for post - procedure sepsis and treatment of mrsa bacteriuria should be considered . Some studies have documented that eradication of asymptomatic bacteriuria is not required before nonurologic procedures, such as arthroplasty but s. aureus is the most pathogenic of all staphylococci and this study did not include patients with mrsa bacteriuria . Our study would suggest that when it comes to urine - colonizing mrsa eradication, or for treating a patient who is not critically unwell, oral therapy with nitrofurantoin or trimethoprim would be a suitable first - line agent, as none of the patients in our cohort were resistant to both trimethoprim and nitrofurantoin . In the unwell or septic patient, vancomycin or teicoplanin interestingly, less than one - third (32.5%) of our mrsa urine samples came from hospital inpatient sources, implying that mrsa bacteriuria diagnosis is more frequently a community - based phenomenon . National recommendations dictate that hospital patients colonized with mrsa should be isolated in single rooms and expert opinion would surmise that there is an increased risk of spread from mrsa - colonized catheterized patients due to increased interventions and manipulations required from staff (e.g. Changing catheter drainage devices). However, within the community, these patients are not recommended to be isolated as would happen within the nosocomial environment and this could contribute to further bacterial spread . The limitations of our study include its retrospective nature which meant that causation was not addressed, and not all key information was available to our research group . Also, acting as a potential confounder is the fact that mrsa bacteriuria is not a common pathology meaning we had only small number of urine isolates (167 from 106 patients) despite the large sample size (425,013). To this author's knowledge, there are no prospective mrsa bacteriuria studies underway and a supra - regional or national study of this type could provide further relevant data in this field.
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Acquired angioedema (aae), an acquired deficiency of c1esterase inhibitor, is a medically treatable condition which can cause severe abdominal pain mimicking an acute surgical abdomen . This disorder is strongly associated with chronic lymphocytic leukemia (cll) and other indolent lymphoplasmacytic disorders . We describe a patient with known cll who developed incapacitating, recurrent severe abdominal pains, culminating in partial bowel resection . Wider appreciation of the possibility of aae, particularly in patients with lymphoproliferative disorders, could lead to preventive therapy and spare unnecessary surgery . Acquired angioedema (aae) is due to acquired deficiency of c1 inhibitor (c1inh), resulting in excessive complement and bradykinin activities . Blood vessel permeability is increased; thus, angioedema occurs . Just as with hereditary angioedema (hereditary c1inh deficiency; hae), common clinical manifestations are skin swelling, laryngeal edema, and/or abdominal pain.1,2 aae often occurs in the context of lymphoplasmacytic disorders, such as monoclonal gammopathy of unknown significance (mgus), non - hodgkin s lymphoma, or chronic lymphocytic leukemia (cll).1,3 among 32 patients with aae, castelli found that 13 (40%) had mgus and 9 (28%) had lymphoproliferative disease.3 therefore, all cases of aae should be evaluated for the possibility of underlying lymphoplasmacytic disorder . Conversely, when patients with known lymphoproliferative disease manifest compatible symptoms, aae should be expeditiously considered . This is important because aae can be effectively treated medically, but delayed diagnosis can lead to unnecessary diagnostic procedures, therapeutic interventions, or life - threatening complications, well - illustrated by our case . A 78-year - old woman with atherosclerotic vascular disease was transferred to our hospital with abdominal pain and underwent emergent laparotomy . One year earlier, she had been diagnosed with rai stage i cll which had been observed without treatment . Two months earlier, she presented with severe abdominal pain, nausea, and vomiting . Over the next 8 weeks, she had six emergency room and/or hospital admissions for identical symptoms . Pains would begin at rest in the lower abdomen, spread to the upper abdomen, described as gas - like, non - radiating, constant . Extensive evaluation included colonoscopy, endoscopic retrograde cholangiopancreatography, magnetic resonance angiography, and abdominal aortography . Benign colon polyps and small gallstones were removed, mesenteric stenoses ruled out, yet pains recurred unabated . There was no dysphagia, change in bowel habits, gi bleeding, fever, or sweats . Physical examination on transfer revealed blood pressure 130/88 mmhg, pulse 88 beats per minute, respiratory rate 20 cycles per minute, pulse oximetry of 95% on ambient air, and temperature 97.0f . Hemoglobin was 18.1 g / dl, hematocrit 55%, platelets 146 10/l, and leukocytes elevated to 34,500 cells/l with 47% neutrophils and 48% lymphocytes . Ct scan of abdomen and pelvis with iv contrast showed multiple abnormal loops of small bowel with contrast - enhanced bowel wall edema (fig . . 1a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema . B a section of small bowel shows massive submucosal edema . There is no infiltration of the wall by lymphocytes a ct scan of abdomen and pelvis with intravenous contrast shows several abnormal loops of small bowel with a target appearance indicating bowel wall edema . B a section of small bowel shows massive submucosal edema . There is no infiltration of the wall by lymphocytes at laparotomy, massively swollen small bowel was encountered and resected . C4 was 3 mg / dl (normal 1746), c3 66 mg / dl (85200), and c1inh activity reportedly 83% (68200%). Serum protein electrophoresis revealed two faint bands immunofixing as monoclonal igm kappa and igg kappa . Lymphocytosis and lymphadenopathy improved and c1inh activity increased to 110% . Over 3 years of follow - up, abdominal symptoms never recurred . Approximately 145 cases have been reported of aae,3 and this is one of four cases that we have diagnosed in the last decade with abdominal pains from aae with associated cll . Table 1 shows characteristics of patients with cll and aae we have seen (some briefly mentioned in a prior report).4 this is our only case to undergo surgery, allowing unique and dramatic demonstration of massive bowel edema visible radiographically, on surgical inspection and on histopathology . An initial c1inh activity was reported low normal, but there can be no doubt about the diagnosis based on radiologic / surgical / histologic findings, further laboratory results, and the clinical course . Characteristic are very low c4 level and low c3; diagnostic recommendations currently add c1q and anti - c1inh antibody levels . Autoantibody is demonstrable in up to 70% with aae.1 our patient had monoclonal gammopathy, which frequently corresponds to the c1inh autoantibody . Patients with the hereditary form (hae) usually manifest before age 20 and give a family history of symptoms . In all our patients with cll and aae, chemotherapy and androgens increased c1inh and produced durable remission.table 1characteristics of patients with aaepatientunderlying diagnosismanifestations of acquired angioedemamonths before diagnosislaboratory valuesinterventions prior to diagnosistreatmentangioeedema episodes post - treatment (years of follow - up)pretreatmentpost - treatment78 years old / fcllrecurrent abdominal pain2c1 inh activity: 83% (68200)c1 inh activity: 110% (68200)exploratory laparotomy with small bowel resectionchemotherapydanazolnone (3 years)c4: 3 mg / dl (1746)ct scanc3: 66 mg / dl (85200)colonoscopysmall monoclonal gammopathyercpmraabdominal aortography74 years old / fsll / cllrecurrent abdominal pain24c1 inh activity: 1% (68200%)c1 inh activity: 117% (68200%)ct scanchemotherapynone (6 years)episodic oropharyngeal swellingc1 inh quantitative: 6.7 mg / dl (> 11 mg / dl)c1 inh quantitative: 21 mg / dl (1025)colonoscopydanazolc4: 13 mg / dl (1647)c3: 70 mg / dl (75161)small monoclonal gammopathy61 years old / mcllrecurrent abdominal pain5c1 inh activity: 4% (68200%)not availablect scanchemotherapynone (10 years)small monoclonal gammopathycolonoscopydanazol67 years old / mcllrecurrent abdominal pain3c1 inh activity: 6%(68200)c1 inh quantitative: 38 mg / dl (2139)colonoscopychemotherapynone (3 months)oropharyngeal swellingc1 inh quantitative: 3 mg / dl (2139)laryngoscopydanazolc1q: 3.6 mg / dl (58.6)c4: <2 mg / dl (1746)small monoclonal gammopathy characteristics of patients with aae angioedema should be borne in mind among medical illnesses that can mimic acute surgical abdomen, along with such other disorders as porphyria, familial mediterranean fever and sickle cell disease (fig . 2). One may need to discern whether a true surgical emergency might supervene even when one of these disorders is present . Our patient had typical symptoms of acute bowel edema, including diffuse abdominal pain, occasionally rebound tenderness and vomiting, with spontaneous resolution within 15 days.1,2 some patients with aae have cutaneous or upper respiratory edema in addition, or instead of, bowel symptoms . Because of cardiovascular comorbidities, there was a high suspicion for ischemic bowel in our patient, but radiography and endoscopy did not support this . In the differential diagnosis, angiotensin - converting enzyme inhibitors rarely precipitate angioedema, but our patient had taken benazepril many years and continued it without incident after aae therapy.fig . 2algorithm for diagnosis and treatment for suspected aae algorithm for diagnosis and treatment for suspected aae in 2009, the us fda approved the c1inh concentrate berinert p and the kallikrein inhibitor ecallantide (kalbitor) for treatment of acute attacks, and the c1inh concentrate cinryze for prophylaxis in severely affected patients.5 fresh frozen plasma can be given when these are unavailable . C1inh concentrates are highly safe and effective; the standard of care for decades in other countries, but us approval was delayed by concerns of virus transmissibility.1,59 doses used in the clinical hae trials may need to be higher for aae because of increased enzyme clearance . A bradykinin b2 receptor antagonist icatibant (firazyr) is under investigation . For long - term control, commonly used for prophylaxis are anti - fibrinolytic drugs or the attenuated androgen danazol, which increases c1inh synthesis at low cost.10 in conclusion, earlier suspicion for aae in our known cll patient could have spared her the morbidities of recurrent abdominal pains, hospitalizations, morbid interventions, and bowel resection . Wider appreciation of this disorder takes on added importance as our ability to effectively treat the problem has grown.
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Distal clavicle fractures associated with partial and complete coracoclavicular ligament disruption are potentially unstable, and non - operative treatment of these fractures is associated with a high non - union rate. [13] partial ligament disruptions (conoid or trapezoid) retain some inherent stability, and should be distinguished from complete dual ligament disruptions that result in an uncommon and highly unstable subgroup . Internal fixation of these fractures necessitates the use of acromioclavicular joint spanning implants (clavicle hook plate, synthes, usa) that overcome distraction forces by subacromial leverage; however, subacromial / acromioclavicular joint encroachment with these implants is associated with subacromial pathology, and this may negatively influence the final outcomes . To overcome the shortcomings of the joint - spanning implants, acromioclavicular joint - sparing implants (2.4 mm lcp distal radius plates, synthes, usa) have been suggested . Additionally, a combination of this implant with some form of coracoclavicular fixation using sutures / anchors / endobutton devices (tightrope, arthrex, fl, usa) has been recently described for use in the highly unstable fracture subgroup . However, comparative clinico - radiological outcomes of the two techniques are lacking in literature . The purpose of this study was (1) to analyze the overall and comparative clinico - radiological outcomes (radiographic and ultrasonographic) of surgical treatment of distal clavicle fractures associated with complete coracoclavicular ligament disruption, and (2) to identify and analyze radiographic patterns of fracture and comminution that are associated with this injury . The hospital records of all patients who were surgically treated for lateral clavicle fractures between 2005 and 2008 at three hospitals were retrospectively evaluated to identify the study group . Inclusion criteria for this study was defined as: (i) acute, isolated, non - comminuted and comminuted fractures of the lateral clavicle, (ii) complete disruption of both coracoclavicular ligaments, confirmed on radiographs and at surgery, (iii) absence of concomitant or pre - existing subacromial pathology (rotator cuff tears, acromial undersurface degeneration), (iv) surgical treatment with either a joint - spanning implant alone (group 1), or combination of a joint - sparing implant and coracoclavicular fixation with either endobutton device, suture anchor, or coracoid cerclage (group 2) [figures 1a and b], and (v) follow - up period of at least 6 months for radiographic evidence of union . Exclusion criteria was defined as: (i) partial disruption of the coracoclavicular ligaments at surgery, (ii) subacromial pathology (iii) concomitant injuries to the ipsilateral shoulder girdle, (iv) surgical treatment with any technique other than those described in the inclusion criteria, and (v) follow - up period inadequate for complete union . Surgical treatment of an unstable distal clavicle fracture with a joint - spanning implant (group 1). (hk: hook plate, cl: clavicle, m: medial fragment, l: lateral fragment, arrow: fracture line, ac: acromion, h: humeral head, co: coracoid process) surgical treatment of an unstable distal clavicle fracture with a joint - sparing implant and coracoclavicular ligament reconstruction using endobutton device (group 2). (rp: locking distal radius plate, arrow: acromioclavicular joint, ac: acromion, cl: clavicle, en: endobutton, h: humeral head) the clinical records and operative notes were analyzed to identify patients that satisfied the criteria described above, and all other lateral clavicle fracture patients were excluded . Pre - operative radiographs of the study group were obtained and analyzed with a graphic analysis software; fracture lines were traced to identify involvement of the acromioclavicular joint, lateral fragment integrity, and presence / absence of comminution . The follow - up clinical protocol included a subjective evaluation of pain and functional status by interview, evaluation of active and passive range of motion by clinical examination, and evaluation of muscle strength in elevation and external rotation using a portable dynamometer . Clinical acromioclavicular joint tests (tenderness and paxinos sign) and rotator cuff tests (lag signs, bear - hug test, impingement signs) were used to evaluate these structures. [811] clinical outcome measures included (i) simple shoulder test, (ii) constant and murley shoulder score, and (iii) walch acromioclavicular joint score . The follow - up radiographic protocol consisted of standardized radiographs that included a true glenohumeral anteroposterior view (neutral rotation, elbow by the side), and a weight bearing comparative cephalic - tilt view; these were analyzed for implant migration, acromioclavicular joint pathology (degeneration, instability), subacromial changes (degeneration, osteolysis), acromiohumeral interval measurement (normal interval = 7 mm or more), peri - coracoid changes (abnormal bone formation), and glenohumeral changes . In addition, in those cases where implant had been previously removed, past radiographs were obtained from the records and individually analyzed as described above . The follow - up ultrasonographic protocol included evaluation of the acromioclavicular joint, subacromial space, glenohumeral joint, bicipital groove and biceps long tendon, and suprascapular and spinoglenoid notches; ultrasound assessment was performed by a senior radiologist experienced in shoulder ultrasound . Statistical analysis was performed using a statistical software (spss inc, chicago, usa) to determine mean values and range of measured parameters of the overall and implant specific groups . Significant differences between clinical and radiological parameters were determined using the non - parametric mann - whitney / wilcoxon rank - sum test for numerical data, and the fisher's exact test for categorical data . The average age of the patients in the study group was 34.5 years (range, 20 to 57 years). The mechanism of injury was a direct impact to the ipsilateral shoulder sustained during a fall; in 8 patients, the fracture was sustained during a cycling - related sporting activity (competitive or recreational). Ten patients were operated using a joint - spanning implant (group 1) and 5 were operated using a joint - sparing implant technique (group 2). The mean follow - up period was 26.1 months (range, 12 to 40 months). The overall and group - specific clinico - radiological outcomes and the types and distribution of fracture patterns are summarized in table 1 . Overall and group.specific clinico.radiological outcomes and fracture patterns clinical outcomes: the mean cs was marginally higher in group 2, while the mean sst score was marginally higher in group 1; none of these differences were statistically significant . Acromioclavicular joint signs were positive in half of the group 1 patients, and in none in group 2; however, the walch scores for the acromioclavicular joint were not significantly different for the two groups . Return to pre - operative level of recreational and competitive sports was seen in approximately two - thirds of the patients of group 1, and all patients of group 2; this difference was not found to be statistically significant . Reoperation rates related to implant removal were significantly higher (p <0.05) in group 1 patients (90%) as compared to group 2 (0%). Radiographic outcomes [figure 2a - d]: radiographic union was observed in 93.3% of fractures (9 in group 1, 5 in group 2) with only one non - union with subsequent fragment resorption (group 1). Radiographic acromioclavicular joint degeneration was present in 3 patients (group 1); all 3 patients had a clinically symptomatic ac joint . Radiographic acromioclavicular joint superior subluxation was present in 4 patients (group 1 = 1 patient, 10%; group 2 = 3 patients, 60%); none of these were clinically symptomatic . Subacromial osteolysis and hook migration were seen in 5 patients (group 1); implant removal was necessary in 4 of these 5 patients, and at final follow - up, resolution of the radiographic subacromial osteolytic lesions was observed . Signs of progressive implant loosening (screws and/or plate disengagement) were seen in 3 patients (group 1). Abnormal bone formation was present in 7 patients (group 1 = 6, group 2 = 1). (b) acromioclavicular joint subluxation (arrows), (c) hook migration and osteolysis of acromial undersurface (arrow), (d) peri - coracoid ossification (arrows). (ac: acromion, cl: clavicle, co: coracoid, p: plate, an: suture anchor, g: glenoid, h: humeral head, hk: hook plate, ahi: acromiohumeral interval) ultrasonographic outcomes [figure 3a and b]: supraspinatus lesions were seen in 3 patients; these included partial articular - side tears (group 1 = 1 patient, 10%; group 2 = 1 patient, 20%), and supraspinatus bursal - side degenerative changes (group 1 = 1 patient, 10%). Acromioclavicular joint screw penetration was observed in 1 patient (group 2); this penetration was not apparent on plain radiographs and the patient was asymptomatic at the final follow - up . No abnormalities were detected in the subacromial bursa on static ultrasound evaluation; dynamic testing revealed abnormal subacromial bursal " bunching " with arm abduction in 2 patients (group 1 = 1, group 2 = 1). (a) partial articular - surface supraspinatus tendon avulsion (black arrows) (b) screw penetration into the acromioclavicular joint (arrows). (gt: greater tuberosity, h: humeral head, ss: supraspinatus, ac: acromion, cl: clavicle, asterix: acromioclavicular joint) radiographic evaluation of fracture comminution revealed 5 non - comminuted fractures (group 1 = 2, group 2 = 3), and 10 comminuted fractures (group 1 = 8, group 2 = 2). Analysis of the fracture lines and fragment cortices revealed repetitive patterns in the fracture geometry, and four types could be identified: (i) type 1 pattern (n = 5, 33%) involved a clean vertical or a short oblique type bicortical fracture line without any comminution [figure 4a], (ii) type 2 pattern (n = 4, 27%) involved a zone of segmental comminution, between intact bicortical lateral and medial fragments [figure 4b], (iii) type 3 pattern (n = 5, 33%) involved propogation of at least one fracture line into the juxta - articular cortex of the ac joint [figure 4c] and (iv) type 4 pattern (n = 1, 7%) consisted of fracture line propogation into the acromioclavicular joint [figure 4d]. Radiographic fracture patterns: (a) type 1, (b) type 2, (c) type 3, and (d) type 4 . (ac: acromion, cl: clavicle, co: coracoid, g: glenoid, h: humeral head, arrows: fracture lines) the present study is the first study that describes overall and comparative outcomes (clinical, radiographic, and ultrasonographic) of surgical treatment of an uncommon lateral clavicular bony - ligamentous injury with joint - spanning and joint - sparing implants . In addition, four radiographic patterns of bone injury were identified to develop guidelines for choice of implants in these injuries . Clinical outcomes and union rates after operative treatment of this injury are satisfactory, and this has been shown in other studies in literature . In the present study, both types of implants resulted in satisfactory and comparable clinical outcome scores, irrespective of the radiographic outcomes . However, a significant difference in the reoperation rate for implant removal (p <0.05) in patients treated with a joint - spanning implant probably implies that a joint - sparing implant could be preferentially used whenever fracture geometry permits . Radiological outcomes, especially those associated with joint sparing techniques, have not been well documented in other studies . Subluxation of the acromioclavicular joint, (present in 60% of group 2 patients) may be related to either concomitant injury to the acromioclavicular ligaments, or to a partial failure of the coracoclavicular ligament reconstruction; transfer of distraction forces to ac ligaments after fracture union may then induce gradual subluxation of this joint . This complication was rare in the other group, probably due to the joint spanning implant design; however, other radiological abnormalities like hook migration, subacromial osteolysis, implant loosening, and new bone formation were frequent . The acromioclavicular joint spanning hook plate is a commonly used implant, and most studies have utilized this as the sole implant of choice . Although clinical outcomes in these studies have been satisfactory, the effect of the subacromial hook on bursal tissues has been debated and use of the distal clavicle plate suggested . By using ultrasonographic evaluation, this study demonstrated the safety of the joint - spanning hook plate in relation to rotator cuff and subacromial bursa . Persistent pain in the post - operative period probably resulted from acromial undersurface irritation; early plate removal after fracture union is recommended, and long - term clinical outcomes seem to be satisfactory after implant removal . An important finding of this study is the grouping of the radiographic fracture patterns into four surgically relevant types . Types 1 and 2 are ideal indications for use of a joint - sparing implant; adequate bone stock in the distal fragment in these types will theoretically permit secure fixation across the fracture site . In types 3 and 4, inadequate distal fragment size may not permit secure bicortical fixation, thereby necessitating use of joint - spanning implants . . The retrospective nature and small sample size does not permit identification of all possible fracture configurations, and statistical analysis may differ with larger numbers . However, the clinical results are similar to other literature studies that have analyzed joint - spanning implants . Also, the current radiographic patterns described provide guidelines for implant choice, and are not intended to be used as a prognostic classification . We attribute the small size of the study group to the stringent inclusion and exclusion criteria that eliminated several other patients with lateral clavicle fractures, and this was necessary to obtain meaningful conclusions . Lateral clavicle fractures with complete disruption of the coracoclavicular ligaments should be regarded as a distinct entity that is perhaps biomechanically and prognostically different from similar fractures with partial ligament disruption . A combination of a locking distal radius plate with coracoclavicular ligament reconstruction resulted in stable fixation and significantly lower reoperation rates, and should be used preferentially in lesser comminuted fractures (types 1 and 2).
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A 68-year - old woman visited the clinic with the chief complaints of pain, swelling, and a warm sensation in her left thigh for over 2 days prior to her visit . The patient also complained about a warm sensation during the nighttime but body temperature was normal when measured in the clinic . She had undergone a left bipolar hemiarthroplasty following a hip fracture 24 days prior to the current visit and was wearing compression stockings to prevent the occurrence of a deep vein thrombosis . She had been taking aspirin 100 mg daily, fexonadine 180 mg daily, and hydrea 500 mg twice a day for a diagnosis of polycythemia vera for 1 year and was phlebotomized in a hemato - oncology setting on an irregular basis . In advance of the operation, her erythrocyte sedimentation rate (esr), and c - reactive protein (crp) levels were 30 mm / hr and 4.99 mg / dl, respectively . Three days after her hip surgery, her esr and crp levels were 23 mm / hr and 6.35 mg / dl, respectively . Medical care was administered in close cooperation with the hemato - oncology department and the level of hemoglobin was maintained at <12 g / dl to prevent a thrombosis prior to her operation . The patient did not have a fever and her skin color seemed normal despite swelling and a slight warm sensation in the left hip and proximal femur . Results of a blood test showed that her complete blood count, electrolyte levels, and liver function were all within normal ranges (white blood cell count [wbc]: 4,700/l, hemoglobin: 10.6 g / dl, hematocrit: 32.3%, platelet: 213,000/l, neutrophil count 67.2%, lymphocyte 24.1%, monocyte 6.8%) whereas the esr and crp level were 38 mm / hr and 8.25 mg / dl, respectively . When compared to tests performed previously, no further observations of additional fractures, signs of infection, or loosening of inserts after the surgery acute or sub - acute inflammation was diagnosed given the perfusion image of soft tissue in the left hip area as well as the blood pool image (fig . The patient was injected intravenously with antibiotics for 1 week while staying in the hospital, however, symptoms were not alleviated . In order to prevent a deep vein thrombosis, the patient started to move with the aid of a wheelchair and exercised her knee joints 3 days after the operation . G / dl, a color doppler ultrasonography was performed due to her history of polycythemia vera . Results revealed a deep vein thrombosis in the common femoral vein as well as in the superficial femoral vein (fig . Symptoms were alleviated following the administration of low - molecular - weight heparin (enoxaparin, 40 mg) and warfarin (5 mg) for 5 days . Additional warfarin was prescribed (2.5 mg daily) for maintenance at discharge . During her first follow - up visit 2 weeks later, no symptoms were noted and all blood test parameters were within normal ranges . It has been reported that patients with polycythemia vera present with various complications related to thrombosis due to excessive blood viscosity1). Several putative reasons have been postulated to explain such complications including hypervolemia, an increase in the volume of red blood cells, telangiectasia due to decreased blood flow velocity as well as vascular elasticity, thrombocytosis, and complex hemostatic disorders attributable to inefficient blood clotting processes2). Thrombosis is the leading cause of death in those with polycythemia vera and has been associated with high morbidity as well . Approximately 12 - 49% of patients with polycythemia vera experience thrombosis and 20 - 40% will die as a result2). Thromboembolism is one of the major complications of hip surgery and contributes to its poor prognosis . Warwick et al.3) reported that the incidence rate for a deep vein thrombosis was about 1.89% if proper medication was not provided after total hip arthroplasty . Therefore, in the case of the patient described in the current case report, it was reasonable to expect that she would be susceptible to the high incidence rate of thrombosis as she underwent the hip surgery based on her history of polycythemia vera . For prophylactic purposes, aspirin was administrated (100 mg daily) and early exercise and active movements were recommended for the patient in order to lower the risk of venous thromboembolism . In the current case report, a patient with polycythemia vera exhibited clinical symptoms and signs of a postoperative infection following bipolar hemiarthroplasty due to femoral neck fractures . There was, however, a deep vein thrombosis which rendered the intravenous administration of antibiotics ineffective in the alleviation of symptoms . Early symptoms of a deep vein thrombosis, including swelling, pain, oppressive pain, and a warm sensation, can mimic those of a postoperative infection . Furthermore, features of systematic inflammatory responses such as increased levels of interleukin-6, interleukin-8, and crp may be observed in patients with a deep vein thrombosis . Collectively, these findings suggest that appropriate clinical examinations are warranted at an early stage in order to avoid making an inaccurate diagnosis4,5). On the first visit after the operation, the patient was suspected to have had a postoperative infection due to the simultaneous increase in esr and crp levels . However, the specificity of esr and cpr levels pertaining to the diagnosis of infections is questionable, as most patients display elevated levels of esr and crp following a hip surgery6). These inflammatory responses stimulate the production of cytokines due to the fractures and operation per se, thus biochemical parameters for inflammation are known to be elevated in a non - specific manner . In cases without postoperative infections, elevated crp levels recover to the normal range within 3 weeks of the operation6,7). In the current case report, the elevated crp level that was maintained for up to 24 days after the operation led us to suspect a postoperative infection . However, the abnormally increased level of acute phase reactants (e.g., crp) may be observed in patients with chronic myeloproliferative diseases (e.g., polycythemia vera) and therefore further specific clinical examinations are required for an infectious diagnosis8). Regarding the diagnosis of a postoperative infection, elevated level of serum procalcitonin may be more valuable than wbcs and crp levels7). Regrettably, the level of serum procalcitonin was not measured in the current case but could have been informative in distinguishing between postoperative infection and a deep vein thrombosis . Despite the presentation of similar clinical symptoms between a postoperative infection and a deep vein thrombosis, their etiological mechanisms are completely distinct . Therefore, different treatments are warranted, as patients receiving inappropriate medical treatments may experience exacerbated symptoms and conditions . The potential for a deep vein thrombosis requires continuous thought and attention regarding relevant examinations for patients at high risk for thrombosis as well as for embolism (e.g., polycythemia vera).
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Brachial plexus surgery using the da vinci surgical robot is a new procedure . To evaluate the advantages and the restrictions of the technique, a cadaveric study of supraclavicular and axillary approaches was conducted . We found that the axillary approach was useful and advantageous for lower roots, particularly for thoracic outlet syndrome (tos). This report will focus on the evaluation of axillary robotic approach as the advantages and disadvantages of supraclavicular robotic intervention have been widely discussed in the literature . A human cadaver was subjected to this experiment in paris university ecole europenne de chirurgie anatomy laboratory and da vinci robot system was used . The left arm was tucked along the side and the right arm was placed in a semiflexed position extending toward the anesthesia location near the head, supported by foam and blankets (figure 1). A 6 cm long incision was made at the right axillar line, lateral to the edge of the pectoralis major muscle (figure 2). A self - retaining chung retractor was placed into the incision to elevate the pectoralis major muscle flap . The robot was docked as a camera; right and left robotic arm were adapted in the incision area (figure 3). A 10 mm 0 downlooking scope, maryland forceps, and a curved scissors the working space was maintained with the self - retaining retractor, without co2 insufflation (figure 4). The subclavian artery was seen in front of the truncus and was positioned to the posterior of the working space . Subclavian artery was dissected from the plexus and truncus of the lower plexus was exposed with blunt dissection . The plexus was exposed thoroughly from t1 to c7 levels . In this surgical setting, the operating surgeon, who has a wide experience in open brachial surgery of the brachial plexus, reported that lower brachial plexus exposure was easier from the axillary working area and a more wide range of motion was achieved to manipulate the robotic tools compared to the supraclavicular exposure for lower part of the brachial plexus . The development of robotic - assisted minimally invasive techniques began in urology, general surgery, and gynecology because of the generally large working spaces available in the abdomen for these types of surgeries [14]. Since then, other surgeons have sought to use robotic devices in other areas, such as the brachial plexus [5, 6]. Brachial plexus dysfunction can be the result of shoulder trauma [7, 8]. It can also occur with tos, which encompasses three separate disorders involving compression of the subclavian artery, subclavian vein, or brachial plexus in the triangular space bordered by the first rib, clavicle, and scalene muscles [9, 10]. Compression of the vessel - nerve package at the thoracic inlet has been treated with soft - tissue (scalene muscle) release and/or bone (first rib) resection . Surgical approaches to first rib resection may be transthoracic, transaxillary, supraclavicular, infraclavicular, or thoracoscopic [9, 10]. However, these approaches are typically associated with incomplete resection of the most medial portion of the first rib and neurovascular complications . Theoretically, a minimally invasive transthoracic approach can obviate these problems, enabling complete resection of the offending portion of the first rib without neurovascular complication ., respectively, reported successful results of robotic en bloc first rib resection for tos treatment via transthoracic and transaxillary approaches [9, 11]. 's techniques were only bony interventions and as being intrathoracic these need to be lung collapsed and lung complication can be waiting risk . Although liverneaux et al . Reported techniques and results of upper brachial plexus injury intervention via robotic surgery with a supraclavicular approach, they described the disadvantages as a narrow working space and difficulty to expose the c7 vertebra [1, 5, 12]. To our knowledge, this report is the first to objectively describe robotic axillar brachial plexus exposure . Thus, we discuss the theoretical and clinical advantages and disadvantages of the axillary approach in the present report . The development of robot - assisted surgery has revealed new perspectives in peripheral nerve microsurgery . Minimally invasive robot - assisted surgery could lead to modification of the classic algorithm for the treatment of traumatic brachial plexus lesions [6, 8]. To date, exploration of these lesions has not been attempted less than 3 months after the traumatic event because clinical examination cannot provide an accurate diagnosis or reliable prognosis in these first weeks . Early intervention may enable initial assessment of the lesion and repair of potentially graftable nerve roots . Several robotics properties are particularly adapted to microsurgery, such as high - resolution three - dimensional (3d) visualization with up to 40 magnification, up to 10-fold magnification of surgical movements, elimination of physiological tremors, and the provision of ergonomic work conditions for otherwise uncomfortable surgery . Robotic surgical systems allow high - definition magnified 3d visualization of the operative field, provide significant instrument maneuverability, even within a confined space, and may overcome the shortcomings of conventional approaches [2, 5]. Axillary (infraclavicular) brachial plexus intervention via robotic surgery has not been described previously . Axillary intervention was previously performed as an open procedure to expose the plexus or resect the first rib for the treatment of tos . Martinez et al . Described first rib resection via robotic surgery but not to address plexus injury without transthoracic exposure, a novel minimally invasive approach to the first rib from inside of the chest . In addition, gharagozloo et al . Reported first rib resection via transthoracic robotic surgery for paget - schroetter disease . 's techniques were considered more useful for lower brachial plexus viewing and assessing according to gharagozloo et al . The experience of the whole surgical team with robotic technology is important for the procedure . During learning curve period, two staff surgeons are required to participate in all procedures to ensure the safety of the program [5, 9]. Martinez et al . Reported importance of the learning curve, not only for the surgeon but also for the entire surgical team and 180 minutes for the initial 10 cases . Reported exclusion criteria include a history of previous incision in the same area and obesity, which present difficulties in robotic surgery initiation . Other drawbacks of this new surgical approach are the increased cost of surgical equipment and longer operating time, especially during the learning curve period . However, we believe that the avoidance of a classic incision leads to significant patient satisfaction for cosmetic reasons and we believe that demand for this procedure from a select group of patients justifies the exploration of alternative ways to avoid classic brachial plexus exposure . This report presents our initial experience with robot - assisted axillary exposure of the brachial plexus region . In our opinion robotic surgery will be used routinely in the future for brachial plexus surgery and particularly for tos that is caused by bone and/or soft tissue . However, newer dedicated surgical instruments need to be developed and further studies should be conducted to evaluate in vivo application and results of this novel approach.
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Older people have particular spiritual needs that are distinct from those of others, particularly in times of ill health and death and dying . It has been reported that spirituality has a positive effect on health outcomes and welfare of the elderly . Nowadays, aging phenomenon and relevant problems with its complexities are important issues that have attracted the attention of social and medical scientists to investigate the spiritual needs of the elderly . Spiritual health is a dimension of health that is portrayed as the validation of life in relation to god, fellow human beings, and the environment . Spirituality is the representative of the basic values that guide a person in searching to find answers to the crucial questions of life, such as the purpose and meaning of life, reality, love, good and bad, disease, and death . Over the past three decades, spirituality, religion, and prayer have turned into a subject of large interest for researchers in medicine, nursing, gerontology, physical health and mental health disciplines, often in conjunction with complementary and alternative medicine . This tendency has been stimulated by research demonstrating physical and psychological benefits of spiritual and religious involvements . In fact, approximately 96% of adults in the united states expressed their belief in god, and 72% of identified religion and spirituality as having the most important influence in their lives . Saying prayers is a regular daily task for muslims which begins at the age of puberty and continues throughout the life . It is a time getting rid of the material world and approaching the world beyond, as well as, to the inner self . In iran, elderly people are more religious than younger ones, and this is a norm of this culture . A recent national survey of the iranians values and attitudes found that over 80% of the iranians practice prayer regularly as a part of their religion . One might argue that this is a true reflection of individual culture where prayer and religious beliefs are a part of people's everyday life . However, prayer can go beyond just performing a religious duty, and based on one's wishes, take different forms of formal and informal practices . It has been shown that disease prevalence, incidence and mortality rates differ from one population to another . This is partly ascribed to their disparities in lifestyle, dietary habits and other characteristics that are somehow affected by culture, especially religious traditions and experiences . During the past two decades some studies have been carried out and the associations between religious beliefs or experiences and various types of physical illnesses, mental health, and mortality have been examined . Spirituality is viewed as personally important matter to the older people and it has been proved that it is highly related to older people's health outcomes . However, there has not been adequate experiential research on the relationship between religious or spiritual factors and healthy lifestyle behaviors, and their impact on the adult's well - being . Scientists believe that probing the effects of religious and spiritual factors as well as healthy lifestyle behaviors may be decisive . Additionally, social science and medical research have concurrently reported that older adults are more inclined to take spiritual chase than younger people are . Kotrotsiou - barboutal et al. (2006) indicated that older adults are remaining members of religious associations without necessarily participating regularly in services, as there are numerous issues that can diminish the active participation in religious events, such as transportation intricacies, bad sound, or the illegibility of small font text in books of prayers available at mosques or religious centers . In spite of those complexities, many elderly manage to find out a way to come up to god through prayer . It is stated that prayer increases the feelings of personal value of an elderly by reducing the feelings of loneliness and desertation, while television and radio provide support for spiritual life . In this regard, researchers have shown that some groups of elderly people with inadequate and difficult livelihood situations communicate their problems by putting their trust in god . Moreover, more than half of the elder patients practiced spiritual needs throughout their hospitalization period, with the most prominent need of being in the search of the meaning of life . Extensive epidemiological studies have shown that higher levels of religiousness are related to lower mortality rates . Studies identified the multidimensional nature of religiousness and spirituality and investigated various religious dimensions, for instance private religious behaviors, devotion, spiritual transcendence, and religious adaptation . For example, it was indicated that some aspects of religiousness predicted lower rates of disability and illness, alcoholism, cardiovascular disease, hypertension, and myocardial infarction . Furthermore, clinical research has suggested that spirituality can play a vital role in the recovery of patients suffering from physical and psychological diseases . It seems that religious obligation has a crucial role in averting physical and mental diseases, recovery promotion, and disease adaptation . Numerous studies have revealed the positive involvement of spirituality and religious indexes on different aspects of health such as cardiac surgery, mortality, immunity system function and self esteem . Above all, religious involvement can be useful for psychological well - being and physical health . Additionally, supporting the effect of spirituality may become more and more prominent in later life as a result of declining health, social and financial sources . Researchers have proposed numerous psychological, social, and physiological interveners that may explain the spirituality - health association . Quantitative measurements and the evaluation of spiritual beliefs are difficult because quantitative studies neither reveal the content, components of the beliefs, and its factors, nor the contexts that might be included . Therefore, implementing qualitative research that explores the personal and experiential aspects of beliefs seems crucial, and may shed light on a range of important issues which can be investigated in detail on larger samples by further quantitative studies in future . Spiritual or religious beliefs may affect the decisions of individuals that make about their health and illness . Thus, recognizing and understanding the spiritual and religious behaviors within the context of cultural influence by a qualitative research is critical for nurse and other health - care providers . The main strengths of the present study are that it focuses on the spiritual life of healthy elderly people . Therefore, the present study aimed to explore the concept of spirituality from the perspectives of iranian healthy elderly people . The central question of the paper was what characterizes the spirituality in the iranian healthy older people? In this research, a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 . It is worth noting that spirituality is underpinned by personal and cultural context in any community; therefore, qualitative research is the best method to study cultural context - bound subjects . Initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data . The study consisted of 17 healthy elders within the age range of 65 - 86 . Criteria for selection were having age above 65, living with family, not having any cognitive problems, not having any physical limitation in adl, and willingness to participate in the study . All participants were shia muslims . In order to achieve the wide range of experiences and perceptions of elderly participants, some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . The first researcher communicated with each of the participants to describe the purpose of research and research questions . Based on the participants preference, the interviews were performed in a private room at the house of elderly, park, worksite, or mosque, using an individual semi - structured interview format and this was primarily the main method for data collection . Interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions, perceptions, and experiences . The participants were asked to describe one day of their life and then to explain their own experiences and perceptions about spirituality in elderly adults . What is your experience concerning the spirituality in later age? And what is the meaning of spirituality in your experience? Each interview was transcribed verbatim and analyzed before the next interview was done, so that each interview supplied bearing for the next . Coding was carried out line by line, and comparative analysis of the quotations was carried out . All interviews were conducted in a single session, at the request and preference of participants, except for two cases that took two sessions . Each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes . Data were collected and analyzed over a six - month--period in 2009 . In the first phase, subcategories and their domains in the data were identified and classified into categories . The coding process was iterative, and categories evolved (inserted, deleted and combined) as re - readings were completed and analyses progressed . In the second phase, the subcategories and domains were regrouped into major categories . Credibility was recognized through prolonged engagement with the participants, field note writing, the participants revisions using member checking procedure, and peer checking . The findings and explanations of this study were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . Also, maximum variation of sampling established the conformability and credibility of the data . This study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability . Permission to conduct this study was obtained from the ethics committee of tarbiat modares university . Other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses . All participants were aware of the purpose of the study, and their participation in the study was optional . Informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki . In this research, a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 . It is worth noting that spirituality is underpinned by personal and cultural context in any community; therefore, qualitative research is the best method to study cultural context - bound subjects . Initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data . The study consisted of 17 healthy elders within the age range of 65 - 86 . Criteria for selection were having age above 65, living with family, not having any cognitive problems, not having any physical limitation in adl, and willingness to participate in the study . In order to achieve the wide range of experiences and perceptions of elderly participants, some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . The first researcher communicated with each of the participants to describe the purpose of research and research questions . The interview was scheduled according to the participant's agreement . Based on the participants preference, the interviews were performed in a private room at the house of elderly, park, worksite, or mosque, using an individual semi - structured interview format and this was primarily the main method for data collection . Interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions, perceptions, and experiences . The participants were asked to describe one day of their life and then to explain their own experiences and perceptions about spirituality in elderly adults . What is your experience concerning the spirituality in later age? And what is the meaning of spirituality in your experience? Each interview was transcribed verbatim and analyzed before the next interview was done, so that each interview supplied bearing for the next . Coding was carried out line by line, and comparative analysis of the quotations was carried out . All interviews were conducted in a single session, at the request and preference of participants, except for two cases that took two sessions . Each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes . Data were collected and analyzed over a six - month--period in 2009 . In the first phase, subcategories and their domains in the data were identified and classified into categories . The coding process was iterative, and categories evolved (inserted, deleted and combined) as re - readings were completed and analyses progressed . In the second phase, the subcategories and domains were regrouped into major categories . Credibility was recognized through prolonged engagement with the participants, field note writing, the participants revisions using member checking procedure, and peer checking . The findings and explanations of this study were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . Also, maximum variation of sampling established the conformability and credibility of the data . This study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability . Permission to conduct this study was obtained from the ethics committee of tarbiat modares university . Other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses . All participants were aware of the purpose of the study, and their participation in the study was optional . Informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki . The participants of the study were 11 males and 10 females, within the age range of 65 - 86 . Also, 15 out of 21 were married, four were widowed, and one was single . Five participants were illiterate, four were primary degree holders, eight had a diploma, two had bachelor degrees, and two had msc degree in human sciences . In terms of their occupation status, two were employees, 13 were pensioners, two was manual worker, and three were housewives . Three main categories emerged from the data and 3 - 4 distinctive subcategories within each category were identified . These categories and their subcategories represent the main factors influencing the spirituality in iranian elderly people . Main categories and subcategories one of the main categories that emerged from data analysis was spiritual health . Four subcategories; saying prayer as a calming factor, beneficence as a way to god, loss of psychological and spiritual support, and faith, a way to happiness, emerged from the participants experiences . Also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them . I want to say that waking up early in the morning, having the spirit for things like prayer, citing quran verses, thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day . (male) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers . They also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives . They said that man would receive god's blessings as a consequence of his / her deeds in the world . Additionally, they believed that the ultimate consequence of good deeds was good in return . If she asks me to fix her air conditioner you can be sure that i ll do it . This is because i think that by doing this i make her happy and she prays for me ., the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support . When i think about my life, i feel that i don't have any support from my children . Also in the street and road we don't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me . (female) the elderly participants lived experiences disclosed the subcategory of faith, a way to happiness in life . One of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs . Also, they stressed that faith will lead them to a sense of physical and mental welfare and happiness . The one who has strong faith and beliefs is always happy and confident . In my opinion worshiping god, praying, fasting, reciting quran, purity and attending mosques are important elements in life, and bring happiness to your life and set your soul free . (male) spiritual beliefs was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including seeking help from god in difficulties doing good deeds is the god's will . Regarding the subcategory of seeking help from god in difficulties, the participants believed that wishing for and seeking help from god can help the elderly people to deal better with life difficulties, disperses and stresses . This desire results in god's attention to elderly and also creates sense of trust in older adults . Some statements said by participants are: i really believe in having trust in god . When i m trapped in a complicated situation, i believe that this is god who helps me and i only trust in god . (male) one participant believed that spirituality was a vital component of older people lives . Also, he believed that god was the ultimate source to affect my life or death and he controls everything . We re all waiting to see when the angel of death arrives and takes us away . (male) concerning the subcategory of doing good deeds is the god's will, the participants believed that trust in god guided them to many positive activities in their lives . They believed that calling the name of god while getting out of house, remembering him throughout the daytime and praying will lead them to prosperity . One participant said that trust in god, submitting to god's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems . When i want to get out of my house for work, e first thing that comes to mind is that my dear god: bestow upon me everything which is good for me and while i m out working, i do my best not to misbehave or do anything bad to anybody . (male) another most important category that emerged was religious practice with three distinctive subcategories including: saying prayers, reciting quran, and going to mosque, religious ceremonies and pilgrimage . The majority of the participants indicated that they had good knowledge of quran, said prayers regularly, and went to mosque for religious and social activities . For example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness . We go to quran classes on friday mornings also. (male) i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced . I go to the religious rites with the my friends and peers, and i want to get out of loneliness . (female) thus, it can be concluded that all of the participants rose early to start their religious acts of devotion, prayers, and worship in their home or mosque . One of the main categories that emerged from data analysis was spiritual health . Four subcategories; saying prayer as a calming factor, beneficence as a way to god, loss of psychological and spiritual support, and faith, a way to happiness, emerged from the participants experiences . Also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them . I want to say that waking up early in the morning, having the spirit for things like prayer, citing quran verses, thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day . (male) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers . They also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives . They said that man would receive god's blessings as a consequence of his / her deeds in the world . Additionally, they believed that the ultimate consequence of good deeds was good in return . If she asks me to fix her air conditioner you can be sure that i ll do it . This is because i think that by doing this i make her happy and she prays for me . (female) moreover regarding loss of psychological and spiritual support, the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support . When i think about my life, i feel that i don't have any support from my children . Also in the street and road we don't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me . (female) the elderly participants lived experiences disclosed the subcategory of faith, a way to happiness in life . One of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs . Also, they stressed that faith will lead them to a sense of physical and mental welfare and happiness . The one who has strong faith and beliefs is always happy and confident . In my opinion worshiping god, praying, fasting, reciting quran, purity and attending mosques are important elements in life, and bring happiness to your life and set your soul free . Spiritual beliefs was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including seeking help from god in difficulties, only god's power over life and death, and doing good deeds is the god's will . Regarding the subcategory of seeking help from god in difficulties, the participants believed that wishing for and seeking help from god can help the elderly people to deal better with life difficulties, disperses and stresses . This desire results in god's attention to elderly and also creates sense of trust in older adults . Some statements said by participants are: i really believe in having trust in god . When i m trapped in a complicated situation, i believe that this is god who helps me and i only trust in god . (male) one participant believed that spirituality was a vital component of older people lives . Also, he believed that god was the ultimate source to affect my life or death and he controls everything . We re all waiting to see when the angel of death arrives and takes us away . (male) concerning the subcategory of doing good deeds is the god's will, the participants believed that trust in god guided them to many positive activities in their lives . They believed that calling the name of god while getting out of house, remembering him throughout the daytime and praying will lead them to prosperity . One participant said that trust in god, submitting to god's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems . When i want to get out of my house for work, e first thing that comes to mind is that my dear god: bestow upon me everything which is good for me and while i m out working, i do my best not to misbehave or do anything bad to anybody . (male) another most important category that emerged was religious practice with three distinctive subcategories including: saying prayers, reciting quran, and going to mosque, religious ceremonies and pilgrimage . The majority of the participants indicated that they had good knowledge of quran, said prayers regularly, and went to mosque for religious and social activities . For example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness . We go to quran classes on friday mornings also. (male) i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced . I go to the religious rites with the my friends and peers, and i want to get out of loneliness . (female) thus, it can be concluded that all of the participants rose early to start their religious acts of devotion, prayers, and worship in their home or mosque . This qualitative study describes the concept of spirituality and its impression on iranian elderly people daily living behaviors . The findings are significant as they reveal that the living experiences of saying prayer as a calming factor, beneficence as a way to god, loss of psychological and spiritual support, and faith, a way to happiness, are the most important domain and subcategory of spiritual health of elderly people, which can positively affect their health . On the other hand spiritual health is an important dimension of human health and it can determine the individual integrity . Also spiritual health is a power that coordinates physical, mental and social dimensions and is necessary in coping with diseases . When spiritual health will is at serious risk, individuals may be stricken by mental disorders such as loneliness, depression, and loss of meaning . There are a number of studies that support the beneficial effects of spirituality on health . Behaviors such as reliance on god, pilgrimage and praying create hope and positive attitudes that lead to internal serenity, peacefulness and meaningful life . Most faithful people describe their relationship with god as a relationship with an intimate friend and believe that they can control the effect of irrepressible situations by having recourse to god . All in all, religious adaptation relies on beliefs and religious activities and help people to control their stress and physical disorders . Having meaning and goal in life, sense of belonging to a sublime source, hope for gods support in time of trouble and social and spiritual support are among the sources that help religious people to suffer less from hardships of life . In the iranian society context, where the majority of the population are muslim, and a religious culture is dominant in the society, it is expected that tendency to spirituality could be affect their health . Praying is the expression of soul and a deep human instinct that arises from human soul and is uttered with profound words . It is neither dependent upon any particular spirituality nor entailed in any time and place . The content of praying includes confession of sins and weakness, pleading for forgiveness, bliss and closeness to god . Praying reduces mental and psychological pains, releases personal emotions, leading individual to attain eternal source of power . Weeping that often comes with praying greatly helps a person to release his or her distress and emotions . Also prayer helped participants to cope with disease and gave them calm, hope and inner strength . In this regard, quran has put it magnificently: those who have faith in god and inundate their hearts with name of god, surely god remembrance ascertains their hearts ((quran, chapter 13: sureh 13, al - rad) verse 28). Many americans, regardless of their health status, rely on their religious and spiritual beliefs to cope with stressful life events . The present study has demonstrated that one of the most important factors that help elderly in confronting with difficulties was seeking help from god . Several studies noted that spirituals beliefs had positive influence on welfare and personal and social life of elderly people . Effective spiritual coping strategies help individuals find meaning and purpose in their life, problems and difficulties . For instance, one study showed that healthy older adults believed that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being . Thus, spirituality plays an important role in the lives of healthy individuals, too . Believing in support from spiritual / religious resource and connectedness with a higher power is beneficial and it can affect issues of control, quality of life, spiritual well - being, coping, depression, decision - making, and possibly health outcomes . For instance, it has been reported that healthy older adults believe that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being . Thus, spirituality plays an important role in the lives of healthy individuals . In the result of our study a sense of god's power over life and death and doing good deeds is the god's will were derived from participants experience around spiritual beliefs . All the participants talked about the importance of god and belief to god's power in their life . Moreover, the findings reveal that god's power was very important in personal and private experiences . In this regard, roff et al. (2009) in a qualitative study through a thematic content analysis identified god's power in the lived experiences of women who were suffering from breast cancer . Also, in another qualitative study by taleghani et al . (2006) belief to god's will was a important key in acceptance of disease . 2002) suggested that personal variables such as the concept of god and perception of others beliefs played important mediating roles in religious coping with stress situations . Religious practices were a subcategory that emerged from the lived experiences of elderly participants in this research . Furthermore, the results of study by keefe et al . (2001) showed that individuals who reported frequent daily spiritual experiences had higher levels of positive mood, lower levels of daily negative mood, and higher levels of all of the social support domains . Another subcategory that emerged from participants experience was going to mosque and religious ceremonies . In this regard, palinkas (1982) found that religious ceremonies are supportive for individuals because liberating and curative friendships relevant to spiritual states brings about psychological health for them . Attending religious ceremonies and shrines also reduces individuals anxieties and sense of loneliness . These results are in accordance with our study findings which show that elderly women who participate in religious ceremonies, are relieved from loneliness . Also koenig (1998) suggested that religious practices, attitudes and coping behaviors are prevalent among hospitalized medically ill older adults and are related to social, psychological and physical health outcomes . Morris (1983) examined the relationship between anxiety and pilgrimage and found that people with religious devotion experienced significantly lower rates of anxiety disorder compared to the non - religious group . Every muslim learns their prayers from a young age and prays five times a day . In addition, those who seek guidance from the holy quran and believe that it has power over every aspect of their lives, expect and welcome any eventualities in their lives (quran, chapter balad, verse 4). It seems that elderly people have more tendencies to religious and spiritual subjects than young people . Koenig et al . (2004) in his study showed spirituality and religion as an important factor in their lives more often than younger ones . Several experimental studies have been conducted that showed positive effects of prayer on the management of physical symptoms of illness . Kwilecki (1986) assessed the effect of prayer on stress and anxiety and found that 42% of participants report that saying prayer can diminish the stress and anxiety . Also salehi (2001) found that individuals who pray regularly, experienced significantly lower rates of anxiety disorder and depression, psychological balance and hopefulness, compared to the non - prayer group . Azizi (1996) found that more than 90% of prayers attained psychological tranquility and relaxation after saying prayer . Several studies noted moderately positive correlations between indicators of physical health and prayer activities . Also several studies have shown that prayer can play an important role in the recovery of patients who suffer from cardiac disease, hiv, arthritis rheumatoid, and cva attack, cancer cases, treatment of addiction, and physical, mental and social well - being . Furthermore, evidence suggests that patients with strong religious beliefs and high levels of religious activity experience lower levels of pain, have better immune function, lower death rates from cancer, fewer incidences of heart disease, lower blood pressure and levels of cholesterol, better health behaviors, and greater compliance with medical treatment . The practical relevance of this study may conduct future work to further explicate and clarify the linkages between spirituality and physical and mental health and life style . Future empirical research and new methods of investigation such as concept analysis, grounded theory, and action research are needed to develop the new models of caring for management of this group . In the life style area, future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects . It is important to conduct such research in different samples, such as unhealthy elderly people (frail, ill, and sick elderly people). Future empirical research and new methods of investigation such as concept analysis, grounded theory, and action research are needed to develop the new models of caring for management of this group . In the life style area, future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects . It is important to conduct such research in different samples, such as unhealthy elderly people (frail, ill, and sick elderly people). In summary, we found that elderly people describe several elements in their illustration of spirituality in healthcare settings . Spiritual health, spiritual beliefs, and religious practice intentionality were the main components of concept of spirituality in this group experiences . Also, the findings of this study made it clear that spirituality has a considerable effect on the health and life of iranian elderly people and is a major supportive resource for their physical and psychosocial health . It can reduce mental distress and induce inner peace and hopefulness . Because of the significant influence of spirituality on all domains of health of elderly people, it is critically important that health care providers understand how spirituality can considerably influence elderly people throughout their life . These findings will assist health professionals such as nurses, physicians, and social workers to recognize the spiritual needs and value the role of spirituality in promoting health and well - being among elderly people from different religions and cultures worldwide.
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According to the national regulations regarding diagnostic criteria of brain death (bd), instrumental confirmatory tests should be used in certain clinical situations, such as intoxications, infratentorial processes, extensive facial damage, children younger than 2 years of age, or any case in which clinical examinations are inadequate . Electrophysiological tests are often unavailable due to limited access to proper equipment and competent specialists . Therefore, two of them, catheter angiography and computed tomographic angiography (cta), are based on the detection of contrast enhancement of cerebral vessels . In these examinations, first is a non - filling phenomenon, which means a complete absence of contrast above the carotid siphons and foramen magnum . Second is a delayed, weak, and persistent opacification of proximal segments of the cerebral arteries not reaching cortical branches . Stasis filling causes a significant problem in interpretation of cta results in the diagnosis of bd . Since 1998, when cta was proposed as the new imaging technique in the diagnostics of bd, a consensus on its interpretation criteria has not been reached . An analysis of stasis filling in a dynamic series of computed tomographic perfusion (ctp) can provide valuable information on the interpretation of cta results and relation of this phenomenon to brain perfusion . The aim of this prospective study was to characterize stasis filling phenomenon in the diagnosis of bd . To achieve this, we performed a dynamic evaluation of contrast enhancement of the cerebral and extracranial arteries in patients with bd and control subjects with the use of the 40-s series of ctp scans . We also confronted stasis filling pattern with ctp findings to assess the relevance of this phenomenon to brain perfusion . We examined 67 patients who fulfilled clinical bd criteria according to the national regulations (coma, apnea, and no brainstem reflexes). The examination protocol consisted of cta, followed by ctp and catheter angiography . Thirty - seven subjects were excluded due to lack of contrast enhancement in the intracranial arteries in ctp series . Finally, 30 (44.8%) patients, in whom analysis of ctp series enabled us to identify contrast - enhanced intracranial arteries, were enrolled in the study . The population consisted of 18 men and 12 women with a median age of 54.5 years (range, 2284 years). Initial causes of coma were intracerebral hemorrhage (n = 16), subarachnoid hemorrhage (n = 11), cerebral edema (n = 2), and ischemic stroke (n = 1). Decompressive craniectomy was performed in 19 (63%) included patients . Among 37 excluded patients, all patients were managed in the intensive care units of two university hospitals and one multi - profile provincial hospital . During all the examinations, the patients were normoventilated and mean arterial blood pressure (mabp) maintained at greater than 80 mmhg . The elapsed time between the onset of bd symptoms and the radiological examination ranged between 6 and 48 h. delay was sometimes caused by a prolonged initial observation due to prior use of sedatives and sometimes the limited availability of an angiographic team . The majority of patients became actual organ donors . In some cases, organ donation was not possible for medical reasons and occasionally because of a failure to obtain permission from relatives . In such situations, families were informed about the termination of futile therapy, and ventilators were legally switched off . The demographic and epidemiological characteristics of bd patients are presented in table 1.table 1demographic and clinical characteristics of bd patientsnumbersexage (years)diagnosiscraniectomyexamined arterystasis filling in angiography1m56ichmca2m71sahmca+3m73ich+mca+4f49sah+aca5f29sahaca6m40isaca7f66ichmca8m84sah+mca9m53ichmca10f67ich+mca+11m63cemca12f58sah+mca13m34sah+aca14m34sah+mca+15m78ichmca+16m39ich+mca+17f38sah+mca18f74sahaca+19f75ichmca+20f40sah+mca+21f22ich+mca+22f34ich+mca23m50ich+aca+24m44sah+mca+25m51ich+pca+26m71ich+mca27f41ce+mca28m59ichmca29m62ich+mca30m56ich+mca+ is ischemic stroke, ce cerebral edema, aca anterior cerebral artery, mca middle cerebral artery, pca posterior cerebral artery demographic and clinical characteristics of bd patients is ischemic stroke, ce cerebral edema, aca anterior cerebral artery, mca middle cerebral artery, pca posterior cerebral artery the control group consisted of 30 patients who underwent surgical clipping of an intracranial aneurysm . The population consisted of 14 men and 16 women with a median age of 53 years (range, 1865 years). Ctp was performed 810 days after neurosurgical clipping of the middle cerebral or anterior communicating artery aneurysms . During all the examinations, mabp stayed above 80 mmhg . None of the control subjects presented any signs of bd, with normal pco2 and po2 . All cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [4, 5]. We used siemens sensation 64 (siemens ag, erlangen, germany) to perform cta . Injection of 80 ml of iodinated contrast medium at a flow rate of 4 ml / s . Then cta was carried out in three phases, which were manually programmed with fixed delays of 25, 40, and 60 s after contrast injection . Siemens sensation 64 (siemens ag, erlangen, germany)to perform ctp in both groups . In bd patients, the time interval between cta and ctp ranged from 1 to 15 min . Administration of 50 ml of the contrast medium at a flow rate of 5 ml / s, a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns . Detailed protocol is presented in table 2.table 2technical parameters of ctp examinationacquisition tube voltage (kvp)80 tube current (mas)270 rotation time (s)1.0 collimation (mm)24 1.2 scan range (mm)28.8 cycle time (s)1.0 scan time (s)40 automatic dose modulationoffreconstruction slice width (mm)3 9.6 fov (mm)220 matrix512 512 recon algorithmh30scontrast injection volume (ml)50 flow rate (ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . In the first part of post - processing, two small circular 0.250.3-cm regions of interest (rois) the second roi covered the cerebral artery in the most distal visible segment (see fig . 1). This artery was visible in all control examinations (contralateral artery to the clipped aneurysm was always chosen) and 23 examinations of bd patients . Among the remaining seven examinations in six cases, the only opacified cerebral artery was aca and in one case pca (see table 1). Rois were automatically propagated over the entire series of scans . For each roi, time calculation was performed using the osirix v.5.5.1 software (pixmeo sarl, bernex, switzerland).fig . 1axial ctp scans in mip reconstruction from 40-s series in a bd patient (a) and in a control subject (b). Rois are positioned in the distal segment of mca (arrowhead) and in the superficial temporal artery (arrow) axial ctp scans in mip reconstruction from 40-s series in a bd patient (a) and in a control subject (b). Rois are positioned in the distal segment of mca (arrowhead) and in the superficial temporal artery (arrow) in bd patients, the same dynamic series of scans were used for measurements of cerebral blood flow (cbf) and cerebral blood volume (cbv) with the multimodality workplace and syngo ve40a software package (siemens ag, erlangen, germany). This software is based on a maximum slope method, which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation . Therefore, the lack of venous outflow in bd patients was not an obstacle, and calculation of cbf and cbv was feasible in all cases . We chose the same cerebral arteries, which were used in the first part of post - processing for calculation of tdcs . Cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal, temporal, occipital lobes, and basal nuclei of both hemispheres with exclusion of major blood vessels . Angiography was performed with a delay of 15 min to 3 h after ctp . We used two angiographic systems: fluorospot top and axiom artis (siemens ag, germany). After positioning a pigtail 45-f catheter in the ascending aorta, 30 ml of contrast was injected at a flow rate of 15 ml / s . We registered 3050 s series with a frequency of 2 f / s visualizing head and neck area with the use of a digital subtraction technique . According to the national guidelines, cerebral circulatory arrest was diagnosed in two situations: non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling delayed, weak, and persistent opacification of the proximal cerebral arterial segments, without opacification of the cortical branches or venous outflow non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling delayed, weak, and persistent opacification of the proximal cerebral arterial segments, without opacification of the cortical branches or venous outflow demographic data recorded for bd patients and control subjects were age and sex . In addition, for bd patients, we registered initial cause of coma and the presence of craniectomy . For each extracranial and cerebral roi in the ctp series, the following parameters were measured: baseline density: ct density on the first scanenhancement at 20, 30, and 40 s: difference between density at the time point of 20, 30, and 40 s and baseline densitypeak enhancement: difference between the highest density and baseline densityc / e peak ratio: ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak: period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak: period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density: ct density on the first scan enhancement at 20, 30, and 40 s: difference between density at the time point of 20, 30, and 40 s and baseline density peak enhancement: difference between the highest density and baseline density c / e peak ratio: ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak: period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak: period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients, the additional recorded data were values of cbf, cbv, and results of catheter angiography classified as non - filling or stasis filling . Whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution (p <0.05, shapiro all cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [4, 5]. We used siemens sensation 64 (siemens ag, erlangen, germany) to perform cta . Injection of 80 ml of iodinated contrast medium at a flow rate of 4 ml / s . Then cta was carried out in three phases, which were manually programmed with fixed delays of 25, 40, and 60 s after contrast injection . We used the same scanner siemens sensation 64 (siemens ag, erlangen, germany)to perform ctp in both groups . In bd patients, the time interval between cta and ctp ranged from 1 to 15 min . Administration of 50 ml of the contrast medium at a flow rate of 5 ml / s, a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns . Detailed protocol is presented in table 2.table 2technical parameters of ctp examinationacquisition tube voltage (kvp)80 tube current (mas)270 rotation time (s)1.0 collimation (mm)24 1.2 scan range (mm)28.8 cycle time (s)1.0 scan time (s)40 automatic dose modulationoffreconstruction slice width (mm)3 9.6 fov (mm)220 matrix512 512 recon algorithmh30scontrast injection volume (ml)50 flow rate (ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . In the first part of post - processing, two small circular 0.250.3-cm regions of interest (rois) were positioned in each ctp series of scans . The second roi covered the cerebral artery in the most distal visible segment (see fig . 1). This artery was visible in all control examinations (contralateral artery to the clipped aneurysm was always chosen) and 23 examinations of bd patients . Among the remaining seven examinations in six cases, the only opacified cerebral artery was aca and in one case pca (see table 1). Rois were automatically propagated over the entire series of scans . For each roi, time density curve (tdc) was plotted . Calculation was performed using the osirix v.5.5.1 software (pixmeo sarl, bernex, switzerland).fig . 1axial ctp scans in mip reconstruction from 40-s series in a bd patient (a) and in a control subject (b). Rois are positioned in the distal segment of mca (arrowhead) and in the superficial temporal artery (arrow) axial ctp scans in mip reconstruction from 40-s series in a bd patient (a) and in a control subject (b). Rois are positioned in the distal segment of mca (arrowhead) and in the superficial temporal artery (arrow) in bd patients, the same dynamic series of scans were used for measurements of cerebral blood flow (cbf) and cerebral blood volume (cbv) with the multimodality workplace and syngo ve40a software package (siemens ag, erlangen, germany). This software is based on a maximum slope method, which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation . Therefore, the lack of venous outflow in bd patients was not an obstacle, and calculation of cbf and cbv was feasible in all cases . We chose the same cerebral arteries, which were used in the first part of post - processing for calculation of tdcs . Cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal, temporal, occipital lobes, and basal nuclei of both hemispheres with exclusion of major blood vessels . Angiography was performed with a delay of 15 min to 3 h after ctp . We used two angiographic systems: fluorospot top and axiom artis (siemens ag, germany). A typical femoral approach was used in all cases . After positioning a pigtail 45-f catheter in the ascending aorta, 30 ml of contrast was injected at a flow rate of 15 ml / s . We registered 3050 s series with a frequency of 2 f / s visualizing head and neck area with the use of a digital subtraction technique . According to the national guidelines, cerebral circulatory arrest was diagnosed in two situations: non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling delayed, weak, and persistent opacification of the proximal cerebral arterial segments, without opacification of the cortical branches or venous outflow non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling delayed, weak, and persistent opacification of the proximal cerebral arterial segments, without opacification of the cortical branches or venous outflow in addition, for bd patients, we registered initial cause of coma and the presence of craniectomy . For each extracranial and cerebral roi in the ctp series, the following parameters were measured: baseline density: ct density on the first scanenhancement at 20, 30, and 40 s: difference between density at the time point of 20, 30, and 40 s and baseline densitypeak enhancement: difference between the highest density and baseline densityc / e peak ratio: ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak: period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak: period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density: ct density on the first scan enhancement at 20, 30, and 40 s: difference between density at the time point of 20, 30, and 40 s and baseline density peak enhancement: difference between the highest density and baseline density c / e peak ratio: ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak: period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak: period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients, the additional recorded data were values of cbf, cbv, and results of catheter angiography classified as non - filling or stasis filling . Whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution (p <0.05, shapiro for cerebral arteries, tdcs in bd patients represented flat curves in contrast to tdcs in the control group, which formed steep and narrow gaussian curves (see fig . 2). Baseline density for cerebral and extracranial arteries was significantly higher in bd patients in comparison to the control group (60.5 vs. 41.5 hu (hounsfield unit); we found significantly longer time to peak enhancement in cerebral arteries in bd patients than in controls (median, 32 vs. 21 s; p <in bd patients, peak enhancement in cerebral arteries occurred with a median delay of 14.5 s to peak in extracranial arteries while practically no delay was noted in controls (it occurred 1 s earlier, which is represented as median 1 s value; p <cerebral arteries in bd patients showed significantly lower peak enhancement in comparison to the control group (34.5 vs. 81.5 hu; p <0.0001) (see fig . Density curves in a bd patient (a) and in a control subject (b). 3distribution of baseline density values in bd patients and controls in cerebral and extracranial arteriesfig . 4distribution of time to peak in cerebral and extracranial arteries in bd patients and controlsfig . 6distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls time density curves in a bd patient (a) and in a control subject (b). Time to peak (ttp) enhancement distribution of baseline density values in bd patients and controls in cerebral and extracranial arteries distribution of time to peak in cerebral and extracranial arteries in bd patients and controls distribution of delay of cerebral peak values in bd patients and controls distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls for extracranial arteries, tdcs represented a similar shape in both groups . However, we observed higher enhancement in bd patients at 20, 30, and 40 s (see table 3). Peak enhancement was significantly higher and earlier in bd patients compared to controls (p = 0.0087 and p <0.0001, respectively) (see figs . 4 and 6). The intensity of peak enhancement in cerebral arteries in relation to extracranial arteries expressed as c / e peak ratio appeared to be significantly lower in bd patients in comparison to the control group (0.31 vs. 0.87; p <0.0001) (see fig . 7).table 3variables calculated from dynamic ctp series in bd patients vs. controlsparameterbd (n = 30)controls (n = 30) p valueenhancement at 20 s (hu) cerebral artery7.5 (24)41 (25)<0.0001 extracranial artery53.5 (40)41.5 (30)nsenhancement at 30 s (hu) cerebral artery18.5 (14)25 (19)ns extracranial artery36 (23)26 (20)0.0225enhancement at 40 s (hu) cerebral artery20 (15)16.5 (8)ns extracranial artery36.5 (18)17 (10)<0.0001values expressed as median (interquartile range) ns not significantfig . 7distribution of cerebral / extracranial peak ratio values in bd patients and controls variables calculated from dynamic ctp series in bd patients vs. controls values expressed as median (interquartile range) distribution of cerebral / extracranial peak ratio values in bd patients and controls analyzing the influence of demographic and clinical features on dynamic ct parameters, we revealed significant differences in time to peak and delay of peak enhancement in cerebral arteries between subgroups of bd patients with and without craniectomy . In the subgroup with craniectomy, time to peak and delay of cerebral peak were shorter in comparison to the subgroup without craniectomy (see table 4). A statistically insignificant trend was found in the analysis of tdcs in bd patients with subarachnoid hemorrhage (sah) and intracerebral hemorrhage (ich). We revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich (see table 5). We did not observe any significant differences between ct parameters in relation to sex or examined artery.table 4variables calculated from dynamic ctp series in bd patients without vs. with craniectomyparameterbd patients without craniectomy (n = 11)bd patients with craniectomy (n = 19) p valuetime to peak in cerebral artery (s)34 (6)26 (9)0.007delay of cerebral peak (s)18 (8)13 (9)0.04values expressed as median (interquartile range)table 5variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhageparameterbd patients with ich (n = 16)bd patients with sah (n = 11) p valuetime to peak in cerebral artery (s)27.5 (10)33 (9)0.34 (ns)delay of cerebral peak (s)13.5 (8)18 (13)0.16 (ns)values expressed as median (interquartile range) ns not significant variables calculated from dynamic ctp series in bd patients without vs. with craniectomy values expressed as median (interquartile range) variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhage values expressed as median (interquartile range) in all 30 bd patients, ctp revealed zero values of cbf and cbv in all rois . Stasis filling pattern was observed in 14 (46.7%) and non - filling in 16 (53.3%) cases . In 37 excluded bd patients, angiography revealed a non - filling pattern in all cases; no stasis filling was noted . The term stasis filling for describing a delayed, weak, and persistent intracranial opacification in patients with bd was used for the first time by kricheff et al . In 1978 . Munari et al . Reported stasis filling in 5% (one out of 20), braun et al . In 11% (15/140), bradac et al . In 12.5% (two out of 16), kricheff et al . In 15% (three out of 20), and savard et al . In 28% (nine out of 32) of cases [2, 69]. Noted stasis filling in 43% (six out of 14) and welschehold et al . In 59% (37/63) of cases [3, 10]. Stasis filling, as specific angiographic pattern of cerebral circulatory arrest, is a consequence of two major factors: raised intracranial pressure (icp) and high cerebrovascular resistance (cvr). They both cause reduction of cbf, according to poiseuille s law: cbf = cpp / cvr = (mabp icp)/cvr, where cpp stands for cerebral perfusion pressure . Increased cvr in extreme cerebral hemodynamic disturbances was revealed in studies using a transcranial doppler (tcd) [11, 12]. This is caused mainly by altered cerebral autoregulation mechanisms, which are sufficient to preserve almost constant cbf when cpp is above 50 mmhg . Cessation of capillary circulation is consistent with cerebral circulatory arrest while proximal arterial segments are still patent . At this stage, tcd frequently shows to and fro or narrow systolic spike patterns, which reflect blood movement [11, 12]. In such circumstances, heartbeat driven slow propagation of contrast column in proximal cerebral arteries is possible . In the present study, recently revealed a potential efficacy of ctp for assessing brain death . In this material, ctp detected complete absence of brain perfusion reflected by zero values of cbf and cbv in all cases . These findings clearly show that stasis filling is not an indicator of any residual brain perfusion but only ineffective propagation of contrast in cerebral vessels; thus, it does not preclude diagnosis of brain death . Ctp results are complementary to the recent report of selcuk et al ., which revealed global reduction of adc consistent with necrosis of neurons using diffusion - weighted mri technique . The findings showed two features of stasis filling phenomenon: delay and weakness of intracranial opacification . Tdcs for cerebral arteries in bd patients were characterized by significantly longer times to peak (median, 32 s) compared to controls (21 s). Moreover, intracranial peak enhancement occurred with a median delay of 14.5 s to extracranial peak . This is consistent with observations in cta studies, in which intracranial filling in bd patients is usually detected in the late phase (performed 60 s after contrast injection) and very rarely in the early phase of scanning [3, 10, 17]. Correlation with ctp results shows that such delayed vascular opacification does not provide any brain perfusion, thus cannot preclude diagnosis of brain death . Typical cerebral tdc in bd patients was a flat curve with a low peak (median value of 34.5 hu); about three times lower compared to the peak in the extracranial artery . In contrast, extra- and intracranial tdcs in the control group represented steep and narrow gaussian curves, and both peaks were of similar height . Such low peak values could be the reason of lower detectability of stasis filling by catheter angiography compared to ct (14/67 = 21% vs. 30/67 = 45% cases) observed in the present study . These findings are consistent with results of others as was presented in the first paragraph of discussion . Also observed underestimation of intracranial filling in catheter angiography compared to cta in four out of seven cases . Although, a larger amount of contrast was used in dynamic ct than in catheter angiography (50 vs. 30 ml), but in angiography, it was injected intra - arterially, which minimizes the effect of dilution . This discrepancy could be a result of advanced reconstruction algorithms, systems of noise reduction, and signal amplification used in ct scanners, which reduce noise, improve spatial resolution, and low contrast detectability . The other explanation could be the time sequence in performing ctp and catheter angiography . Angiography was carried out with a delay from 15 min to 3 h after ctp . During this period, slow rising icp could stop the propagation of contrast at the level of skull base . Analyzing the influence of demographic and clinical features on tdc s parameters, we found a statistically significant trend towards shorter time to peak and shorter delay of peak in the cerebral arteries of bd patients with craniectomy compared to those without it . Moreover, the incidence of craniectomy was much lower in bd patients excluded from the study due to lack of intracranial opacification (four out of 37 = 11%) compared to included patients who presented intracranial filling (19/30 = 63%). Several authors previously reported a relationship between skull defect and preserved intracranial filling in bd patients [7, 18, 19]. This can be explained by a decreased icp enabling propagation of contrast in the cerebral vessels . A statistically insignificant trend was found in the analysis of tdcs in bd patients with sah and intracerebral hemorrhage . We revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich . This difference was probably caused by vasospasm after sah, which independently increases cvr and additionally contributes to slowing down the propagation of contrast . The major drawback of this study is a consequence of performing ctp shortly after cta in bd patients . The influence of residual contrast injected for cta was reflected by higher baseline density in the cerebral and extracranial arteries, higher and earlier peak enhancement, and shorter time to peak in the extracranial vessels in bd patients compared to controls . However, this contrast contamination should not significantly change values of delay of cerebral peak or c / e peak ratio as they were calculated on the basis of both cerebral and extracranial tcds . In this study, we assessed the characteristic features of intracranial filling in bd patients delay and weakness of cerebrovascular opacification . It led to the conclusion that delayed and weak opacification of cerebral arteries do not necessarily mean the presence of cerebral perfusion, thus does not preclude diagnosis of bd.
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Gastric cancer is the second most frequent neoplasm of the alimentary tract after the large intestine . 5,103 people in poland were affected by it in 2008 . The case - to - death ratio of around 1 indicates unfavourable prognosis as to recovery from this disease [1, 2]. This poor result is determined by the fact that it is rarely (only in around 8%) detected in the form of early gastric cancer, in the mildly symptomatic or asymptomatic phase . In most patients it is diagnosed at a higher degree than the 1st degree of disease progression and its classic symptoms are weight loss, continuous and dull pain in the epigastrium, loss of appetite, nausea, vomiting and chronic bleeding [3, 4]. The existence of gastric cancer metastasis to the ureter has been described twice in the literature to date . A female patient, age 67, was diagnosed at the district hospital (22.04 - 30.04.2010) because of intensified symptoms of left - sided renal colic . Based on the conducted usg and single - phase computed tomography tests of the abdominal cavity and the pelvis, dilation of the ureter was found because of its infiltration by a pathological focus with the dimensions of 28 mm 15 mm . Another lesion was located nearby at the level of the left iliac muscle 30 mm 27 mm 20 mm, adhering to the sigmoid colon . Tissue infiltration of the pelvis minor wall was found descending in the direction of the left appendages . Colonoscopy was conducted and in this test the large intestine was described without pathology, while the gynaecological usg test confirmed the presence of fluid in the pelvis . During her stay the patient was treated with analgesic and diastolic medication and then referred for further treatment at the regional oncology centre . Because of reported pain complaints, she was immediately admitted to the department of oncological surgery on the day of her visit to the outpatient clinic (04.05.2010). The urologist consulting the patient indicated the possibility of kidney damage due to ureteral obstruction with recommendation for an accelerated operation . Therefore, pre - operative diagnostics were not extended beyond the tests received from the district hospital . The patient underwent surgery on 10.05.2010 and intraoperatively, besides the expected neoplastic tumour of the left ovary with infiltration of the ureter, numerous neoplastic foci were also found: a sigmoid colon tumour, a caecum tumour, a tumour of the body of the stomach and two single tumours in the omentum . Because of the resectability of the neoplastic foci described above, the operation plan adopted earlier was changed and the following were performed in succession with palliative intention: partial gastrectomy by the rydygier method, right - sided hemicolectomy, left ovariectomy and sigmoid resection . After restoring the continuity of the alimentary tract, the tumour occluded the lumen, which is why segmental ureterectomy was performed with end - to - end anastomosis over a pigtail catheter . The patient passed the post - operative period without complications, except a two - day fever . Because of the expected alimentary tract failure, parenteral nutrition was included on the 1st day after the procedure and blood deficits were supplemented with 2 units of erythrocyte mass and 7 units of plasma . On the 9th day after the operation, the patient was discharged home in a good general condition with recommendations for further treatment . The received postoperative histopathological protocol indicated that the stomach was the origin of the neoplastic process . To lauren), type i (acc . To goseki), of a stomach adenocarcinoma with a g2 malignancy degree, with occupation of the whole thickness of the stomach wall, with the following immunohistochemical characteristics: ck7(+++), ck20(++), ca125(+), mucicarmine(+). Neoplastic infiltrations along nerves and neoplastic embolisms of blood vessels, as well as metastatic foci in the greater omentum, were observed in the specimen . Because of the palliative partial gastrectomy type, only 3 lymph nodes were described in this specimen and all of them contained neoplastic cells . Besides the above, histopathological confirmations were obtained of the metastatic character of the foci in the ovary, caecum and sigmoid colon . The cancer occupied large and small intestine walls without infiltration of the mucous membrane and the immunohistochemical characteristics were identical as for the primary lesion . According to the above protocol, excision within tissues with a healthy margin of these organs was achieved . The widening of the left ureter is visible (marked by arrow) focus of metastatic carcinoma in the ureter wall . Microscopic section, he staining, magnification 40 neoplastic invasion in the ureter wall . Microscopic section, he staining, magnification 100 it is interesting biologically and constitutes the basis for this paper that the ureteral tumour was described by the pathologists not as a neoplastic infiltration encroaching on the ureter, but as a metastatic focus to the ureteral wall . After the operative treatment, the patient was qualified for palliative chemotherapy and received it from 28.07.2010 . The first treatment course was according to the eox regimen (oxaliplatin and capecitabine), but because of the occurrence of neutropenic fever the regimen was changed to pf (cisplatin and 5-fluorouracil). She again received only one course and the treatment was changed once more because newly formed metastatic foci in the liver were located in imaging tests . Next, three chemotherapy courses were administered according to the folfiri regimen (irinotecan, leucovorin and 5-fluorouracil), but only until 11.11.2010 because due to progression of changes in the liver described in examinations the chemotherapy was discontinued and the patient was qualified for symptomatic treatment . According to our knowledge treatment of gastric cancer in the disseminated phase of the disease aims at extension of life and achieving a good palliative effect . The recommended methods, allowing the above to be achieved, are chemotherapy or combination radiochemotherapy . In an advanced stage, palliative operations on this organ are performed for life reasons and serve to eliminate complications such as bleeding, perforation or obstruction of the organ . It is indicated that the above complications forced surgical intervention in 1/4 of the patients previously disqualified from operative treatment because of the presence of metastatic foci . According to literature data, patients operated on by palliative resection in the disseminated phase of the disease achieve a survival time of between 9 and 15 months . The survival time is limited by the number of metastatic foci; when their number is higher than two foci, no statistically significant differences are observed in this scope . The value of these procedures is increasing because of the reported low perioperative mortality and the observed significant improvement in the further quality of life for these patients [513]. It seems that the operative procedure conducted in the presented patient allowed a good palliative effect to be achieved along with loss of severe colic and maintenance of kidney function . The literature often presents, as characteristic of gastric cancer, blood - borne metastasis to the ovary, termed a krukenberg tumour . This is a term generally defining metastasis to the ovary, mainly gastric cancer and next colon cancer . Metastases from other organs, such as the lungs, the mammary gland and the uterus, are also possible, though much rarer . Krukenberg tumours are encountered in the course of 2 - 4% of disseminated neoplastic processes . This is a negative prognostic factor, with varying median survival time after its diagnosis: 12 - 13 months in the course of gastric cancer compared to 17 - 29 months in the course of large intestine cancer . This undoubtedly results from biological differences in the course of these neoplasms [1423]. In summary, the available literature sources have described this only twice to date (in 1976 and 2000). However, nephroureterectomy was conducted in these quoted cases [24, 25]. In the presented patient the kidney was spared and healing of the ureter after end - to - end suture was achieved . The dissemination of a neoplasm with extremely rarely encountered symptomatology described above is an interesting experience which we wanted to share . The beginning of the symptomatic disease as a left - sided renal colic is, in itself, a previously unreported case.
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Eosinophilic gastritis is an extremely rare disease that is characterized by eosinophilic infiltration of the various layers of the gastrointestinal tract in the absence of any definite causes of eosinophilia . Patients with eosinophilic gastritis have diverse symptoms, including abdominal pain, emesis, abdominal distension, and weight loss . These symptoms are associated with eosinophilic infiltration of the various layers of the gastrointestinal tract . The disease has to be distinguished from generalized eosinophilic disorder presenting with involvement of other organs . A 52-year - old female patient visited another hospital because of epigastric pain and tenderness . She had no history of allergic diseases such as eczema or atopy, or food or drug allergies ., she had undergone esophagogastroduodenoscopy (egd), which revealed multiple focal ulcerative lesions with diffuse discoloration and edematous change of the rugae in the gastric fundus, cardia, and upper body (fig . The symptoms had been recalcitrant to treatment with proton pump inhibitors, fasting, and fluid . She had reported no nausea, vomiting, hematemesis, or melena . On admission, her blood pressure was 110/60 mm hg, pulse rate 76 beats per minute, respiratory rate 22 breaths per minute, and body temperature 36.2. physical examination revealed tenderness of the epigastric area . Her white blood cell count was 22,770/mm with markedly increased eosinophils (5,009/mm, 22%). The c - reactive protein concentration was 13.95 mg / dl . Her other blood chemistry test results were normal . The tests for viral markers included hepatitis a, b, and c virus and human immunodeficiency virus; those for autoimmune antibodies included anti - nuclear, anti - double stranded dna, and anti - neutrophilic cytoplasmic antibodies; and those for tumor markers included -fetoprotein, carbohydrate antigen 19 - 9, and carcinoembryonic antigen . The serological test for toxocara antibodies (igg) was positive, whereas those for echinococcus, paragonimus westermani, sparganumi, and trichinella antibodies were negative . The chest and abdomen radiographic examinations were normal . To investigate the cause of the epigastric pain and tenderness, abdominal computed tomography was performed, which revealed severe edematous wall thickening with focal localized low attenuation of the fundus and cardia of the stomach (fig . She underwent a repeated egd, which showed diffuse necrotic change in the fundus, cardia, and upper body (fig . A biopsy specimen was obtained during egd; a rapid urease test (clotest) revealed no evidence of helicobacter pylori . On histopathologic evaluation, the gastric surface was found to be eroded and the underlying lamina propria showed dense eosinophilic infiltration . She was treated with empirical intravenous antibiotics (cefoperazone and metronidazole) immediately after her transfer because infectious gastritis was not ruled out . Although she was treated with a broad - spectrum anthelmintic (albendazole), antibiotics, proton pump inhibitor, and prokinetic agent during the next 5 days, her symptoms became worse . Eosinophilic gastritis was diagnosed according to the clinical pictures, laboratory findings, and endoscopic findings . She was immediately started on methylprednisolone (62.5 mg / day; this dose was maintained for 7 days). Therefore, she underwent a repeated short - term follow - up egd that showed regenerative epithelial tissue with peeling off, of the necrotic tissue (fig . She underwent a repeated egd that showed the replacement of white color scar tissue (fig . Currently, she is being treated with 30 mg / day prednisone and is showing considerable clinical improvement . Eosinophilic gastroenteritis is an uncommon and rarely reported disorder characterized by eosinophilic infiltration of the various tissue layers of the digestive tract in the absence of any definite causes of eosinophilia, without eosinophilic infiltration in other organs . The diagnosis of eosinophilic gastroenteritis is based on the following criteria: the presence of gastrointestinal symptoms, histological presentation of eosinophilic infiltration in tissue layers of the digestive tract, presence of high eosinophil count in ascites, and no evidence of parasitic infection or eosinophilic involvement of extraintestinal organs . Eosinophilic gastroenteritis can involve any portion of the gastrointestinal tract from the esophagus to the rectum, and the stomach is the most commonly involved organ, especially the antrum . In eosinophilic gastritis, the endoscopic features are rather extensive: rugal fold thickening, erythema, friability, nodularity, gastric outlet obstruction, gastric ulcer, and even a normal mucosa . Although not clearly defined, the common pathophysiological mechanism of this disease is associated with eosinophilic infiltration and degranulation in specific tissue layers of the digestive tract . This eosinophilic recruitment and activation regulated by diverse cytokines is a part of the host immune mechanism in the gastrointestinal mucosa; however, it can be a type of serious allergic or inflammatory reaction in the deeper tissue layers of the gastrointestinal tract . Although there is no treatment consensus on eosinophilic gastroenteritis, several studies report good results with steroids in dosages from 20 to 40 mg / day, for 6 to 8 weeks [7 - 10]. In some case studies, leukotriene modifiers such as montelukast or mast cell stabilizers such as sodium cromoglycate have been proposed to be helpful for symptomatic improvement . Antihistamines such as ketotifen or immunosuppressants such as mycophenolate mofetil are also used in the treatment of eosinophilic gastroenteritis; however, their therapeutic effects are not clear and would require more studies . Although rare, eosinophilic gastritis should be considered in the differential diagnosis of patients with gastrointestinal symptoms and peripheral blood eosinophilia . Lymphoma of the stomach, gastric cancer, and crohn disease involving the stomach may demonstrate endoscopic features similar to those of eosinophilic gastritis . Gastrointestinal parasitic infection should be considered in patients with abdominal discomfort, weight loss, and peripheral eosinophilia . In particular, infestations by hookworms, ascaris, strongyloides, toxocara, trichuris, and intestinal capillaria should be considered in patients from endemic areas . In this case, the concentration of the antigen - specific ige to the a. simplex was 0.57 moreover, the serology for toxocara antibodies (igg) was positive . However, this is not clinically significant in korea because koreans often consume raw fish, which causes repeated exposures to anisakis . In addition, she had not had contact with any animals, including dogs and cats, at least for several years . Moreover, she was treated with albendazole for 5 days, and her symptoms had become worse . After steroid treatment, the symptoms disappeared and the eosinophil count decreased to the reference range . Moreover, the follow - up egd showed regenerative epithelial tissue with peeling off, of the necrotic tissue . She had undergone several egds that showed diffuse necrotic change in the fundus, cardia, and upper body . The etiology includes thromboembolism and occlusion of major arterial supply, ingestion of corrosive agents, volvulus of the stomach, endoscopic hemostatic injections, and infectious gastritis . In this case thus, the possible cause of gangrene could be infection, and she was treated with empirical intravenous antibiotics (cefoperazone and metronidazole). However, she was treated with broad - spectrum antibiotics during the next 5 days, and her symptoms became worse . The necrotic portion of the gastric high body is very vulnerable site of retching injury . This retching injury is called prolapse gastropathy syndrome, a clinical syndrome involving the invagination of part of the gastric mucosa into the lower esophagus . Direct trauma to the mucosa occurs when the gastric mucosa becomes incarcerated through the lower esophageal sphincter . In addition, the endoscopic findings and histopathologic results were not compatible to prolapse gastropathy syndrome . On the basis of the clinical picture, laboratory findings, and therapeutic results, we concluded the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis in our patient . This case highlights the reality of eosinophilic gastritis presenting as necrotizing gastritis, and that endoscopy and histopathological examination of the biopsies are the most useful tools for the diagnosis of eosinophilic gastritis presenting as necrotizing gastritis . Eosinophilic gastritis should be considered in the differential diagnosis in patients with necrotic gastritis who do not respond to empirical treatment.
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The skin is the largest organ of the body and protects the organism against external physical, chemical, and biological insults such as wounding, uvb radiation, and microorganisms . This major barrier resides in the upper layers of the epidermis (for review see segre, 2006). The epidermis is the upper part of the skin that is continuously renewed . The basal layer, or stratum basale, of the epidermis contains proliferating keratinocytes (fig . 1). Upon withdrawal from the cell cycle, these basal keratinocytes detach from the basement membrane and undergo a terminal differentiation program to become corneocytes in the outer layers of the epidermis . The cells reinforce their cytoskeletal keratin filament network, and adjacent cells interact via many desmosomes, a specialized type of cell junction, to resist physical trauma . In the stratum granulosum, the keratinocytes become more flattened and express certain proteins such as profilaggrin and loricrin, which aggregate to form the typical keratohyalin granules of the stratum granulosum . In addition, lipids are produced and stored in lamellar bodies . At the final stage of differentiation, the keratinocytes lose their organelles, including the nucleus, and become the dead, flattened corneocytes of the stratum corneum . During cornification, proteins are cross - linked at the inner side of the cytoplasmic membrane to form a cornified envelope (for review see candi et al ., 2005). In the transitional layer between the stratum granulosum and the stratum corneum, lipids are extruded to form a water - repelling envelope around the cornified envelope, thereby assuring an adequate permeability barrier function of the mammalian epidermis . Improper formation of these envelopes results in an impaired epidermal barrier that cannot protect against dehydration, uvb, and infection . The signaling cascades involved in epidermal barrier formation are largely unknown, but the many proteases that seem to be involved are currently being intensively studied . See introduction for details . Since the cloning of caspase-14 in the late nineties, it has become clear that this protease is a unique member of the caspase family . Unlike apoptotic caspases, which evolved in common ancestors such as hydra, echinodermata, insects, nematodes, and chordates, caspase-14 has so far been found only in terrestrial mammals (lamkanfi et al ., 2002). In contrast to the ubiquitously expressed other members of the caspase family, caspase-14 is expressed and activated mainly in the epidermis and is absent from most other adult tissues (eckhart et al . Recently, caspase-14 was found to be involved in epidermal barrier formation (denecker et al ., 2007). In this review, we discuss current knowledge of the expression, regulation, and function of caspase-14 . The expression pattern of caspase-14 is unique among the caspases, as it is present mainly in cornifying epithelia, such as the epidermis, the hassall's bodies of the thymus, and the forestomach of rodents (lippens et al ., 2000, 2005; in skin, caspase-14 is expressed only in the differentiating and cornifying layers of the epidermis and the hair follicle (lippens et al ., 2000; this is consistent with the observation that, in vitro, caspase-14 is only expressed in differentiating but not in proliferating keratinocytes (lippens et al . Remarkably, nail matrix keratinocytes that differentiate into specialized nail corneocytes, the building blocks of the nail plate, do not express caspase-14 (jager et al . In addition, caspase-14 is not expressed in the noncornifying keratinocytes of the sweat gland or the mouth epithelium (lippens et al ., 2000; ultrastructural analysis demonstrated that spatial distribution of caspase-14 in the epidermis and hair follicles is strongly conserved among several mammalian species (alibardi et al . Caspase-14 was found to be associated with the nucleus, the keratohyalin granules, and the desmosomes, whereas in corneocytes, caspase-14 was found in the cytoplasm and was associated with corneodesmosomes (a modified version of desmosomes) and nuclear remnants . These observations suggested a role for caspase-14 in nuclear degradation during cornification, but nuclear degradation was not affected in caspase-14deficient mice (denecker et al ., 2007). The expression of caspase-14 in the hassall's bodies of the thymus and in the forestomach of rodents is somewhat expected, as they are cornifying structures and express the typical late differentiation markers, such as profilaggrin and loricrin, which are also found in the epidermis (laster and haynes, 1986; favre, 1989; jarnik et al ., 1996). Protein expression of caspase-14 has also been reported in several noncornifying tissues (lippens et al ., 2003; krajewska et al ., 2004, 2005; kam et al ., 2005 however, these observations should be interpreted carefully, as we have recently shown that the reported expression of caspase-14 in such tissues can be the result of aspecific staining (denecker et al ., 2007). Remarkably, so far caspase-14 has been found only in terrestrial mammals but not in birds or reptiles . Whereas birds and reptiles have a stiff, dry, scaly epidermis, mammals have a soft stratum corneum because of the larger amounts of histidine - rich late differentiation markers (e.g., profilaggrin; alibardi, 2003). Interestingly, profilaggrin is a direct substrate of caspase-14 (denecker et al ., 2007). This could indicate that the occurrence of a soft stratum corneum and the caspase-14 gene are associated during evolution . Although the expression of caspase-14 is very restricted, little is known about the transcriptional regulation of its gene . In vitro, caspase-14 is only expressed when keratinocytes are forced to differentiate by growing them postconfluently or in suspension or by adding vitamin d3 (eckhart et al . In contrast, adding ca at high concentrations to the medium, a method frequently used to induce differentiation, did not induce caspase-14 expression (eckhart et al . Retinoids, which suppress keratinocyte differentiation, down - regulate caspase-14 expression (rendl et al ., these results indicate that transcription factors that are specifically active during terminal differentiation are required to regulate caspase-14 expression . Whether caspase-14 expression levels can be regulated at the posttranscriptional level is not known . Down - regulation of several differentiation - associated genes by retinoids has been shown to be mediated by the transrepression of activator protein 1 (ap-1)mediated gene activation (fisher and voorhees, 1996). Indeed, the caspase-14 promoter contains at least two potential ap-1binding sites (unpublished data). The green tea phenol ()-epigallocatechin-3-gallate (egcg) is a potent activator of ap-1 and has been shown to up - regulate caspase-14 in a p38- and jnk - dependent way (hsu et al ., 2005, 2007). Ap-1 alone is probably not sufficient to drive caspase-14 expression because tnf and 12-o - tetradecanoyl - phorbol 13-acetate, two potent activators of ap-1 in keratinocytes (arnott et al ., 2002), did not induce caspase-14 expression in keratinocytes (lippens et al ., 2004). Differentiation - dependent expression of caspase-14 in keratinocytes could also result from a strong transcriptional repression in proliferating keratinocytes . This possibility is supported by the observation that mice deficient in nuclear receptor corepressor hairless (hr) had 510-fold higher levels of caspase-14 and profilaggrin mrna, starting from postnatal day 6 and progressing during development (zarach et al ., 2004). These alterations in gene expression were detected mainly in the keratinocytes of the utricle, an abnormal pouch - like structure at the upper part of the hair follicle . Increased gene expression occurred before the morphologically distinct utricle could be identified, indicating that the up - regulation was probably a cause rather than a consequence of utricle formation . It would be interesting to analyze both caspase-14 activation and filaggrin processing in these mice . Multiple mutant hr alleles in mice and in humans show phenotypic variations that include congenital hair loss, skin wrinkling, and papular rash (cichon et al ., 1998; 1998). Whether caspase-14 overexpression is important for the observed phenotypes could be addressed by generating epidermis - specific caspase-14 transgenic mice or by crossing the hr mice with caspase-14deficient mice . Procaspases consist of a prodomain, a large subunit (p20), and a small subunit (p10). Activation of caspases is induced by dimerization, (auto-)proteolytic cleavage at asp residues, and/or conformational changes (lamkanfi et al ., 2003). So far, maturation of caspase-14 by proteolytic cleavage into p20 and p10 subunits has been consistently observed only in cornifying epithelia such as the epidermis and the rodent forestomach (lippens et al ., 2000; although some investigators suggested that caspase-8 and -10 can activate caspase-14 in vitro (ahmad et al ., 1998; van de craen et al ., 1998), this could not be confirmed by others (lippens et al . Furthermore, caspase-14 is probably not proteolytically activated by a caspase in vivo, as other caspases are not activated during epidermal differentiation (eckhart et al . 2000; raymond et al ., 2007), and caspase-14 is not processed at an aspartate residue like other caspases but is processed at ile in man and presumably at leu in the mouse (chien et al ., 2002). Alignment of the protease - sensitive loop between the p20 and p10 subunits of the known mammalian procaspase-14 amino acid sequences (fig . 2) reveals a conserved hydrophobic patch that is n terminal of the caspase-14 cleavage site . This patch contains p1-preferred amino acids of elastase - like serine proteases, such as val, ala, leu, and ile (mallory and travis, 1975; vered et al ., 1985; takahashi et al ., 1989), suggesting that a serine protease with elastase - like properties could be involved in caspase-14 activation (unpublished data). All together, these data indicate that during skin homeostasis, the caspase-14activating protease is not a caspase, separating caspase-14 activation from the apoptotic and inflammatory caspase cascades that could be detrimental to epidermal integrity . The precise epidermal layer in which caspase-14 is processed and activated is unknown because antiserum specifically recognizing activated caspase-14 is not yet available . However, the following findings indicate that activation of caspase-14 occurs at the interface between the granular and cornified layers of the epidermis or early during cornification . First, both the proform and activated form of caspase-14 can be found in total epidermal extracts, whereas in the cornified layer only activated caspase-14 is found (fischer et al ., 2004). Second, caspase-14 activation coincides with stratum corneum formation both during embryonic development and in organotypic skin cultures (eckhart et al . This implies that the main biological function of caspase-14 is exercised in the stratum corneum, as proven by the phenotype of the caspase-14deficient mice (see the next section). Sequence analysis indicates that a hydrophobic patch in the protease - sensitive loop is conserved . Only part of the alignment is shown here, including the c - terminal part of the p20 subunit, the protease - sensitive loop, and the n - terminal part of the p10 subunit . The darker the yellow, the more the amino acids are conserved between species . The catalytic qacrg box is delineated with a green box . The conserved hydrophobic patch is delineated with a red box, and the cleavage site in human caspase-14 is indicated with a red arrow . The alignment was performed using clustalw (mega version 3.1; kumar et al ., 2004) and identification of caspase-14 substrates has been hampered for a long time by the unavailability of enzymatically active caspase-14 . However, it was recently shown that proteolytically processed caspase-14 requires high concentrations of kosmotropic salt to be active in vitro, such as sodium citrate, in addition to proteolytic cleavage between the p20 and p10 subunit (mikolajczyk et al ., 2004). These salts induce both dimerization and ordering of active site loops by partial desolvation of the protein to a more compact, catalytically active protease . The cellular environment of the stratum corneum of the epidermis probably favors caspase-14 activity in a similar way . Indeed, the water content decreases from 45% at the transitional layer to 1525% at the skin surface (warner et al ., 1988; caspers et al ., 2001). Human caspase-14 preferentially accommodates tryptophan or tyrosine in the s4 subsite, whereas mouse caspase-14 is more tolerant, with almost equal preferences for -branched and aromatic amino acids (mikolajczyk et al ., 2004). For example, both human and mouse caspase-14 efficiently cleave the fluorescent peptide substrate wehd - amc, but only mouse caspase-14 cleaves ietd - amc as efficiently (fischer et al ., 2004; mikolajczyk et al ., these substrate preferences would classify human caspase-14 as an inflammatory caspase and mouse caspase-14 as an inflammatory and apoptotic initiator caspase (thornberry et al ., 1997; thornberry, 1998). However, human caspase-14 cannot proteolytically activate the inflammatory cytokines pro 2004), and there are no data supporting a direct role for caspase-14 in apoptosis (lippens et al ., 2000; denecker et al ., 2007). Whether the substrate preferences of human and murine caspase-14 observed in vitro on peptide substrates also occur in vivo is not clear . Importantly, profilaggrin, a major structural protein in the differentiating epidermis, has been shown to be a physiological substrate of caspase-14 (denecker et al ., 2007). Identification of additional substrates and determination of the cleavage sites will provide more insight into the preferred recognition sequence of caspase-14 in the context of a protein . Keratinocytes can die by two different processes: apoptotic cell death induced by damaging agents such as uvb, chemicals, and cytotoxic cytokines or by a continuous process of differentiation leading to the formation of corneocytes . These processes are clearly distinct pathways executed by different players (for review see lippens et al ., 2005), but the role of caspases in these two cell death programs has long been controversial . Although almost all procaspases are constitutively expressed in the epidermis, only caspase-14 has consistently been shown to be activated during epidermal cornification (eckhart et al ., 2000b; lippens et al ., 2000; raymond et al ., 2007). In addition, knockouts for apoptotic caspases were not reported to have a phenotypic skin anomaly except for caspase-3deficient mice, in which keratinocyte differentiation is delayed in the embryo but normalized at birth (okuyama et al ., 2004). However, others could not confirm the activation of caspase-3 during embryonic epidermal development (fischer et al . Although they are not activated during cornification, apoptotic caspases, in contrast to caspase-14, become activated during uvb-, staurosporine-, tnf-, and tnf - related apoptosis - inducing ligand induced apoptosis of keratinocytes and, thereby, play a role in the apoptotic cell death of keratinocytes (for review see lippens et al ., 2005). Thus, we conclude that apoptotic caspases are not involved in the physiological keratinocyte cell death program leading to cornification . Furthermore, caspase-14deficient epidermal cells can undergo classical apoptosis, which genetically demonstrates that caspase-14 is dispensable for the apoptosis of keratinocytes . During development, caspase-14 protein expression is detectable from embryonic day (e) 15.5 on, and its processing is observed from e17.5 (hu et al ., 1998; van de craen et al ., 1998; fischer et al ., 2005), which coincides with stratum corneum formation and establishment of the epidermal barrier . However, no differences in outside - in barrier formation of the skin of caspase-14deficient mice during embryogenesis were observed (denecker et al ., 2007). Furthermore, caspase-14deficient mice were born at the expected mendelian ratios, were fertile, and had long survival rates . Detailed analysis of caspase-14deficient mice indicated that caspase-14 has an important role in cornification, hydration, and uvb protection . The skin of caspase-14deficient mice was shinier, characterized by deeper skin lines, and had larger scales (denecker et al ., 2007) even though the shape and size of the cornified envelopes themselves were not altered . Biochemical analysis indicated that caspase-14 was responsible for the correct processing and degradation of (pro)filaggrin, as epidermis lacking caspase-14 was characterized by an altered profilaggrin processing and staining pattern (fig . 3) and by the presence of aberrant keratohyalin granules, the profilaggrin storage granules . Profilaggrin is a large, insoluble protein consisting of a calcium - binding a domain, a b domain, and several tandem repeats of filaggrin units . In the transitional layer 4), which aid in the bundling of keratin intermediate filaments and formation of the cornified envelope (for review see candi et al ., 2005). Subsequently, filaggrin is deiminated (conversion of arginine to citrulline by elimination of the imino group of arginine by peptidylarginine deiminases), causing its release from keratin and allowing its degradation into free hygroscopic amino acids that act as natural moisturizing factors of the stratum corneum (scott and harding, 1986; rawlings and matts, 2005). Therefore, filaggrin plays an important role in skin hydration . Although the filaggrin unit was detected in caspase-14deficient epidermis by western blot analysis, lower molecular weight filaggrin fragments were also present (denecker et al ., 2007). Immunofluorescence analysis showed that in these mice, filaggrin immunoreactive fragments accumulated in the upper layers of the stratum corneum (fig . This indicates that the correct degradation of filaggrin into free amino acids was affected in caspase-14deficient skin . Interestingly, caspase-14 was found to be associated with keratohyalin granules in the stratum granulosum and to remain cytoplasmic in the stratum corneum (alibardi et al ., 2004), which could correlate with its possible involvement in the generation of free amino acids . Expression of caspase-14 and (pro)filaggrin in wild - type and caspase-14deficient skin . Immunofluorescence staining for caspase-14 (red) and (pro)filaggrin (green) on paraffin sections of 5.5-d - old skin of both wild - type (+ /+) and caspase-14deficient (/) mice (denecker et al ., 2007). Fluorescence microscopy was performed on a cellm system (olympus) with an upright microscope (bx61; olympus). A specific dapi emission band - pass filter (450470 nm) and a gfp emission band - pass filter (510550 nm) were used . Image acquisition and processing were performed with the cellm software using a cooled ccd camera with a 1,344 1,024 pixel resolution . Image intensity scaling and color conversion were completed in imagej (national institutes of health). Caspase-14 is expressed mainly in the spinous, granular, and cornified layers of wild - type mice and is absent in caspase-14deficient mice . (pro)filaggrin is expressed in the granular layer and in the lower cornified layer in wild - type skin . In caspase-14deficient skin, additional filaggrin caspase-14 protects the skin against uvb photo damage and water loss and is involved in the processing of (pro)filaggrin . Caspase-14 expression starts in the spinous layer (indicated in shades of red), and cleavage into its p20 and p10 subunits occurs at the transition of the granular to the cornified layer . Caspase-14 is active in the dehydrating environment of the cornified layer, where it has an important function in formation of the epidermal barrier leading to protection against uvb and water loss (denecker et al ., 2007). Profilaggrin is a large structural molecule consisting of an n - terminal a domain and a b domain followed by multiple filaggrin repeats and a unique c - terminal sequence (for review see candi et al ., 2005). Profilaggrin undergoes many posttranslational modifications, eventually leading to release from the keratin intermediate filaments (see the section on the function of caspase-14 for details). In the lower stratum corneum these amino acids compose 40% of the natural moisturizing factors present in the stratum corneum and are important for maintaining epidermal hydration (rawlings and matts, 2005). In caspase-14deficient skin, accumulating filaggrin fragments are present (denecker et al ., 2007), indicating that an unidentified protease (asterisk) cleaves the filaggrin monomer into these fragments and that caspase-14 is responsible for the further processing and degradation of these fragments into free amino acids . As it is very unlikely that caspase-14 is directly responsible for degradation of the filaggrin fragments into free amino acids, we propose two possible mechanisms: (1) caspase-14 could first cleave these filaggrin fragments, leading to further degradation into free amino acids by another endo- and/or exopeptidase; or (2) caspase-14 could directly or indirectly (by inactivating an inhibitor) activate an endo- and/or exopeptidase that further processes the smaller filaggrin fragments . Kg, keratohyalin granule; kif, keratin intermediate filament; nmf, natural moisturizing factors; sb, stratum basale; sc, stratum corneum; sg, stratum granulosum; ss, stratum spinosum; tg, transglutaminase . These results, together with the finding that caspase-14 can directly cleave (pro)filaggrin in vitro, demonstrate that caspase-14 has a critical role in the correct processing of (pro)filaggrin during cornification . Whether the degradation of other differentiation - associated proteins is also affected in caspase-14deficient mice first, caspase-14 may cleave the filaggrin fragments and, thereby, expose cleavage sites that can be recognized by other endo- and/or exopeptidases for further degradation . Second, caspase-14 may be the activator of an endo- and/or exopeptidase that cleaves and degrades filaggrin . Direct degradation of filaggrin fragments into free amino acids by caspase-14 can be ruled out, as caspases only cleave after aspartate residues . The lack of filaggrin processing into free hygroscopic amino acids in caspase-14deficient mice may lead to the reduced epidermal hydration and increased trans - epidermal water loss observed in these mice (denecker et al ., 2007). These results point to an important function of caspase-14 in the maintenance of epidermal hydration . Although a profilaggrin - deficient mouse has not been generated, it has been demonstrated that flaky tail (ft / ft) mice, which have an autosomal recessive mutation in the flaky tail gene (probably the profilaggrin gene), lack a functional filaggrin monomer (presland et al ., 2000). These mice have been proposed as a model for the filaggrin - deficient skin disease ichthyosis vulgaris because they have dry, flaky skin and irregular scales of variable size . The importance of filaggrin has been underscored recently by human genetic studies demonstrating that loss - of - function mutations in the profilaggrin gene are the primary cause of the skin disease ichthyosis vulgaris (smith et al ., 2006), which is characterized by silvery scales on the abdomen and palmar hyperlinearity . 2006), possibly as a result of a defect in epidermal barrier function that allows the increased entry of allergens and infectious agents . Two of the important functions of the skin are prevention of water loss and protection against environmental stress, such as protection against uvb radiation, which are essential for terrestrial life . The development of caspase-14deficient mice revealed that the absence of caspase-14 enhances sensitivity toward uvb - induced photo damage and apoptosis of the skin (denecker et al ., 2007). Importantly, this is not caused by cell - autonomous differences in dna damage sensitivity and apoptosis between wild - type and caspase-14deficient keratinocytes . Instead, the uvb - filtering capacity of the stratum corneum is severely reduced in caspase-14deficient skin, as higher levels of cyclobutane pyrimidine dimers are detected immediately after uvb irradiation . This indicates that caspase-14 has an indispensable role in the photoprotective function of the stratum corneum . Interestingly, topical application of egcg, an inducer of caspase-14 expression, has been shown to be photoprotective (elmets et al ., 2001). How caspase-14 alters the structural and biochemical properties of the stratum corneum is currently under investigation . Caspase-14 was shown to be expressed at the protein level in several cancer cell lines (pistritto et al ., 2002; koenig et al ., 2005; krajewska et al ., 2005). In addition, caspase-14 mrna, together with keratin 1 and profilaggrin mrna, was decreased in murine uvb - induced squamous cell carcinoma, possibly reflecting reduced differentiation in the tumor (rundhaug et al ., 2005). Furthermore, in some cases, caspase-14 protein expression was associated with highly differentiated cornified areas of lung squamous cell carcinoma and cervix carcinoma (koenig et al ., 2005). However, caspase-14 activation in tumors has not been shown, and so it might not be responsible for the tumor phenotype . Presumably, the ectopic caspase-14 expression is caused by the changed transcriptional activity in these epithelial tumors . Mutations in the caspase-14 gene have not been found in human carcinomas except very rarely in colorectal tumors, which most probably were not the cause of altered caspase-14 expression (koenig et al ., 2005; we as well as other investigators demonstrated that caspase-14 expression is substantially down - regulated in psoriatic lesions but is unaffected in the nonlesional epidermis (lippens et al ., 2000, 2004; psoriasis is an autoimmune disease characterized by the uncontrolled proliferation of keratinocytes and impaired cornification, which results in the aberrant presence of nuclei in the cornified layer, also called parakeratosis . Although caspase-14 is absent in these parakeratotic regions, this is probably not the cause of the development of parakeratotic plaques, as caspase-14deficient mice did not show spontaneous parakeratosis (denecker et al ., 2007). More likely, caspase-14 down - regulation results from the impairment of terminal differentiation or up - regulation of transcriptional repressors . The absence of caspase-14 in psoriatic plaques may lead to the formation of a defective barrier and, therefore, to the aggravation of psoriatic lesions . Treating the parakeratotic plaques of patients with a vitamin d3 analogue results in the up - regulation of caspase-14 and likewise, in the flaky skin (fsn / fsn) mouse model of psoriasis, topical egcg treatment causes the up - regulation of caspase-14 and the amelioration of psoriasis (hsu et al ., 2007). Interestingly, the expression of junb is strongly down - regulated in psoriatic lesions, and inducible epidermal deletion of both junb and c - jun in mice results in a psoriatic phenotype (zenz et al ., 2005). Because caspase-14 might be regulated by these transcription factors, it would be interesting to elucidate whether caspase-14 is down - regulated in junb / c - jun deficient mice . Recent evidence sheds light on the crucial role of caspase-14 in the skin, but several major questions remain . Activation of caspase-14 occurs most probably at the interface between the granular and cornified layer, implicating a role for caspase-14 in the stratum corneum . It is now clear that the caspase-14activating protease is not a caspase but probably an epidermis - specific serine protease with elastase - like properties . This is not surprising, as it has been known for a long time that serine proteases are of major importance in epidermal homeostasis . Determination of the in vitro conditions for caspase-14 activity that mimic stratum corneum conditions, together with the generation of caspase-14deficient mice, led to the identification of (pro)filaggrin as the first known physiological caspase-14 substrate . Importantly, caspase-14 seems to be involved in the correct processing of filaggrin preceding its degradation into free hygroscopic amino acids, which might explain its role in the prevention of water loss from the epidermis . Proteomic approaches could lead to the identification of additional caspase-14 substrates, which would contribute to understanding the role of caspase-14 in the skin . Caspase-14 also protects against uvb - induced damage, which means that it is involved in the establishment of the biochemical or structural properties of the stratum corneum as a uvb filter . How caspase-14 establishes the uvb - filtering capacity of the corneum is not completely understood . An extensive biochemical analysis of caspase-14deficient epidermis could reveal these mechanisms . Whether caspase-14, its activating protease, or its substrates could be used as therapeutic agents or as targets to improve formation of the epidermal barrier is a challenging research goal.
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Gossypiboma (retained surgical sponge) describes a mass of cotton or sponge that is left behind in the body cavity during a surgical operation . These foreign bodies can often mimic tumors or abscesses when detected clinically or with radiation . In fact, surgical sponges without opaque markers are the leading cause for the difficulty of a correct diagnosis . Here, we present a case in which a foreign body, gauze without radiopaque markers, was left behind during a procedure that involved the removal of an intrapelvic tumor . After a computed tomography (ct) scan had revealed information leading to an inaccurate diagnosis, an accurate diagnosis of gossypiboma was successfully made using endoscopic ultrasound - fine - needle aspiration (eus - fna). A 60-year - old female patient was referred with an unexpected detection of an intrapelvic tumor during a routine ct scan . The patient's medical history included a hysterectomy and a left side ovariectomy at the age of 27 . The ct scan revealed a 45 mm tumor in the patient's pelvis [figure 1a and b], which was diagnosed as a gastrointestinal stromal tumor (gist), a chronic expanding hematoma or leiomyoma . A forward - view eus (tgf - uc260j, olympus optical co., tokyo, japan) identified a low echoic lesion when scanned from the sigmoid colon . Fna was performed with a 22-gauge needle (ezshot2, olympus optical co., tokyo, japan) by using the forward - view eus [figure 2]. Examination of the fna specimen revealed a foreign body (string of gauze) displaying macrophage cells and necrotic tissue [figure 3; hematoxylin and eosin, original magnification 40]. During the surgery, histological examination of the tissue adjacent to the foreign body revealed granuloma formations with fragmented silken threads [figure 4; hematoxylin and eosin, original magnification, 100 and 200]. A: sagittal section (black arrow); b: horizontal section (black arrow). Endoscopic ultrasound (eus)-fine - needle aspiration using forward - view eus a foreign body (string of gauze) obtained using the 22-gauge needle, with macrophage cells and necrotic tissue granuloma formations with fragmented silken threads gossypiboma describes a mass of cotton or a sponge that is retained in the body after a surgery . Gossypiboma causes foreign body reactions of the surrounding tissue since they are inert and show no specific decomposition . In this case, radiopaque sponges were not used, and there were no adverse symptoms in the 30 years postsurgery . The typical appearance of gossypiboma shows a spongiform pattern with gas bubbles . In typical cases, however, it can be difficult to diagnose the disorder with ct scans in cases without gas bubbles . In the case described in this paper, radiologists using findings from a ct scan, initially and incorrectly diagnosed the lesion as gist with fibrosis . However, the disorder was correctly diagnosed by a team of gastroenterologists using eus - fna . The eus - fna procedure revealed a segment of string in a fixed foreign body in the pelvis . The application of eus using curved linear array (cla) endoscopes in the lower gi tract has been limited to the rectum and distal sigmoid colon because of the oblique - viewing optics . There were case reports using oblique - viewing cla echoendoscopes to evaluate lesions proximal to the sigmoid colon, but advancement of the cla echoendoscope beyond the sigmoid required the use of an overtube or a guide wire previously placed with a colonoscope . Binmoeller et al . Have reported the feasibility and safety of the front - view forward - array echoendoscope for evaluating right colon lesions . Thus, we used a front - view forward - array echoendoscope to safely evaluate the intrapelvic tumor and sample the tissue . A correct diagnosis was obtained using this method . In summary, this is the first known case report diagnosing gossypiboma by eus - fna.
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To demonstrate the usefulness of enhanced depth imaging optical coherence tomography (edi - oct) in investigating choroidal lesions inaccessible to ultrasound sonography . In a 60-year - old woman with an asymptomatic choroidal nevus, normal oct was used to observe the macula and edi - oct to image the choroidal nevus that was inaccessible to ultrasound . The exact location of the lesion in the choroid and the dimensions of the nevus were measured . The lesion was located in the superior macula, and the nevus was homogeneous in its reflectivity . We observed a thickened choroid delineated by the shadow cone behind it, measuring 1,376 325 m in the larger vertical cut and 1,220 325 m in the larger horizontal cut in an image with a 1:1 pixel mapping and automatic zoom . Edi - oct appears to be an excellent technique for measuring choroidal nevi and all choroidal lesions accessible to oct imaging by depicting their exact location in the choroid, their dimensions, and their demarcation from the surrounding healthy tissue, thus allowing for a more efficient and accurate follow - up . Choroidal nevus is the most common tumor of the ocular fundus, present in 7% of the white population [1, 2]. Despite its benign nature, it presents a risk of visual loss and visual field loss, and can rarely transform into malignant melanoma . Optical coherence tomography (oct) and ultrasonography have been used for imaging choroidal nevi . Recently, enhanced depth imaging oct (edi - oct) has been used in order to measure choroidal thickness in normal and pathologic eyes [3, 4]. Here, we report a case of a flat choroidal nevus inaccessible to ultrasound sonography that was evaluated by edi - oct . A 60-year - old woman was referred to our department with an asymptomatic choroidal nevus in her left eye . Her best - corrected visual acuity was 20/20 in both eyes . Slit lamp and fundus examinations of the right eye were normal . In the left eye, the anterior segment was normal; however, on fundoscopy a choroidal nevus located in the superior macula was observed (fig . Spectral - domain (sd) oct (heidelberg engineering, heidelberg, germany) revealed normal foveal thickness (fig . 2). The flat nevus was inaccessible to ultrasound sonography, and edi - oct was used to image its posterior surface (fig . Edi - oct is a new technique that consists of positioning a heidelberg sd - oct close enough to the eye in order to obtain an inverted image, averaged for 100 scans . The advantage of this technique is that the sensitivity of the imaging in deeper tissue layers is increased, and thus the obtained measurements are more accurate . We employed this technique to image the location of the nevus in the choroid and obtain its dimensions as a reference measure for the follow - up examination (fig . 3). Heidelberg sd - oct provides an exact correlation between the oct image and the infrared photograph . As infrared light is absorbed by melanin, the nevus appears brighter . In the edi - oct image, the lesion appears homogeneous and hyperreflective at the level of the large choroidal vessels, masking the underlying choroidal vasculature . The choroidal thickness at the level of the nevus appears greater than the neighbor choroid (fig . The diameter of the nevus was calculated based on the presence of a shadow cone created by the lesion's lateral borders . The choroidal thickness was measured based on the hyperreflectivity between the bruch's membrane and the beginning of the shadow cone (fig . The choroid measured 1,220 325 m in the larger horizontal cut and 1,376 325 m in the larger vertical cut in an image with a 1:1 pixel mapping and automatic zoom (fig . Edi - oct appears to be an excellent technique for evaluating flat choroidal nevi that are inaccessible to ultrasound sonography and accessible to oct imaging, displaying simultaneously the lesion and the retinal changes . It is a reproducible examination that allows depiction of the exact location of the nevi in the choroid, their dimensions and their demarcation from the surrounding healthy tissue . To date, the software available for sd - oct does not allow for objective measures . The subjective measures obtained are useful upon comparison with the follow - up images . In order to limit the possibility of bias, these follow - up images must be obtained and compared with the same sd - oct machine . We believe that this technique is clinically useful and, together with color fundus photography, can provide a more efficient and accurate follow - up of choroidal nevi.
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Kelch - like ech - associated protein 1 (keap1)-nuclear factor e2-related factor 2 (nrf2) is a major cellular pathway that protect normal cells against oxidative and xenobiotic damage.1 nrf2 is an essential transcription factor for antioxidant and detoxification genes and is crucial for the chemopreventive effect of various phytochemicals against carcinogenesis . Representative chemopreventive agents that induce nrf2 include carotenoids, curcumins, cyclic lactones, diterpenes, dithiolethiones, epithionitriles, flavonoids, indoles, isothiocyanates, organosulfides, and phenols.2,3 accumulating evidences have demonstrated that phytochemicals can protect cells from oxidative stress related diseases including inflammatory, cardiovascular, and neurodegenerative diseases and cancer, through nrf2-dependent responses . Paradoxically, recent researches demonstrated the dark side of nrf2.4 cancer cells acquire a growth advantage by eliminating keap1-mediated negative control of nrf2, which leads to activation of the nrf2-dependent defense response.4 constitutive upregulation of nrf2 has been found in many types of cancers, including skin, breast, prostate, lung, head and neck, and endometrial cancer.5 overexpressed nrf2 provides a growth advantage for cancer cells by protecting those cells from oxidative stress and anticancer agents, thus contributing to chemoresistance.5 in this review, we summarize the therapeutic development of nrf2 inhibitors that enhance the efficacy of the current cancer treatments . Nrf2 is a basic leucine zipper transcription factor with 7 domains from neh1 to neh7 . Neh1 has dna binding motifs.6 neh3, neh4, and neh5 have domains involved in the transactivation of nrf2 target genes by binding coactivators . Neh2 has a major regulatory domain with 2 binding sites for keap1 named etge and dlg (fig . Keap1 has 5 functional domains including kelch, intervening region (ivr), broad complex / tramtrack, bric - a - brac domain (btb), an n - terminal region (ntr), and a c - terminal region (ctr).3 cysteine residues of ivr play as a sensor for reactive oxygen species (ros). The btb domain has a cys151 residue and is responsible for nrf2 ubiquitination through binding to cullin e3 ubiquitin ligase (cul3)-based ubiquitin e3 ligase (fig . 1a). Under unstressed conditions, nrf2 forms a complex with keap1, leading to polyubiquitination and subsequent degradation by the 26s proteasome (fig . 2a).7 upon exposure to a variety of stressors, including ros, toxicants, and carcinogens, nrf2 is released due to modification of the cysteine residues of keap1 (fig . 2b).8 two mechanistic models have been proposed for the regulation of nrf2: the hinge and latch model and the keap - cul3 dissociation model.810 in the hinge and latch model, 2 binding motifs of the neh2 domain in nrf2 have different affinities.9 for the ubiquitination, both the dlg and etgf motifs should be occupied by keap1 proteins . Ros modify the cysteine residues of keap1, leading to the release of the dlg motif (latch) without changing the etgf motif (hinge) on neh2 . Another model is the keap - cul3 dissociation model in which oxidative stress disrupts the keap1-cul3 e3 ligase interaction without changing the conformation of keap1.8 nrf2 stabilization with de novo synthesis increases the cytoplasmic level of nrf2 . Nrf2 translocates into the nucleus and binds to an antioxidant response element (are) as a heterodimer with musculo - aponeurotic fibrosarcoma (maf) to recruit a transcriptional coactivator and promote thetranscription of various cytoprotective genes that are associated with antioxidant and detoxification enzymes . Upon recovery of cellular redox homeostasis, keap1 translocates to the nucleus to dissociate nrf2 from the are resulting in degradation of nrf2.11 nrf2 binds to ares in promoter regions of various genes that regulate the cellular response to oxidative and xenobiotic stress . Nrf2 target genes include antioxidant genes and phase ii enzymes such as heme oxygenase-1 (ho-1), nad(p)h: quinone oxidoreductase 1 (nqo1), glutathione s - transferase (gst), and glutathione peroxidases . The are - bearing genes that are regulated by nrf2 in humans have been studied using microarray analysis of genes induced by chemopreventive agents or knockout of keap1 in human cell lines.2 these genes can be categorized into 5 groups: antioxidant genes (gclc, gclm, gpx2, gsr, slc7a11, srxn1, and txnd1), nadph - generating enzymes (g6pd, me1, and pgd), metal - binding proteins (fth1, ftl, mt1, and mt2), drug - metabolizing enzymes and drug transporters (akr1b1, akr1b10, akr1c1, akr1c2, akr1c3, akr1c4, cbr1, gstm3, mrp2, nq1, and ptgr1), and stress response proteins (gadd45, hmox1, hsp40, and hsp70).2 knockdown of keap1 in human keratinocytes resulted in upregulation of 23 mrnas while akr1b1, akr1b10, and akr1c1 were induced to the greatest extent, showing increases of between 12- and 16-fold indicating that aldo - keto reductases could be useful biomarkers for nrf2 activation.12 as keap1 is responsible for inhibitor kappa b (ib) kinase beta (ikk-) ubiquitination, upregulation of some of these genes could be attributable to the increase of nuclear factor - kappa b (nf-b) activity.13 expression profiling in mice has revealed that nrf2 regulates approximately 100 genes and that two - thirds of nrf2-target genes are not involved in detoxication or antioxidation . Instead, many of the target genes are associated with inflammation and immunity proteins.2 the aryl hydrocarbon receptor (ahr) is a transcription factors that mediates the biological effects of its xenobiotic ligands including dioxin.14 the ahr activates the transcription of nqo1 and other genes through the xenobiotic - responsive element (xre).15 ahr induces the direct binding of nrf2 to promoters or indirectly produces reactive intermediates that trigger the nrf2 signaling pathway.16 the cross - talk between nf-b and nrf2 is more complex . Several chemopreventive agents, such as sulforaphane, epigallocatechin-3-gallate, and curcumin, induce nrf2 signaling with concomitant repression of nf-b.14 the nf-b p65 subunit re - presses the nrf2-are pathway by preventing creb binding protein (cbp) from binding nrf2 through competitive interaction or by promoting the recruitment of histone deacetylase 3 (hdac3), a corepressor, to the are.17 in chondrocyte apoptosis, shear - induced cyclooxygenase-2, an nk-b target gene, can suppress phosphatidyl inositol 3-kinase (pi3k) activity, causing a reduction in thenrf2-mediated transcriptional response.18 in contrast, nrf2 inhibits the activity of nf-b by attenuating phosphorylated ib . Nrf-2 target genes, including ho-1 have been reported to negatively affect nf-b activation.19,20 p53 has been reported to suppress the nrf2-dependent transcription of antioxidant response genes by directly interacting with are - containing promoters . Previous studies have suggested that the function of this negative regulation is to prevent the formation of antioxidant environment, which could hinder the induction of apoptosis by p53.21 in contrast, direct targets of p53 could upregulate nrf2 activity . Studies have shown that p21 inhibits the degradation of nrf2 by interacting with the dlg motif, leading to upregulation of antioxidant genes.22 these dual functions of p53 toward nrf2 indicate that p53 may decide the fate of damaged cells whether to repair the damaged cells or apoptosis . Nrf2 is also involved in autophagy by inducing p62 which is a receptor for autophagic degradation . As p62 could block binding between nrf2 and keap1, p62 contributes to the activation of nrf2 target genes in response to oxidative stress creating a positive feedback loop.23 the association of nrf2 overexpression and chemoresistance has been reported in many cancers including non - small cell lung cancer, stomach cancer, endometrial cancer, and osteosarcoma.2427 overexpression of nrf2 target genes was also found in a variety of solid tumors.28 ho-1 overexpression was found in brain cancer, prostate cancer, and renal cancer.29,30 nqo1 is known to be overexpressed in hepatoblastoma, colon cancer, breast cancer, and non - small cell lung cancer.31 several mechanisms have been proposed for the activation of keap1-nrf2 signaling in cancer . Mutation of keap1 can result in accumulation of nrf2 in nucleus through decreased degradation or inhibition of nuclear export.28 in non - small cell lung cancer, heterozygous mutation of keap1 (8% of patients) was reported to be sufficient for nrf2 overexpression.32 in addition to lung cancer, mutations in the keap1 gene were found in various cancers including breast (2%), colon (8%), gastric (11%), liver (9%), ovary (19%), and prostate (8%).3336 mutations in the neh2 domain of nrf2 were found in lung (11%), cancer (6%), and head and neck cancers (25%).37,38 all of these mutations were somatic mutations . Mutation of egfr, kras, braf, myc, and the bcr - abl fusion can activate nrf2, resulting in enhancement of ros detoxification and other oncogenic roles of nrf2, including chemoresistance.3941 furthermore, posttranslational modifications can activate the keap1-nrf2 signaling pathway . Hypermethylation of the keap1 promoter was found in 47% of lung cancer patients and this feature was associated with poor outcome.42 epigenetic regulation of keap1 was also found in malignant glioma, colon cancer, and breast cancer.4345 in addition to the upregulation of cytoprotective genes, constitutive expression of nrf2 may confer a survival advantage to cancer cells by promotion of cell proliferation, chemoresistance and inhibition of apoptosis . An active pi3k - akt pathway augments the nuclear accumulation of nrf2 which then re - directs glucose into the anabolic pathway to increase metabolism, indicating the reinforcement of metabolic reprogramming by nrf2.46 nrf2 could mediate cell proliferation with dual regulation through epidermal growth factor receptor (egfr) signaling and keap1 interactions.39 in cells with the keap1 gene mutation (a549 cells), activated nrf2 promotes cell proliferation independent of egfr signaling . Therefore, egfr tyrosine - kinase inhibitors are intrinsically ineffective in these types of non - small cell lung cancer.39 cancer metastasis and tumor progression requires the epithelial - mesenchymal transition (emt) and the loss of e - cadherin is considered to be a main event in emt . In hek293 cells, complex of e - cadherin and beta - catenin could bind to c - terminus of nrf2 preventing nuclear translocation of nrf2.47 as e - cadherin inhibits nrf2-mediated transcription, loss of e - cadherin could promote nrf2 translocation and confer an additional survival advantage to cancer cells.47 the keap1-nrf2 pathway is involved in the inhibition of apoptosis by interacting with p53 and b cell lymphoma-2 (bcl-2). P53 inhibits the activation of nrf2 target genes by direct interacting with are - containing promoters or activating p21.21 considering that p53-induced apoptosis requires the accumulation of ros, increased activity of antioxidant genes by nrf2 in cancer cells can inhibit p53 dependent apoptosis . Bcl-2 can repress cell death by dimerization with bcl-2-associated x protein (bax) and the bcl-2 homology 2 (bh2) domain of bcl-2 is required for this heterodimerization.48 it was found that keap1 binds to the bh2 domain and facilitates the ubiquitination of bcl-2 leading, to bax accumulation and enhanced apoptosis . Thus, mutations in the keap1 binding site for bcl2 are responsible for the anti - apoptotic effect as well as overexpression of nrf2.49 moreover, it has been reported that nrf2 can directly activate the transcription of bcl-2 and bcl - xl.50,51 many studies have reported the association between nrf2 upregulation and chemoresistance in various cancers, including gastric cancer, osteosarcoma, non - small cell lung cancer, endometrial cancer, bladder cancer, and neuroblastoma.2427,52 platinum drugs generate electrophilic molecules that damage cancer cells . Chemoresistance to these drugs can be explained by high expression of antioxidant nrf2 target genes.4 another mechanism of chemoresistance is the induction of the drug efflux pump family, which includes the mdr, by nrf2.53 some drugs, including hdac inhibitors, oxaliplatin, and proteasome inhibitors can induce nrf2 and thus decrease the effectiveness of chemotherapy.5456 in light of the data presented in this review, nrf2 is an attractive molecular target for the inhibition of cancer . In contrast to nrf2 activators, including numerous phytochemicals, only a small number of nrf2 inhibitors have been identified (table). Trigonelline, a coffee alkaloid, efficiently decreased basal and tertiary butylhydroquinone (tbhq)-induced nrf2 activity in chemoresistant pancreatic carcinoma cell lines (panc1, colo357, and miapaca2) which have high nrf2 activity . Along with inhibiting nrf2 the sensitivity of all cell lines to anticancer drugs and tumor necrosis factor - related apoptosis inducing ligand (trail)-induced apoptosis was enhanced by trigonelline.57 chrysin (5,7-dihydroxyflavone) is a natural flavonoid that is found in many plant extracts . In doxorubicin resistant hepatocellular carcinoma cell line (bel-7402/adm), chrysin significantly reduced nrf2 expression by downregulating the pi3k - akt and erk pathways.58 in that study, chrysin restored chemosensitivity by downregulating the nrf2-downstream genes such as ho-1, akr1b10, and mrp5 . Apigenin (4,5,7-trihydroxyflavone) is a natural flavone that is present in many fruits and vegetables and has various biological activities, such as anti - inflammatory and antioxidant properties . In a study using bel-7402/adm cell lines, apigenin reduced nrf2 expression as well as the expression of nrf2-downstream genes . When cells were treated with doxorubicin, apigenin showed a synergistic anti - tumor effect on bel-7402/adm cell lines, resulting in reduced cell proliferation and more substantially induced apoptosis.59 brusatol is a quassinoid that is extracted from brucea javanica . Brusatol reduced nrf-2 protein level without changing the keap1 level and sensitized various cells including hela, mda - mb-231, and a549 to chemotherapeutic agents such as carboplatin, 5-fluorouracil, etoposide, and paclitaxel.60 the anticancer effect of brustalol mediated by nrf2 was confirmed in a xenograft model indicating that brusatol could be used to combat intrinsic resistance . Ascorbic acid is an antioxidant that can suppress the level of nrf2 . In the imatinib - resistant chronic myelogenous leukemia cell line kcl22/sr, binding of nrf2 to the are of the gamma - glutamylcysteine synthetase gene promoter was much stronger than in the parental imatinib - sensitive cell line kcl22.61 furthermore, addition of ascorbic acid to kcl22/sr cells resulted in a decrease in nrf2-dna binding and partial restoration of imatinib sensitivity to kcl22/sr.61 luteolin (3,4,5,7-tetrahydroxyflavone) is a polyphenolic flavonoid found in high concentrations in celery, green pepper, and parsley . In a study using a cell - based are - reporter assay, luteolin was found to be a potent nrf2 inhibitor.62 in that study, luteolin significantly sensitized a549 cells to the anticancer drugs oxaliplatin, bleomycin, and doxorubicin indicating the potential application of luteolin as a natural sensitizer in chemotherapy . Constitutively expressed nrf2 can promote cancer cell proliferation and protect cells against oxidative stress and therapeutic agents . It must be kept in mind that chemopreventive agents including various phytochemicals can induce chemoresistance and tumor progression by activating the keap1-nrf2 pathway . However, it will be necessary to understand the molecular regulation of nrf2 and identifying the individualized status of nrf2 expression.
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A 3-year - old male, known to have isolated speech delay, presented to the emergency department with severe body aches and inability to walk . Few days prior to his presentation, the child started to have flu - like symptoms in the form of rhinorrhea and low - grade fever . This was followed by irritableness, hypoactivity, and inability to move his legs and arms . He was seen in a private hospital where computed tomography of the brain and a lumber puncture were done that showed no abnormalities . In the next day, his condition continued to worsen . He developed difficulty in swallowing and dark - colored urine, so he was transferred to our institution . Upon physical examination, the laboratory tests showed elevated creatine kinase level (1 778 856 u / l; normal range 20 - 200 u / l), elevated serum transaminases (serum glutamic - pyruvic transaminase [sgpt] = 1857 u / l [normal range 0 - 50 u / l], serum glutamic - oxaloacetic transaminase [sgot] = 8626 u / l [normal range 0 - 50 u / l]), and creatinine 0.72 mg / dl (0.3 - 0.7 mg / dl). The child started on intravenous hydration and received 1 dose of intravenous immunoglobulin . On the second day, the creatine kinase and serum transaminase levels improved, with creatinine reaching 0.94 mg / dl, the highest level reached . The creatine kinase and serum transaminase levels continued to improve gradually over the following days, with slow improvement in the clinical condition . He received another 2 doses of intravenous immunoglobulin on days 13 and 14 of admission . Muscle biopsy showed endomysial inflammatory infiltrates, mostly composed of cd8 and cd4 lymphocytes; myofibers necrosis mostly ventral portion of myofibers; residual granular microcalcifications; and terminal complement complex deposits that outline necrotic myofibers, all features compatible with necrotic myopathy and early rhabdomyolysis (figure 2). The arrows showed the degenerated myofibers . Upon discharge, the child had mild muscle aches, was able to sit, and move his arms and feet . He had persistent weakness in the lower limbs, but was unable to walk, with a follow - up examination 1 month after discharge, revealing that the child was able to walk normally with positive deep tendon reflex . Two months after discharge, the creatine kinase level dropped to 193 iu / l . Rhabdomyolysis results from skeletal muscle breakdown due to various causes . Viral myositis, trauma, and inherited disorders are the most common causes in the pediatric age group . The breakdown releases the normal cell contents into the bloodstream, including creatine kinase, myoglobin, phosphorus, and potassium . Rhabdomyolysis is suspected clinically when muscle aches, muscle weakness, and tea - colored urine are present . The diagnosis is based on the high creatine kinase level, which is the most sensitive marker . Although there is no established cutoff levels, a concentration 5 to 10 times higher than the upper limit of normal reference range (ie, 500 - 1000 other laboratory indicators include serum and urine myoglobin concentrations that may be useful but not essential for the diagnosis . The most common complication of rhabdomyolysis is kidney damage . In recent large pediatric studies reported by mannix et al and wu et al, it was shown that the rate of acute renal failure in the pediatric patients with rhabdomyolysis ranged from 5% to 8.7% . The risk factors that predispose a patient with rhabdomyolysis for developing acute renal injury are creatine kinase level concentration more than 5000 u / l, creatine kinase value upon admission, and slower decline in the serum creatine kinase level . Other factors include high myoglobin level, persistent or abrupt increase in the potassium or calcium level, as well as persistent metabolic acidosis . On the other hand, a recent study by fernandez et al showed that the most reliable predictor of acute renal failure and the need for dialysis is the creatinine level above 1.7, despite the peak creatine kinase . The mainstay for the prevention and treatment of acute kidney injury is early and aggressive volume resuscitation . Other management options include alkalization of the urine, forced diuresis with mannitol, and loop diuretics . In severe cases or in our case, the child developed extremely high creatine kinase levels that were not reported in the literature . A thorough literature review was done searching for high creatine kinase levels in patients with acute rhabdomyolysis . In 2014, a case report described a pediatric patient with mcardle disease who developed rhabdomyolysis with a high 6-digit creatine kinase level reaching 500 000 2 pediatric cases were reported . In both, the creatine kinase levels reached were 60 000 a case report of an adolescent with rhabdomyolysis due to undiagnosed hypothyroidism was reported with a creatine kinase level that reached around 34 000 a research article in 2013 examined the clinical spectrum of patients with rhabdomyolysis presenting to the pediatric emergency department, in which the peak serum level of creatine kinase was 9825.1 23 079.1 the only risk factor for developing acute renal injury that our patient had was a high peak creatine kinase level upon admission . On the other hand, he did not develop metabolic acidosis and had a normal albumin and electrolytes levels throughout his hospitalization . He responded very well to volume resuscitation with rapid decline in the creatine kinase level . Although he received intravenous immunoglobulin for the fear of guillain - barre syndrome or other underlying immune - mediated diseases, neither the creatine kinase level decline nor the clinical status improvement was affected by the intravenous immunoglobulin administration . The sharp decline in the creatine kinase level there are no clues in the family history or the patient s personal history that may suggest an underlying inherited metabolic, muscular, or genetic disease ., this could be the first presentation of lipin-1 mutation that causes recurrent rhabdomyolysis in children . Lipin-1 gene encodes the muscle - specific phosphatidic acid phosphatase, a key enzyme in triglyceride and membrane phospholipid biosynthesis . The episodes of rhabdomyolysis are mostly triggered by intercurrent infections and fever and to a lesser extent by fasting or exercise . The prognosis of lipin-1 deficiency is poor, with up to one - third of patients dying during an episode of rhabdomyolysis . In about 60% of patients with recurrent rhabdomyolysis,
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The study was approved by the institutional ethics committee, and informed consent was obtained from all patients in accordance with the tenets of the declaration of helsinki . The donors were selected based on these inclusion criteria: myopic spherical refractive error ranging from 0.5 d to 10.00 d with no astigmatism or astigmatism less than 0.5 d; healthy eyes free from significant systemic and ocular diseases; and ability to understand the content of the consent form and willingness to undergo serological testing . Eligible candidates underwent the relex smile procedure with the standard technique11 by the same surgeon (s.g .) Using the visumax fs laser . The cap thickness was 100 m, and the optical zone varied from 6 to 6.5 mm . After dissection of both anterior and posterior planes, the lenticule was extracted through a superior 2-mm incision . Bites were taken from one end to the diagonally opposite end, with the knot tied on the superior aspect of the lenticule to determine the side at the time of subsequent implantation . The lenticule was immediately transferred to a sterile plastic vial containing phosphate - buffered solution, labeled with the patient's identification number, eye, and date, and sent to the cryobank for processing and cryopreservation . Two types of solutions (washing solution and cryopreservation solution) were prepared . The washing solution comprised 10 ml of phosphate - buffered saline (1), 200 l of penicillin streptomycin (100), and 200 l of amphotericin b (100). The cryopreservation solution comprised 9 ml of tissue culture media (cryobank wash media) and 1 ml of 20% dimethyl sulfoxide (dmso). The tissue culture medium is a commercially available medium used for semen processing in reproductive biology (cryocell india pvt ltd, new delhi, india). The medium contains sodium chloride, potassium chloride, magnesium sulfate, anhydrous potassium phosphate monobasic, calcium chloride, anhydrous sodium bicarbonate, hepes, glucose, sodium pyruvate, sodium lactate, phenol red, and human albumin in company - specified compositions . Extracted lenticules were washed twice for 10 minutes each in the washing solution (fig . The lenticules were then transferred into a cryovial and resuspended in 500 l medium containing 10% tissue culture media . Using a liquid dropper, a stock freezing solution containing 10% human tissue culture medium and 20% dmso (sigma - aldrich, st louis, mo) was added slowly to make a final volume of 1 ml freezing solution containing 10% human tissue culture medium and 10% dmso . The cryovials containing the lenticules were transferred in canisters and frozen in liquid nitrogen at a controlled cooling rate that gradually brought down the temperature from 4c to 196c and stored in long - term storage containers (ibp; indian oil corporation limited, nasik, india)., the tissue was transported from the cryobank to the hospital in a liquid nitrogen container . The vials containing frozen tissue were gradually thawed by rubbing them between the palms or keeping them at room temperature for 5 to 10 minutes until the frozen medium inside the vial was liquefied . The lenticule was then transferred into a petri dish and washed twice with balanced salt solution for 5 minutes each to remove the cryoprotectant agents . A visumax fs laser was used to create a 7.5 mm diameter pocket at a depth of 160 m and 4 mm superior incision . The incision was opened with a seibel spatula and the plane of the pocket was dissected . The cryopreserved lenticule (which was matched for the refractive error after correcting for back vertex distance) was placed on the patient's cornea with its center marked with gentian violet dye . After insertion, the lenticule was spread out, and the center was aligned with the pupillary center . A depth of 160 m was chosen for implantation in all patients due to uncertainty in refractive outcomes, the novelty of the nomograms, and so the surgeon could have adequate tissue in the cap for later enhancement with surface ablation if required . Postoperatively, a topical steroid (1% prednisolone acetate; allergan, irvine, ca) was prescribed for 3 months in a tapering dosage . In addition, 5% hypertonic saline drops (hypersol-5; jawa pharmaceuticals, india) were prescribed 6 times per day for 1 week to reduce endothelial stress and to aid in lenticule clearing . Patients were examined postoperatively on days 1 and 15 and at 1, 3, and 6 months . A slit - lamp examination was performed to check for lenticule clarity, position, and wound healing . Postoperative corneal haze and folds were graded at every follow - up time point using the scale described by nakamura et al and the corneal folds grading atlas.12,13 on postoperative day 15 and onward, the following assessments were performed: uncorrected visual acuity, best - corrected visual acuity (bcva), retinoscopy for residual refractive error, topography using orbscan(bausch & lomb - technolas, munchen, germany) and sirius (schwind eye - tech solutions, kleinostheim, germany), anterior segment optical coherence tomography (optovue, fremont, ca), clinical photography, specular microscopy (tomey, japan), dry eye assessment (schirmer's i), and aberrometry (itrace; tracey technologies, houston, tx). Two types of solutions (washing solution and cryopreservation solution) were prepared . The washing solution comprised 10 ml of phosphate - buffered saline (1), 200 l of penicillin streptomycin (100), and 200 l of amphotericin b (100). The cryopreservation solution comprised 9 ml of tissue culture media (cryobank wash media) and 1 ml of 20% dimethyl sulfoxide (dmso). The tissue culture medium is a commercially available medium used for semen processing in reproductive biology (cryocell india pvt ltd, new delhi, india). The medium contains sodium chloride, potassium chloride, magnesium sulfate, anhydrous potassium phosphate monobasic, calcium chloride, anhydrous sodium bicarbonate, hepes, glucose, sodium pyruvate, sodium lactate, phenol red, and human albumin in company - specified compositions . Extracted lenticules were washed twice for 10 minutes each in the washing solution (fig . The lenticules were then transferred into a cryovial and resuspended in 500 l medium containing 10% tissue culture media . Using a liquid dropper, a stock freezing solution containing 10% human tissue culture medium and 20% dmso (sigma - aldrich, st louis, mo) was added slowly to make a final volume of 1 ml freezing solution containing 10% human tissue culture medium and 10% dmso . The cryovials containing the lenticules were transferred in canisters and frozen in liquid nitrogen at a controlled cooling rate that gradually brought down the temperature from 4c to 196c and stored in long - term storage containers (ibp; indian oil corporation limited, nasik, india). On the day of surgery, the tissue was transported from the cryobank to the hospital in a liquid nitrogen container . The vials containing frozen tissue were gradually thawed by rubbing them between the palms or keeping them at room temperature for 5 to 10 minutes until the frozen medium inside the vial was liquefied . The lenticule was then transferred into a petri dish and washed twice with balanced salt solution for 5 minutes each to remove the cryoprotectant agents . A visumax fs laser was used to create a 7.5 mm diameter pocket at a depth of 160 m and 4 mm superior incision . The incision was opened with a seibel spatula and the plane of the pocket was dissected . The cryopreserved lenticule (which was matched for the refractive error after correcting for back vertex distance) was placed on the patient's cornea with its center marked with gentian violet dye . The lenticule was held with 2 forceps and inserted into the pocket . After insertion, the lenticule was spread out, and the center was aligned with the pupillary center . A depth of 160 m was chosen for implantation in all patients due to uncertainty in refractive outcomes, the novelty of the nomograms, and so the surgeon could have adequate tissue in the cap for later enhancement with surface ablation if required . Postoperatively, a topical steroid (1% prednisolone acetate; allergan, irvine, ca) was prescribed for 3 months in a tapering dosage . In addition, 5% hypertonic saline drops (hypersol-5; jawa pharmaceuticals, india) were prescribed 6 times per day for 1 week to reduce endothelial stress and to aid in lenticule clearing . Patients were examined postoperatively on days 1 and 15 and at 1, 3, and 6 months . A slit - lamp examination was performed to check for lenticule clarity, position, and wound healing . Postoperative corneal haze and folds were graded at every follow - up time point using the scale described by nakamura et al and the corneal folds grading atlas.12,13 on postoperative day 15 and onward, the following assessments were performed: uncorrected visual acuity, best - corrected visual acuity (bcva), retinoscopy for residual refractive error, topography using orbscan(bausch & lomb - technolas, munchen, germany) and sirius (schwind eye - tech solutions, kleinostheim, germany), anterior segment optical coherence tomography (optovue, fremont, ca), clinical photography, specular microscopy (tomey, japan), dry eye assessment (schirmer's i), and aberrometry (itrace; tracey technologies, houston, tx). Table 2 provides the details of donors and lenticules used for fili in the 9 eyes treated . Demographic details of patients who underwent fili (7 patients, 9 eyes) details of donors and lenticules used for fili (n = 9) on day 1, in the first 2 consecutive eyes, the implanted lenticules had mild to moderate descemet membrane folds that resolved spontaneously within 1 week . Subsequently, the treated eyes were prescribed hypertonic saline drops, which showed clear lenticules within 48 hours and until the last follow - up time point . Figure 2 shows serial clinical photographs and anterior segment optical coherence tomography of the eye treated with fili at day 15 and at 6 months post - fili showing clear lenticules with good centration . A, serial digital clinical photographs (16) of 32-year - old woman operated for + 6.5 d hyperopia in the right eye with fili . Distance from edge of the lenticule to limbus was measured at 3 points and verified at every visit to check for centering and any shift in position . B, six - month postoperative anterior segment optical coherence tomography of an eye treated for + 6.5 d hyperopia showing a clear and well - centered lenticule . All eyes had an uncorrected visual acuity equal to or better than the preoperative bcva . The refractive predictability for hyperopia was fairly accurate, and all eyes had a residual spherical equivalent within 1.0 diopter (d), although in the aphakic eye, the residual spherical equivalent was + 4.1 d. there was an average of 0.75 to 1.00 d against - the - rule astigmatism in all eyes due to the superior 4 mm incision, which reduced and stabilized over time . Postoperative results of eyes treated with fili (n = 9) table 4 provides changes in anterior and posterior curvature, pachymetry and asphericity (q value) following fili . All eyes had central corneal steepening with a mean change in anterior keratometry of 3.5 d in the central 3-mm zone . There was an average 0.33 d flattening of posterior corneal curvature in all eyes, which was seen more in thicker lenticules compared with the thinner ones . Figure 3 illustrates changes in anterior and posterior curvature post - fili in an eye, over time . Changes in anterior and posterior topography, pachymetry, and asphericity post - fili (n = 9) orbscan anterior corneal surface topography (top) and sirius posterior elevation (bottom) over the 6-month postoperative course after fili for eye with + 6.5 d hyperopia . The postoperative q values in all eyes became more negative, suggesting a hyperprolate shift . Table 5 provides analysis of the epithelial thickness profile, which suggested mild epithelial thinning in the 5-mm zone in all hyperopic eyes, except for the aphakic eye, which showed mild epithelial thickening in the corresponding zone . Figure 4 illustrates the epithelial thickness map of an eye treated with fili at 6 months . Epithelial thickness profile on anterior segment optical coherence tomography in the central 5-mm zone epithelial thickness profile with anterior segment optical coherence tomography of an eye 6 months post - fili for + 6.5 d hyperopia . Higher order aberrations (hoa) did not show a significant increase in root mean square values postoperatively in the 8 hyperopic eyes (p> 0.05). Endothelial cell count and schirmer i test scores were normal and the changes were not significant at the last follow - up (p> 0.05) (table 6). Total hoas at 5-mm pupil size (n = 8), ecd, and schirmer's i scores, pre- and post - fili (n = 9) the potential advantages of cryopreservation of refractive lenticules have been largely described for use in the same patient, in the event of future ectasia or presbyopia by restoring corneal volume.3 with the increasing number of eyes undergoing relex smile for myopia, and thus, the extracted lenticules as a by - product, it may be a novel idea to preserve these lenticules on a long - term basis using cryopreservation for potential future use and research . Following establishment of one such tissue bank, we wanted to explore the possibility of use of such cryopreserved refractive lenticules for potential treatment of hyperopia and study the feasibility, safety, efficacy, and reproducibility of this new treatment . Initially, the amblyopic eyes were studied mainly for changes in refraction and topography following tissue addition . There was also the goal of evaluating any loss of bcva and complications, such as haze and tissue rejection . Even these patients benefited from the surgery, as their refractive error was corrected to their best spectacle - corrected visual acuity and reduced their dependence on eyeglasses . After reviewing the results for 6 months, the indications were extended to normal eyes and bilateral surgery . Unlike myopia, the correction of hyperopia involves creation of a furrow - like ring zone in the paracentral zone of the cornea . Both photorefractive keratectomy and lasik are subtractive procedures that steepen the cornea by ablating the midperipheral tissue.14,15 this creates an abnormal hyperprolate shape of the cornea that is associated with significant regression, especially in higher degrees of refractive errors.16 it potentially leads to the induction of hoas like coma and spherical aberrations and loss of bcva.17 fili is an innovative technique based on barraquer's law of thickness,18 whereby the cornea is made steeper by addition of a lenticule of known thickness and power into a pocket created in a patient's cornea using the fs laser . The concept is similar to epikeratophakia, used to treat aphakia in the past, which had its own disadvantages.19 a fs laser overcomes these problems by creating precise lenticules and incisions at an accurate depth . The lenticular tissue processing technique described by mohamed - noriega et al10 was modified in this study by replacing bovine serum albumin with human tissue culture media to eliminate the risk of transmission of zoonotic diseases or unexpected adverse reactions due to bovine serum . The protocols of gradual staged thawing, which are essential for the whole cornea to prevent endothelial cell injury due to sudden thawing, were not strictly followed in this study . Microbiological and cellular cultures were not deemed necessary, as the tissue processing and preservation were conducted under strict sterile conditions, and the tissue did not contain vital endothelial cells . Also, previous studies have suggested that stromal keratocytes within the extracted lenticules remain viable and can be stimulated to proliferate in appropriate cell culture conditions, even after a prolonged period of cryopreservation.9,10 the change in asphericity after fili suggests an increase in the prolateness of the cornea, which is expected after tissue addition, although the observed change was not seen as much as it is after hyperopic lasik.20 the shape of the cornea is more natural after tissue addition compared with tissue subtraction, which does not lead to significant induction of hoas.21 the total hoas after fili remain within normal acceptable values and may be better than hyperopic lasik / photorefractive keratectomy.22,23 also, tissue addition may be more forgiving for mild decentration, which is not the case with hyperopic lasik . The rate of enhancement following hyperopic lasik can be up to 10% or more,24 especially in younger patients . After tissue addition, significant changes in topography or regression are not expected; thus, retreatment rates may be lower . Epithelial thickness profile is more favorable, which, unlike hyperopic lasik, does not cause midperipheral epithelial hyperplasia to compensate for loss of stromal tissue, hence reducing chances of regression.25 problems of haze and flap - related issues like delayed healing, infection, and dislocation are eliminated, thereby leading to greater patient comfort and faster recovery . A fairly good refractive predictability was observed with this technique in the treatment of moderate hyperopia, although results were not highly accurate for correcting very high hyperopia (eg, aphakia). In a case study reported by pradhan et al,4 a 10 d lenticule was implanted to achieve correction of aphakia but had a high residual refraction of + 5 d. there are possible explanations for this result . First, the technique of pradhan et al of pocket creation was different from the technique used in this study . In the said study, a smile lenticule was first created, followed by dissection of the anterior plane to create a pocket, thus making 2 planes that could possibly cause weakening of the cornea . Second, the optical zone of the lenticule in the study of pradhan et al was 5.7 mm compared with 6 mm in the current aphakia case, causing comparatively less volume of tissue addition . The correction is expected to be relatively higher in larger lenticules because there is more volume of the tissue added . In this hyperopic series, the lenticule size ranged from 6.3 to 6.8 mm . Also, the posterior curvature changes as described in the case study of pradhan et al reduced the effect on anterior curvature . This was also probably the cause of the high + 4.1 d residual refraction in the current study . After compensation, implantation of lenticule of approximately 12.5 d is required, and this is currently not possible with visumax . The biomechanics of corneas in these patients were not studied due to lack of equipment such as the corvis (oculus optikgerte gmbh, wetzlar, germany). However, because most of the bowman layer is preserved and tissue is being added, it is assumed that the biomechanics are more stable than lasik, where a flap is cut and tissue is subtracted from the periphery.26 the procedure certainly amounts to corneal transplantation and thus, theoretically carries the risk of future rejection . However, unlike traditional full thickness grafts, which have a graft host junction proximal to host limbus, in this technique, the lenticule was well protected in the host corneal pocket and did not come in direct contact with the host's limbal vasculature and immune system, making the risk of allogeneic graft rejection remotely possible.27 also, the lenticule was mainly stromal collagen tissue; the incidence of rejection is significantly low in such a scenario compared with endothelial rejection . Because no sutures are applied, there are no suture - related complications such as loosening, vascularization, or infection that would potentially trigger graft rejection.28 nevertheless, there is a need to monitor for signs of rejection and treat it like allogeneic graft rejection should rejection occur . Because it is a reversible procedure, the lenticule can always be explanted in the event of any adverse effects or rejection in future . The preliminary results of fili in hyperopia indicate that this may be a better technique than hyperopic lasik for moderate hyperopia in terms of stability, regression, aberrations, and postoperative dry eye . Although it is a bit early to exactly predict the refractive outcomes, initial data indicate that the cryopreservation technique used is safe in heterologous individuals . Further studies with a larger cohort and modification of the surgical technique with a goal of reducing postoperative astigmatism and refining nomograms are suggested to determine the long - term safety and refractive effects on the cornea and establish the technique as a valid option to treat hyperopia.
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Sepsis is the major cause of death in the intensive care unit . Despite improvement of antibiotics treatment and supportive techniques, the mortality of septic shock increases to approximately 60% ., it not only helps doctors to make an early diagnosis of sepsis, but also predicts outcomes . There have been some biomarkers and cytokines used in both the clinical practice and laboratory including soluble triggering receptor expressed on myeloid cells-1 (strem-1), procalcitonin (pct), n - terminal probrain natriuretic peptide (nt - pro - bnp), c - reactive protein (crp), interleukin-6 (il-6), and interleukin-10 (il-10). Trem-1 is a recently discovered member of the immunoglobulin superfamily of receptors that is specifically expressed on the surfaces of neutrophils and monocytes . Strem-1 is a soluble form of trem-1 and is upregulated when exposed to infectious diseases . Pct is a polypeptide consisting of 116 amino acids and is the precursor of calcitonin; it was proven useful to identify nonsystemic inflammatory response syndrome and was firstly used in sepsis . Nt - pro - bnp is a biologically inactive form that is cleaved from the prohormone probrain natriuretic peptide (pro - bnp) by proteolytic enzymes before secretion . Crp is a widely used biomarker to discriminate the inflammatory response to sepsis . Il-6 and il-10 are important proinflammatory and anti - inflammatory cytokines during sepsis course . . Indicated that strem-1 was more accurate than pct and crp in the diagnosis of sepsis, but others showed that the prognostic utility of serum strem-1 in septic shock was inferior to that of pct . The purpose of the study was to compare the prognostic value of biomarkers and cytokines versus clinical severity scores and improved death risk prediction . A total of 102 patients with sepsis from single centre hospital intensive care unit were enrolled from december 2010 to august 2012 according to the 2001 international sepsis definition conference . Exclusion criteria included: age younger than 18 years, preexisting thyroid disease and lung cancer that influence procalcitonin levels, patients with acute coronary syndromes and renal dysfunction, and patients staying in icu less than 24 hours . The study was approved by the hospital's ethics committee and either the patients or their relatives provided informed consent . Demographic and disease data of patients included age, gender, chief complaints for admission, vital signs, length of stay in icu, infection sites, microorganisms, routine blood test results, liver and kidney functions, coagulation indicators, blood gas analysis, acute physiologic assessment and chronic health evaluation (apache) ii scores, and sequential organ failure assessment (sofa) scores . These were recorded on 3 days (days 1, 3, and 5). Serum was collected at these same time points and pct, strem-1, nt - pro - bnp, crp, il-6, and il-10 levels were determined in the end . Pct was measured using an enzyme - linked fluorescence analysis kit (elfa, vidas brahms pct kit, biomerieux sa, france). Strem-1 was determined using a double antibody sandwich elisa (quantikine human trem-1 immunoassay elisa kit, r & d systems, minneapolis, mn, usa). Nt - pro - bnp was measured with an available immunoassay analyzer (elecsys 2010; roche diagnostics, mannheim, germany). Crp was determined using scattering using a nephelometric assay (dade - behring, sa paris, france). Il-6 and il-10 were determined using elisa (immulite; diagnostics products corporation, los angeles, ca). Quantitative data with normal distributions are given as means standard deviations (sd). Quantitative data that were not normally distributed were summarized as medians (interquartile ranges) and compared by nonparametric tests (mann - whitney u test). We made a logarithmic conversion for the nondistribution data when we did dynamic comparison in figure 2 . Proportions were used to express qualitative data and the differences in proportions between groups were compared using a chi - square test . We compared the characteristics of survivors versus nonsurvivors using univariate analysis and used receiver operating characteristics (roc) curves to evaluate prognostic value of the biomarkers and cytokines predicted 28-day mortality . Those variables with p values less than 0.05 on univariate analysis were then entered into a multivariate logistic regression analysis to further identify the independent predictors of 28-day mortality . There were no significant difference in age and sex of these two groups (p> 0.05). The apache ii and sofa scores of patients in the nonsurvival group were higher than those of patients in the survival group (p = 0.000, p = 0.000, resp . ), (table 1). Serum pct, strem-1, il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1 (p <0.001). There were no differences in nt - pro - bnp and crp, il-10 levels between the two groups (p> 0.05) (table 2). The roc analysis showed that the accuracy of the pct, strem-1, il-6, apacheii, and sofa scores on day 1 for the prediction of 28-day mortality was moderate (auc> 0.7, p <0.01), whereas the accuracy of nt - pro - bnp, crp, and il-10 was low (auc <0.7, p> 0.05) (figure 1). Comparing auc of pct, strem-1, and il-6, we found that there was no significant difference of auc between strem-1 and pct (p = 0.2910), and the auc of the two markers were higher than that of il-6 (p> 0.05). Meanwhile, there was no significant difference of auc between apacheii and sofa scores (p = 0.3753). The auc of apacheii and sofa scores were higher than those of strem-1 and pct (p <0.05) (table 3). The baseline day 1 variables that were found to be significantly different between survivors and nonsurvivors on univariate analysis (pct, strem-1, il-6, apacheii, and sofa scores) were entered into a logistic regression model . Among these variables, three variables remained independently associated with 28-day mortality: strem-1, pct, and sofa score (table 4). Median serum biomarkers and cytokines levels were determined on days 1, 3, and 5 and were compared between the survival and nonsurvival groups . Serum pct, strem-1, and il-6 levels in the nonsurvival group were higher than those in the survival group on days 1, 3, and 5 (p <0.01). There was no difference in nt - pro - bnp levels on day 1 (p> 0.05), but later the nt - pro - bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5 (p <0.05). There were no differences in crp and il-10 levels on days 1, 3, and 5 . Serum pct, strem-1, il-6, and nt - pro - bnp levels showed a decrease trend in the survival group (p <0.05), but there was no decrease tendency in the nonsurviving group for these four biomarkers; strem-1 even had a increase trend (p <0.05). Serum crp levels in both surviving and nonsurviving groups had decrease tendency (p <0.05) (figure 2). Recently, pct, strem-1, crp, and nt - pro - bnp cytokines were widely used to diagnose sepsis and reflect the severity, but the results were not the same . Meanwhile, there were few studies to put so many biomarkers in one study, particularly how to combine the biomarkers, and clinical severity scores remained unclear . The present study showed that the serum levels of strem-1 and pct in nonsurvival group were higher than those in the survival group; meanwhile, they decreased in survival group, but stayed in high levels even increased in the nonsurvival group during sepsis time course . Many previous studies have shown that dynamic changes in strem-1 levels could predict survival and mortality of patients at the early stage of sepsis [10, 11]. Strem-1 is widely used to diagnose sepsis [7, 12]. In the present study, serum strem-1 levels of patients in the nonsurvival group were significantly higher than those in the survival group on day 1; it decreased in survival group, but it even increased in the nonsurvival group . Some studies failed to find the association between strem-1 and poor outcome [8, 13]. At a cutoff of 252.05 pg / ml, strem-1 measurements yielded a sensitivity of 85.7%, specificity of 75.7%, positive predictive value of 70.6%, negative predictive value of 88.2%, and an accuracy of 79.4% for differentiating nonsurvivors from survivors . Pct is normally produced in the c cells of the thyroid gland; plasma pct levels in healthy humans are approximately 550 pg / ml in normal state; its half - time is about 2233 hours in serum . Many tissues and cells except thyroid gland produce and release that pct on systemic inflammation . Several previous studies reported pct could serve as a useful tool to distinguish sepsis from systemic inflammatory response syndrome [15, 16]. On the other hand, pct could reflect the severity of sepsis and outcome . A study by christophe clec'h and coworkers found that serum pct on day 1 was significantly higher in patients with than without septic shock . Meanwhile, among patients with sepsis, pct concentrations were significantly higher in those who died than in the survivors, at all four measurement time points . Similar results were drawn from other investigations [16, 18]. Very few studies failed to find the prognostic value . At a cutoff of 10.64 ng / ml, procalcitonin measurements yielded a sensitivity of 76.2%, specificity of 81.7%, positive predictive value of 53.5%, negative predictive value of 67.8%, and an accuracy of 61.8% for differentiating nonsurvivors from survivors . Nt - pro - bnp has been found to be a useful markers in the diagnosis, management, and prognosis of patients with congestive heart failure and was secreted into blood in response to atrial or ventricular wall stretch . A recent meta - analysis suggested that an elevated nt - pro - bnp level may prove to be a powerful predictor of mortality in septic patients . In our study, there was no difference in nt - pro - bnp level between groups on day 1, but the nt - pro - bnp levels in the nonsurvival group were higher than those in the survival group on days 3 and 5 . Meanwhile, serum nt - pro - bnp level showed a decreased trend in the survival group, but there was no decrease tendency in the nonsurvival group . We concluded that nt - pro - bnp may predict sepsis 28-day mortality in different stages . One research demonstrated that elevated serum nt - pro - bnp value represented an independent predictor for poor icu outcome in the presence of clinical severity scores; the cut - off in admission nt - pro - bnp that best predicted outcome was 941 pg / ml . Crp is an acute phase protein and a sensitive systemic marker of inflammation and tissue damage . The secretion of crp begins within 46 h after stimulus, doubles every 8 h thereafter, and peaks at 3650 h . The role of crp in sepsis prognostic value seemed different . In our study, there was no significant difference between survivors and nonsurvivors during the three measures, similar to previous study, indicating that crp was just an inflammatory biomarker and failed in reflecting sepsis severity . Have reported that crp levels in severe sepsis were lower than those in sepsis, suggesting that crp levels did not reflect the severity of sepsis . Il-6 and il-10 are important proinflammatory and anti - inflammatory cytokines in sepsis . In our study, serum il-6 levels of patients in the nonsurvival group were significantly higher than those in the survival group on days 1, 3, and 5 . The above results showed that il-6 had the prognostic utility for sepsis, whereas il-10 did not show the power . Surez - santamar and coworkers enrolled 253 hospitalized septic patients; they found that il-10 and il-6 were the best predictors, whereas pct showed only moderate predictive value for mortality . Another study investigated the prognostic value of il-6, pct, and crp in critically ill patients during the first increase of fever; only il-6 levels were significantly higher in nonsurvivors compared with survivors, in which prognostic value was superior to pct and crp . In contrast, kawczyski and polakowska indicated that the predictive value of il-10 plasma concentration was better than that of il-6 . To sum up, strem-1, pct, and il-6 serum values attribute to the prognosis of sepsis during the time course . The dynamic changes of biomarkers and cytokines were more meaningful for predicting the sepsis procession . Schneider and coworkers retrospectively analyzed the relationships between serum pct, il-6, and apacheii score and prognosis of 220 patients on the first day after operation . They found that pct was the sole independent predictor of 28-day mortality, in which prognostic ability was superior to those of il-6 and apacheii score . Zhang et al . Suggested that serum strem-1 levels reflected the severity of sepsis more accurately than those of pct and crp and were more sensitive for dynamic evaluations of sepsis prognosis . Facing the results, we wonder which was the best predictor and how to combine them together and which was more valuable compared to clinical severity scores . Apache ii and sofa scores have been widely used to validate mortality risk stratification . In our study, we used roc and logistic regression model to search for the best predictor . Based on roc analysis, strem-1 and pct showed the equal prognostic ability (0.792 for pct, 0.862 for strem-1, p = 0.291), whereas their prognostic utility was inferior to that of apacheii and sofa scores which had equal power to predict outcome (0.923 for apacheii score, 0.953 for sofa score, p = 0.375). Logistic regression model showed that serum strem-1, pct, and sofa score were the independent predictors of 28-day mortality, which was supported by other result . As far as we know, the interrelationship between strem-1, pct, nt - pro - bnp, cytokines, and clinical severity scores for mortality prediction in general icu patients has not been previously evaluated . Our research firstly discovered that strem-1 and pct had the equal prognostic ability for sepsis mortality and were superior to other parameters . Previous study has indicated that the iteraction of trem-1 and interact adaptor protein dap12 can stimulate neutrophil and monocyte - mediated inflammatory response via the triggering and release of pro - inflammatory cytokines and chemokines . Strem-1 increases quickly when exposued to infection, and its half - time is short . In bacterial infections, serum pct levels start to rise at 4 h after the onset of systemic infection and peak at between 8 and 24 h; it decreased 50% every 24 hours along with therapy . In contrast, crp rises slowly and peaks 36 h after an endotoxin challenge . The mechanism of nt - pro - bnp release in sepsis is complex, and kinetics characteristic is unknown . Il-6 and il-10 rise quickly and peak at 24 hours and maintain a short time . The patients admitted to icu often delayed more than 24 hours, either crp or cytokines serum concentration was unable to reach the peak at the period of sepsis . Of course, the exact roles of biomarkers and cytokines in sepsis process are not clear, and need to be further studied . Firstly, our study chosen a part of sepsis biomarkers and did not put all biomarkers in the research . Of course, it was a costly and unnecessary task to do so . Secondly, every biomarker has its own dynamic characteristics; meanwhile the patients were not in the same sepsis stages in the study; thus the explanation for the results would be influenced . Thirdly, we excluded patients with previous heart diseases history, but we did not perform the ucg to evaluate cardiac function . Finally, the sample size of the study was small and larger studies are needed . In summary, elevated serum strem-1 and pct levels provide superior prognostic accuracy to other biomarkers . Combination of serum strem-1 and pct levels and sofa score can offer the best powerful prognostic for sepsis mortality . In the future, in order to improve the accuracy of the prognosis of sepsis, the combination of novel biomarkers and traditional markers of sepsis, reflecting different aspects of sepsis, is an attractive advice and is worthy of further investigation.
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Nine male competitive cyclists participated in this study . Their mean sd age, height, and body mass were 33.3 7.5 yr, 184 4 cm, and 77.3 7.0 kg, respectively . They had a maximal oxygen consumption (vo2max) of 4.8 0.2 lmin (oxycon pro; viasys healthcare, germany) with a peak power output of 418 16 w (protocol, 25-w increase every minute from 100 w until exhaustion), corresponding to a performance level of 4 (i.e., well - trained cyclist (7)). All cyclists were experienced with performing tt in cold - to - temperate conditions (<25c) and provided their written informed consent to participate in this study . The protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee . The participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study (i.e., november to march). They trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization . They were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated (table 1). The cyclists performed 3 tt in hot ambient conditions (tth, see following section). The first tt in hot conditions (tth-1) was not preceded by any outdoor riding in hot conditions; tth-2 was preceded by 5 d in hot ambient conditions, and tth-3 was preceded by 13 d in the heat . The participants spent a minimum of 4 hd outside (average temperature, 34c 3c; relative humidity, 18% 5%) but slept, rested, and ate indoors in an air - conditioned facility . Two additional tt were performed in a cool condition (ttc, see following section) before and after the heat intervention and were averaged to represent ttc (no difference in power output between them). The outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain (maximal elevation difference, 10 m). Cyclists had 36 h (i.e., two nights) of rest after arrival in qatar before performing tth-1, allowing for recovery from travel fatigue . The time difference between denmark and qatar is only 2 h, which should not induce significant jet lag (34). On the day of the tt, the riders performed a self - paced warm - up (approximately two laps on the tt course). The cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . During tt, the riders had access to hr, speed, distance, and power output data . Wind speed was 6.0 ms during ttc, and 6.8, 5.8, and 4.4 ms during tth-1, -2, and -3, respectively . Environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c, 37.4c 0.8c, and 36.2c 1.6c during tth-1, -2, and -3, respectively . Relative humidity was 30% 8% during ttc and 13% 1%, 16% 2%, and 12% 3% during tth-1, -2, and -3, respectively . On the basis of the ideal gas law (pv = nrt) adapted to a mixture of ideal gases (dry air and humid air), the air density (number of mol (n)/volume (v)) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135, 1.127, and 1.131 kgm during tth-1, -2, and -3, respectively . Power output and speed during the tt were measured with powertap wheel sets (powertap, madison, wi) logged continuously on garmin devices (garmin 705 edge) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt . All powertap wheel sets were measured within 10 w from a power2max power meter (power2max, berlin, germany), and each rider used the same equipment during the tt . Hr was continuously recorded during all tt via a chest strap (polar team system 2; polar electro, kempele, finland). Rectal temperature was measured at the end of each tt by a clinical thermometer (precision, 0.1c; depth, approximately 2 cm). In addition, rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor (precision, 0.01c; vitalsense; mini mitter, respironics, herrsching, germany) inserted the length of a gloved index finger beyond the anal sphincter . Body mass losses were estimated from the changes in body weight from before to after tth (seca 769; seca, hamburg, germany) (precision, 0.1 kg). Continuously recorded data (power, hr, and temperature) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova (four conditions 10 times). In addition, total time was analyzed via one - way repeated - measures anova . Anova assumptions were verified preceding all statistical analyses; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . In case of post hoc comparisons, reported p values were adjusted for multiple comparisons using a bonferroni correction (i.e., correction for six potential comparisons between conditions). Effect sizes are described in terms of partial eta - squared (; with 0.06 representing moderate difference and 0.14, large difference). Data are presented as mean sd along with the mean differences (95% confidence interval). Nine male competitive cyclists participated in this study . Their mean sd age, height, and body mass were 33.3 7.5 yr, 184 4 cm, and 77.3 7.0 kg, respectively . They had a maximal oxygen consumption (vo2max) of 4.8 0.2 lmin (oxycon pro; viasys healthcare, germany) with a peak power output of 418 16 w (protocol, 25-w increase every minute from 100 w until exhaustion), corresponding to a performance level of 4 (i.e., well - trained cyclist (7)). All cyclists were experienced with performing tt in cold - to - temperate conditions (<25c) and provided their written informed consent to participate in this study . The protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee . The participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study (i.e., november to march). They trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization . They were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated (table 1). The cyclists performed 3 tt in hot ambient conditions (tth, see following section). The first tt in hot conditions (tth-1) was not preceded by any outdoor riding in hot conditions; tth-2 was preceded by 5 d in hot ambient conditions, and tth-3 was preceded by 13 d in the heat . The participants spent a minimum of 4 hd outside (average temperature, 34c 3c; relative humidity, 18% 5%) but slept, rested, and ate indoors in an air - conditioned facility . Two additional tt were performed in a cool condition (ttc, see following section) before and after the heat intervention and were averaged to represent ttc (no difference in power output between them). The outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain (maximal elevation difference, 10 m). Cyclists had 36 h (i.e., two nights) of rest after arrival in qatar before performing tth-1, allowing for recovery from travel fatigue . The time difference between denmark and qatar is only 2 h, which should not induce significant jet lag (34). On the day of the tt, the riders performed a self - paced warm - up (approximately two laps on the tt course). The cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . During tt, the riders had access to hr, speed, distance, and power output data . Wind speed was 6.0 ms during ttc, and 6.8, 5.8, and 4.4 ms during tth-1, -2, and -3, respectively . Environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c, 37.4c 0.8c, and 36.2c 1.6c during tth-1, -2, and -3, respectively . Relative humidity was 30% 8% during ttc and 13% 1%, 16% 2%, and 12% 3% during tth-1, -2, and -3, respectively . On the basis of the ideal gas law (pv = nrt) adapted to a mixture of ideal gases (dry air and humid air), the air density (number of mol (n)/volume (v)) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135, 1.127, and 1.131 kgm during tth-1, -2, and -3, respectively . Power output and speed during the tt were measured with powertap wheel sets (powertap, madison, wi) logged continuously on garmin devices (garmin 705 edge) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt . All powertap wheel sets were measured within 10 w from a power2max power meter (power2max, berlin, germany), and each rider used the same equipment during the tt . Hr was continuously recorded during all tt via a chest strap (polar team system 2; polar electro, kempele, finland). Rectal temperature was measured at the end of each tt by a clinical thermometer (precision, 0.1c; depth, approximately 2 cm). In addition, rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor (precision, 0.01c; vitalsense; mini mitter, respironics, herrsching, germany) inserted the length of a gloved index finger beyond the anal sphincter . Body mass losses were estimated from the changes in body weight from before to after tth (seca 769; seca, hamburg, germany) (precision, 0.1 kg). Continuously recorded data (power, hr, and temperature) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova (four conditions 10 times). In addition, total time was analyzed via one - way repeated - measures anova . Anova assumptions were verified preceding all statistical analyses; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . In case of post hoc comparisons, reported p values were adjusted for multiple comparisons using a bonferroni correction (i.e., correction for six potential comparisons between conditions). Effect sizes are described in terms of partial eta - squared (; with 0.06 representing moderate difference and 0.14, large difference). Data are presented as mean sd along with the mean differences (95% confidence interval). Nine male competitive cyclists participated in this study . Their mean sd age, height, and body mass were 33.3 7.5 yr, 184 4 cm, and 77.3 7.0 kg, respectively . They had a maximal oxygen consumption (vo2max) of 4.8 0.2 lmin (oxycon pro; viasys healthcare, germany) with a peak power output of 418 16 w (protocol, 25-w increase every minute from 100 w until exhaustion), corresponding to a performance level of 4 (i.e., well - trained cyclist (7)). All cyclists were experienced with performing tt in cold - to - temperate conditions (<25c) and provided their written informed consent to participate in this study . The protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee . The participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study (i.e., november to march). They trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization . They were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated (table 1). The cyclists performed 3 tt in hot ambient conditions (tth, see following section). The first tt in hot conditions (tth-1) was not preceded by any outdoor riding in hot conditions; tth-2 was preceded by 5 d in hot ambient conditions, and tth-3 was preceded by 13 d in the heat . The participants spent a minimum of 4 hd outside (average temperature, 34c 3c; relative humidity, 18% 5%) but slept, rested, and ate indoors in an air - conditioned facility . Two additional tt were performed in a cool condition (ttc, see following section) before and after the heat intervention and were averaged to represent ttc (no difference in power output between them). The outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain (maximal elevation difference, 10 m). Cyclists had 36 h (i.e., two nights) of rest after arrival in qatar before performing tth-1, allowing for recovery from travel fatigue . The time difference between denmark and qatar is only 2 h, which should not induce significant jet lag (34). On the day of the tt, the riders performed a self - paced warm - up (approximately two laps on the tt course). The cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . During tt, the riders had access to hr, speed, distance, and power output data . Wind speed was 6.0 ms during ttc, and 6.8, 5.8, and 4.4 ms during tth-1, -2, and -3, respectively . Environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c, 37.4c 0.8c, and 36.2c 1.6c during tth-1, -2, and -3, respectively . Relative humidity was 30% 8% during ttc and 13% 1%, 16% 2%, and 12% 3% during tth-1, -2, and -3, respectively . On the basis of the ideal gas law (pv = nrt) adapted to a mixture of ideal gases (dry air and humid air), the air density (number of mol (n)/volume (v)) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135, 1.127, and 1.131 kgm during tth-1, -2, and -3, respectively . Power output and speed during the tt were measured with powertap wheel sets (powertap, madison, wi) logged continuously on garmin devices (garmin 705 edge) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt . All powertap wheel sets were measured within 10 w from a power2max power meter (power2max, berlin, germany), and each rider used the same equipment during the tt . Hr was continuously recorded during all tt via a chest strap (polar team system 2; polar electro, kempele, finland). Rectal temperature was measured at the end of each tt by a clinical thermometer (precision, 0.1c; depth, approximately 2 cm). In addition, rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor (precision, 0.01c; vitalsense; mini mitter, respironics, herrsching, germany) inserted the length of a gloved index finger beyond the anal sphincter . Body mass losses were estimated from the changes in body weight from before to after tth (seca 769; seca, hamburg, germany) (precision, 0.1 kg). Continuously recorded data (power, hr, and temperature) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova (four conditions 10 times). In addition, total time was analyzed via one - way repeated - measures anova . Anova assumptions were verified preceding all statistical analyses; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . In case of post hoc comparisons, reported p values were adjusted for multiple comparisons using a bonferroni correction (i.e., correction for six potential comparisons between conditions). Effect sizes are described in terms of partial eta - squared (; with 0.06 representing moderate difference and 0.14, large difference). Data are presented as mean sd along with the mean differences (95% confidence interval). There was a large (= 0.88) and significant condition effect on the time to complete the tt (table 1) (p <0.001). The time to complete ttc (66 min 13 s 3 min 26 s) and tth-3 (65 min 37 s 3 min 44 s) was not significantly different (0.6 (2.5 to + 1.4) min, p> 0.999) but was significantly shorter than tth-1 (77 min 17 s 6 min 26 s) and tth-2 (69 min 25 s 4 min 37 s) (all p <0.01). Speed (table 1) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc, followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 (all p <0.001). Speed during tth-3 was similar to that during ttc (+ 0.4 (0.5 to + 1.7) kmh, p = 0.797). Average power output was significantly lower in tth-1 than that in ttc (48 (67 to 30) w, p <0.001). This decrement was partly restored after 1 wk of acclimatization (tth-2 vs ttc, 24 (40 to 9) w, p = 0.003) and further restored after the second week (tth-3 vs ttc, 11 (21 to 0) w, p = 0.042) (table 1). 1) (= 0.90, p <0.001) and showed a large (= 0.51) and significant (p <0.001) time condition interaction . The post hoc analysis revealed that there was no effect of condition during that first 20% of the tt (fig . 1) (all p> 0.05). However, power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward (p <0.01) and lower than that during tth-2 from 80% onward (fig . Power output during tth-2 became lower than that during ttc from 50% of the distance covered onward (fig . Power output during tth-3 was lower than that during ttc in one segment of the tt only (i.e., 70%, fig . Power output during a 43.4-km cycling tt in ttc (plain line) and in tth-1 (long dashed line), tth-2 (short dashed line), and tth-3 (dotted line). Ttc was significantly (p <0.05) higher than tth-1, tth-2, and tth-3, respectively . 2, hr significantly increased during the tt (= 0.67, p <0.001) relative to testing conditions (= 0.24, p <0.001). The post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt (all p <0.05). Consequently, hr was different between conditions (= 0.41, p = 0.024), without pairwise differences reaching significance (e.g., tth-1 vs ttc, + 7 (2 to + 15) bpm, p = 0.127; tth-2 vs ttc, + 4 (4 to + 12) bpm, p = 0.616; tth-3 vs ttc, + 5 (2 to + 13) bpm, p = 0.254). Hr (upper panel) and rectal temperature (lower panel) during a 43.4-km cycling tt in ttc (plain line) and in tth-1 (long dashed line), tth-2 (short dashed line), and tth-3 (dotted line). * rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt (= 0.93, p <0.001) and was higher during the first 80% of tth-1 than that during tth-3 (fig . 2), leading to an overall higher temperature during tth-3 than that during tth-1 (+ 0.3c (0.1c0.5c), = 0.63, p = 0.019). However, final rectal temperature (table 1) was significantly higher after the tth than that after ttc (all p <0.001) (fig . 2) but without differences between tth (all p> 0.999). As displayed in table 1, the differences in body mass loss (p = 0.071, = 0.28) and fluid consumption (p = 0.095, = 0.30) between the tth did not reach significance . There was a large (= 0.88) and significant condition effect on the time to complete the tt (table 1) (p <0.001). The time to complete ttc (66 min 13 s 3 min 26 s) and tth-3 (65 min 37 s 3 min 44 s) was not significantly different (0.6 (2.5 to + 1.4) min, p> 0.999) but was significantly shorter than tth-1 (77 min 17 s 6 min 26 s) and tth-2 (69 min 25 s 4 min 37 s) (all p <0.01). Speed (table 1) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc, followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 (all p <0.001). Speed during tth-3 was similar to that during ttc (+ 0.4 (0.5 to + 1.7) kmh, p = 0.797). Average power output was significantly lower in tth-1 than that in ttc (48 (67 to 30) w, p <0.001). This decrement was partly restored after 1 wk of acclimatization (tth-2 vs ttc, 24 (40 to 9) w, p = 0.003) and further restored after the second week (tth-3 vs ttc, 11 (21 to 0) w, p = 0.042) (table 1). 1) (= 0.90, p <0.001) and showed a large (= 0.51) and significant (p <0.001) time condition interaction . The post hoc analysis revealed that there was no effect of condition during that first 20% of the tt (fig . 1) (all p> 0.05). However, power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward (p <0.01) and lower than that during tth-2 from 80% onward (fig . Power output during tth-2 became lower than that during ttc from 50% of the distance covered onward (fig . Power output during tth-3 was lower than that during ttc in one segment of the tt only (i.e., 70%, fig . Power output during a 43.4-km cycling tt in ttc (plain line) and in tth-1 (long dashed line), tth-2 (short dashed line), and tth-3 (dotted line). Ttc was significantly (p <0.05) higher than tth-1, tth-2, and tth-3, respectively . 2, hr significantly increased during the tt (= 0.67, p <0.001) relative to testing conditions (= 0.24, p <0.001). The post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt (all p <0.05). Consequently, hr was different between conditions (= 0.41, p = 0.024), without pairwise differences reaching significance (e.g., tth-1 vs ttc, + 7 (2 to + 15) bpm, p = 0.127; tth-2 vs ttc, + 4 (4 to + 12) bpm, p = 0.616; tth-3 vs ttc, + 5 (2 to + 13) bpm, p = 0.254). Hr (upper panel) and rectal temperature (lower panel) during a 43.4-km cycling tt in ttc (plain line) and in tth-1 (long dashed line), tth-2 (short dashed line), and tth-3 (dotted line). * rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt (= 0.93, p <0.001) and was higher during the first 80% of tth-1 than that during tth-3 (fig . 2), leading to an overall higher temperature during tth-3 than that during tth-1 (+ 0.3c (0.1c0.5c), = 0.63, p = 0.019). However, final rectal temperature (table 1) was significantly higher after the tth than that after ttc (all p <0.001) (fig . 2) but without differences between tth (all p> 0.999). As displayed in table 1, the differences in body mass loss (p = 0.071, = 0.28) and fluid consumption (p = 0.095, = 0.30) between the tth did not reach significance . There was a large (= 0.88) and significant condition effect on the time to complete the tt (table 1) (p <0.001). The time to complete ttc (66 min 13 s 3 min 26 s) and tth-3 (65 min 37 s 3 min 44 s) was not significantly different (0.6 (2.5 to + 1.4) min, p> 0.999) but was significantly shorter than tth-1 (77 min 17 s 6 min 26 s) and tth-2 (69 min 25 s 4 min 37 s) (all p <0.01). Speed (table 1) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc, followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 (all p <0.001). Speed during tth-3 was similar to that during ttc (+ 0.4 (0.5 to + 1.7) kmh, p = 0.797). Average power output was significantly lower in tth-1 than that in ttc (48 (67 to 30) w, p <0.001). This decrement was partly restored after 1 wk of acclimatization (tth-2 vs ttc, 24 (40 to 9) w, p = 0.003) and further restored after the second week (tth-3 vs ttc, 11 (21 to 0) w, p = 0.042) (table 1). 1) (= 0.90, p <0.001) and showed a large (= 0.51) and significant (p <0.001) time condition interaction . The post hoc analysis revealed that there was no effect of condition during that first 20% of the tt (fig . 1) (all p> 0.05). However, power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward (p <0.01) and lower than that during tth-2 from 80% onward (fig . Power output during tth-2 became lower than that during ttc from 50% of the distance covered onward (fig . Power output during tth-3 was lower than that during ttc in one segment of the tt only (i.e., 70%, fig . Power output during a 43.4-km cycling tt in ttc (plain line) and in tth-1 (long dashed line), tth-2 (short dashed line), and tth-3 (dotted line). Ttc was significantly (p <0.05) higher than tth-1, tth-2, and tth-3, respectively . 2, hr significantly increased during the tt (= 0.67, p <0.001) relative to testing conditions (= 0.24, p <0.001). The post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt (all p <0.05). Consequently, hr was different between conditions (= 0.41, p = 0.024), without pairwise differences reaching significance (e.g., tth-1 vs ttc, + 7 (2 to + 15) bpm, p = 0.127; tth-2 vs ttc, + 4 (4 to + 12) bpm, p = 0.616; tth-3 vs ttc, + 5 (2 to + 13) bpm, p = 0.254). Hr (upper panel) and rectal temperature (lower panel) during a 43.4-km cycling tt in ttc (plain line) and in tth-1 (long dashed line), tth-2 (short dashed line), and tth-3 (dotted line). * rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt (= 0.93, p <0.001) and was higher during the first 80% of tth-1 than that during tth-3 (fig . 2), leading to an overall higher temperature during tth-3 than that during tth-1 (+ 0.3c (0.1c0.5c), = 0.63, p = 0.019). However, final rectal temperature (table 1) was significantly higher after the tth than that after ttc (all p <0.001) (fig . 2) but without differences between tth (all p> 0.999). As displayed in table 1, the differences in body mass loss (p = 0.071, = 0.28) and fluid consumption (p = 0.095, = 0.30) between the tth did not reach significance . The current study is the first to determine the effects of acute heat exposure and heat acclimatization on performance and pacing during outdoor cycling tt in experienced cyclists . The cyclists initiated all tt in the heat with a similar power output as maintained during the first 20% of ttc . However, while maintaining similar hr, they subsequently experienced a marked decrease in power output and speed in the heat . These decrements were progressively restored with heat acclimatization, despite core temperature in all tt in the heat increasing significantly more than that in cool conditions . Our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists . This decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . Even if this decrement is not linear, as the effect of an absolute increase in air temperature is more important in warm than in cold environments (13), this rate is comparable with the decrements reported during laboratory tt (0.3% to 0.9% per 1c (12,23,24,32). In addition, the current study quantified the effects of heat stress on performance at different acclimatization stages . To date, most studies investigating the effects of heat on performance have examined unacclimatized participants . These have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory (18,21) and that natural heat acclimatization increases physical performance during sporting activities in hot environments (25,26,33). However, a comparison of the magnitude of improvement in performance after heat acclimatization, relative to the initial decrement in performance associated with the first exposure to heat stress, has yet to be examined . Our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . From an average power decrement of 16% 5% on the first day of heat exposure (tth-1) relative to ttc, the decrement was reduced to 8% 4% after 1 wk of training in the heat (tth-2) and to 3% 4% after 2 wk (tth-3). Despite the cooling effect of air movement, our data showed that the average final temperature of the riders was above 40c during tth, irrespective of heat acclimatization (table 1). One rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . Despite an average final temperature of 40.2c (range, 39.6c41.0c), this confirms that well - prepared athletes reach high core temperatures while exercising in the heat, asymptomatic of heat illness (4), and that there is no absolute critical temperature threshold set at 40c (11). In the current study, despite the decrease in power output (fig . 1) and the possibility to drink ad libitum on the bike, participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions (fig . Our data showed a decrement in absolute intensity (i.e., power output) during the tt but the likely maintenance of a similar relative intensity . Indeed, it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate (1,36,37) and self - paced (24) exercise mediates a decrease in maximal aerobic capacity, resulting in an increase in relative intensity for a given absolute work rate . Although power output decreases during prolonged self - paced exercise in the heat, it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr (24). It therefore seems that despite a decrease in power output, hr remained elevated and stable (fig . Moreover, the pacing pattern was not dependent on the environmental conditions or the acclimatization level, which is in line with recent reports that pacing strategies are not affected by environmental temperature (23), thermal perception (2), or the presence of previous muscle fatigue (6). Rather, it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity (manifested by hr). Given the progressive reduction in vo2max as hyperthermia develops, sustainable power output is reduced, owing to a reduction in work rate for a given relative exercise intensity (24). Furthermore, it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization . Notwithstanding, it has previously been reported that tt are initiated at the same power output in hot and cool conditions (11,24,31). This similar work rate adopted seems to correspond to a critical power (16). Given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat (27,29,35), athletes seem to adopt a relative intensity associated with this critical power output . As vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat, the maintenance of a similar relative intensity requires reduction in power output (24). However, given the role of previous experiences on pacing (30), one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 . Initial power output was similar between trials and decreased thereafter in relation to acclimatization state . Indeed, early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation (19), allowing normalization of the relation between physiological responses and work intensity (10). Consequently, the cyclists seem to have finished all tt at a similar relative intensity, as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . Of note, the slightly higher hr in tth may be attributable to higher skin blood flow, as suggested by the higher core temperature, although the cyclists were wearing thermal clothing in ttc, which would also have lead to increase in skin temperature and blood flow . The current study is the first to investigate cycling performance during outdoor tt in a hot environment . . However, the evaporative capacity of the environment is improved with higher air velocities, reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments (28). In the current study, the cyclists wore thermal clothing in ttc including long tights, long sleeves, and gloves, which limited the evaporative and convective power of the environment, whereas they wore short tights and a jersey with short sleeves in the heat (except one cyclist who was consistently using white long sleeves). Therefore, it cannot be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study . Interestingly, the detrimental effect of hot ambient conditions on speed was not as important as that on power output (table 1). The different relations between the environment and speed and power output could be partly related to a temperature effect on air density, as the aerodynamic drag of a cyclist is related to air density and temperature (3,9,17). For example, at an air temperature of 11c, the temperature reported to optimize laboratory cycling capacity (14), air density is approximately 1.245 kgm at sea level . In contrast, air density drops to approximately 1.165 kgm at a temperature of 30c, reducing the drag force by approximately 6% . Consequently, the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . In the current study, air density was 9.4% lower during tth-3 (1.131 kgm) than that during ttc (1.249 kgm), representing a power economy of almost 8% (22). Thus, despite the slightly lower power output in tth-3 than that in ttc (3%), the average speeds were not significantly different . Our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists . This decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . Even if this decrement is not linear, as the effect of an absolute increase in air temperature is more important in warm than in cold environments (13), this rate is comparable with the decrements reported during laboratory tt (0.3% to 0.9% per 1c (12,23,24,32). In addition, the current study quantified the effects of heat stress on performance at different acclimatization stages . To date, most studies investigating the effects of heat on performance have examined unacclimatized participants . These have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory (18,21) and that natural heat acclimatization increases physical performance during sporting activities in hot environments (25,26,33). However, a comparison of the magnitude of improvement in performance after heat acclimatization, relative to the initial decrement in performance associated with the first exposure to heat stress, has yet to be examined . Our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . From an average power decrement of 16% 5% on the first day of heat exposure (tth-1) relative to ttc, the decrement was reduced to 8% 4% after 1 wk of training in the heat (tth-2) and to 3% 4% after 2 wk (tth-3). Despite the cooling effect of air movement, our data showed that the average final temperature of the riders was above 40c during tth, irrespective of heat acclimatization (table 1). One rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . Despite an average final temperature of 40.2c (range, 39.6c41.0c), this confirms that well - prepared athletes reach high core temperatures while exercising in the heat, asymptomatic of heat illness (4), and that there is no absolute critical temperature threshold set at 40c (11). In the current study, despite the decrease in power output (fig . 1) and the possibility to drink ad libitum on the bike, participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions (fig . Our data showed a decrement in absolute intensity (i.e., power output) during the tt but the likely maintenance of a similar relative intensity . Indeed, it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate (1,36,37) and self - paced (24) exercise mediates a decrease in maximal aerobic capacity, resulting in an increase in relative intensity for a given absolute work rate . Although power output decreases during prolonged self - paced exercise in the heat, it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr (24). It therefore seems that despite a decrease in power output, hr remained elevated and stable (fig . Moreover, the pacing pattern was not dependent on the environmental conditions or the acclimatization level, which is in line with recent reports that pacing strategies are not affected by environmental temperature (23), thermal perception (2), or the presence of previous muscle fatigue (6). Rather, it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity (manifested by hr). Given the progressive reduction in vo2max as hyperthermia develops, sustainable power output is reduced, owing to a reduction in work rate for a given relative exercise intensity (24). Furthermore, it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization . Notwithstanding, it has previously been reported that tt are initiated at the same power output in hot and cool conditions (11,24,31). This similar work rate adopted seems to correspond to a critical power (16). Given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat (27,29,35), athletes seem to adopt a relative intensity associated with this critical power output . As vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat, the maintenance of a similar relative intensity requires reduction in power output (24). However, given the role of previous experiences on pacing (30), one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 . Initial power output was similar between trials and decreased thereafter in relation to acclimatization state . Indeed, early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation (19), allowing normalization of the relation between physiological responses and work intensity (10). Consequently, the cyclists seem to have finished all tt at a similar relative intensity, as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . Of note, the slightly higher hr in tth may be attributable to higher skin blood flow, as suggested by the higher core temperature, although the cyclists were wearing thermal clothing in ttc, which would also have lead to increase in skin temperature and blood flow . The current study is the first to investigate cycling performance during outdoor tt in a hot environment . However, the evaporative capacity of the environment is improved with higher air velocities, reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments (28). In the current study, the cyclists wore thermal clothing in ttc including long tights, long sleeves, and gloves, which limited the evaporative and convective power of the environment, whereas they wore short tights and a jersey with short sleeves in the heat (except one cyclist who was consistently using white long sleeves). Therefore, it cannot be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study . Interestingly, the detrimental effect of hot ambient conditions on speed was not as important as that on power output (table 1). The different relations between the environment and speed and power output could be partly related to a temperature effect on air density, as the aerodynamic drag of a cyclist is related to air density and temperature (3,9,17). For example, at an air temperature of 11c, the temperature reported to optimize laboratory cycling capacity (14), air density is approximately 1.245 kgm at sea level . In contrast, air density drops to approximately 1.165 kgm at a temperature of 30c, reducing the drag force by approximately 6% . Consequently, the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . In the current study, air density was 9.4% lower during tth-3 (1.131 kgm) than that during ttc (1.249 kgm), representing a power economy of almost 8% (22). Thus, despite the slightly lower power output in tth-3 than that in ttc (3%), the average speeds were not significantly different . Our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists . This decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . Even if this decrement is not linear, as the effect of an absolute increase in air temperature is more important in warm than in cold environments (13), this rate is comparable with the decrements reported during laboratory tt (0.3% to 0.9% per 1c (12,23,24,32). In addition, the current study quantified the effects of heat stress on performance at different acclimatization stages . To date, most studies investigating the effects of heat on performance have examined unacclimatized participants . These have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory (18,21) and that natural heat acclimatization increases physical performance during sporting activities in hot environments (25,26,33). However, a comparison of the magnitude of improvement in performance after heat acclimatization, relative to the initial decrement in performance associated with the first exposure to heat stress, has yet to be examined . Our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . From an average power decrement of 16% 5% on the first day of heat exposure (tth-1) relative to ttc, the decrement was reduced to 8% 4% after 1 wk of training in the heat (tth-2) and to 3% 4% after 2 wk (tth-3). Despite the cooling effect of air movement, our data showed that the average final temperature of the riders was above 40c during tth, irrespective of heat acclimatization (table 1). One rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . Despite an average final temperature of 40.2c (range, 39.6c41.0c), this confirms that well - prepared athletes reach high core temperatures while exercising in the heat, asymptomatic of heat illness (4), and that there is no absolute critical temperature threshold set at 40c (11). In the current study, despite the decrease in power output (fig . 1) and the possibility to drink ad libitum on the bike, participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions (fig . Our data showed a decrement in absolute intensity (i.e., power output) during the tt but the likely maintenance of a similar relative intensity . Indeed, it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate (1,36,37) and self - paced (24) exercise mediates a decrease in maximal aerobic capacity, resulting in an increase in relative intensity for a given absolute work rate . Although power output decreases during prolonged self - paced exercise in the heat, it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr (24). It therefore seems that despite a decrease in power output, hr remained elevated and stable (fig . Moreover, the pacing pattern was not dependent on the environmental conditions or the acclimatization level, which is in line with recent reports that pacing strategies are not affected by environmental temperature (23), thermal perception (2), or the presence of previous muscle fatigue (6). Rather, it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity (manifested by hr). Given the progressive reduction in vo2max as hyperthermia develops, sustainable power output is reduced, owing to a reduction in work rate for a given relative exercise intensity (24). Furthermore, it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization . Notwithstanding, it has previously been reported that tt are initiated at the same power output in hot and cool conditions (11,24,31). This similar work rate adopted seems to correspond to a critical power (16). Given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat (27,29,35), athletes seem to adopt a relative intensity associated with this critical power output . As vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat, the maintenance of a similar relative intensity requires reduction in power output (24). However, given the role of previous experiences on pacing (30), one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 . Initial power output was similar between trials and decreased thereafter in relation to acclimatization state . Indeed, early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation (19), allowing normalization of the relation between physiological responses and work intensity (10). Consequently, the cyclists seem to have finished all tt at a similar relative intensity, as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . Of note, the slightly higher hr in tth may be attributable to higher skin blood flow, as suggested by the higher core temperature, although the cyclists were wearing thermal clothing in ttc, which would also have lead to increase in skin temperature and blood flow . The current study is the first to investigate cycling performance during outdoor tt in a hot environment . However, the evaporative capacity of the environment is improved with higher air velocities, reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments (28). In the current study, the cyclists wore thermal clothing in ttc including long tights, long sleeves, and gloves, which limited the evaporative and convective power of the environment, whereas they wore short tights and a jersey with short sleeves in the heat (except one cyclist who was consistently using white long sleeves). Therefore, it cannot be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study . Interestingly, the detrimental effect of hot ambient conditions on speed was not as important as that on power output (table 1). The different relations between the environment and speed and power output could be partly related to a temperature effect on air density, as the aerodynamic drag of a cyclist is related to air density and temperature (3,9,17). For example, at an air temperature of 11c, the temperature reported to optimize laboratory cycling capacity (14), air density is approximately 1.245 kgm at sea level . In contrast, air density drops to approximately 1.165 kgm at a temperature of 30c, reducing the drag force by approximately 6% . Consequently, the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . In the current study, air density was 9.4% lower during tth-3 (1.131 kgm) than that during ttc (1.249 kgm), representing a power economy of almost 8% (22). Thus, despite the slightly lower power output in tth-3 than that in ttc (3%), the average speeds were not significantly different . The novel findings of this investigation are that competitive cyclists performing an outdoor tt undertake their effort at the same power output, irrespective of the environmental conditions or previous experience . Consequently, non heat - acclimatized cyclists are incapable of sustaining this absolute effort . However, the decrement in power output is partly recovered after 1 wk of heat acclimatization and almost fully restored after 2 wk . Furthermore, our data seem to confirm that sustainable power output is related to a given relative intensity (24), which is partly reflected in the maintenance of hr within a certain range . Finally, because of the reduction in air density associated with cycling in hot ambient conditions, speed was not different between tth-3 and ttc.
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Worksites in glass and metal works, foundries, rolling mills, in the vicinity of hardening furnaces, rotary kilns in the cement industry, high - temperature laboratory furnaces, as well as firefighting, are associated, in addition to common mechanical hazards (e.g., due to splashes of molten metals and slag), sparks and direct contact with open fires, with hazards caused by emission of harmful optic radiation within the visible (vis) and infrared (ir) spectrum range . Excessive exposure to ir radiation leads to dryness of the mucous membranes, burns of the eyeball and development of cataracts . Protection against thermal hazards due to excessive amounts of ir radiation reaching the eyes is provided by protective optic filters installed in spectacles, goggles or face protections . Besides protecting the user against hazardous ir radiation, such filters should ensure that the object / area of work can be seen clearly . If the level of ir radiation is very high, the use of filters reflecting radiation is recommended, as it allows the temperature increase in the filter itself to be reduced . The filters currently used for the protection of the face and eyes against hazardous ir radiation are of metallic reflective type, made of glass or organic material (mainly polycarbonate) bases coated with a single metallic layer for reflectance of ir radiation or absorption filters . Absorption filters are manufactured from dyed glass obtained in a mass staining process by introduction of a factor modifying the radiation within the vis and near infrared (nir) spectrum range passing through the filter . The effect of a protective optical filter obtained as a result of this process is based on absorption of a large proportion of optic radiation, whereas only a small proportion of radiation the filter is exposed to is reflected . This is an undesirable phenomenon, contributing to deterioration of the filter user's comfort . Reflective metallic filters are obtained by deposition of a metallic layer of copper (cu) or gold (au) on mineral or organic bases . Such filters take advantage of the characteristic properties of the metals used in their production, i.e., significant reflection coefficient for ir radiation and significant transmittance for the vis spectrum . Despite the fact that the technique of deposition of interference layers has been known for many years the application of thin film ir filters in eye protections to block the previous papers published by the authors of this study contain descriptions of the structure of interference filters designed for application in eye protections highly effective in reduction of ir radiation with high level of transmission of the vis spectrum . The authors investigated also the optical and mechanical parameters of the newly developed protective optic filters manufactured with the use of interference technologies . The analysis of the results of the conducted tests demonstrated numerous advantages of interference technology application in the construction of protective filters, including: improved resistance to damage of the external protective filter surface (cracking, scratching etc .) As well as high reflection coefficients for the nir spectrum . The aim of the present study was to compare the optic properties of protective filters manufactured by taking advantage of the interference technology and those coated with a single metallic layer reflecting ir radiation . The coefficients characterizing their optic parameters, allowing to assess the protective properties within the vis and ir spectrum range were compared . The following coefficients were analysed: luminous transmittance (v),ir transmittance within the 7801400 nm range (a),ir transmittance within the 7802000 nm range (n),ir reflectance within the 7802000 nm range (r n),ir absorptance within the 7802000 nm range (a n),shade number (n). Luminous transmittance (v), ir transmittance within the 7801400 nm range (a), ir transmittance within the 7802000 nm range (n), ir reflectance within the 7802000 nm range (r n), ir absorptance within the 7802000 nm range (a n), the level of protection (shade number) is determined on the basis of luminous transmittance (the higher protection level correlates with the higher level of ir radiation blocked and appropriately lower vis light transmittance). Protection level consists of a code number (e.g., 4) for ir filters and a shade number (e.g., 3, 5, 7) calculated according to the following equation: (1) where v = luminous transmittance . For the particular level of protection, the mean ir transmittance within the 7801400 and 7802000 nm ranges as well as the ir reflectance and absorptance within the 7802000 nm range are determined . For comparative analysis of the metallic reflective filters available in the market versus the interference filters developed by the authors, the following samples were prepared for the tests: off the shelf metallic reflective filters on a polycarbonate base (flat parallel plate of 50 mm diameter and 2 mm thickness with a metallic layer of copper (cu) protection levels: 4 - 3, 4 - 5, 4 - 7);interference filters on a polycarbonate base (flat parallel plate of 50 mm diameter with interference coating made up of the following materials: aluminium, h4 latio3 substance and silicon dioxide). Off the shelf metallic reflective filters on a polycarbonate base (flat parallel plate of 50 mm diameter and 2 mm thickness with a metallic layer of copper (cu) protection levels: 4 - 3, 4 - 5, 4 - 7); interference filters on a polycarbonate base (flat parallel plate of 50 mm diameter with interference coating made up of the following materials: aluminium, h4 latio3 substance and silicon dioxide). The protection levels, luminous transmittance and symbols used for marking the tested filters are presented in table 1 . Characteristics of the filters.symbolluminous transmittance v (%) protection levelfilter typecu 4 - 314.1334 - 3metallic reflectiveitf 4 - 39.0824 - 3interferencecu 4 - 52.0214 - 5metallic reflectiveitf 4 - 51.4704 - 5interferencecu 4 - 70.3534 - 7metallic reflectiveitf 4 - 70.1604 - 7interferencenote: protection level consists of code number (4) for infrared filters and shade number (3, 5, 7) calculated according to equation (1). Note: protection level consists of code number (4) for infrared filters and shade number (3, 5, 7) calculated according to equation (1). Optical properties of the filters were measured with a cary 5000 spectrophotometer (varian, australia). The spectrophotometer enabled transmittance or reflectance of optical radiation within the vis and nir spectrum to be recorded with 1 nm steps and 0.001% resolution level . Spectral transmittance and reflectance measurements were carried out within the wavelength range of 7802000 nm . The quartz halogen lamp was used as a source of radiation . To detect transmitted or reflected radiation the photomultiplier tube and lead sulphide detector were used for vis and nir spectrum, respectively . Spectral transmittance was measured with incident radiation falling normally on the filter surface in the visual centre (defined as in). For spectral reflectance the measurements were taken every second owing to the high absorption of investigated filters within the nir region . Since the minimum averaging time of the cary 5000 spectrophotometer was 0.0125 s, the values of transmittance or reflectance were measured 125 times for a given wavelength . The aforementioned coefficients were calculated from the following equations [1012]: luminous transmittance (2) where () = spectral transmittance, v() = spectral visibility function of the average human eye for daylight or night vision, s d65() = spectral energy distribution of standard illuminant d65; ir transmittance within the 7801400 nm range (3) where () = spectral transmittance; ir transmittance within the 7802000 nm range (4) where () = spectral transmittance; ir reflectance within the 7802000 nm range (5) where () = spectral reflectance; ir absorptance within the 7802000 nm range (6) where r n = ir reflectance within the 7802000 nm range, n = ir transmittance within the 7802000 nm range . Luminous transmittance (2) ir transmittance within the 7801400 nm range (3) ir transmittance within the 7802000 nm range (4) ir reflectance within the 7802000 nm range (5) ir absorptance within the 7802000 nm range (6) the factor before integrals corresponding to the value of the measurement step at the measurement of spectral characteristics . Transmittance and reflectance characteristics of the studied filters.note: cu 4 - 3 = metallic reflective filter; itf 4 - 3 = interference filter; cu 4 - 5 = metallic reflective filter; itf 4 - 5 = interference filter; cu 4 - 7 = metallic reflective filter; itf 4 - 7 = interference filter . Table 2 presents the values of coefficients determined on the basis of equations (3)(6). Infrared transmittance within the 7801400 and 7802000 nm range, infrared reflectance within the 7802000 nm range and infrared absorptance within the 7802000 nm range . Infrared transmittance a and n (%) infrared reflectance rn (%) infrared absorptance an (%) symbola 7801400 (nm)n 7802000 (nm)rn 7802000 (nm)an 7802000 (nm)cu 4 - 31.4860.94283.51615.542itf 4 - 31.0030.56382.08417.353cu 4 - 50.2160.13487.31912.547itf 4 - 50.0380.00691.0378.957cu 4 - 70.0040.02388.30811.669itf 4 - 70.0040.03994.3125.649 optical radiation within the 7801400 nm spectrum range passes through the anterior compartment of the eye, the lens and reaches the retina . Exposure to an excessive amount of radiation from this range can result in burns of the retina and, in extreme cases, its permanent damage ., chronic long - term exposure may lead to the development of cataracts, referred to as ir or glass worker's cataracts . In view of the above the results presented on the graphs in figure 1 and the determined transmittance values (see table 2) indicate unequivocally more effective blocking of ir radiation provided by interference filters . The values of a and n for interference filters are lower even by an order of magnitude than those obtained for the metallic reflective filters . For instance, the mean value of ir transmittance within the 7802000 nm range for interference filters with protection level 4 - 5 (one of the most common protection levels used in the metallurgic industry) equals 0.006% and is significantly lower than the maximum acceptable value specified in, which is 10.6% . For all the investigated filters, the values of r n exceed 60%, which classifies them in the group of filters with increased ir reflectance . The levels of the ir absorptance for metallic reflective filters and interference filters of 4 - 3 and 4 - 5 protection class are similar . For the highest of the investigated protection levels 4 - 7 the above findings are important for selection of a filter appropriate for the radiation source . The selection of filter protection level is dependent on the temperature of the radiation source . The 4 - 7 level is used for radiation sources whose temperature exceeds 1600 k (e.g., occurs in the metallurgical industry). With such high temperatures, reduction of filter heating as a result of exposure to ir radiation is important . As it follows from comparative analysis of the optic properties of filters taking advantage of the interference technology and filters coated with a single metallic layer reflecting ir radiation, the interference filters provide a more effective blocking of ir radiation in comparison with the currently used protective filters.
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This retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network (visn 16), one of the largest of the 23 visns in the veterans health administration (vha). The vha is a national integrated health care system providing a set of comprehensive services to veterans . As of 2010 the visn 16 data warehouse is an integrated, de - identified, individual - level database representing 7.8% of u.s . Veterans and covers a geographic region of 170,000 square miles, including the states of arkansas, louisiana, mississippi, and oklahoma, and parts of alabama, florida, missouri, and texas . It includes records for> 445,000 veterans from 10 medical centers and 40 outpatient clinics, with information regarding demographics, vital signs, laboratory results, diagnoses, procedures, inpatient and outpatient services (e.g., admission date, length of stay, and emergency room visits), drug prescriptions, and database enrollment history . As in the national vha population, patients in visn 16 are predominantly male (90.1%). The study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems . Adult patients (18 years of age) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 . Patients had at least one measurement of hba1c and ldl - c within 30 days of each other (paired measurements) after the first diabetes diagnosis; the earlier date of the hba1c or ldl - c measurement was defined as the index date . All patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date, irrespective of the gap between the measurements . The final sample included patients who were enrolled in the database for at least 12 months after the index date . Average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method (24,25). For each cycle, these estimated averages were used to stratify patients into one of four goal achievement categories: dual goal (average hba1c <7% [53 mmol / mol] and average ldl - c <100 mg / dl), hba1c only (average hba1c <7% [53 mmol / mol] and average ldl - c 100 mg / dl), ldl - c only (average ldl - c <100 mg / dl and average hba1c 7% [53 mmol / mol]), or no goal (average hba1c 7% [53 mmol / mol] and average ldl - c 100 mg / dl). Patient characteristics as of the first cycle were summarized for the overall population, as well as stratified according to goal achievement status . Demographic information included age on index date, sex, race, bmi, and year of index date . The history of diabetes - related complications (microvascular, macrovascular, and other), comorbidities, and surgical procedures was identified as of the first cycle using icd-9, clinical modification (icd-9-cm) codes . Medication use during the first cycle was categorized by drug therapeutic class; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits . Characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and tests for categorical variables . Clinical outcomes were selected a priori and comprised 1) a composite cardiovascular - related end point (cerebrovascular disease [stroke], acute myocardial infarction, or cardiovascular death [defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death]), 2) a composite end point for microvascular complications (diabetic retinopathy, nephropathy, or neuropathy), 3) acute coronary syndromes (acs; acute myocardial infarction or unstable angina), and 4) cardiovascular procedures (percutaneous coronary intervention [pci] or coronary artery bypass graft [cabg]). For each clinical outcome, goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle . The time to the first clinical event was evaluated using a cox proportional hazards model, with goal achievement status as a time - dependent variable, controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history, resource utilization, and medication use . All clinical outcomes were measured from the start of the second cycle until the first event, death, or end of data; for the analyses of specific clinical outcomes, patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle . The numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle . Diabetes - related medical service costs were measured in each cycle using the average cost method (26). Dollars according to the medical care services component of the consumer price index (27,28). Utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following: diabetes, macrovascular complications, or microvascular complications . The associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models (glms) with a poisson distribution; results are reported as adjusted incidence rate ratios with 95% cis . The associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a distribution, and adjusted results are reported as annualized incremental cost differences . All longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history, resource utilization, and medication use . Sas software version 9.2 was used to conduct statistical analyses, and a two - tailed level of 0.05 was used to determine statistical significance . This retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network (visn 16), one of the largest of the 23 visns in the veterans health administration (vha). The vha is a national integrated health care system providing a set of comprehensive services to veterans . As of 2010 the visn 16 data warehouse is an integrated, de - identified, individual - level database representing 7.8% of u.s . Veterans and covers a geographic region of 170,000 square miles, including the states of arkansas, louisiana, mississippi, and oklahoma, and parts of alabama, florida, missouri, and texas . It includes records for> 445,000 veterans from 10 medical centers and 40 outpatient clinics, with information regarding demographics, vital signs, laboratory results, diagnoses, procedures, inpatient and outpatient services (e.g., admission date, length of stay, and emergency room visits), drug prescriptions, and database enrollment history . As in the national vha population, patients in visn 16 are predominantly male (90.1%). The study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems . Adult patients (18 years of age) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 . Patients had at least one measurement of hba1c and ldl - c within 30 days of each other (paired measurements) after the first diabetes diagnosis; the earlier date of the hba1c or ldl - c measurement was defined as the index date . All patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date, irrespective of the gap between the measurements . The final sample included patients who were enrolled in the database for at least 12 months after the index date . Average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method (24,25). For each cycle, these estimated averages were used to stratify patients into one of four goal achievement categories: dual goal (average hba1c <7% [53 mmol / mol] and average ldl - c <100 mg / dl), hba1c only (average hba1c <7% [53 mmol / mol] and average ldl - c 100 mg / dl), ldl - c only (average ldl - c <100 mg / dl and average hba1c 7% [53 mmol / mol]), or no goal (average hba1c 7% [53 mmol / mol] and average ldl - c 100 mg / dl). Patient characteristics as of the first cycle were summarized for the overall population, as well as stratified according to goal achievement status . Demographic information included age on index date, sex, race, bmi, and year of index date . The history of diabetes - related complications (microvascular, macrovascular, and other), comorbidities, and surgical procedures was identified as of the first cycle using icd-9, clinical modification (icd-9-cm) codes . Medication use during the first cycle was categorized by drug therapeutic class; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits . Characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and tests for categorical variables . Clinical outcomes were selected a priori and comprised 1) a composite cardiovascular - related end point (cerebrovascular disease [stroke], acute myocardial infarction, or cardiovascular death [defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death]), 2) a composite end point for microvascular complications (diabetic retinopathy, nephropathy, or neuropathy), 3) acute coronary syndromes (acs; acute myocardial infarction or unstable angina), and 4) cardiovascular procedures (percutaneous coronary intervention [pci] or coronary artery bypass graft [cabg]). For each clinical outcome, goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle . The time to the first clinical event was evaluated using a cox proportional hazards model, with goal achievement status as a time - dependent variable, controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history, resource utilization, and medication use . All clinical outcomes were measured from the start of the second cycle until the first event, death, or end of data; for the analyses of specific clinical outcomes, patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle . The numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle . Diabetes - related medical service costs were measured in each cycle using the average cost method (26). Dollars according to the medical care services component of the consumer price index (27,28). Utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following: diabetes, macrovascular complications, or microvascular complications . The associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models (glms) with a poisson distribution; results are reported as adjusted incidence rate ratios with 95% cis . The associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a distribution, and adjusted results are reported as annualized incremental cost differences . All longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history, resource utilization, and medication use . Sas software version 9.2 was used to conduct statistical analyses, and a two - tailed level of 0.05 was used to determine statistical significance . Of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010, a total of 75,646 patients met the selection criteria and were included in the analysis . As shown in table 1, as of the index date, most patients were older than 55 years (84.1%; mean age 64.7 years) and had an average bmi of 31.6 kg / m . Almost all patients were men (97.4%), and approximately two in three were white (67.4%). During the first cycle, 35.1% of patients achieved both goals (dual - goal achievers), whereas 21.6% achieved only the ldl - c goal (ldl - c achievers), 24.6% achieved only the hba1c goal (hba1c achievers), and 18.6% did not achieve either goal (no - goal achievers) (table 1). Compared with all other groups, dual - goal achievers were older (67.1 vs. 61.464.4 years). Rates of microvascular complications were lower for dual - goal (24.1%) and hba1c achievers (22.4%) than for ldl - c (33.0%) and no - goal achievers (30.2%); similar differences were observed for the usage of insulin (10.4 and 7.5% vs. 34.6 and 30.1%, respectively) and oral antidiabetic drugs (67.4 and 64.0% vs. 82.2 and 82.7%, respectively). A higher rate of macrovascular complications was observed among dual - goal (47.4%) and ldl - c achievers (45.6%) than among hba1c (33.8%) and no - goal achievers (33.9%) (table 1). Patient baseline characteristics, demographics, comorbidities, complications, medications, and resource use the median duration of follow - up time was 4.5 years from the index date . After adjusting for demographics, diabetes - related complications, comorbidities, surgical procedures, diabetic medication use, and health care utilization, dual - goal achievement, when compared with achievement of ldl - c goal alone, was associated with a significantly reduced risk of microvascular complications (adjusted hazard ratio 0.79 [95% ci 0.760.82]), acs (0.88 [0.810.96]), pci (0.78 [0.670.90]), and cabg (0.74 [0.600.92]), but not of the composite cardiovascular - related end point (1.00 [0.941.06]) (fig . Dual - goal achievement was associated with a lower risk of cabg (0.62 [0.490.79]) and the composite cardiovascular end point (0.87 [0.810.93]). Dual, patients achieving both ldl - c and hba1c goals; hba1c, patients achieving only the hba1c goal; ldl - c, patients achieving only the ldl - c goal; none, patients achieving neither goal (see text for details). Compared with no - goal achievers, dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events, and a 4549% lower hazard of undergoing pci or cabg . In addition, relative to no - goal achievers, both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures, except microvascular complications among ldl - c achievers . After controlling for demographics, diabetes - related complications, comorbidities, surgical procedures, diabetic medication use, and health care utilization, dual - goal achievers generally used less health care resources compared with single- or no - goal achievers (fig . 2). In particular, compared with ldl - c achievers, dual - goal achievers had significantly fewer hospitalization days (adjusted incidence rate ratio 0.93 [95% ci 0.871.00]) and outpatient visits (0.88 [0.870.89]). Compared with hba1c achievers, dual - goal achievers had significantly fewer outpatient visits (0.98 [0.971.00]), but there was no statistical difference in the number of hospitalization days (0.98 [0.891.07]). Compared with no - goal achievers, dual - goal achievers had significantly fewer hospitalization days (0.81 [0.720.90]) and outpatient visits (0.86 [0.850.87]). Similarly, both groups of single - goal achievers also had significantly fewer hospitalization days (ldl - c achievers, 0.87 [0.780.96]; hba1c achievers, 0.83 [0.730.94]) and outpatient visits (ldl - c achievers, 0.98 [0.960.99]; hba1c achievers, 0.87 [0.860.89]) than patients who did not achieve either goal . Dual, patients achieving both ldl - c and hba1c goals; hba1c, patients achieving only the hba1c goal; ldl - c, patients achieving only the ldl - c goal; none, patients achieving neither goal . * p <0.05; * * p <0.01; * * * p <0.001 . After adjusting for demographics, diabetes - related complications, comorbidities, surgical procedures, diabetic medication use, and health care utilization, dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ($130.89; p = 0.016), but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ($56.17; p = 0.404) (fig . Diabetes - related medical service costs were significantly lower for dual - goal ($376.50; p <0.001), ldl - c ($245.61; p <0.001), and hba1c achievers ($320.32; p <0.001) compared with those who did not achieve either goal . Dual, patients achieving both ldl - c and hba1c goals; hba1c, patients achieving only the hba1c goal; ldl - c, patients achieving only the ldl - c goal; none, patients achieving neither goal . Of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010, a total of 75,646 patients met the selection criteria and were included in the analysis . As shown in table 1, as of the index date, most patients were older than 55 years (84.1%; mean age 64.7 years) and had an average bmi of 31.6 kg / m . Almost all patients were men (97.4%), and approximately two in three were white (67.4%). During the first cycle, 35.1% of patients achieved both goals (dual - goal achievers), whereas 21.6% achieved only the ldl - c goal (ldl - c achievers), 24.6% achieved only the hba1c goal (hba1c achievers), and 18.6% did not achieve either goal (no - goal achievers) (table 1). Compared with all other groups, dual - goal achievers were older (67.1 vs. 61.464.4 years). Rates of microvascular complications were lower for dual - goal (24.1%) and hba1c achievers (22.4%) than for ldl - c (33.0%) and no - goal achievers (30.2%); similar differences were observed for the usage of insulin (10.4 and 7.5% vs. 34.6 and 30.1%, respectively) and oral antidiabetic drugs (67.4 and 64.0% vs. 82.2 and 82.7%, respectively). A higher rate of macrovascular complications was observed among dual - goal (47.4%) and ldl - c achievers (45.6%) than among hba1c (33.8%) and no - goal achievers (33.9%) (table 1). The median duration of follow - up time was 4.5 years from the index date . After adjusting for demographics, diabetes - related complications, comorbidities, surgical procedures, diabetic medication use, and health care utilization, dual - goal achievement, when compared with achievement of ldl - c goal alone, was associated with a significantly reduced risk of microvascular complications (adjusted hazard ratio 0.79 [95% ci 0.760.82]), acs (0.88 [0.810.96]), pci (0.78 [0.670.90]), and cabg (0.74 [0.600.92]), but not of the composite cardiovascular - related end point (1.00 [0.941.06]) (fig . Dual - goal achievement was associated with a lower risk of cabg (0.62 [0.490.79]) and the composite cardiovascular end point (0.87 [0.810.93]). Dual, patients achieving both ldl - c and hba1c goals; hba1c, patients achieving only the hba1c goal; ldl - c, patients achieving only the ldl - c goal; none, patients achieving neither goal (see text for details). Compared with no - goal achievers, dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events, and a 4549% lower hazard of undergoing pci or cabg . In addition, relative to no - goal achievers, both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures, except microvascular complications among ldl - c achievers . After controlling for demographics, diabetes - related complications, comorbidities, surgical procedures, diabetic medication use, and health care utilization, dual - goal achievers generally used less health care resources compared with single- or no - goal achievers (fig . 2). In particular, compared with ldl - c achievers, dual - goal achievers had significantly fewer hospitalization days (adjusted incidence rate ratio 0.93 [95% ci 0.871.00]) and outpatient visits (0.88 [0.870.89]). Compared with hba1c achievers, dual - goal achievers had significantly fewer outpatient visits (0.98 [0.971.00]), but there was no statistical difference in the number of hospitalization days (0.98 [0.891.07]). Compared with no - goal achievers, dual - goal achievers had significantly fewer hospitalization days (0.81 [0.720.90]) and outpatient visits (0.86 [0.850.87]). Similarly, both groups of single - goal achievers also had significantly fewer hospitalization days (ldl - c achievers, 0.87 [0.780.96]; hba1c achievers, 0.83 [0.730.94]) and outpatient visits (ldl - c achievers, 0.98 [0.960.99]; hba1c achievers, 0.87 [0.860.89]) than patients who did not achieve either goal . Dual, patients achieving both ldl - c and hba1c goals; hba1c, patients achieving only the hba1c goal; ldl - c, patients achieving only the ldl - c goal; none, patients achieving neither goal . * p <0.05; * * p <0.01; * * * p <0.001 . After adjusting for demographics, diabetes - related complications, comorbidities, surgical procedures, diabetic medication use, and health care utilization, dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ($130.89; p = 0.016), but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ($56.17; p = 0.404) (fig . Diabetes - related medical service costs were significantly lower for dual - goal ($376.50; p <0.001), ldl - c ($245.61; p <0.001), and hba1c achievers ($320.32; p <0.001) compared with those who did not achieve either goal . Dual, patients achieving both ldl - c and hba1c goals; hba1c, patients achieving only the hba1c goal; ldl - c, patients achieving only the ldl - c goal; none, patients achieving neither goal . * the study results showed that, compared with the achievement of only the ldl - c goal, achievement of both hba1c and ldl - c goals is associated with a lower risk of microvascular complications, acs, and cardiovascular surgeries (pci or cabg), lower utilization of health care resources, and lower costs of care, but no additional effect was observed for the composite cardiovascular - related end point . In addition, dual - goal achievement relative to hba1c goal achievement is associated with a lower risk of the composite cardiovascular - related end point, cabg, and outpatient visits . To our knowledge, this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . The results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . In steno-2, a danish open - label, randomized, parallel - group study of patients with established t2 dm, patients assigned to receive an intensive treatment targeting tighter goals for blood pressure (systolic <130140 mmhg; diastolic <8085 mmhg), hba1c (<6.5% [48 mmol / mol]), total cholesterol (<175190 mg / dl), and triglycerides (<150 mg / dl) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years, compared with patients assigned to antidiabetic treatment in accordance with national guidelines (21). A 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death (20). In the steno-2 study, lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction, whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications (29). These results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement, while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement . In contrast to the steno-2 study (21), we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c . The blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes (<130/80 mmhg) may be difficult to achieve, and the benefits of achieving this blood pressure goal are unclear (30). We decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements . Current lipid and glycemic goals, however, are relatively easy to achieve, as can be seen from our study, and their effects are therefore easier to take into consideration . Our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals . The 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease (31). This lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials, such as action in diabetes and vascular disease: preterax and diamicron modified release controlled evaluation (advance) (4), action to control cardiovascular risk in diabetes (accord) (2,3), and veterans affairs diabetes trial (vadt) (32), except in the case of patients with newly diagnosed diabetes (5,33). Our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers . Our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes (610). In our study, achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries, which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg (3436) and pci (37,38) often have elevated hba1c levels, and that lowering ldl - c levels is also associated with lower risks of coronary events (6,7,39). This is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes . Our results suggest that dual - goal achievement provides additional economic benefits . Over ldl - c goal alone, but not over hba1c goal alone . Our results are consistent with previous studies that show that control of either ldl - c (40) or hba1c (15) is associated with cost savings . For example, a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s . Patients with diabetes and ldl - c levels> 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl, the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $480950 and $5901,920, respectively (16). A 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm, compared with patients who remained at hba1c levels of 7% (53 mmol / mol) or 6.5% (48 mmol / mol), found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . Would result in $3572 billion savings over the subsequent 10 years (15). First, the data used for this study include laboratory measurements for patients over time . Second, this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements . This allowed for better estimation of the association between goal achievement and risk of complications over time, compared with a simple cross - sectional design, which would use baseline laboratory values in regression models . First, due to the retrospective observational design, the analysis may have been affected by unobserved factors that were not taken into account in the model . Second, the electronic medical records did not include information on disease severity, disease duration, lifestyle modifications, or other interventions . Third, although patients enrolled in the vha do not typically use services outside of the system, any health care services that were administered by a provider outside of the vha were not included in the electronic records . Fourth, because we used vha data, the patients in our study were predominantly male . Although our sample was representative of the vha population, gender imbalance may limit the generalization of our findings . As the vha population may have characteristics that are distinct from those in the general population, similar studies in the general population should be performed . Finally, studies on the clinical and economic benefits associated with triple goal achievement of hba1c, ldl - c, and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . In conclusion, veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits, compared with the achievement of either goal alone . These findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes . To our knowledge, this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . The results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . In steno-2, a danish open - label, randomized, parallel - group study of patients with established t2 dm, patients assigned to receive an intensive treatment targeting tighter goals for blood pressure (systolic <130140 mmhg; diastolic <8085 mmhg), hba1c (<6.5% [48 mmol / mol]), total cholesterol (<175190 mg / dl), and triglycerides (<150 mg / dl) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years, compared with patients assigned to antidiabetic treatment in accordance with national guidelines (21). A 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death (20). In the steno-2 study, lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction, whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications (29). These results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement, while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement . In contrast to the steno-2 study (21), we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c . The blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes (<130/80 mmhg) may be difficult to achieve, and the benefits of achieving this blood pressure goal are unclear (30). We decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements . Current lipid and glycemic goals, however, are relatively easy to achieve, as can be seen from our study, and their effects are therefore easier to take into consideration . Our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals . The 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease (31). This lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials, such as action in diabetes and vascular disease: preterax and diamicron modified release controlled evaluation (advance) (4), action to control cardiovascular risk in diabetes (accord) (2,3), and veterans affairs diabetes trial (vadt) (32), except in the case of patients with newly diagnosed diabetes (5,33). Our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers . Our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes (610). In our study, achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries, which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg (3436) and pci (37,38) often have elevated hba1c levels, and that lowering ldl - c levels is also associated with lower risks of coronary events (6,7,39). This is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes . Our results suggest that dual - goal achievement provides additional economic benefits . Over ldl - c goal alone, but not over hba1c goal alone . Our results are consistent with previous studies that show that control of either ldl - c (40) or hba1c (15) is associated with cost savings . For example, a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s . Patients with diabetes and ldl - c levels> 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl, the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $480950 and $5901,920, respectively (16). A 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm, compared with patients who remained at hba1c levels of 7% (53 mmol / mol) or 6.5% (48 mmol / mol), found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . Would result in $3572 billion savings over the subsequent 10 years (15). First, the data used for this study include laboratory measurements for patients over time . Second, this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements . This allowed for better estimation of the association between goal achievement and risk of complications over time, compared with a simple cross - sectional design, which would use baseline laboratory values in regression models . First, due to the retrospective observational design, the analysis may have been affected by unobserved factors that were not taken into account in the model . Second, the electronic medical records did not include information on disease severity, disease duration, lifestyle modifications, or other interventions . Third, although patients enrolled in the vha do not typically use services outside of the system, any health care services that were administered by a provider outside of the vha were not included in the electronic records . Fourth, because we used vha data, the patients in our study were predominantly male . Although our sample was representative of the vha population, gender imbalance may limit the generalization of our findings . As the vha population may have characteristics that are distinct from those in the general population, similar studies in the general population should be performed . Finally, studies on the clinical and economic benefits associated with triple goal achievement of hba1c, ldl - c, and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . In conclusion, this retrospective study among u.s . Veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits, compared with the achievement of either goal alone . These findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes.
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Endotracheal intubation is one of the most important skills for anesthesiologists in securing the airway during general anesthesia and resuscitation . Failure to secure the airway can cause anesthesia - related life - threatening morbidity and mortality . However, the difficult laryngoscopy and tracheal intubation rate still remains at 1.513% due to poor reliability of traditional protocols, algorithms, and combinations of screening tools in identifying a potentially difficult airway . Due to the portable, noninvasive characteristics, point - of - care ultrasound (us) technique has been widely used in the operating room for ultrasound - guided nerve block, central venous access, and pneumothorax diagnosis . With improved visualization of airway structures, prasad et al . First found that us can reliably image all of the structures visualized by ct, and the infrahyoid airway structure parameters measured by ultrasound agree well with the parameters measured by ct . Adhikari et al . Further demonstrated that the anterior neck soft tissue thickness measured by ultrasound at hyoid bone and thyrohyoid membrane levels can be used as an index to predict difficult laryngoscopy, but only the anterior neck soft tissue thickness at thyrohyoid membrane levels can be used as an independent predictor of difficult laryngoscopy . Interestingly, they did not find a correlation between us measurements and clinical screening tests . . Also found that an abundance of fat tissue at the anterior neck region as detected by ultrasound in israeli obesity patients was a very good independent predictor of difficult laryngoscopy, being a much better predictor than body mass index (bmi) per se . However, a similar study in the unites states showed that us quantification of anterior soft tissue and general bedside screening tests failed to predict difficult laryngoscopy in american obese patients, suggesting that racial or body shape differences might exist . To define the role of airway us in predicting difficult laryngoscopy, we evaluated the feasibility of ultrasound in predicting difficult laryngoscopy in a chinese han population . After approval of the research protocol by the local hospital ethics committee for human studies and obtaining personal informed consent, 203 american society of anesthesiologists (asa) grade i and ii adult patients undergoing elective surgeries and receiving general anesthesia were included in this study . Exclusion criteria included patients who had facial, cervical, pharyngeal and epiglottis surgical or trauma history, patients with most teeth lost, and patients with arthritis . The modified mallampati score (mms) was specified according to the visibility of pharyngeal structures with the patient in an upright sitting position, head in neutral position, mouth wide open, and tongue protruding to its maximum without phonation (figure 1a). Class i is visualization of the hard palate, soft palate, fauces, uvula, and pillars . Class ii is visualization of the hard palate, soft palate, fauces, and base of uvula . Thyromental distance (tmd) was measured from the mental prominence to the thyroid cartilage with the patient s neck extended fully . Interincisor gap (iig) was measured from the upper central incisors to the lower central incisors while the patient s mouth was fully opened . Ultrasound measurements were performed by the primary investigator with the patient supine and the head and neck in neutral position . The thicknesses of anterior neck soft tissue at hyoid bone, thyrohyoid membrane, and anterior commissure levels were obtained transversely across the anterior surface of the neck with a 136 mhz hfl38 linear array ultrasound probe attached to a sonosite s - nerve machine (sonosite inc ., the minimal distance from the hyoid bone to skin surface (dshb) was measured (figure 2a, 2b). At thyrohyoid membrane level, the distance from skin to epiglottis midway (dsem) between the hyoid bone and thyroid cartilage was measured (figure 2c, 2d). At anterior commissure level, the minimal distance from skin to anterior commissure (dsac) was obtained (figure 2e, 2f). After anesthesia induction with midazolam 0.04 mg / kg, propofol 22.5 mg / kg, fentanyl 24 g / kg, and succinylcholine 2 mg / kg, endotracheal intubation was carried out by anesthesia providers with a minimum of 2 years of endotracheal intubation experience . The macintosh blades were used to expose the target larynx, and no external laryngeal pressure was used to facilitate this process . Classification of laryngoscopic views was based on the method described by cormack and lehane (figure 1b). Continuous data are expressed as means (standard deviation [sd]); categorical data are expressed as numbers of occurrences (percents). Comparison analysis was performed using the t - test for continuous variables and chi - square or fisher exact tests, as appropriate, for non - continuous variables . The best fit lines of dshb, dsem, dsac, mms, iig, and tmd were determined using a least - squares regression technique . Receiver operating characteristic (roc) analyses were used to calculate the comparable threshold values of dshb, dsem, dsac, tmd, iig, and mms . Optimal cutoff values were calculated using the youden index (calculated as: sensitivity + specificity 100). Statistical analyses were performed using medcalc for windows, version 12.6.1.0 (medcalc software, ostend, belgium). The level of statistical significance was p<0.05, and p<0.0001 was considered to be very statistically significant . Continuous data are expressed as means (standard deviation [sd]); categorical data are expressed as numbers of occurrences (percents). Comparison analysis was performed using the t - test for continuous variables and chi - square or fisher exact tests, as appropriate, for non - continuous variables . The best fit lines of dshb, dsem, dsac, mms, iig, and tmd were determined using a least - squares regression technique . Receiver operating characteristic (roc) analyses were used to calculate the comparable threshold values of dshb, dsem, dsac, tmd, iig, and mms . Optimal cutoff values were calculated using the youden index (calculated as: sensitivity + specificity 100). Statistical analyses were performed using medcalc for windows, version 12.6.1.0 (medcalc software, ostend, belgium). The level of statistical significance was p<0.05, and p<0.0001 was considered to be very statistically significant . A total of 203 eligible patients (120 females, 83 males) were included in this study . There were 28 of 203 patients (13.8%) categorized as difficult laryngoscopy, and 50% of patients with difficult laryngoscopy in our study were males . No differences were noted in sex, age, and height, but bw and bmi values in the difficult laryngoscopy group were higher . The bmi value was 25.632.80 kg / m for the difficult laryngoscopy group, and 23.613.43 kg / m for the easy laryngoscopy group (p<0.05). Airway evaluation parameters, including mms, tmd, iig, dshb, dsem, and dsac, are shown in table 2 . The thicknesses of anterior neck soft tissue measured by us were greater in the difficult laryngoscopy group compared to the easy laryngoscopy group at the level of the hyoid bone, thyrohyoid membrane, and anterior commissure . However, no statistically significant difference was found between easy and difficult laryngoscopy groups for tmd and iig . A strong positive correlation existed between dsem and dshb; moderate positive correlations between dsem and dsac, dshb and dsac; small positive correlations between mms and dshb, mms and dsem, mms and dsac; and very small positive correlations between tmd and iig . The correlation coefficients along with 95% confidence interval (ci) between dsem and dshb, dsem and dsac, dshb and dsac, mms and dshb, mms and dsem, mms and dsac, and between tmd and iig were 0.74 (0.670.80, p<0.0001), 0.60 (0.500.68, p<0.0001), 0.69 (0.610.75, p<0.0001), 0.32 (0.190.44, p<0.0001), 0.27 (0.140.40, p=0.0001), 0.32 (0.190.44, p<0.0001), 0.18 (0.05 to 0.31, p=0.0089), respectively (figure 3a3 g). Small negative correlation were found between iig and mms (r=0.27, p=0.0001); and very small negative correlations were found between mms and tmd (r=0.20, p=0.0042), iig and dsac (r=0.18, p=0.0113), and iig and dshb (r=0.15, p=0.0344) (figure 3h3k). No correlations were found between tmd and dsem, tmd and dshb, and tmd and dsac (data not shown). To further assess the roles of mms, iig, tmd, dshb, dsem, and dsac in predicting difficult laryngoscopy, the roc curves (figure 4) were drawn using medcalc software with laryngoscopy grade over ii as the threshold of difficult laryngoscopy . As determined by the youden index, the optimal cutoff values (with sensitivity and specificity in parentheses) for mms, iig, tmd, dshb, dsem, and dsac to predict difficult laryngoscopy were over 2 (50.0%, 82.3%), 3.8 cm (75.0%, 48.0%), 5.9 cm (21.4%, 93.7%), 1.28 cm (85.7%, 85.1%), 1.78 cm (100.0%, 66.3%), and 1.1 cm (75.0%, 80.6%), respectively . The areas under the roc curve (aucs) were significantly different from the area under the reference line (area=0.5) for mms, dshb, dsem, and dsac; and the p values of mms, dshb, dsem, and dsac were all less than 0.0001 . No difference between the aucs of tmd, iig, and the area under the reference line were identified, suggesting that mms, dshb, dsem, and dsac might be better than tmd and iig in predicting difficult laryngoscopy (table 3). With the rapid expansion of portable us in anesthesia for guiding the placement of central venous catheters and the performance of regional block [1113], some preliminary results exploring the feasibility of portable us in airway evaluation and management have been reported, even though the air attenuates us transmission and thereby creates artifacts [35,14,15]. In the present study, we compared the role of the us measurements and traditional screening tests, including iig, tmd, and mms, in predicting difficult laryngoscopy in a chinese han population . We found that strong positive linear correlations existed among the thicknesses of anterior neck soft tissue measured by us at hyoid bone, thyrohyoid membrane, and anterior commissure levels . The aucs of mms, dshb, dsem, and dsac are all over 0.7, indicating they are all good parameters in predicting difficult laryngoscopy . The aucs of tmd and iig were less than 0.7, suggesting that tmd and iig were poor parameters in predicating difficult laryngoscopy . To clearly visualize the glottis and smoothly finish the intubation procedure, anesthesiologists need to place the laryngoscope blade to the base of the epiglottis, and then lift it up therefore, in addition to the performer s skills and experience, many factors, including dental status, mouth opening, oropharyngeal space, mandibular space, and neck motility, are all involved this complicated procedure . Mms mainly reflects the size of the tongue relative to the oral cavity, or the oropharyngeal space [1820], iig means the mouth opening, and tmd can reflect the length of the neck . The mallapati score was first introduced by mallampati et al . In 1985, and then modified by samsoon and young in 1987 . Due to the simple and clear evaluation standards, mms has become one of the most commonly used tools to predict a difficult airway . However, mms mainly depends on patient cooperation and body position, which can greatly affect the accuracy of airway evaluation . Therefore, mms has been reported to be a good predictor by many, but was found to be of limited value by others . A recent meta - analysis involving 177 088 patients demonstrate that mms is inadequate as a stand - alone test to predict difficult laryngoscopy or tracheal intubation, but it may well be a part of a multifactorial model for the prediction of a difficult tracheal intubation . Iig has been repeatedly reported to be associated with difficult intubation, but our results showed that there was no significant difference in mean iig between difficult and easy laryngoscopy groups (3.740.43 vs. 3.860.47 cm). Furthermore, the auc of iig was 0.580.06, which was far less than 0.7, suggesting that iig might be not a good screening parameter for predicting difficult laryngoscopy in our study . Our iig conclusion was similar to the previous report by savva, but different from results of other studies . Whether this difference was really caused by racial factors requires further investigation because the measurements of iig need patients to fully cooperate . Tmd is also used as a screening test to predict difficult airway, but our results suggest that tmd is not a useful screening test . However, we could not determine whether this was because of limited patient numbers or because tmd was not useful in predicting difficult airway due to the small sample size . Ct, mri, and other imaging techniques can accurately measure the thickness of anterior neck soft tissue, but they are expensive and unavailable in many operating rooms . Portable us is inexpensive, rapid, and convenient to perform in the operating room, and most importantly, it can quantify the neck fat thickness as accurately as mi . However, the results of 2 prior studies focusing on soft tissue thickness at and below the level of the vocal cords in a specific group of obese patients are inconsistent . Further measured the anterior neck soft tissue at the hyoid bone and thyrohyoid membrane levels, which is important to displace the glottis by the laryngoscopic blade, and found that us measurements of anterior neck soft tissue thickness at the level of hyoid bone and thyrohyoid membrane can be used to predict difficult laryngoscopies, and a 2.8-cm us measurement at the thyrohyoid membrane was a good independent predictor of difficult laryngoscopy . Our results show that the thicknesses of anterior neck soft tissue at the level of the hyoid bone (1.51 [95% ci=1.401.61] cm vs. 0.98 [95% ci=0.941.02] cm), the thyrohyoid membrane (2.39 [95% ci=2.172.62] cm vs. 1.49 [95% ci=1.431.55] cm), and the anterior commissure (1.30 [95% ci=1.181.42] cm vs. 0.92 [95% ci=0.870.94] cm) were greater in the difficult laryngoscopy group and were significantly correlated . Furthermore, the ranges of anterior neck soft tissue for those with difficult laryngoscopy were mutually exclusive from those patients with an easy laryngoscopy, indicating that they are independent predictors of difficult laryngoscopy, even though the ranges of these 3 parameters were smaller than in a previous report . Glottis exposure by place laryngoscope is a very complicated procedure, and many subjective and objective factors such as the provider s skills and experience, airway secretions, and abnormalities of anatomical structures are involved in this procedure . The investigators were not totally blinded to the study purpose, and some clinical signs might indicate the possibility of difficult laryngoscopy, which can cause some bias during us measurements . Due to the low incidence of difficult laryngoscopy, it is impossible for us to randomly assign an equal number of patients to both groups, thus there were 28 patients in the difficult group and 175 patients in the easy group . Anterior neck soft tissue thicknesses measured by us at the hyoid bone, thyrohyoid membrane, and anterior commissure levels are independent predictors of difficult laryngoscopy . The commonly used screening tests for difficult intubation have only poor - to - moderate predictive power when used alone . Combinations of these screening tests or risk factors with us measurements might increase the ability to predict difficult laryngoscopy.
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Badana grupa skadaa si z 96 pacjentw (77 mczyzn i 19 kobiet) w wieku rednio 58,1 9,8 roku . Pacjenci zostali losowo przydzieleni do jednej z trzech grup: grupy pacjentw poddanych perikardiotomii tylnej (grupa i, n = 30), grupy kontrolnej (grupa ii, n = 33) oraz pacjentw, u ktrych zastosowano tylny drena osierdziowy, ktrzy jednak nie zostali poddani zabiegowi perikardiotomii tylnej (grupa iii, n = 33). Czas trwania hospitalizacji pooperacyjnej (p = 0,03; 11,56 10,64) oraz czsto reoperacji z powodu tamponady (p = 0,019; 12,1%) byy znaczco wysze w grupie ii . Znaczny wysik osierdziowy wykryto u jednego pacjenta (grupa ii, n = 1) w pierwszym dniu po operacji, u jednego pacjenta w pitym dniu po operacji (grupa iii, n = 1) oraz u jednego pacjenta w 30 dniu po operacji (p = 0,028) w grupie i odnotowano regresj wysiku osierdziowego . I stwierdzono rwnie wyszy wspczynnik pooperacyjnego migotania przedsionkw, jednake w tym wzgldzie midzy grupami nie byo znaczcych rnic . Pacjenci, ktrzy nie zostali poddani perikardiotomii tylnej lub u ktrych nie zastosowano tylnego drenau klatki piersiowej, wykazywali szcztkowy wysik osierdziowy, wymagali duszej hospitalizacji i musieli by ponownie operowani z powodu tamponady . Zarwno perikardiotomia tylna, jak i tylny drena klatki piersiowej okazay si skuteczne we wczesnym okresie pooperacyjnym . Pericardial effusion, which is a common complication after coronary artery surgery (cas), may be the cause of significant morbidity . It is often benign and small in volume, but can lead to life - threatening events, such as cardiac tamponade . Delayed postoperative cardiac tamponade following an open - heart operation is an infrequent, but potentially serious complication . Although previous reports have dealt with the clinical features and management of this problem, an optimal approach to prevent postoperative pericardial effusion and related problems has not been clearly defined [3, 5]. This study aims to investigate the effects of posterior pericardiotomy (pp) on the development of pericardial effusion, tamponade, and atrial fibrillation following cas . This prospective randomized case - controlled study was carried out on 96 patients (77 men and 19 women; age: 35 - 78 years; mean: 58.1 years). The patients, undergoing cas performed by the same surgical team between march 2012 and january 2013 in istanbul mehmet akif ersoy thoracic and cardiovascular surgery research and training hospital, were assigned to 3 groups . After the study had received the approval of the institutional review board, informed consent was obtained from all patients . The treating physician did not know the groups to which individual patients had been assigned . Patients with renal failure, hyperthyroidism, emergency cas, history of cardiac operations associated with valvular heart disease, low ejection fraction (<35%), and preoperative atrial fibrillation or other rhythm disorders, as well as patients who did not provide research authorization, were excluded from the study . The study groups included patients after pp (group i, n = 30), a control group (group ii, n = 33), and patients without pp who received posterior pericardial tubes (the tube was placed behind the heart and sutured to the adjacent pericardial tissue to avoid tube - induced ventricular arrhythmias) (group iii, n = 33). I, a 4-cm longitudinal incision was made parallel and posterior to the left phrenic nerve, extending from the left inferior pulmonary vein to the diaphragm, as described by mulay et al . . In group i, the relationship between the location of the distal part of circumflex anastomosis and pp was also carefully considered . Moreover, all patients underwent pleurotomy and lateral pericardiotomy (approximately 3.5 - 4.5 cm in size) in which the internal thoracic artery (ita) was used for anastomosis and positioned medially to the lung, which allowed the ita to run without kinking or twisting . The inferior part of the pericardium was left open (2 cm) in our study . A straight tube was placed in the anterior mediastinum and an angled tube was placed in the left hemithorax in all patients . The same postoperative pain management protocol was provided for all patients . After a routine closure of the chest, drains were frequently milked and stripped to ensure tube patency during the intensive care unit stay . The chest tubes were removed on the 2 postoperative day when the drainage became serous; otherwise, they remained in use for one or more days . Pericardial effusion was defined as the accumulation of fluid in the pericardial space determined by transthoracic echocardiography (tte). The presence of pericardial effusion on 2-dimensional tte was assessed using the criteria previously described by bakhshandeh et al . . Two - dimensional echocardiography was performed on postoperative days 1 and 5, as well as 1 month after surgery . The maximum diastolic separation between the pericardium and epicardium was measured at the tip of the mitral valve leaflet . When the diastolic echo - free space between the left ventricular posterior wall and the pericardium was <10 mm, the pericardial effusion was classified as small . When the space was 10 - 20 mm, the effusion was classified as moderate, while for spaces> 20 mm, the effusion was classified as severe . If the diagnosis of evident pericardial effusion (moderate or severe) was established, tte was performed daily, and the volume of the effusion was observed closely . We compared the groups with regard to the volume of effusion on postoperative days 1, 5, and 30 . Patients with no or small pericardial effusion and an uneventful postoperative course were discharged from the hospital on postoperative day 5 . Patients with moderate and severe pericardial effusion were treated with non - steroidal anti - inflammatory drugs . During hospitalization, all patients were continuously monitored with the use of a portable electrocardiogram (ecg, telemetry) to detect atrial fibrillation . Atrial fibrillation was considered to be persistent and clinically significant when it lasted for more than 5 min . Statistical analysis was performed with the ncss (number cruncher statistical system) 2007 statistical software (utah, usa). One - way anova and tukey hsd tests were used for the statistical analysis of the groups parameters . The differences were also analyzed using tests and independent t - tests where applicable . The hospital mortality in the study was 2% (n = 2); both patients were in group ii . One of these patients experienced low cardiac output during and following reoperation due to cardiac tamponade and died on postoperative day 12 . The other patient had an uneventful postoperative course, but was readmitted to the hospital on postoperative day 11 due to cardiac tamponade and acute kidney failure . Pericardial drainage was performed, but the patient died of sepsis on postoperative day 45 . The groups did not differ in terms of demographic parameters, which are summarized in table i. the average total volume of drainage (mediastinal and thoracic) during the first 24 hours was similar in all groups (536.67 237.873 cc, 656.97 407.634 cc, and 577.27 374.349 cc, respectively; p = 0.383) (table ii). In group i, the volume of the fluid evacuated by mediastinal drainage was lower than in groups ii and iii, but the difference was not statistically significant (table ii). Two patients in group ii required reoperation due to excessive mediastinal hemorrhage and cardiac tamponade . The incidence of cardiac tamponade was higher in group ii (n = 4, 12%), and the differences between the groups were statistically significant (p = 0.019). Two of the patients with pericardial tamponade were managed by subxiphoid pericardial drainage alone; 400 to 1000 ml of fluid was removed . Two patients in group iii were reoperated due to excessive hemorrhage in the early postoperative period . Demographic variables in groups differential drainage volume from each chest tube the volume of pericardial drainage was added to the volume of mediastinal drainage for group iii . The average volume of pericardial effusion significantly decreased with time in groups i and ii (fig . Approximately 50% of the patients had small pericardial effusion on the first postoperative day; no difference between the groups was found . Moderate pericardial effusion was detected in 8 patients (group i, n = 1; group ii, n = 3; group iii, n = 4) on the first postoperative day, in 11 patients (group ii, n = 7; group iii, n = 4) on the fifth postoperative day, and in 7 patients on the 30 postoperative day (group ii, n = 6; group iii, n = 1) during the study period . Severe pericardial effusion was detected in one patient (group ii, n = 1) on the first postoperative day, in one patient (group iii, n = 1) on the fifth postoperative day, and in one patient (group iii, n = 1) on the 30 postoperative day . The incidence of moderate to severe pericardial effusion in group i was significantly lower than in the other groups on the 30 postoperative day (n = 0, p = 0.028). Figure 1 shows a comparison of the groups with regard to the volume of pericardial effusion . Four of these patients (group ii, n = 4) underwent drainage of severe pericardial effusion on postoperative days 0, 1, 11, and 17; one patient (group ii) underwent sternal dehiscence repair . Comparison of the groups with regard to the volume of pericardial effusion atrial fibrillation was detected in 24 of the 96 patients (25%: 6 patients in group i, 11 patients in group ii, 7 patients in group iii). No statistically significant difference between the groups (p = 0.392) was present in this respect . The length of hospital stay was significantly shorter in group i (6.63 2.71, p = 0.03). There are a number of articles favoring the use of posterior pericardiotomy, but no comprehensive comparison has been conducted between the use of a pericardial tube and pp in patients undergoing cas . Pericardial effusion and the associated complications are common after all kinds of cardiac surgery due to postoperative bleeding or post - cardiotomy syndrome . However, clinically significant pericardial effusion occurs rarely and can be a crucial risk factor for cardiac tamponade (1%). Posterior pericardiotomy has been reported to reduce the prevalence of pericardial effusion from 40% to 8% with a simultaneous reduction in the prevalence of svt (supraventricular tachycardia). The semi - sitting position helps thoracic drainage in the early postoperative period by supporting the patient's back . Despite all the above - mentioned measures, the posterior wall of the pericardial sac forms an unprotected space that may cause pericardial effusion to develop septae . This leads to the appearance of adhesions preventing blood drainage, leading to cardiac compression . Moreover, the collection of fluid may persist due to bleeding resulting from the use of anticoagulants . In fact, anti - coagulants (low - molecular - weight heparin) were used postoperatively in our patients with atrial fibrillation . Posterior pericardial effusion can be evacuated by placing a tube in the posterior pericardial space . The placement of the pericardial tube is simple to perform, but it may have certain drawbacks . The tube may come into contact with coronary grafts during cardiac activity or movement . However, the patients included in the study (in group iii as well as the other groups) did not exhibit any arrhythmias (apart from af) or ischemic changes . This may be due to the fact that we fixed the pericardial tube to the surface of the diaphragmatic pericardium with polypropylene . Pp or the use of posterior tubes may lessen the volume of pericardial effusion after cas . Moreover, patients in group i exhibited regression of pericardial effusion during 30 postoperative days . This proved that pp had a lasting effect on pericardial effusion, as presented by figure 1 . What stands out in our results is that pericardial effusion may occur if no intervention is performed on the posterior part of the pericardium . This technique is suggested in both cas and valve surgery, but it is not routinely used [13, 14]. What is more, pp is recommended by the american college of chest physicians guidelines and by some authors as a preventive technique for postoperative atrial fibrillation [15, 16]. Supraventricular tachyarrhythmias, mainly in the form of atrial fibrillation, occur in up to 40% of patients undergoing cas . Although af is usually benign, it can cause hemodynamic instability, prolonged hospital stay, and increased cost and, in rare cases, predispose to cerebrovascular accidents . The antiarrhythmic effect of pp may, however, be a theoretical issue, as we observed similar results regarding atrial fibrillation in all the groups . In fact, the results of our study were different than expected with regard to atrial fibrillation compared to other researchers [4, 6, 16]. Additionally, asimakopoulos et al . Also found that the prevalence of atrial fibrillation was not significantly reduced (20%) in comparison to the conventional technique (26%). Another issue is that humans have increasingly longer life spans, which may result in higher numbers of reoperations . Ellman et al . Found that patent coronary grafts were the most commonly injured structures during reoperation (46%). We routinely cover the aorta and proximal anastomoses of grafts with thymic fat tissue and the anterior wall of the pericardial cavity . We also place an anterior mediastinal tube over the thymic fat tissue before closing the sternum in all patients . Behind the heart, adhesions between the inferior surface of the heart and the diaphragm can cause difficulties when dissecting the tissue layers during reoperation . Mulay et al . Demonstrated that pp provides an effective drainage pathway from the left pleural cavity, thereby reducing the prevalence of pericardial effusion . For this reason, pp may be advantageous for reoperated patients in terms of reducing adhesions related to pericardial effusion . Echocardiography can be useful in confirming and identifying the presence of pericardial effusion, hematoma, and tamponade . According to the results of our study, it is important to diagnose pericardial effusion at an early stage . Hemodynamic deterioration due to pericardial fluid can be divided into four stages: pericardial fluid, hemodynamic tamponade, echocardiographic tamponade, clinical tamponade . Echocardiographic follow - up of patients with pericardial effusion might be a factor extending the duration of hospital stay . We recommend the pp technique and routine echocardiographic monitoring during the postoperative period in patients undergoing cas . There is not sufficient research regarding the effects of pp on long - term results . The present study also demonstrated that patients undergoing pp required a significantly shorter hospital stay and experienced lower rates of pericardial effusion and cardiac tamponade than the other groups . This research was carried out on a small set of patients (n = 96); therefore, there was an imbalance between men and women enrolled in this trial . Group ii had a 2-fold elevation in the number of patients going into af postoperatively . In conclusion, pp may be a useful technique in the prevention of pericardial effusion and cardiac tamponade after cas because it is simple, safe, and effective . . Oral presentation at the 22 annual meeting of the asian society for cardiovascular and thoracic surgery that will be held in istanbul on april 3 - 6, 2014.
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Cancer places a huge burden on society and has been identified as the leading cause of death in both more and less economically developed countries . Projections based on the globocan 2012 estimates predict a substantive increase to 19.3 million new cancer cases per year by 2025, due to growth and ageing of the global population . South africa, like other developing countries, is also experiencing an increase in the overall burden of disease attributable to cancer, with the number of new cancer cases predicted to increase by 46% by 2030 . This is likely to result in an increase in the use of cancer chemotherapy agents, which assist in preventing the proliferation, invasion, and metastases of the cancer cells . The basis for chemotherapy is anticancer drugs containing platinum, that is, cisplatin (cis - diamminedichloroplatinum ii) and carboplatin (cis - diammine 1,1-cyclobutane dicarboxylatoplatinum ii). Other chemotherapy drugs include nitrogen mustard, amino - nicotinamide, dichloromethotrexate, bleomycin, and 5-fluorouracil [5, 6]. The first of these drugs, that is, cisplatin, consists of a divalent pt (ii) central atom and four ligands of cis - positioned pairs of chlorine atoms or amine groups . Since its discovery in the 1970s, cisplatin continues to be hailed as one of the most potent cancer chemotherapeutics in children and adults, as it is unique and unmatched in its effectiveness against many cancers, namely, osteogenic sarcoma, medulloblastoma, testicular, cervical, and ovarian cancers . Similarly, its toxicity profile is expansive, involving the gastrointestinal, hematologic, renal, and auditory systems . While the use of saline hydration and mannitol diuresis may prevent nephrotoxicity, neurotoxicity is still not curable or preventable . Ototoxicity refers to the hearing disorder that results from the temporary or permanent inner ear dysfunction after treatment with an ototoxic drug . Other drug classes known to have ototoxic properties include aminoglycosides, loop diuretics, quinine, nonsteroidal anti - inflammatory drugs, and antiretroviral therapy (art). This is of concern in south africa, as it is estimated that 12.2% of the population (6.4 million persons) were hiv positive in 2012, which is 1.2 million more people living with hiv than in 2008 (10.6%, or 5.2 million). Resultantly, art exposure had almost doubled from 16.6% in 2008 to 31.2% in 2012 . Not only will many infected people be at risk for ototoxicity due to arts, but a large number will also be susceptible to hiv - related cancers, such as kaposi's sarcoma, non - hodgkin's lymphoma, and cervical cancer, as well as infectious diseases such as tuberculosis, conditions that often require pharmacological therapy with the adverse side effect of ototoxicity . It is possible that their treatments could consist of simultaneous use of more than one ototoxic drug, increasing the likelihood of ototoxicity . All health care professionals managing patients with cancer should therefore be knowledgeable about the ototoxic properties of cisplatin . However, malhotra indicated that most oncologists in india do not make referrals for audiological evaluations of patients receiving cisplatin, while a study in south africa revealed that the effects of ototoxicity, the role of audiologists, and need for their expertise were not fully realized by the oncologists sampled . This is further supported by evidence from the south african study of khoza - shangase and jina which indicated that most general practitioners sampled also do not appear to carry out ototoxicity monitoring strategies, despite being aware of their own role within an ototoxicity monitoring programme . This review therefore aims to serve as resource for health professionals to enhance their understanding of ototoxicity as well as their roles within an ototoxicity monitoring programme by providing an overview and description of this condition in patients diagnosed with cancer and receiving cisplatin chemotherapy . The review identified peer - reviewed articles available from january 1975 to july 2015 on the topic of cisplatin - associated ototoxicity and ototoxicity monitoring and included english articles only . The same researcher conducted the literature search and reviewed the abstracts and articles for inclusion in the study . Studies were identified using keyword and mesh term searches of electronic databases depicted in table 1 . A manual search of relevant authors and journals the references cited by each publication, review, or book chapter were reviewed in order to locate additional potential publications . In order to be selected, the article had to present data on either cisplatin ototoxicity and/or ototoxicity monitoring in human participants, and no research designs were excluded . Running these searches yielded a total of 2106 records, of which 1581 were excluded based on the title and/or abstract as well as duplication . Eighty - five relevant articles, comprising six national and 79 international articles, were selected . A perusal of narrative reviews of other auditory pathologies was conducted in an attempt to determine areas of significance for an overview of cisplatin ototoxicity . This resulted in the following eight areas being included: the mechanisms of cisplatin ototoxicity, clinical presentation, risk factors, incidence rates in adults and children, the effect on quality of life, ototoxicity monitoring, otoprotective strategies, and management of an ototoxic hearing loss . One such mechanism, the antioxidant model, involves the formation of reactive oxygen species (ros) within the cochlea and consequent reduction in antioxidant enzymes following exposure to cisplatin chemotherapy [1620]. Another mechanism of cisplatin ototoxicity involves the significant contribution of nicotinamide adenine dinucleotide phosphate oxidase 3 isoform (nox3) to the generation of reactive oxygen species within the cochlea, when activated by cisplatin [17, 21], while a third mechanism relates to the activation of transient receptor potential vanilloid 1 channel (trpv1) [2224]. The molecular mechanisms of cisplatin ototoxicity therefore include the following:creation of reactive oxygen species, depletion of antioxidant glutathione and its regenerating enzymes, increased rate of lipid peroxidation, oxidative modifications of proteins, nucleic acids damage by caspase system activation ands - nitrosylation of cochlear proteins .with the cellular mechanisms of cisplatin - associated ototoxicity including damage to the outer hair cells, supporting cells, marginal cells of the stria vascularis, spiral ligament, and the spiral ganglion cells, it is evident that the structures of the inner ear are most susceptible to damage by cisplatin chemotherapy, with apoptotic degeneration of the hair cell in the organ of corti being most prominent . The outer hair cells in the basal turn of the cochlea are most affected [27, 28]. This leads to an initial elevation of high frequency audiometric thresholds, followed by a progressive loss into the lower frequencies with continued therapy [27, 28]. Knowledge of the different mechanisms of cisplatin ototoxicity is important for health care professionals as it will create an awareness of its complexity and the resulting clinical presentation . Creation of reactive oxygen species, depletion of antioxidant glutathione and its regenerating enzymes, increased rate of lipid peroxidation, oxidative modifications of proteins, nucleic acids damage by caspase system activation and s - nitrosylation of cochlear proteins . Cisplatin - associated ototoxicity usually manifests as irreversible, progressive, bilateral, high frequency sensorineural hearing loss with tinnitus . The latter may occur with or without a hearing loss and may be permanent or transient, sometimes disappearing a few hours after treatment or alternatively persisting a week after treatment . While most of the hearing loss is permanent, there is sometimes sporadic and partial recovery . In addition, rare cases of unilateral hearing loss have been reported, which are usually explained by tumour location and surgical or therapeutic intervention on the affected side . Moreover, the hearing loss is not always symmetrical [33, 34], with jenkins et al . Finding that 75% of women on cisplatin chemotherapy displayed an asymmetry of hearing thresholds of at least 10 db between ears posttreatment . Schmidt et al ., in their investigation of 55 children on cisplatin chemotherapy, found that the high frequency hearing thresholds were slightly elevated in the left ear and that males had a greater degree of hearing loss than the females . The degree of hearing loss is often variable and is related to the dose; that is, the higher the cumulative dose, the greater the ototoxic effect [35, 36]. The duration, number of cycles administered, and method of administration also influence cisplatin - associated ototoxicity . Additional factors that may increase ototoxicity include exposure to concomitant noise, chemicals, and other ototoxic medications . Furthermore, evidence also shows that melanin content is related to an increased risk of cisplatin - associated ototoxicity . Individuals with dark eyes and therefore a higher melanin content in the cochlear are at greater risk of ototoxic damage, as the melanin causes retention of the platinum within the cochlear and subsequently increases the risk of damage [41, 42]. Individuals presenting with renal insufficiency, that is, high levels of serum creatinine, are at a greater risk for cisplatin - associated ototoxicity . Genetic risk factors, such as megalin and glutathione s - transferases gene polymorphism, have also been reported to influence cisplatin ototoxicity, as do physiological factors such as age, with younger children and older adults (older than 46 years) presenting with a greater severity of hearing damage . Awareness of these risk factors may assist health care professionals with informational counselling of the patient receiving cisplatin chemotherapy . The incidence of cisplatin ototoxicity is variable in adults (table 2) and children (table 3). The variations may be due to a number of factors, such as differences in the dose, both within a cycle and the total amount administered over multiple cycles, time interval between courses, method of administration, and treatment duration, as well as differences in patient population . Ototoxicity poses a major problem to the cancer patient, as the quality of life after receiving cisplatin chemotherapy may be negatively affected due to hearing loss, resulting in social, emotional, and vocational difficulties, as effective communication is often hindered . Tasks that normal hearing persons take for granted may become challenging and frustrating . In addition, an individual's safety may be compromised due to the hearing loss, as appropriate response to alarms and warning signals may be delayed . Furthermore, a hearing loss may also result in psychosocial and physical health problems, as well as depression and social isolation . Hence, hearing loss, often referred to as the invisible condition, has serious visible ramifications on the quality of life of a hearing impaired individual . This is particularly relevant if the individual has already experienced the hearing world, as the hearing function is never restored to normal, even though patients may benefit from the use of assistive listening devices, such as hearing aids and cochlear implants . The impact of an ototoxic hearing loss may be more profound for infants and young children who are at a critical stage of their speech and language development . Furthermore, the high frequency nature of an ototoxic hearing loss may result in speech recognition and comprehension being compromised, resulting in possible neurocognitive and psychosocial delays . There is also an elevated risk for academic learning problems and psychosocial difficulties in school - aged children and adolescents . Literature indicated that childhood survivors of neuroblastoma had twice the rate of difficulties, as indicated by parent reports, with reading and math skills, and/or attention and a higher risk of a general learning disability than those without a hearing loss . There was also poorer self - reported quality of life scores in these children with regard to school functioning . Hence, cisplatin - associated ototoxicity further complicates the morbidity of cancer patients, as it would also isolate them from family members and significant others at a time when they require the greatest support . Advancements in medical knowledge and technology, such as screening and early detection of several cancers, have resulted in notable improvements in relative five - year survival rates for cancer [64, 65]. Therefore, improving the quality of life after cisplatin - based chemotherapy becomes increasingly important, and resulting comorbidities such as ototoxicity can be managed appropriately and immediately if adequate monitoring is in place . The nature of ototoxicity is such that it often goes undetected until speech intelligibility is affected and is usually detected when a communication problem becomes evident . Communication problems, such as constantly asking for repetition or not responding when spoken to, signify that the hearing loss has progressed to the frequencies important for understanding speech . In this case, an audiological monitoring programme can avert, to a large extent, the reduced quality of life as a result of hearing loss, as patients on cisplatin chemotherapy can be identified early, counselled, monitored, and managed appropriately through interventions in a logical, systematic, and coherent manner . Audiological monitoring should aim to identify the hearing loss early and reduce its impact on the individual's life by means of proper medical and hearing intervention . Prospective audiological evaluations remain the only reliable method for detecting ototoxicity before it becomes symptomatic . An ototoxicity monitoring programme should involve a health care team comprising of an oncology nurse, oncologists, audiologist, and pharmacist to ensure effective sustainability of such a programme, if implemented, with the patient being the central focus . The audiologist is involved in identifying an ototoxic hearing loss, informing the oncologist of such a development, counselling the patient and their family, and prescribing amplification devices, such as hearing aids and cochlear implants . Early identification of an ototoxic hearing loss provides oncologists with an opportunity to adjust the chemotherapy regimen in order to reduce or prevent further deterioration of hearing . The oncologist and nurses should also counsel patients on the side effects of cisplatin, including ototoxicity, in an attempt to prepare them for treatment outcomes and help them set realistic expectations . Pharmacists who have access to a patient's medication list may also alert the oncologists and audiologists to those who are on other ototoxic medication and therefore at a greater risk for cisplatin - induced ototoxicity . Effective management of such patients using evidence - based practices may improve management of those with cancer, ensuring that they and their families are counselled and appropriate interventions are timeously implemented . The principles of early identification and early intervention are a part of ototoxicity monitoring, and the audiologist can manage such a programme . In countries without ototoxicity management guidelines, the guidelines for the audiological management of individuals receiving cochleotoxic drug therapy developed by the american association of speech - language - hearing association may, consequently, guide the audiologist in the implementation of an ototoxicity monitoring programme within a local, regional, or national setting . For widespread acceptance and use, ototoxicity monitoring programmes need to incorporate efficient and cost - effective ototoxicity identification techniques, while considering the health care system and demographics of the patient population being managed . For any population receiving ototoxic medication, the following should be considered: (1) the patient's level of alertness or ability to respond reliably; (2) the most appropriate times during the treatment protocol for test administration, and; (3) the test should comprise the baseline, monitoring and post - treatment evaluations . Appropriate time intervals for audiological assessments may differ depending on the type of cancer as well as the frequency and dose of cisplatin (figure 2). The audiological assessments should incorporate a detailed case history, otoscopic examination, immittance audiometry, speech audiometry, dpoaes, and conventional and extended high frequency audiometry (i.e., up to 20 000 hz) (hfa) [69, 73]. These procedures are all conducted for the baseline assessment and the six - month follow - up evaluation [69, 73]. While auditory brainstem response may be used, it is not considered a standard procedure for monitoring ototoxicity . Monitoring audiological evaluations during treatment and the one- and three - month follow - up evaluations include case interview, otoscopy, and immittance audiometry as well as air conduction pure tone and objective testing . However, full - frequency threshold testing is impractical for many patients on cisplatin chemotherapy, as these individuals are often extremely ill and easily fatigued . The use of abbreviated threshold monitoring procedures that are clinically practical for these patients is therefore recommended . One such method involves the use of the sensitive range for ototoxicity (sro). This is the highest frequency with a threshold at or below 100 db spl followed by the next six lower adjacent frequencies in 1/6-octave steps or the one octave range near the highest audible frequency . Sro is usually determined during the baseline evaluation and is dependent on each patient's hearing threshold configuration . During monitoring evaluations however, full - frequency testing should be conducted within the same session if an asha significant hearing change is noted within the sro . If a patient on cisplatin chemotherapy is still responsive and alert, the protocol presented above would be suitable . However, a patient who has limited responsiveness may be required to undergo the same audiological evaluations, except speech audiometry . Patients who are responsive as well as those who have limited responses can undergo both behavioural and objective testing . However, those patients who are too ill or too young to respond should undergo only objective testing, such as otoscopy, tympanometry, acoustic reflexes, and dpoaes or abrs . While pure tone audiometry in the conventional frequency range is suitable for evaluating hearing in the range responsible for speech understanding, as well as for differential diagnosis, it is less sensitive to detecting early ototoxic change [11, 70]. The two tests identified as being the most important for the early detection of cisplatin ototoxicity are hfas and oaes, each also having limitations (see table 4). Therefore, using each test in isolation may not be as effective as utilizing a test battery approach, as it increases the chances of obtaining reliable audiologic monitoring data over time . In addition, these two tests could be used to complement one another in every cycle of chemotherapy to ensure the earliest detection of ototoxicity . The ototoxicity monitoring protocol proposed by asha represents an aggressive and ideal approach for monitoring ototoxicity and is dependent on a country's infrastructure and resource constraints . The asha guidelines may therefore not be generalized to a country without considering the contextual factors that may influence its applicability to that country . However, it does provide guidance towards creating a roadmap that countries, such as south africa, may aspire towards in implementing an ototoxicity monitoring programme . Similar to india, no programmes have been formally implemented to identify and monitor ototoxicity in patients on cancer chemotherapy in south africa . As a result, there is no contextually relevant research to steer the implementation of an accountable and effective ototoxicity monitoring program in the country . This is probably one of the main reasons for ototoxicity monitoring programmes not being commonplace in local hospitals and clinics . In addition, the health of south africans is characterized by a quadruple burden of disease, encompassing the occurrence of infectious diseases, the rise of noncommunicable diseases, and perinatal and maternal disorders, as well as injuries and violence, which may result in cancer receiving low priority for health care services . However, the creation of an audiological monitoring programme allows for better control of cancer related comorbidities . Over the years, a number of studies have investigated the use of otoprotectants with cisplatin, their purpose being to protect the inner ear from any injury while not interfering with the antitumor effects of cisplatin . Otoprotective strategies include reducing the formation of free radicals by maintaining glutathione levels and antioxidant activity . Three mechanisms may provide protection against cisplatin, these being endogenous molecules, exogenous agents, or a combination of exogenous agents that trigger endogenous protective mechanisms . However, endogenous agents are not effective against cisplatin when the dose exceeds a certain threshold [17, 81]. Nearly all of the otoprotective agents are sulfur- or sulfhydryl - containing compounds (thio compounds), known as antioxidants, and potent heavy metal chelators . The numerous otoprotective agents utilized in clinical and animal studies include amifostine, d - or l - methionine, methylthiobenzoic acid, lipoic acid, tiopronin, glutathione ester, sodium thiosulfate, melatonin, vitamin e, n - acetylcysteine, dexamethasone, and resveratrol . However, none of these agents have been found to be unequivocally beneficial in preventing cisplatin ototoxicity and no agent is currently recommended for routine use . Further research is therefore needed to find new methods and optimize old ones to prevent and/or treat hearing loss during cisplatin therapy . In addition, intratympanic administration of medication together with gene therapy needs to be further explored . Intratympanic administration involves the diffusion of the otoprotective agent across the round window into the inner ear, where its therapeutic effect is exerted . An advantage of this method of administration is that there are higher concentrations of the otoprotective agent in the inner ear, this being in comparison to the use of oral or parenteral administration, without potentially reducing the efficacy of the cisplatin treatment [89, 90]. The disadvantage of this procedure, however, is that each ear would have to be treated with a moderately invasive procedure . Alternatively, gene therapy may prove to be beneficial in protecting an individual against cisplatin - induced hearing loss as several genes, namely, megalin, glutathione - s - transferases, thiopurine s - methyltransferase, and catechol - o - methyl transferase, may be responsible for susceptibility to hearing loss . If a cisplatin - associated hearing loss results in communication difficulties, it is the audiologist's ethical responsibility to begin or recommend aural rehabilitation . However, this intervention should not only occur once hearing loss has been detected but before the patient begins the cisplatin chemotherapy . Aural rehabilitation techniques such as speech reading and counselling on compensatory communication strategies should be conducted . The counselling should include spouses and significant other, as hearing loss may not only impact the person with cancer but also frequent communication partners . Patients with sensorineural hearing loss due to the use of cisplatin may also benefit from the use of assistive listening devices such as hearing aids or cochlear implants . Children with ototoxic hearing loss may also require the use of personal frequency modulated systems in the classroom . Furthermore, with the recent developments in hearing aid technology, a patient with an ototoxic hearing loss is more likely to receive the desired amplification benefit . These hearing aids are able to amplify sounds at and above 8000 hz . However, there is limited data indicating significant improvements in speech recognition with the use of this technology .hearing aids with frequency lowering technology achieved by linear frequency transposition, nonlinear frequency compression, or spectral envelope warping . Frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear, by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound . While there are no published studies suggesting one approach to be superior to another, frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds . Commercially available types of frequency lowering signal processing include frequency transposition (widex), nonlinear frequency compression (phonak), and frequency translation (starkey). These processors are commercially labelled as audibility extender, soundrecover, and spectral iq, respectively . These hearing aids are able to amplify sounds at and above 8000 hz . However, there is limited data indicating significant improvements in speech recognition with the use of this technology . Hearing aids with frequency lowering technology achieved by linear frequency transposition, nonlinear frequency compression, or spectral envelope warping . Frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear, by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound . While there are no published studies suggesting one approach to be superior to another, frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds . Commercially available types of frequency lowering signal processing include frequency transposition (widex), nonlinear frequency compression (phonak), and frequency translation (starkey). These processors are commercially labelled as audibility extender, soundrecover, and spectral iq, respectively . This review has highlighted that cisplatin ototoxicity is a frequent adverse event of cisplatin chemotherapy that may negatively affect the quality of life of patients with cancer . The different molecular and cellular mechanisms involved in cisplatin - associated ototoxicity highlight the complexity of this condition and the consequent difficulty in identifying an effective otoprotective agent . The varying incidence rates reported in both adults and paediatrics may be due to the different audiological tests employed in the monitoring of the cancer patient's hearing status and therefore highlight the importance of the use of extended high frequency audiometry and dpoaes in ototoxicity monitoring . An audiological monitoring programme comprising a team of health care professionals, knowledgeable about cisplatin ototoxicity, may therefore serve to improve evidence - based service delivery to these patients.
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The synthetic modification of proteins enables the construction of biomolecular hybrids that can be used to study protein function, deliver potent therapeutics to cellular targets, and build new materials . The synthesis of these constructs requires a suite of chemoselective bioconjugation reactions that proceed under mild, aqueous conditions in the presence of the native functional groups that are present on protein surfaces . The most common methods for protein modification target the nucleophilic side - chains of lysine and cysteine . However, these strategies can result in complex product mixtures, as lysine is typically found in high abundance on the protein surface and uniquely reactive cysteine labeling sites can be difficult to install in many instances (such as thiol proteases and proteins produced via the eukaryotic secretory pathway, for example). Many newer approaches for the site - selective modification of proteins involve the introduction of artificial amino acids with reactivities that are orthogonal to those of the native amino acids . Along these lines, a number of powerful methods have been developed for the selective modification of azide, alkyne, alkene, carbonyl, and aniline moieties . However, the difficulty of introducing a non - canonical amino acid can limit the application of these methods . Complementary approaches rely on the site - selective modification of native amino acids by enzymes . In addition, a reliable method for the modification of c - terminal thioesters with n - terminal cysteines, termed native chemical ligation, has been developed by kent and co - workers . This method has been used for the semi- and total synthesis of complex protein substrates, including the chemical synthesis of a single glycoform of human erythropoietin . As an alternative strategy, we and others have developed methods for the selective modification of the n - terminal amino group . Methods that target the n - terminus can offer significant advantages for bioconjugate preparation, as they can be used for a wide range of protein targets produced by virtually any expression system . Conceptually powerful as they are, however, these methods can be hampered by long reaction times, often require large excesses of reagent, and/or involve at least two - steps for the attachment of synthetic molecules . We have therefore sought to develop new techniques that can achieve n - terminal modification with similarly high positional selectivity, but with significantly improved efficiency . Herein, we report an oxidative coupling pathway that can preferentially modify the n - terminus of proteins with fast kinetics . Peptide substrates were first used to screen reaction conditions and identify the site of modification . A peptide panel with varying n - terminal residues was then evaluated to determine the sequence specificity of the reaction, leading to the identification of proline as the optimal n - terminal amino acid . The reaction was next applied to protein substrates, showing similarly high levels of conversion when an n - terminal proline residue was present . This mild bioconjugation reaction enables the facile, rapid modification of proteins to create a well - defined and stable linkage in a single position, and thus should be useful for many different applications in chemical biology and the construction of biomolecular materials . We have previously reported the chemoselective coupling of aniline moieties on proteins to electron - rich aromatic rings, such as o - aminophenols, at slightly acidic ph (6.06.5). These reactions require the addition of naio4 or k3fe(cn)6 as a terminal oxidant, with the latter reagent exhibiting improved compatibility with glycoproteins and substrates with free sulfhydryl groups . The use of ferricyanide as the oxidant also yields a single reaction product (1), whereas periodate leads to the formation of a ring contracted species as a competing pathway . The ferricyanide - based reactions are presumed to involve an o - iminoquinone as the reactive intermediate, as suggested in scheme 1, or could involve the corresponding o - quinone after imine hydrolysis . Taken together, the oxidative coupling strategies have demonstrated excellent functional group compatibility and the ability to join large unprotected biomolecules at low concentrations, as demonstrated for the coupling of peptides, polymers, and nucleic acids to specific locations on viral capsids and antibody fc domains . While these coupling reactions were found to be highly aniline - selective under the conditions used, several studies have reported the reaction of o - aminophenols and o - catechols with native amino acids, dating back to 1949 . In addition, recent work by messersmith has shown the ability of proteins to be coupled to o - quinone moieties present on polydopamine - coated surfaces . These reports suggested that secondary coupling pathways could be developed to achieve the modification of native amino acids with o - aminophenols (scheme 1), and thus initial experiments were designed to identify the optimal reaction conditions for achieving this with complex molecules . In our previous work, we noted that low amounts of background reactivity could be observed in aniline - based oxidative coupling reactions when higher ph conditions (> ph 6.5) were used . In an initial effort to characterize this alternative reaction pathway, conditions and reaction times were first screened to increase the reaction yields for peptides that did not contain aniline groups . Angiotensin i and melittin were used as substrates, as they contain many reactive amino acids, including lys, arg, his, trp, and tyr . The peptides were exposed to 2-amino - p - cresol using k3fe(cn)6 as the oxidant . The reaction ph was varied from 5.5 to 8.5, and the reaction mixtures were analyzed using maldi - tof ms (supporting information figure s1). The level of modification increased with the basicity of the reaction, with near quantitative modification of angiotensin i after 20 min at ph 7.5 and higher . Throughout these initial investigations, it was noted that angiotensin and melittin showed significant differences in reactivity, with angiotensin consistently demonstrating better conversion . Ms / ms analysis of the angiotensin product was used to identify the participating residue, and revealed that the n - terminal amino group was responsible for the observed reactivity (supporting information figure s2). As further confirmation of the site - selectivity, several peptide substrates were screened for reactivity (figure 1a and supporting information figure s3). Consistent with the n - terminal reaction selectivity, the only peptide that did not react had a pyroglutamate in this position, and therefore no free amino group . (a) modification of commercially available peptides was monitored by maldi - tof ms . (b) a positional scan of the n - terminal amino acid was evaluated . Peptides with the sequence xadswag were tested for reactivity with 2-amino - p - cresol . The reactions were run with 100 m peptide, 200 m aminophenol, and 5 mm ferricyanide at ph 7.5 and analyzed by lc ms . Shown is the average percent modification with error bars representing the standard deviation of three reactions . The same peptides were used for a screen of coupling partner equivalents (c) and a time screen (d). Most buffers did not alter the reactivity, but imidazole and buffers containing a morpholine or piperazine ring (pipes, hepes, hepps, and mops) significantly impeded the reaction (supporting information figure s4). This is possibly due to small amounts of buffer impurities that react competitively with the oxidized intermediate . The time course of the reaction was also investigated (supporting information figure s5). In addition, it was found that the peptides could be modified using naio4 as the oxidant, or 4-methylcatechol as the coupling partner (supporting information figures s67). Both of these reactions showed the same dependence on ph; however, moderate levels of modification were still observed at acidic ph (5.56.5) when using these alternative coupling conditions . These observations suggested that the peptides were reacting with the o - quinone intermediate formed in situ from either the catechol or the iminoquinone precursor (after imine hydrolysis). Given the differential reactivity observed on peptide substrates, we synthesized peptides with varied n - terminal residues (xadswag) to determine the specificity of the reaction . The base sequence was selected to increase the mass of the peptide, impart water solubility, and include a tryptophan residue for quantitation using uv monitoring . The peptides were synthesized on the solid phase using standard fmoc synthesis, cleaved from the resin, and purified by hplc . After purification, the peptides were resuspended in phosphate buffer, ph 7.5, adjusted to a concentration of 1 mm, and stored at 20 c until use . To assess the effect of the n - terminal residue on reactivity, the peptides (100 m) were reacted with 2 equiv of 2-amino - p - cresol (200 m) in the presence of k3fe(cn)6 (5 mm) in phosphate buffer, ph 7.5 (figure 1b). Ms (see supporting information figure s8 for representative ms data for the modified peptides). Most n - terminal amino acids showed good - to - high levels of conversion (6090%), but proline stood out as the only residue that showed nearly complete modification (90100%). A second observation of this screen was the fact that tryptophan, tyrosine, and methionine residues were not oxidized by the ferricyanide reagent, consistent with our previous report of oxidative coupling with this oxidant (see supporting information figure s8). However, free cysteine residues can be oxidized to various species, potentially including disulfides and sulfenic acids, and thus it is recommended that they be protected as disulfides before oxidative coupling is attempted (vide infra). To optimize the reagent ratios (specifically the equivalents of o - aminophenol), the peptides (100 m) were reacted with 110 equiv of the o - aminophenol (1001000 m) in the presence of ferricyanide (10 mm). After 30 min, the reactions were quenched with excess tris(2-carboxyethyl)phosphine (tcep). It was demonstrated that conversion was highest using 25 equiv of the coupling partner (figure 1c). Using more than 5 equiv of this was most likely due to the ability of the aminophenol to react with itself at higher concentrations (1 mm). Consistent with this, when using 10 equiv of the o - aminophenol, a byproduct was observed with a mass that corresponded to the condensation of 3 aminophenols (344 da). We also investigated the differences in coupling rates for representative n - termini . The reaction of 2-amino - p - cresol with three different peptides was monitored over the course of 1 h (figure 1d). The peptides (100 m) were reacted with 2 equiv of the o - aminophenol (200 m) in the presence of ferricyanide (5 mm), and aliquots were quenched with excess tcep at the indicated time points . The proline terminal peptide not only reached the highest level of conversion, but also did so in a significantly shorter time than the other termini . Despite efforts to optimize conditions for all n - termini the reaction of n - terminal amines with o - aminophenols was characterized using small molecule mimics . The methyl esters of phenylalanine (h - phe - ome) and proline (h - pro - ome) were coupled to 2-amino - p - cresol using ferricyanide at ph 7.5 . The crude products were characterized using two - dimensional nmr and high - resolution mass spectrometry . The primary amine of h - phe - ome formed p - iminoquinone product 2, which was analogous to the one formed with aniline coupling partners (scheme 1, supporting information figure s9). However, the secondary amine of proline prevented the formation of the p - iminoquinone tautomer, and thus favored o - quinone product 3 (scheme 1, supporting information figure s10). Given the different linkage obtained with proline, we verified the stability of the product to a variety of conditions . The proline terminal peptide, padswag, was first modified with 2-amino - p - cresol . After purification, the modified peptide (100 m) was exposed to reductants, nucleophiles and acidic and basic ph (10 mm additives or buffer). No loss of product was observed under any of the conditions tested, demonstrating the hydrolytic stability of the product (supporting information figure s11). The ability of the linkage to withstand these conditions renders this method quite useful for the construction of biomolecular materials for a variety of applications . With a view toward in vivo applications, current efforts are examining the stability of the linkage in blood plasma, as well as evaluating the intrinsic immunogenicity of the o - quinone group . In the process of characterizing the reaction products, it was observed that the colored products absorbed light at wavelengths greater than 500 nm (with max between 505 and 525 nm depending on the amine coupling partner). As the starting coupling partners and ferricyanide did not absorb at these wavelengths, this unique absorbance provided a means to monitor the reaction progress . The different amine coupling partners (p - toluidine, h - pro - ome, and h - phe - ome) were reacted with 4-methylcatechol in the presence of 10 mm ferricyanide, and the absorbance of the resulting solution was monitored at 520 nm to determine the relative rates of reactivity (figure 2a; for unnormalized data see supporting information figure s12). The catechol substrate was used for these studies to simplify the reaction pathway by eliminating the imine hydrolysis step . The reactions were run under pseudo - first order conditions with 0.1 mm catechol and 1 mm amine coupling partner . When the reaction was carried out at ph 6.0, the reaction with the proline analogue reached completion nearly as rapidly (2 min), but the reaction with the primary aliphatic amine of phenylalanine required longer reaction times (10 min). (a) amine coupling partners were reacted with 4-methylcatechol as a model substrate . Reactions were run under pseudo - first order conditions with 100 m catechol, 1 mm amine, 10 mm ferricyanide in 50 mm phosphate buffer . (b) a peptide containing both an n - terminal proline and a p - aminophenylalanine residue (pad(paf)swag) was tested for reactivity with 2 equiv of 2-amino - p - cresol at ph 6 . The remainder of the reaction was purified and then reacted with 2 equiv of the aminophenol at ph 7.5 and analyzed by lc ms . By quantifying product formation by absorbance, we were also able to measure the second - order rate constant for the proline - based coupling (supporting information figure s13). The reaction of 1 equiv of h - pro - ome (100 m) with 1 equiv of 4-methylcatechol (100 m) and 100 equiv of k3fe(cn)6 (10 mm) was performed in triplicate at 25 c . The second - order rate constant for the coupling was determined to be 44 4 m s. while proline reacted rapidly with the electron - rich coupling partner, the small molecule studies indicated that aniline should react faster . The rate for the aniline reaction was too fast under these conditions to determine the second - order rate constant accurately . Given the differences in reactivity observed at ph 6.0 and 7.5, we hypothesized that it would be possible to modify the aniline side chain of p - aminophenylalanine (paf) and the n - terminal proline amine sequentially . To test this hypothesis, we synthesized a peptide containing both reactive moieties (pad(paf)swag). Only one modification was observed when the peptide was reacted with 2-amino - p - cresol at ph 6.0 (figure 2b). Ms / ms analysis of the modified peptide confirmed that the single modification occurred on the aniline side chain (supporting information figure s14). After this step, the peptide was purified and subsequently reacted with 2-amino - p - cresol at ph 7.5 . Reaction at the higher ph enabled a second modification of the peptide substrate, at the n - terminal proline residue . The differential reactivity was investigated by comparing the reactivity of a protein containing a paf residue to proteins without the artificial amino acid . The paf residue was introduced into the coat protein of bacteriophage ms2, which self - assembles into a spherical, hollow protein shell . Myoglobin and a mutant of the tobacco mosaic virus (tmv) coat protein were used as native protein substrates . Reactions with 2-amino - p - cresol were either performed on the isolated, individual proteins or with the aniline containing protein mixed with the native protein substrate (supporting information figure s15). Addition of the aniline containing protein to the native protein decreased the n - terminal reactivity, indicating that the aniline residues react more rapidly than n - terminal residues with the o - aminophenols . In addition, ms2 showed significantly higher reactivity at all of the phs tested, confirming preference for aniline residues . The oxidative coupling reaction with n - terminal amino groups was first tested on proteins with native n - termini . Several proteins were reacted with o - aminophenol - functionalized 5 kda peg under the optimized reaction conditions (figure 3). The native proteins showed moderate levels of reactivity, which could be attributed to inaccessible n - termini or simply to the less reactive n - terminal residues . To test if proline terminal proteins were more reactive, a proline residue was introduced to the n - terminus of gfp and the tobacco mosaic virus (tmv) coat protein . The n - terminus of tmv was also slightly extended from the native sequence (addition of pag). The proline - gfp was treated with a variety of conditions to determine the specificity of the reaction (figure 4a). Only at basic ph in the presence of both the o - aminophenol substrate and the oxidant was modification observed . Additionally, the proline - terminal variant showed significantly improved reactivity compared to that of the wild - type n - terminus . These high levels of modification were maintained even when only 12 equiv of the o - aminophenol peg was used . The site of modification was confirmed to be the n - terminal proline by lc ms / ms analysis of a tryptic digest of proline - gfp modified with 2-amino - p - cresol (supporting information figure s16). (a) the n - terminus of several proteins was pegylated using o - aminophenol - functionalized 5 kda peg and ferricyanide . (b) modification of wild type proteins with 5 kda o - aminophenol - peg was monitored by sds - page . (a) a proline was introduced to the n - terminus of gfp . The proline terminal variant showed much higher levels of modification than the wild - type protein . The band doubling is due to a gel artifact, and appears in all lanes . (b) mutants of tmv were reacted with 5 equiv of 2-amino - p - cresol and 1 mm k3fe(cn)6 for 30 min and analyzed by lc ms . Reaction conditions for both native and proline terminal proteins were optimized by evaluating reactivity with myoglobin and proline - gfp . The reaction time, buffer, and ph were screened (supporting information figures s17 and s18). Similar to the results obtained with peptide substrates, the reaction reached its highest level of conversion after about 1530 min . In addition, most buffer salts tested were compatible with the reaction with the exception of buffers containing morpholine (mops) or piperazine moieties (hepes), as was observed with peptide substrates . These buffers decreased the level of modification slightly, but did not completely inhibit reactivity . The effect of reaction ph was tested using both k3fe(cn)6 and naio4 as the oxidants . Little reactivity was observed at acidic ph, and the level of conversion increased between ph 7.0 and 8.0 . At higher reaction ph (8.0) a second modification was observed, indicating that lysines may also participate in the reaction . However, it is also possible that under more forcing conditions, such as higher reaction ph or increased concentration of aminophenol substrate, the aminophenol reacts with both itself and the n - terminal amino group resulting in double modification of the n - terminus . N - terminal mutants of tmv were also evaluated for their reactivity with o - aminophenols . The tmv monomers assemble into well - known double disk structures, displaying 34 copies of the n - terminal groups on their peripheries . Two n - terminal mutants (pag and ag) were reacted with 5 equiv of 2-amino - p - cresol (100 m) and 1 mm k3fe(cn)6 for 30 min . Ms demonstrated that the proline terminal mutant reached nearly complete conversion, while the alanine terminal mutant showed low levels of modification under these coupling conditions (figure 4b, see supporting information figure s19 for wider mass range and ion series). The compatibility of the reaction with cysteine residues was also tested using tmv . A single cysteine residue (s123c) was introduced into the tmv coat protein with a proline n - terminus (pag s123c tmv). This mutant was reacted with 2-amino - p - cresol and analyzed by lc the cysteine residue also reacted with the o - aminophenol, resulting in two modifications . However, it was found that the n - terminal proline could be modified selectively if the cysteine was first capped (figure 5a, b, see supporting information figures s2122 for wider mass range and ion series). To do this, the cysteine residue was protected as a disulfide bond by reaction with 5,5-dithiobis(2-nitrobenzoic acid) (dtnb, ellman s reagent). After the oxidative coupling step the disulfide bond was readily reduced by tcep, leaving the free cysteine and the modified n - terminus . Alternatively, the cysteine residue was modified with a maleimide, followed by modification at the n - terminus with an o - aminophenol reagent . A) pag s123c tmv was reacted with small molecule substrates and analyzed by lc cysteine residues were protected as a disulfide using ellman s reagent (dtnb) before oxidative coupling . Subsequent reduction of the disulfide resulted in selective modification of the n - terminus . The n - terminal proline was then modified with a rhodamine - functionalized o - aminophenol . This strategy allowed for the direct, dual modification of the protein at both the cysteine residue and the n - terminus . Two fluorophores paired for frster resonance energy transfer (fret, alexa fluor 488 c5-maleimide and o - aminophenol functionalized rhodamine b) were thus conjugated to tmv using this strategy to create a templated array of chromophores for light harvesting applications . The free cysteine was first quantitatively labeled with an alexa fluor maleimide (supporting information figure s22). The n - terminal proline was then coupled to a fluorescent o - aminophenol resulting in 50% modification of the tmv monomers with both fluorphores (figure 5b). Complete modification of the n - terminus was not observed as tmv precipitated from solution with increasing levels of modification with the rhodamine dye . In current experiments, we are using this dual - labeling strategy to introduce more soluble chromophores . The oxidative coupling reaction was also compared to the reaction of protein amines with activated esters . This acylation methodology is commonly employed, and can be targeted to the n - terminus by controlling the reaction ph in some cases . The reactions were compared on creatine kinase, a protein with a native proline n - terminus . Reaction with 15 equiv of o - aminophenol peg resulted in good levels of modification of creatine kinase (5060%), while reaction with 15 equiv of n - hydroxysuccinimide (nhs) peg resulted in low levels of modification (525%, figure 6). Only when a vast excess of the nhs peg was used were moderate levels of modification achieved . As was the case with proline - gfp, some over modification was observed under the oxidative coupling conditions when using five or more equivalents of aminophenol . This could result from dimerization of the oxidized species before protein coupling, but has yet to be characterized due to the low abundance of this product . In any case, lowering the reaction ph slightly or using fewer equivalents of the o - aminophenol substrate prevented the over modification from occurring . The modification of the n - terminus of creatine kinase with aminophenol peg was compared to the reaction of creatine kinase with nhs peg . In this study, we have identified conditions for the oxidative coupling of o - aminophenols to n - terminal amino acids . Proline residues work particularly well with this strategy, and are therefore strongly recommended when using it . These groups can be introduced readily in n - terminal positions using site - directed mutagenesis and escherichia coli expression, especially since the methionine residue resulting from the start codon is cleaved when proline is in the second position . The fast kinetics of the reaction allow it to be successful even at low reagent and substrate concentrations, and suggest that it can be used for sterically demanding bioconjugations . The oxidative coupling strategy reported here offers two distinct advantages over other n - terminal labeling methods . First, the modification occurs in a single step and does not require initial oxidation of the n - terminus . Second, the fast second - order kinetics allow for low concentrations of the coupling partners to be used . However, to achieve high levels of modification on protein substrates, proline was required as the n - terminal residue . This new protein modification strategy is currently being explored in our lab for the generation of protein - based materials . In the larger context, new techniques for the introduction of a single functional group in a specific position on a protein surface are always in demand . The ability of the n - terminal oxidative coupling method to achieve this in a single, brief reaction step is highly advantageous, and the fact that it can be combined with cysteine modification chemistry provides new opportunities for complex bioconjugate synthesis.
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Alcohol withdrawal syndrome (aws) is characterized by varied symptoms that range from mild to severe intensity depending on several factors including the quantity, frequency and duration of alcohol intake, and the number of prior withdrawal episodes, as well as individual differences in the vulnerability [14]. Symptoms usually present themselves within 6 to 24 hours after cessation of alcohol intake [5, 6]. Subtyping of the aws has been attempted in the past, as gross conceptualized and proposed 3 constellations of alcohol withdrawal symptoms: factor 1 hallucinogenic that consisted of nausea, tinnitus, visual disturbance, pruritus, parasthesia, muscle pain, agitation, sleep disturbance, tactile hallucinations, and hallucinations which are auditory or visual or both; factor 2 affective and physiological that consisted of anxiety, depression, tremor, and sweats; and factor 3 delirium that consisted of clouding of the sensorium, impairment of consciousness, and impairment of contact with the observer . A cluster analytic study identified three different symptoms clusters of alcohol withdrawal, namely, cns excitation, adrenergic hyperactivity, and delirium . Several rating instruments have been used to measure severity of alcohol withdrawal . Among them, the most commonly used observer - rated scale is the 10-item clinical institute withdrawal assessment - alcohol, revised (ciwa - ar). It has been proposed that alcohol withdrawal symptoms in ciwa - ar appear multidimensional . A pubmed search supplemented with the study by pittman et al . Was to explore the relationship between awsc and ciwa - ar, for which they carried out study on 127 male inpatients of alcohol dependence with principle components factor extraction with varimax rotation of ciwa - ar, a self - rated alcohol withdrawal symptoms checklist (awsc) and on combined items of ciwa - ar and awsc . They found three, five, and seven factor solution, respectively, for ciwa - ar, awsc, and combination of ciwa - ar and awsc . The analysis of ciwa - ar was done on 7 items as 3 items had zero variance . The first factor (variance 23.9%) was tension / anxiety which consisted of anxiety, agitation, and tactile disturbances . The second factor (variance 22.9%) was autonomic arousal which consisted of paroxysmal sweats, tremor, and headache or fullness in head, whereas the third factor (variance 17.4%, eigenvalue less than 1) was nausea and vomiting which consisted of a single item, nausea, and vomiting . In our setup, we use ciwa - ar as part of the measure for the management of alcohol withdrawal symptoms . It is generally observed that alcohol withdrawal symptoms fluctuate in presentation and severity across time . The present study was carried out to explore the dimensionality of this scale in an attempt to identify a set of underlying factors that exist and can explain the interrelationships among various manifestations of acute alcohol withdrawal symptoms . The knowledge of these underlying factors may enhance our understanding of aws and better prediction of complications thus management plans . This was a cross - sectional hospital - based study, conducted at centre for addiction psychiatry, central institute of psychiatry, ranchi, india, a tertiary care referral centre during may 2005 to june 2006 . Study sample included 201, only male fulfilling icd-10 dcr (world health organization) for alcohol dependence with currently withdrawal state, aged between 18 and 60 years, admitted within 24 hours of abstinence and patient himself or his guardian consenting for the study . Information on patient's demographics, treatment history, past history, and family history was obtained from interviews with patients and accompanying person . Detailed physical and neurological examinations were done to exclude any comorbid general medical condition, comorbid other psychiatric disorder, and any other comorbid substance use disorders except caffeine and tobacco . The sociodemographic data sheet included age, marital status, religion, community, education, and economic status . Whereas clinical variables recorded were age of onset of drinking alcohol, duration of dependence, past history of detoxification, number of previous detoxification, past history of withdrawal seizure, past history of delirium tremens, family history of alcohol or substance dependence, degree of relationship if family history of alcohol or substance dependence is present, and family history of mental illness . It is the most widely used and studied 10-item alcohol withdrawal monitoring scale, which excludes vital sign abnormalities . It was developed from the 18-item clinical institute withdrawal assessment for alcohol and it has been studied across various geographic locations . It has a good reliability, validity, and it is considered as one of the most widely used alcohol withdrawal assessment scale for symptom - triggered therapy . Each sign and symptom item of ciwa ar is evaluated on a 07 point likert scale except for one item orientation and clouding of sensorium, which is scored on a 04 point likert scale . A score of 8 points or less indicates mild withdrawal and patients scoring less than 10 do not usually need additional medication for withdrawal . Patients admitted with alcohol dependence syndrome with acute withdrawal were evaluated with ciwa - ar immediately after admission then every six hours, as a routine protocol of the ward . Detailed physical examination, mental status examination, and planned screening laboratory investigation were done to ensure conformity of study criteria . The patients get admitted with varying lengths of abstinence, ranging from hours to days, so we initially took all the ratings of all the patients and arranged them as rating at first 6 hours of abstinence and at every six hours like at 6, 12, 18, 24, 30, 36, 42, 48, 52, 58, and 64 hours till ciwa - ar scoring reaches below 10 . The averages of each rating of ciwa - ar scores were computed to see the severity of withdrawal symptoms across time span of abstinence . Many patients were admitted after overnight, 12 to 18 hours of abstinence, so we included the patients who were admitted with at least 24 hours of abstinence . Meanwhile management and medications were continued as per ward protocol and no adjustment for study purpose was done . The standard detoxification protocol included thiamine supplementation, benzodiazepines either lorazepam or diazepam, and correction of fluids and electrolytes if any and other symptomatic treatment of associated conditions like dyspepsia or concurrent injury, wound, and infections . The collected data on 201 patients was statistically analyzed, using statistical package for social sciences (spss, inc ., exploratory factor analysis (maximum likelihood method) was carried out to identify factor structure on all items of ciwa - ar for day three . Kaiser - meyer - olkin measure of sample adequacy and bartlett's test of sphericity were also done to assess appropriateness of conducting factor analysis . Two criteria for retaining the number of components were considered: kaiser's criterion to retain eigenvalues greater than unity and cattell's scree plot inspection for the point of inflexion . The mean age of the group was 37.18 (sd 9.35, range 1869) years . Most of them were married (83.3%), educated (90%), residing in urban background (64.7%), belonging to middle socioeconomic status (60.3%), and earning a livelihood (80.4%). The mean age of starting alcohol was 21.63 (sd 4.99) years, whereas mean duration of dependence on alcohol was 6.49 (sd 5.06) years . 22.5% had history of withdrawal seizures, in their course of alcoholism, and only 2% reported history of delirium tremens in the past . Family history of substance dependence was present in 33.3% of the total sample, out of which alcohol dependence was present in 56.9% of subjects and cannabis dependence in 8.3% . The item frequency and mean of all six hourly ciwa - ar ratings were calculated; the mean scores of ciwa - ar at 24 hours and at 36 hours are shown in table 2 . The mean ciwa - ar score at 24 hours was 13.32 (sd 9.27) and 20.4 (sd 9.09) at 36 hours . Based on the frequency variance and total ciwa - ar score we decided to carry out factor analysis with the 10 items of ciwa - ar as on scoring at 36 hours of abstinence . Bartlett's test of sphericity was significant (2 = 1.044, df = 45, p <.001), indicating that a factor analysis is appropriate . Factor analysis (extraction method - maximum likelihood) with the 10 items of ciwa - ar for day three, resulted in initial three factors with eigenvalues greater than unity . The scree plot was also showing clear inflexion, supporting three factors (figure 1). Therefore, three factors were retained, which captured 68.74% of variance . Following varimax rotation with kaiser's standardization, the factors and their item loadings, with absolute values greater than 0.1, are shown in table 3 . None of the items loaded on more than one factor . The first factor named as delirious factor, which had highest loading from tactile disturbances (.999), followed by auditory disturbances, orientation and clouding of sensorium, and agitation . It explained 34.34% of variance and showed good internal consistency (cronbach's alpha = .91). The second factor named as autonomic factor reflected four - item loading, highest from anxiety, followed by paroxysmal sweats, tremor, and headache or fullness in head . It explained 24.25% of variance and showed moderate internal consistency (cronbach's alpha = .66). The third factor named as nonspecific factor reflected two - item loadings, nausea, and visual disturbances that explained 10.04% of variance and a cronbach's alpha of .26 . We examined the factor structure of the ciwa - ar in a population of adult men hospitalized to a tertiary psychiatric institute for treatment of alcohol dependence . The ideal study of aws could have been the assessment before starting medication, but this was practically not possible for many reasons . Firstly, many of the patients come for admission after 12 to 18 hours of abstinence and severe withdrawal, so keeping them drug free was ethically not possible . Thus natural aws presentation and its severity were masked by routine benzodiazepam administration and thiamine supplement . Secondly, many other patients were referred from primary care centers with initial management, including long acting benzodiazepines like diazepam that masks the aws . Thirdly, many other patients came even before onset of withdrawal and in a state of intoxication; the aws was not fully evolved in terms of range of symptoms and severity . For all these reasons, the mean ciwa - ar score for the initial 24 hours of abstinence (first day) of admission was only 13.32 (sd 9.27). Later the sequential rating found more prominent withdrawal symptoms reaching highest mean score of 20.4 at thirty - six hours then gradually decreased . As drug free aws was not possible, we consider that the higher mean score of ciwa - ar represents the aws better than low score . Also as both aws medications and alcohol itself are cns depressant and act in a similar way, either medication or alcohol intake should not make much difference in clinical picture . So we decided to proceed for factor analysis with highest mean ciwa - ar scoring at the 36th hour . The severity of withdrawal symptoms and appearance of complete sets of withdrawal symptoms at the 36th hour may have been influenced by plasma half - life of benzodiazepams being used for detoxification . In this study, choice of medication was with treating team of the hospital; however, only either intermediate acting lorazepam or long acting diazepam was used for this purpose . There was no use of short acting benzodiazepines, which causes varying and rebound withdrawal symptoms across different time frame with its dosing and changing plasma concentration . Another more important influencing reason for varying withdrawal symptoms across different time frame could have been the dose of benzodiazepines, but we did not interfered with any medications or dosing and it was continued as per ward protocol to ensure naturalistic conditions . However, the equivalent benzodiazepines mean dose at 36 hour was 30 mg of diazepam per day . But this equivalent dose may not be accurate as many patients were on oral medications and others were on parenteral benzodiazepines . We excluded any other comorbid substance use disorders or polysubstance dependence but a few patients may also have undisclosed benzodiazepine or organic inhalant abuse or addiction . We also excluded any comorbid general medical condition especially epilepsy for that reason, patients on antiepileptic either taking it regularly or skipping will modify the alcohol withdrawal symptoms . For that matter any psychotropic drugs causing cns depression or any effecting stimulants will alter the withdrawal symptoms . Most of the patients needed proton pump inhibitor drugs like pantoprazole or omeprazole for the alcohol induced dyspepsia, peptic ulcer disease, or gastroesophageal reflux disease, but these medications do not impose any effect on alcohol withdrawal symptoms . In a previous study by pittman et al ., the mean ciwa - ar score for day one was 13.2 (sd 3.7) which was similar to our study, 13.32 (sd 9.27) at 24 hours; they continued with analysis on data collected on the first study day to exclude medication effects . But we analyzed the data as of 36 hours of abstinence, showing higher mean ciwa - ar score and all ten items had variance above 10%, thus representing fully developed withdrawal syndrome . However we could not control the medication effect used to control the withdrawal symptoms for ethical reasons, the used medications were benzodiazepine and thiamine supplementation for all the patients . Two items of ciwa - ar, namely, auditory disturbances and tactile disturbances were very infrequent in our sample (9.5 and 10%) on day one which increased to 55.1 and 65.7% at 36 hours, other items scoring raised on day two like agitation, orientation and clouding of sensorium, visual disturbances, and paroxysmal sweats; few other items remained unchanged on day one and two like anxiety and tremor; however scoring of only this item headache or fullness in head improved on day two from 28.9 to 12.9% (table 2). This indicates the importance of time duration of abstinence to study the alcohol withdrawal symptoms . We found a three - factor solution based on rotated eigenvalues and scree plot analysis . The first factor explained 34.34% variance and consisted of four items, namely, tactile disturbances, auditory disturbances, orientation and clouding of sensorium, and agitation . This factor appears to represent perceptual abnormality and delirium and may be considered as subclinical spectrum of delirium . However, the only other factor analytic study of ciwa - ar by pittman et al . Found these items (except agitation) were infrequent in their sample and were not included in analysis . The difference may be due to the time span of withdrawal on which data was collected . Study analyzed ciwa - ar for day one and also our data on day one showed very low variance for these items . This suggests that ratings on alcohol withdrawal symptoms on the very first day may miss certain set of symptoms, which appears on and around day two and are characterized by perceptual abnormality and delirium like picture . Further progression of alcohol withdrawal, we could not found beyond 36 hours, may be due to effects of continued medications for withdrawal suppression . Surprisingly, visual disturbances item did not have high loading on this factor, whereas tactile and auditory disturbances had maximum factor loading (0.999 and 0.873, resp . ). The etiological basis for these two disturbances includes cns rebound excitation which alters the perceptual quality . Furthermore, some contribution of nutritional and specific vitamin deficiencies, such as thiamine and folate, and associated peripheral neuropathy probably add to these perceptual disturbances . The other two items, orientation and clouding of sensorium and agitation with factor loadings of 0.851 and 0.777, respectively, represent delirium . The perceptual alteration and delirium being loaded in a single factor may have some predictive association and hence need to be studied for management plan . The second autonomic factor explained 24.25% of variance and consisted of four items: anxiety, paroxysmal sweats, tremor, and headache and fullness in head with factor scores of .998, .660, .528, and .245 respectively . Cronbach's alpha for this factor was 0.66 showing adequate internal consistency . Within this factor this factor may be a result of the practice of not using adrenergic medication on routine basis at our institute . All items (paroxysmal sweats, tremor, and headache or fullness in head) were loaded similarly . We had additional anxiety to this factor; hence, we share the name of this factor with pittman et al . As autonomic . The third nonspecific factor explained 10.04% of variance and consisted of two items: nausea and vomiting and visual disturbances, with low factor scores of .51 and .27, respectively . There are three proposed physiologic bases for the symptom manifestation of alcohol withdrawal symptoms: cns excitation, adrenergic hyperactivity, and delirium, which may be attributed to different neurotransmitters and they respond selectively to different pharmacotherapy . The cns excitation may be secondary to deficiency in gaba activity, whereas increase in cns epinephrine level causes adrenergic hyperactivity . The nmda receptor hypersensitivity and overactivity of certain subtypes of nmda receptors are associated with delirium . Though we did not find a factor structure of aws in accordance with very strict pathophysiological manifestation of either autonomic, or cns excitation or psychological / affective, or perceptual / hallucinogenic in our sample, the obtained factors suggest existence of subgroups of patients with different set of symptoms . This was expected as alcohol withdrawal manifests by simultaneous involvement of all mechanisms rather than any mutually exclusive mechanism . This simultaneous involvement of several other neurotransmitters besides gaba and nmda, like noradrenaline, acetylcholine, and dopamine as well as hormones and electrolytes [17, 18] will affect the symptom presentation . Additionally our data analysis had ciwa - ar ratings of day two (36 hours) of admission with suppression of withdrawal symptoms from detoxification medications, that is, benzodiazepines . This would have differential effect on withdrawal symptoms manifestation in terms of gaba suppression only and selective unopposed action on other neurotransmitters or lacking adrenergic activity . Though avoiding pharmacological suppression or modification was not possible on ethical grounds to understand completely natural unmodified withdrawal manifestation . There may also be possible influence of different types of alcoholic beverage and percent content of alcohol . Strength of our study includes large sample size and not interfering with any medications or management strategies thus providing setting of naturalistic conditions . The use ciwa - ar is the most widely accepted alcohol withdrawal assessment scale and selection of abstinent hours was important to allow time for full appearance of symptoms, even though under cover of detoxification medication . There was for better coverage and inclusion of withdrawal symptoms at 36 hours as indicated by total ciwa - ar score and item frequency . These results have wider implications for the recognition and management of aws, particularly, for better understanding and identification of the symptom profiles for and differential management plans across subtypes of aws . The use of adrenergic antagonists may have a valuable role in addition to benzodiazepines, in a set of patients with autonomic features . One of the limitations in our study is that it includes male only patients; however, gender can be an important issue in aws presentation and its severity . Even studies found that sex hormone affects the aws by modulating the function of the gaba - a receptor . Also low levels of testosterone are associated with symptoms like indecision, excessive worrying, fatigability, and lassitude . Another limitation of this study is patients sample with severe aws requiring inpatient management, thus limiting generalizability of our findings across gender and to mild to moderate severity cases . Another limitation was that diagnosis was made clinically with icd-10 dcr criteria for alcohol dependence with currently withdrawal state, but not by a validated clinical interview . It is known that autonomic arousal is an important mechanism in aws; thus, other physiological measures and biological markers for objective assessment may be included in future studies . Further studies may also be carried out including cases of mild to moderate severity and in both sex to uncover the differences . Also, factor analysis depends heavily on the population studied; therefore, studies on different population may be required to generalize our findings . For the clinical practice, it is advisable not to overdepend on rating scales and it must not replace a thorough clinical evaluation of the patient's medical status in prediction of those at risk of severe alcohol withdrawal . The acute alcohol withdrawal symptoms was most severe at 36 hours of abstinence in our sample . This study finds multidimensionality of alcohol withdrawal symptoms as measured with ciwa - ar; we found three factors explaining 68.74 percentage of variance and named as delirious, autonomic and nonspecific.
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This included only workers who had worked more than 50% of each day's working period and had worked for at least 10 days of the follow - up period . Information regarding the amount of liquid intake expressed (in litres, l) and daily productivity output (in tons) were recorded . The rehydration strategies were re - structured by decision makers participating in the project and the author of this paper . For an eight working hour schedule, all workers were instructed to drink 1 l of the rehydration solutions or tap water at least 30 minutes before they began to work . They were also encouraged to drink water and rehydration solutions (250 ml) every 3045 minutes . In total, they were each asked to drink 10 l of liquid (tap water and rehydration solutions) per day . In order to facilitate the monitoring of basic heat stress indexes during the 2007/2008 harvesting season, temperature, humidity and dew point were measured by using a portable temperature monitoring device (easylog, model el - usb-2). Variables such as heart rate and weight were evaluated at the beginning and at the end of the working day . Neither the amount of liquid intake nor productivity was validated . Wet bulb globe temperature (wbgt) values during the 2007 harvest season at montelimar farm descriptive statistics were used to analyse climate data (temperature (c); relative humidity (%); dew point (c)); liquid intake and output production . Chi - squared test was used to evaluate liquid intake versus output production by using spss version 13 . Descriptive statistics were used to analyse climate data (temperature (c); relative humidity (%); dew point (c)); liquid intake and output production . Chi - squared test was used to evaluate liquid intake versus output production by using spss version 13 . 1 shows in situ hourly climate variation direct from the sugarcane fields while workers harvested the crop . As can be seen, temperature and relative humidity values oscillated from 23.5 to 34.5c and from 40% to 64%, respectively, reaching maximum values as early as 810 am . This meant that water distribution had to be started at around that time on an hourly basis . Climate measurements during 160 working hours of follow - up for 22 sugarcane workers at the montelimar farm . Table 2 shows the amount of liquid intake (including both regular tap water and specially formulated rehydration drinks). What is of great concern is that, although temperature increased to maximum values early in the morning, many workers did not follow the rehydration measures and drank less than 6 l, a potentially dangerously low volume . Comparison between temperature measurements and daily water intake for 22 sugarcane workers at the montelimar farm . This showed that 13 workers who had the highest production output (range 68 tons, average 7.45 tons) were those who drank more than 6 l of liquid (regular tap water and/or specially formulated rehydration solutions) in comparison to those who drank less liquid and had a smaller production output . Relationship between production output and daily water intake for 22 sugarcane workers at the montelimar farm . Measurements of heart rate and body weight at the start and end of the working day showed that workers experienced increases in heart rate and loss of body weight as they worked in these hot conditions . Historically, monitoring of toxins in the work environment has been the primary focus for identifying risks . Some potential biomarkers linked to cell injury are immunological factors, lymphokines, growth factors, prostaglandins, endothelins, collagen, adhesion molecules, thromboxanes, leukotrienes, platelet activating factors and heat shock proteins (10). As mentioned earlier, heat illness is a major cause of preventable morbidity worldwide (1) and although human beings possess considerable ability to compensate for naturally occurring heat stress, many occupational environments and/or physical activities expose workers to heat loads which are so excessive as to threaten their health and productivity (11). It is important to remember that the normal human body contains approximately 60% of water, the present project evaluated climatic conditions at sugarcane plantations located at sea level, on the southwest coast of nicaragua . Monitored climate indices (temperature and relative humidity) values varied from 23.5 to 34.5c and from 40% to 64%, respectively, reaching maximum values as early as 8 am . According to the nicaraguan institute of territorial studies (ineter) (13), relative humidity values for the whole month of april varied between 69 and 79% all along nicaragua's pacific coastline from san juan del sur (southern region) to chinandega (western region). Managua lies between the two regions; the montelimar sugar farm is located 62 km from managua . However, the ineter humidity data are quite different from the relative humidity registered on the farm located in western managua . Temperature data were not available at ineter's website . Although only 22 subjects were followed - up for a short period of time in this study, important results were obtained . Other authors have shown the relationship between heat stress health effects and the ability to perform different tasks, as well as the increased risk of suffering work - related injuries (14). In this study, the workers drank more liquid as temperature values increased to maximum peaks . This was part of a rehydration process which was well planned in advance by the company's decision makers . Unlike during past harvesting seasons, when water and rehydration solutions were distributed quite randomly, during this harvesting season cool water and specially formulated drinks were distributed or intended to be distributed to workers, who received 1 l just before they began their working day and then 500 ml every 30 minutes . The basis of this principle is that drinking to satisfy thirst is not enough to keep a person well hydrated . Most of the people become aware of thirst once they have lost 12 l of body water and persons highly motivated to perform hard work may incur losses of 34 l before serious thirst forces them to stop and drink . Since dehydration reduces the capacity for absorption from the gut, workers must be educated regarding the importance of drinking enough water during work and continuing generous rehydration during off - duty hours (14). There was a significant increase of production, with up to 8 tons per worker during the follow - up period compared to the normal 5.5 tons per worker prior to the change in rehydration measures . This important change in rehydration policies and increase in production output is the result of various efforts of training workers, foremen and managers on heat stress prevention, proper hydration measures and quality of (working) life carried out by occupational health and safety professionals (physicians and engineers), human resources departments and top management at sugarcane farms . . Often workers rejected the new rehydration measures, most of the time because it was difficult for them to understand thoroughly the dehydration and physiological compensatory mechanisms . Some of the reasons for this can be attributed to their low educational level, and feeling that nothing bad has ever happened to me before, etc . Certainly more effort in terms of intervention strategies and scientific investigation needs to be carried out among workers in nicaragua who perform jobs in which they are exposed to high ambient temperatures . These include farm workers, construction workers, miners and fishermen, especially those employed in the informal sector, which occupies about half of nicaragua's economically active population . More funds should also be designated by companies decision makers for improving basic working conditions, in order to increase overall productivity (and workers satisfaction in terms of better wages). This would also translate into safer and healthier workers, less absenteeism from sick leave, fewer accidents and other incidents . The author has not received any funding or benefits from industry to conduct this study.
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Choroid plexus papilloma (cpp) is a rare benign tumor accounting for 0.5% of all intracranial tumors that is more common in childhood, consisting of 1.5 to 6.4% of pediatric intracranial tumors.1 2 the majority of cpps occur during the first 2 years of life, with 12.5 to 20% (10 to 12%) reported in infants under 1 year of age.1 3 there have been some reports of calcified cpp, cpp in an adult, cpp in the cerebellopontine angle (cpa), and atypical form of cpp.4 5 6 7 however, a case combining with these four features adult, dense calcification, atypical form, and cpa is considered to be extremely rare; such a case has never been encountered among the 8,540 skull base tumors on which the senior author (t.f .) Has operated in the past 20 years . We report an adult case of densely calcified, atypical cpp originated from the cpa without connection to the forth ventricle . A 41-year - old man presented with a chief complaint of headache, dizziness, and difficulty with gait that had been gradually worsening for 8 months . Neurological examination revealed blurred vision, nystagmus with lateral gaze, and mild right facial weakness . Intermittent difficulty with balance and gait were also present; however, no motor weakness was observed . Computed tomography (ct) of the head without contrast revealed a high - density multilobular mass in the right cpa causing mass effect and slight ventricular enlargement (fig . Magnetic resonance (mr) images demonstrated a trilobed solid mass that measured 4.1 cm in maximum diameter (fig . The mass was extra - axial in location and revealed isointensity on both t1-weighted images (t1-wi) and t2-weighted images (t2-wi). Ct and mr images did not reveal any connection between the tumor and the fourth ventricle . The surface of the tumor was roughly irregular and looked grayish pink . Since the tumor was friable, gelatinous, vascular, and adherent to surrounding structures, it was hard to separate from cranial nerves . Cranial nerves vi, vii, viii, ix, and x were enveloped in the tumor mass, which included calcification . The patient was discharged after recovery from the dysphagia by appropriate rehabilitation . Computed tomography without contrast revealed a high - density multilobular mass in the right cerebellopontine angle . Magnetic resonance images demonstrated a trilobed solid mass that was in an extra - axial location . The tumor revealed isointensity on both t1-weighted images (upper left) and t2-weighted images (upper right). Homogeneous intense enhancement was identified (axial in lower left, sagittal in lower right). Cpp reported in this article primarily occurred in the cpa with close proximity to the foramen of luschka lacking continuity of the fourth ventricle . The majority of choroid plexus tumors (cpts) originate in the ventricular system, 43 to 67% in the lateral ventricle, 24 to 39% in the fourth ventricle, and 9.5 to 11% in the third ventricle, respectively.8 9 the common site of origin differs in each pediatric and adult group . In the pediatric age group, 60 to 80% of such tumors occur in the lateral ventricle, especially in the trigone, because the vascular stalk of the choroid plexus in the lateral ventricle is usually attached to the inferior portion of the trigone . On the other hand, most cpp in adults occurs within the fourth ventricle.9 10 11 a small number of the tumors have been reported to be located in extraventricular regions, the cpa, or the cerebellar hemisphere or the suprasellar cistern.8 10 12 cpp within the cpa was first described by cushing in 1917.13 to date, cpp within the cpa has been reported in 7 to 9% of all cpts and is most exclusively found in adults.9 11 14 15 it is divided into two categories according to the origin of the tumor; one is primary extraventricular cpts and the other is a result of a direct extension of fourth ventricular tumors through the foramen of luschka.15 because the tumor of our case revealed no connection from the fourth ventricle, it is considered to originate from small choroid tufts that normally project from each recess at the foramen luschka into the cpa or from ectopic choroidal islets unconnected with the choroid plexus.16 the cpp of our case indicated dense calcification with ventricular enlargement . The bulbous tufts extended from the fourth ventricular choroid plexus through the foramen of luschka have been known as the bochdalek flower basket, which could be identified in 75% on ct and 96% on mr imaging.17 calcification of the region was found in 38% on normal imaging anatomy.17 in pathological states, calcification of cpp was reported in 4.1% on plain skull radiographs and 23.8% on ct examinations in patients of all 9 the feature of the calcification was commonly described as a stippled or patchy configuration that may include some degrees of hemorrhage.8 9 10 dense calcification was exceptionally reported by sarkar et al.2 the rate of hydrocephalus in association with cpp is 90% and the rate increases up to 100% in malignant cpps.8 the cause of hydrocephalus was considered a combination of cerebrospinal fluid (csf) overproduction (four to five times that in healthy persons), obstruction of csf pathways by the tumor mass, impaired csf absorption, increased protein content of csf around the tumor, subarachnoid scarring or granulations related to recurrent bleeding from the tumor, elevated intraventricular pulse pressure, and adhesions around the exit foramina of the fourth ventricle caused by highly proteinaceous or hemorrhagic csf.2 10 18 the finding of tumor calcification in the cpa generally required consideration of differential diagnosis with papillary or psammomatous meningioma, melanotic schwannoma, and myxopapillary ependymoma . Among these tumors, association of ventricular enlargement with the finding of calcification might suggest the possible presence of cpts . Gross total removal was performed in our case of atypical cpp, although the tumor adhered to adjacent structures . In the 2007 world health organization classification of tumors of the central nervous system, cpts are classified into cpp as grade i, atypical cpp as grade ii, and choroid plexus carcinoma (cpc) as grade iii . Among cpts, total removal of cpts is recommended and seems to be feasible since a significant prognostic factor was relevant to the extent of tumor removal, and no definitive evidence of the beneficial effect of radiotherapy has been reported6 18 19 20 . However, it is not always possible because of tumor bleeding and firm adhesion to adjacent structures, especially cranial nerves . The rate of complete resection of benign cpp was 80% and the 5-year survival rate indicated is nearly 100% . The majority of survivors are neurologically intact.19 because of the good prognosis, the preservation of cranial nerve functions should be primarily considered in surgery for benign cpp . As for the atypical cpp, the rate of total removal was decreased to 50 from 63%.11 21 atypical cpp showed a recurrence rate of 29%, which was much higher than the 6% seen in cpp . In addition, seeding within the central nervous system, spontaneous pulmonary metastasis, or rapid regrowth of atypical cpp were also reported.7 22 23 total or gross total removal should be advocated, especially in malignant cpts even though the tumor encases the cranial nerves . The finding of calcification in the cpa should suggest the possible presence of cpp, especially if ventricular enlargement is present.
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Multipath signals occur in numerous microwave and rf applications when an unwanted portion of the original transmission propagates along any alternate path and ultimately couples to the receiver distorting the amplitude and phase of the desired signal [14]. If the amplitude of the multipath signal is sufficiently large, its impact can be considerable . In multistatic radar and communication systems, these types of interference are most often caused by reflections of either the main beam or side lobes with objects near or actually in the beam path . Classic examples include main beam propagation close to the earth's surface with associated reflections off of the ground or water (figure 1(a)). Various approaches can be used to filter or compensate for these reflections through time - gating and signal time synchronization . The potential for interference from multipath signals increases substantially in near - field applications (figure 1(b)), especially in situations where the receiving and transmitting hardware are integrated . Commercially available multichannel network analyzers (e.g., zvt8 by rohde & schwarz; munich, germany) utilize robust strategies to minimize these signal coupling problems . We are developing multichannel transceiving arrays for medical microwave imaging which exploit near - field concepts to produce electrical property maps (permittivity and conductivity) of tissues of interest [8, 9] and have addressed the issue by incorporating (a) dedicated mixers for each channel, (b) additional solid state switches for isolation, (c) double- and triple - braided coaxial cables, and (d) compartmentalized rf circuitry . The implementation has proven effective for our application achieving channel / signal isolation greater than 130 db . An alternative data acquisition strategy integrates a commercial, 2-port network analyzer with an electronic switching network to feed an array of antennas [1012] which is effective but also has limitations because (i) dynamic range is constrained by the provisions of the network analyzer, (ii) two - way signal loss is incurred through the network, and (iii) the switching matrix has relatively poor cross - channel isolation . Equally important in near - field imaging is the multiple paths a signal can take within the imaging zone . Figures 2(a) and 2(b) show a photograph of our clinical breast imaging tank and a schematic diagram of the antenna configuration, respectively . In this situation, the array of 16 monopole antennas surrounds the breast and can be moved to multiple vertical positions . The antennas and target are submerged in a solution of glycerin and water which is lossy over the operating frequency range (700 mhz3 ghz). Early empirical tests have indicated that reflections off the tank side walls do not impact the desired signals for an array with the antennas mounted on a 15.2 cm diameter circle . Likewise, analysis of the monopole beam patterns as a function of frequency has shown that artifacts are minimal when the array approaches the liquid interface at the top of the tank . With respect to reflections off the bottom surface, the base of the tank was at least 1.8 wavelengths (at the lowest frequency) below the active sections of the antennas in our initial clinical installation when the array was located at its lowest position during an exam . Since minimal multipath signals resulted from reflections off the top liquid surface, symmetry would suggest that the same should be true for the base of the tank (figure 1(b)). However, surface waves can cause multipath signals that can be especially difficult to eliminate in near - field systems . Their excitation can be complex, but their propagation characteristics along two material interfaces, whether planar or along cylindrically shaped structures, have previously been studied in depth [1520]. Surface waves can readily propagate at the interface of two dielectric materials or one conductor juxtaposed directly with a dielectric . Their propagation and attenuation characteristics are nominally determined by the electrical properties of the two materials . In addition, their amplitude decays exponentially away from the interface in the perpendicular direction as a function of the lossiness of the complementary materials . It should be noted that these investigations have stemmed partially from our efforts to perform microwave tomographic images on patients in an actual mr scanner for the purpose of exploiting the refined spatial resolution of the mr along with the more specific nature of the tissue dielectric properties . The mr bore is quite small and places a significant constraint of space for the microwave system . This was where we first encountered multipath signal corruption which subsequently led to this study . In the following sections, we discuss the theoretical underpinnings of these modes for the geometries present in our system . We demonstrate cases from our current imaging system, where the measurements indicate corruption of the desired signals from multipath signals associated with the base of the tank . We then show experiments that allow us to partially isolate the effects to surface waves propagating along other pathways . We realize that there are a number of propagation modes around the antennas, their feedlines and the tank surfaces, of which the surface waves are only one possible contributor, but understanding these contributions is important . We present an initial strategy for minimizing the effects of these signals which may be instructive for designing other near - field imaging systems, including simulations confirming the earlier theoretical discussion and validating our feedline design strategy . A challenging situation occurs when a portion of the transmitted signal propagates along an unwanted path and recombines with the original signal at the receiver . Time - gating strategies can sometimes be effective when the nature of the multipath signal is well understood [1, 2]. The potential influence of a multipath signal when the original transmission is a continuous wave can be written as (1)resultant signal = acombcos(t+comb), where (2)comb = acombtan1(b1a1),acomb = a12+b12,a1=[adecosde+ampcosmp],b1=[adesinde+ampsinmp]. Here, ade, amp, de, and mp are the desired and multipath signal amplitudes and phases, respectively, is the operating frequency, and t is time . For example, if the magnitude of the multipath signal is 25 db below that of the desired signal, the maximum possible amplitude and phase errors in the resultant signal would be 0.48 db and 3.22, respectively . For a 15 db multipath signal, these values increase to 1.42 db and 10.24. clearly, the resultant phase and amplitude errors can become very significant for multipath signals that are on the same order of magnitude as the desired signals . It should be noted that there can be many multipath contributions with a range of amplitude and phase contributions . This single contributor analysis serves to give a flavor that the unwanted effects can be significant and is generalizable to multiple sources . The first surface mode to be considered involves propagation along the interface between the tank base and coupling liquid . Following the analysis by stratton, figure 3 shows a plane wave in region 1 impinging on an interface (x = 0) with region 2 . In this case, the magnetic field (hy) is only oriented in the y - direction (out of the page). The complex relative dielectric properties in the two regions are 1 = 1 j1 and 2 = 2 j2, respectively . The classic surface wave solution occurs when the reflection coefficient is zero (at the brewster angle), which is complex - valued in this instance . If a is the amplitude of the incident plane wave, then the magnetic component of the incident and transmitted waves can be represented as (3)hy = ae[jh1xjz], x>0,hy = ae[jh1xjz], x<0, where h1 + = k1 and h2 + = k2 are needed to satisfy the wave equation, k1 and k2 are the wave numbers for the two regions, and is the propagation constant . The wave impedances for the two regions are given by (4)z1=h1k1z1=h1k1z011j1,z2=h2k2z2=h2k2z012j2, where z1 and z2 are the free space impedances in the corresponding regions, and z1 and z2 are the associated wave impedances [21, 22]. This yields (5)h1=h2, where (6)=21=2j21j1. From these relationships, we can solve for (7)h1=k01(1)12,h2=k012(1)12,=k01(1)12 . In this situation, we are primarily interested in surface waves propagating along the outside of a coaxial cable, and their associated attenuation as a function of distance after the mode has been sufficiently established . For this analysis, we will consider the case of a coaxial line which is abruptly terminated by an open end (figure 4). A coaxial cable supports a tem - mode electromagnetic field which is incident on the cable opening . Part of the signal is partially reflected into the cable, while a second portion is transmitted as a surface wave propagating along the outside of the surrounding cable . The fields transmitted into the surrounding space can be determined from the distribution at the coaxial opening which can be found by solving the integral equation for the radial component of the electric field over the opening: (8)14+jcabe(,0)kc(,)d = j1abe(,0)kc(,)d0cosejkrrd, where c and 1 are the complex - valued permittivity of the coaxial cable insulator and the surrounding dielectric materials, respectively, a and b are inner and outer coaxial radii, respectively, is the operating frequency in radians, and k is the wavenumber in the coupling liquid, where 0 is the free space magnetic permeability . And are the radial cylindrical coordinates within and outside of the coaxial cable, respectively, is the angular coordinate within the coaxial cable, and r is defined as r=2+2 - 2cos. The variable kc(,) is represented as (9)kc(,)=jn=0n()n()an2n, where (10)n()=y0(na)j1(n)j0(na)y1(n),n={kc2n2,kc>njn2kc2,kc>n, an2=22n2[j02(na)j02(nb)1], n>0,a02=lnba . The eigenvalue n are solutions of the characteristic equation: (11)y0(na)j1(nb)=j0(na)y1(nb), where jn and yn are bessel functions of the first and second kind of order n, respectively, and kc is the wavenumber inside the coaxial line . Once e(, z = 0) is determined, then electric and magnetic fields can be found at any point (, z) in the surrounding medium from (12)e(,z)=1abe(,0)0(jk+1r) zcosejkrrdd, or (13)e(,z)=1abe(,0) 0[1(jk+1r)]cosrejkrrddh(,z)=j1abe(,0) 0cosejkrrdd, where (14)r=z2+2+22cos. From these equations it follows that the electromagnetic fields inside the surrounding medium decay approximately as e. figure 2(a) shows the illumination tank used in our current clinical breast imaging system . Each monopole antenna consists of an exposed length (3.8 cm) of 2.2 mm diameter semirigid coaxial cable with only the center conductor and insulating teflon layer intact . For mechanical robustness, the coaxial feed line is enclosed in a 6.4 mm diameter rigid stainless steel tube, and the active section of the antenna is covered with an accompanying length of a delrin cylinder acting as a protective radome . The space between the copper coaxial outer conductor and the stainless steel sleeve is sealed at either end with silver epoxy to eliminate wave propagation along the gap . The antennas have a nominal return loss of 10 db over the bandwidth of 7003000 mhz . The black delrin fittings at the antenna / tank base contain hydraulic seals through which the antenna feeds pass to allow vertical motion of the array while eliminating any coupling fluid leakage . The 16 antennas are positioned on a 15.2 cm diameter circle, and both sets of 8 interleaved antennas are supported by individual mounting plates under the tank which provide independent motion of the array in groups of 8 . The tank is fabricated out of plexiglas with an inner wall diameter of 27.3 cm and thickness of 1.3 cm, and the base has a thickness of 2.5 cm . All connecting cables are double - braided to minimize stray radiation . In these experiments, the antennas were positioned at heights close to the tank base (that were not used in any clinical exams) to study the multipath phenomenon in detail . Figures 5(a), 5(b), and 5(c) show three illumination tanks with different heights, arrays of monopole antennas, and coaxial feed lines that were fabricated from the same plexiglas and had identical diameters and wall / base thicknesses as figure 2(a) tank . The feed lines passed through holes in the base of each tank and were fastened to sma flange connectors which were bolted to the tank floor . The tanks were filled with an 80: 20 glycerin / water mixture with the liquid level 1.5 cm above the antenna tips . In figures 5(a) and 5(c), the feed lines were both 10 cm long; however, the latter was bent in a serpentine shape such that the top height of the feed line was only 5 cm above the tank floor . The feed line in figure 5(b) was straight and was only 5 cm long . In these experiments, we used plexiglas for the tank materials with a dielectric constant of r = 2.7 that was effectively lossless in this frequency range . The dielectric properties of the 80: 20 glycerin / water bath are plotted in figure 6 as a function of frequency . Utilizing the clinical system described in section 2.2, a + 5 dbm signal was transmitted at multiple frequencies over the 7002500 mhz range from a single antenna and received at the remaining 15 antennas . This sequence was repeated for the array positioned at multiple heights above the tank base . Receive antenna amplitudes are plotted for representative frequencies in figure 7 . At 900 mhz, the measured levels are high for the receivers closest to the transmitter (relative receiver numbers 1, 2, 14, and 15) compared to the rest of the array and do not change dramatically with changes in antenna height . However, the amplitudes are considerably lower for the more distant receive antennas . For antenna heights 7 cm or greater (above the tank floor), the attenuation follows a smooth curve hitting a maximum at antenna 8 (which is furthest away from the transmitter being located on the opposite side of the array). At antenna heights 5 cm and lower, the behavior is consistent with the three frequencies shown in figure 7 . Given that the distance from the antennas to the tank side walls did not change during the experiments, and that the antennas were sufficiently far from the liquid / air interface at all times for any array heights (10 cm in the worst case), the corruption of these most distantly received signals appears to be caused by multipath propagation associated with antenna tip proximity to the base of the tank most probably due to reflections off of the tank base or surface wave propagation along the dielectric interfaces . In this set of experiments figures 8(a) and 8(b) show the received signals for a single transmitter over a range of frequencies for straight feed line lengths of 10 cm and 5 cm (tanks in figures 5(a) and 5(b)), respectively . For the longer (10 cm) lines in figure 8(a), the field patterns appear well - behaved like those in the previous section, when the array was positioned at the largest heights above the tank floor . However, the patterns for the shorter (5 cm) line lengths in figure 8(b) exhibit corruption of the signals resulting from the longer propagation distances similarly to when the array heights were closest to the base of the tank for the clinical system (in figure 7). These corrupted signals are nominally between 50 to 70 dbm and occur in uneven patterns relative to the same signals from the longer feed lines which reach 80 to 90 dbm at the furthest antenna . For the shorter propagation distances (i.e., the signals received at antennas 14 and 1215), the attenuation patterns from the shorter and longer feed lines are similar (figure 8(b) versus 8(a)). These results suggest that the unwanted multipath signal effect is the same in both the experimental tanks and the clinical system tests in the previous section and depends on the antenna height from the base of the tank . However, in figure 8(c), the antenna feed length is exactly the same as in figure 8(a), but is curved such that the antenna tip is the same height above the tank floor as the antennas in figure 8(b) (which led to signal corruption); yet, the measurement results emulate those in figure 8(a) (which are not corrupted). Here, the active part of the antennas is still positioned on the same 15.2 cm diameter circle as in the other two tanks . These findings show that the principle signal corruption observed in figure 8(b) is not due to reflections off the tank floor; otherwise it would have appeared in figure 8(c) since the antennas are at the same position above the base for both figures 8(b) and 8(c). It should be noted that the signal disruptions for this situation appear more substantial than that for the antennas used in the clinical system as discussed in section 3.1 . This is very likely due to differences in the designs of the hydraulic seals, feedline shielding, and antenna radome for the other system . More likely, the multipath signals in figure 8(b) (with short feed lines) result from surface waves traveling along the outside of the coaxial lines, across the plexiglas / liquid and/or plexiglas / air interface and back up the outside of the receiver coaxial feed . The theoretical considerations in section 2.2 indicate that the attenuation along the coaxial lines is far more substantial than from the planar tank - base surface wave modes . Figure 9(a) shows a plot of attenuation as a function of frequency for 15.2 cm of plexiglas / liquid surface waves and indicates that very little attenuation of a surface wave propagating along this interface occurs at the frequencies we used . Thus, in our experiments, the real source of surface wave attenuation comes from propagation along the outside of the coaxial lines . Figure 9(b) shows plots of the attenuation that results from single 5 and 10 cm lengths of feed line in the coupling fluid . The theoretical predictions of attenuation along the two 5 cm antenna feed lengths (transmit and receive) and the path along the plexiglas base / liquid interface are approximately 70 db ({2 34 db} + 2 db) for the 1 ghz case (values interpolated from figures 9(a) and 9(b)). Given a transmit power level of + 5 dbm, the resultant 65 dbm multipath signal for the shorter line would easily corrupt the desired 81 dbm signal (figure 8(a)). Only when the feed lines are nearly doubled in length, and the associated surface wave attenuation increased accordingly is the corruption of the desired signals reduced to an acceptable level . Along with the analytical discussion in sections 2.1 and 2.2, we have also performed simulations of the configurations described in section 2.4 . Figures 10(a), 10(b), and 10(c) show the 900 mhz electric field magnitude distributions for the long and short, straight antenna feed and the longer, serpentine structure, respectively . These simulations were computed using ansys (burlington, ma, usa) hfss version 13.0 . For all cases, there is a reasonably broad antenna pattern emanating outwards from the active part of the antennas, and this feature is reasonably similar for all feed line types . For the straight feeds especially the longer one, it is clear that there are considerable surface currents generated along the coaxial lines . For the shorter straight one, there is a high degree of field strength along the coaxial line within the plexiglas volume below the horizontal interface . The fields along the interface generally agree with our previous notion that the surface waves preferentially propagate within the lower - loss medium (in this case the plexiglas) as can be seen by the substantially greater amplitudes directly under the line . The results for the serpentine feedlines are consistent with previous results in that the field strength in the plexiglas is lower than that for the short, straight line case . Figure 11 shows a plot of the field strength just below the liquid interface from a point directly underneath the antenna extending 140 cm to the right for all three corresponding plots . The field values are considerably less for the longer straight line but are also less for the serpentine cases compared to the short, straight cases . For the serpentine case, there seems to be some signal coupling between the lower feedline bend and the plexiglas . As discussed in section 1, surface waves do decay exponentially from the surfaces, and given the proximity of the feedlines and the liquid / plexiglas interface, some coupling is expected . The potentially debilitating effects of unwanted multipath signals is a critical consideration in translating near - field microwave imaging approaches into clinical and commercial systems . For our noncontacting antenna approach, surface waves (relative to signal reflections from the imaging tank walls) appear to cause the biggest effects as they propagate along the outside of the transmitting coaxial line across the illumination tank floor and back up the coaxial feed lines of the receivers . When the imaging tank is deep and the transmitting / receiving antenna tips are sufficiently far above the tank base, the surface wave signals are adequately suppressed relative to the transmissions through tissue . The results presented here indicate that 9 - 10 cm of distance along the feed line is adequate . However, reducing the tank depth is of interest for practical reasons and is essential in some settings and appears possible because reflections from the floor of the tank are still too small to degrade the measured signals propagating through tissue . Indeed, we found that antenna tip distances as little as 5 cm from the tank floor maintain receiver signal fidelity across the array provided the surface wave contributions are attenuated through an equivalent feedline length approaching 10 cm . These findings are significant because they indicate that the antenna array and imaging tank geometry can be altered substantially by manipulating the shape of the antenna feed line, which can be exploited to ensure sufficient surface wave attenuation . There are certainly other mechanisms for multipath propagation including coupling of fields from the feedlines directly to portions of the breast tissue outside the immediate plane of propagation and are certainly good topics for further investigation.
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Enucleation generally induces bone regeneration within a cystic lesion less than 3 cm in diameter and eliminates the lesion; however, it is difficult to postoperatively predict lesion recurrence and bone regeneration in cases of larger cysts . For this reason, a surgeon can choose between conservative and aggressive treatments for a large, aggressive, cystic tumor12 . It has been reported that marsupialization and decompression, both of which are conservative treatments that create an opening to reduce pressure within a cystic cavity and induce bone formation, generate few postoperative complications and produce great therapeutic effects3456 . These procedures not only provide good surgical access to smaller lesions, but they also preserve intraoral tissues, maintain pulp vitality, and reduce recurrence rates . For this reason, many surgeons prefer these methods for treating large cysts . This study aimed to analyze and assess the therapeutic effects of decompression for patients who underwent decompression followed by enucleation . We reviewed the charts of patients who visited the department of oral and maxillofacial surgery at chosun university dental hospital (gwangju, korea) and who had undergone enucleation after decompression . We collected data on their age and gender, location and size of the lesions, preoperative and postoperative histopathological findings, gap in time between decompression and enucleation, and variation in the size of the lesions, complications, and recurrence according to the time gap . Patients were excluded from group review if they had no radiological data, if they had cured soft tissues due to the failure to maintain the window, or if they had been observed for less than six months after enucleation . We measured the sizes of lesions in pre- and post - decompression panoramic photographs for each patient, measured width and length with a caliper, and multiplied them together to determine the lesion index (li) with the aim of simplifying measurement of irregularly sized lesions . To determine the efficacy of decompression, lis were divided into initial and final lis, which were then graded on the basis of nakamura et al . 's formula7 . To assess the extent of reaction to decompression for each lesion, the difference between initial and final lis was divided by the decompression period to measure and analyze the reduction rate in each period . Before conducting a biopsy, clinical and radiographic data were used to determine if patients should undergo decompression . In decompression, the window was formed in the buccal alveolar bone region of the jaw, and a biopsy was conducted after excising some portion of the cyst wall . 1) after the treatment, patients were educated on how to wear and remove the device, and they were instructed to clean the device twice a day (morning and night) using saline solution . Panoramic radiographs were taken every two months for comparison with the baseline panoramic radiograph, and cyst enucleation was conducted when the size of the lesion was determined to no longer be decreasing . We reviewed the charts of patients who visited the department of oral and maxillofacial surgery at chosun university dental hospital (gwangju, korea) and who had undergone enucleation after decompression . We collected data on their age and gender, location and size of the lesions, preoperative and postoperative histopathological findings, gap in time between decompression and enucleation, and variation in the size of the lesions, complications, and recurrence according to the time gap . Patients were excluded from group review if they had no radiological data, if they had cured soft tissues due to the failure to maintain the window, or if they had been observed for less than six months after enucleation . We measured the sizes of lesions in pre- and post - decompression panoramic photographs for each patient, measured width and length with a caliper, and multiplied them together to determine the lesion index (li) with the aim of simplifying measurement of irregularly sized lesions . To determine the efficacy of decompression, lis were divided into initial and final lis, which were then graded on the basis of nakamura et al . 's formula7 . To assess the extent of reaction to decompression for each lesion, the difference between initial and final lis was divided by the decompression period to measure and analyze the reduction rate in each period . Before conducting a biopsy, clinical and radiographic data were used to determine if patients should undergo decompression . In decompression, the window was formed in the buccal alveolar bone region of the jaw, and a biopsy was conducted after excising some portion of the cyst wall . 1) after the treatment, patients were educated on how to wear and remove the device, and they were instructed to clean the device twice a day (morning and night) using saline solution . Panoramic radiographs were taken every two months for comparison with the baseline panoramic radiograph, and cyst enucleation was conducted when the size of the lesion was determined to no longer be decreasing . In total, 17 patients (7 males and 10 females) were reviewed, and their average age was 33.1 years (range, 13 - 76 years). One patient was diagnosed with nevoid basal cell carcinoma syndrome (nbccs) and showed multiple keratocystic odontogenic tumors (kcot) in the maxilla and mandible . Two of these cysts were treated with marsupialization. (table 1) jaw cysts were observed in 10 patients (58.8%) in their teens or 20s, which was a higher rate compared to other age groups, and the sex ratio was 1:1.4 with seven males (41.2%) and 10 females (58.8%). (fig . 2) radiographic results showed that all lesions were unilocular and were either pushing or intruding into the surrounding structures (maxillary sinus or mandibular canal) in 15 patients (88.2%). Based on the results of the histopathologic examination, 10 patients were diagnosed with kcot (58.8%), five patients with dentigerous cysts (dc) (29.4%), one patient with radicular cyst (rc) (5.9%), and one patient with nasopalatine duct cyst (5.9%). Only one patient (5.9%) was treated with marsupialization only, and 16 patients (94.1%) were treated with marsupialization followed by enucleation . There was no case where the preoperative histopathologic results changed after the operation . The average time of decompression was 8.13.4 months, with 5.54.8 months for a cyst in the mandible and 8.33.1 months for the maxilla . The average time of decompression based on lesion type was 8.83.2 months for kcot and 6.02.7 months for dc, suggesting that kcots require approximately three more months of treatment . The li was measured at an average of 1,349 mm before decompression and 463 mm after decompression, showing an average size decrease of 64% after decompression . According to nakamura's formula, five patients (29.4%) showed a greater than 80% decrease in size, nine patients (52.9%) showed a 50%-80% decrease in size, and three patients (17.6%) showed a less than 50% decrease in size. (fig . 3) the mandible decreased by 10% more in size than the maxilla, and it had a 1.7-fold higher reduction rate in the same period . Also, when comparing size reduction based on patient age, the decrease was greater in patients in their teens or 20s compared to the other age groups, but the reduction rates were not significantly different . Males were observed to have a smaller final lesion size, while the reduction ratio was higher in females; however, no significant differences were observed . Additionally, larger lesions reduced faster that smaller ones, and kcots showed more positive results for decompression than other lesion types. (table 2) one patient's recovery was complicated by hypoesthesia after marsupialization, but this naturally recovered over time . The total follow - up period was 1 to 8 years, and 3 out of 17 patients were suspected recurrence . However, one patient was diagnosed with scar tissue after re - examination, and one patient was diagnosed with periapical cyst due to re - infection of an incomplete root canal treatment . A true recurrence appeared on one nbccs patient's left maxilla and is currently being followed - up after re - operation . Since decompression was introduced as a conservative treatment for odontogenic cyst, many cases have been treated with decompression, and various studies have reported high success rates89 . However, the reaction varies depending on the cyst . Anavi et al.10 conducted decompression on 57 patients who were diagnosed with kcot, dc, or rc, and no statistically significant differences in reduction rate were reported based on the histologic diagnosis . In present study, the reduction rate in kcot was higher than the other cysts, with an average of 131.84 mm / month, with the final reduction being the highest . This is because kcots are aggressive, so the size of the lesion tends to be greater than that of other cystic lesions . According to this study, cases of li larger than 1,000 mm showed twice the sensitivity of li smaller than 1,000 mm . Therefore, in this study, kcot patients seemed to have a higher sensitivity to decompression since the li was larger than 1,000 mm . There are no formal criteria for the decompression period or change in size; however, according to previous studies, a 65% reduction was reported when maintained for an average of 8.4 months . Additionally, an 81% reduction was observed when decompression was maintained for an average of 17.5 months911 . Another study reported that it is preferable to perform enucleation if the size of the lesion after decompression decreases more than 50%-60%8 . In this study, the average decompression period was 8.1 months, and the lesions showed an average reduction of 64% . The effects of decompression based on age are controversial, as it was reported that younger patients had higher reduction rates10, but it also was reported that decompression is not correlated with age12 . Therefore, even at older ages, if a large cystic lesion exists within the jaw, postoperative complications can be reduced by decreasing size via decompression . Brndum and jensen13 performed enucleation after decompression in 12 patients with kcot; during 7 to 17 years of follow - up, no recurrence was reported . Such low recurrence rates were reported in many studies, and some of them reported biopsy results indicating that the histological characteristics of the residual cyst epithelium changed from parakeratinization to orthokeratinization or were no longer observed14 . Based on these results, august et al.11 reported that histological changes are likely to appear when decompression is maintained for longer than nine months, and he proposed a decompression period of at least nine months . This study used a panoramic radiograph to two - dimensionally evaluate the lesion by measuring the maximum vertical and horizontal widths of the lesion . This method is widely used because of ease, but it has a disadvantage in accuracy, as it two - dimensionally evaluates a three - dimensional lesion . Therefore, a recent method of measuring volume via three - dimensional analysis using a computer program was introduced15 . However, this method requires a special program and skilled technique, so it can be difficult to apply clinically . Although plane analysis was performed in the present study due to lack of data for three - dimensional analysis, a more accurate analysis is expected once sufficient data is obtained . This study was limited in that statistical analysis was not performed due to lack of samples . Thus, when a lesion is large, reducing its size via decompression not only allows for conservative treatment, but it can also minimize complications . According to this study, all patients treated with decompression were reported to have shown a reduction in lesion size and higher sensitivity with a larger lesion size . There was no difference in the effect of decompression based on age, and only one patient experienced recurrence of the cyst.
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For the first time, the presented data suggest that a variant in the retn gene plays a significant role in acne pathogenesis . We also demonstrate an excess transmission of mutant allele g at retn-420 from 130 trio families using tdt analysis . Therefore, we suggest that inherited variation in retn at -420 appears to be associated with heritable acne vulgaris.
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Many studies over the past 15 years have focused on discovering solvents that help promote efficient cc bond formation under benign reaction conditions . New solvents in dielsalder(2) and barbier reactions have been sought to help streamline the one - pot transformations further, reducing synthetic steps and organic waste . Polar media often accelerate reactions of this type which has led to the use of water as an acceptable solvent for such organic conversions . Recent contributions from our laboratory to this field have centered on coupling reactions between propargyl aldehydes and allyl or propargyl halides under barbier conditions to form homoallylic and homopropargylic propargyl alcohols . These compounds find wide use as synthetic templates and show a propensity for oxy - cope rearrangements (scheme 1). The use of -chloropropargylphenyl sulfide (4), coupled with a propargyl aldehyde should offer an easy route into formation of enediyne and epoxydiyne skeletal structures (scheme 2). Previous studies revealed that formation of 2 or 3 under indium - promoted barbier conditions is solvent dependent . Use of an organic solvent for the coupling reaction results in a mixture of 2 and 3 while the use of water as a solvent yields 2 as the sole product . It is possible that water protonates the coupled product to form 2 quickly, quenching the alkoxide intermediate, thereby suppressing the possible oxy - cope pathway shown in scheme 1 . It may also be possible that the increased polarity of the solvent itself stabilizes the anion formed upon coupling, thus raising the relative rate of cope rearrangement in this system . With a less polar, aprotic solvent, the anion will not be quenched, nor will it be stabilized to the same extent as witnessed in water . A combination of these phenomena may be why an oxy - cope rearrangement occurs under less polar conditions, forming product 3 (scheme 1). A coupling reaction between 1 and 4 (scheme 2) was conducted under aqueous conditions to form 2 (y = sph), with good regioselectivity; however, the rate of this reaction is surprisingly slow . Although reactions of the type shown in scheme 1 are reported in the literature to be accelerated by polar solvents such as water, coupling reactions between 1 and 4, as shown in scheme 2, are quite slow . Indeed, when water is used as the solvent, reactions often require 1036 h for completion . This oil has a density greater than that of water so it is presumed to be an organometallic species formed by interaction of the indium metal with the halide species . Isolation of the oil followed by workup in an acidic thf / h2o mixture leads to isolation of propargylphenyl sulfide and, to a smaller extent, the aldehyde . We surmise that low solubility of this organometallic complex leads to poor reagent mixing, slowing the barbier coupling under aqueous conditions . Second, indium - promoted coupling reactions conducted in water become more acidic as the reaction proceeds . We believe it is possible that the aldehyde is in equilibrium with its corresponding hydrate species, reducing the electrophilicity of the carbon center . A combination of these two reasons would lead to a decrease in the rate of reaction between 1 and 4 when conducted in an aqueous solvent . The reaction proceeds more quickly (812 h) in dmf, a less polar solvent, due to better solubility of all reagents, however, a mixture of 2 and 3 was isolated . It was originally expected that a mixture of water and dmf would lead to faster reaction times while still maintaining regiocontrol of product formation . The use of a water / dmf solvent system, however, resulted in a mixture of products with water constituting up to 50% v / v of the solvent system . When water exceeds 50% v / v of the solvent system, only product 2 is isolated, but the rate of reaction slows perceptively once again . To help expedite this reaction sequence, we began a search for solvent with polarity similar to, or greater than that of water . It was expected that an extremely polar organic solvent would promote the same type of acceleration normally witnessed in barbier couplings carried out under aqueous conditions . An organic solvent could also help solubilize the intermediate and reduce possible formation of the hydrate species, thus allowing for a faster coupling reaction to occur . If increased polarity stabilizes the anion formed by coupling of 1 and 4, oxy - cope rearrangement should also be suppressed . With a dielectric constant of 186.9 at 25 c, significantly greater than that of water (78.37(7)), and dmf (38.3(7b)), n - methylformamide (nmf) has found occasional use in previous reactions requiring polar organic solvents; however, it has historically found more widespread use as an organic reagent . With a pka of approximately 24,(7d) nmf is reactive under a range of organometallic reagent conditions, limiting its use as a solvent . Indium - promoted coupling reactions, however, have been shown to be accelerated under aqueous conditions,(1) and the relative acidity of nmf, being higher than that of water, should not hinder the proposed barbier couplings proposed within this paper . We were especially intrigued by the fact that the dielectric constant of nmf increases to 220 as the temperature is lowered from 25 to 0 c . We were interested in examining what effect the increase in dielectric constant at lower temperature could have on a barbier - style reaction of the type shown in scheme 2 among others . This paper reports on the use of nmf in barbier - style reactions with a focus on rates of reaction, stereochemistry, and its use to form enediyne and epoxydiyne skeletal structures. (9) although nmf has characteristics similar to those of dmf, it also has characteristics similar to water, such as a polar xh bond. (7) thus, having found a very polar solvent with properties similar to both water and organic systems, we hypothesized that the 1,2-coupling product could be formed in high yield under short reaction times . A series of reactions was set up to test this hypothesis . As shown in table 1, use of either water or nmf resulted in formation of the 1,2-product (2) solely with no trace of 3 detected in the product mixture . Although the yield of product was moderate to high in either solvent, product formation in reactions conducted in nmf proceeded at a much greater rate . We believe that there are two possible reasons behind the rate increase of coupling reactions carried out in nmf . The ratio of reagent was as follows: 1.0:1.5:1.1 (aldehyde / halide / indium). The reaction was run at 0.1 m with respect to indium . In the lewis acid catalyzed reaction, 10 molar% of the lewis acid was used . All species completely dissolved in nmf, unlike what was observed for reactions in water . As such, we believe mixing is much more efficient, allowing the reaction to proceed at a faster rate . Has also been noted in many literature examples to lead to rate enhancement in indium - promoted barbier reactions . We believe a mixture of better solubility coupled with greater solvent polarity results in an additive effect toward increasing the rate of reactions conducted in nmf when compared to water . H - bonding capabilities of nmf may explain the greater regioselectivity for reactions conducted in nmf when compared to those observed in dmf and thf; however, the h - bonding capacity of nmf is much lower than that of water. (7d) increased solvent polarity, thus increasing the stability of the intermediate anion and raising the relative energy of the oxy - cope rearrangement, may seem more likely at this time . In an effort to exclude the solvent as a direct participant in intermediate formation when nmf is used, nmr spectroscopy studies were performed to determine if a possible hemiaminal or imminium species was forming in situ (figure 1). The imminium functionality carries a positive charge and would increase the electrophilicity of the reactive carbon . Mixing of 1 and nmf under sonication for periods of up to 24 h, however, revealed the presence of only 1 and nmf by nmr spectroscopy . Possible formation of hemiaminal or imminium species . The presence or absence of a lewis acid has shown to help control stereoselectivity in indium - promoted cc bond - forming reactions . Previous studies in our laboratory have shown that the addition of indium(iii) chloride as a lewis acid favors formation of the syn - product in indium - promoted cc bond - forming reactions between 4 and aldehyde functional groups . In the absence of the lewis acid, the anti - product is favored (figure 2). (5b) it was our hope that similar effects would be witnessed in the present system, especially while using nmf as the reaction solvent, and we were pleased to see this trend continue . Absence of a lewis acid in the reagent mixture favored a nonchelated pathway, leading to formation of anti-2 . Use of indium chloride to control stereochemistry. (5b) the presence of a lewis acid in the reaction mixture favored a chelated pathway, leading to formation of syn-2 (figure 3). Diastereoselectivities were determined either by direct measurement of vicinal coupling constants in the hydroxyl sulfide or by corresponding conversion to the epoxide compounds as shown in figure 4. (11) general j values for hydroxyl sulfides and epoxide structures . Entries 1, 5, and 13 (table 1) describe reactions run in nmf in the absence of a lewis acid . Good anti - selectivity is witnessed with ratios as high as 15:85 (syn / anti). In all cases, with the exception of r = phenyl, systems, with and without incl3, run in nmf gave better selectivity than systems run in water . Both systems showed poor selectivity when r = tms for reasons of which we are not quite sure . When incl3 was added as a lewis acid, the chelated route was favored in ratios as high as 90:10 (syn: anti, entry 15). N - methylformamide was shown to increase the rate of reaction compared with water and offered better stereocontrol in the systems studied . Enediyne and epoxydiyne functionalities contain either a (z)-3-ene-1,5-diyne or (z)-3-epoxy-1,5-diyne unit which leads to their noted activity. (9) these unique functional groups cyclize to form a p - benzyne- or p - benzyne - like intermediate that interacts with dna by abstracting hydrogen. (9) the dna diradical then either couples with itself, or cleaves, stopping replication . Natural product enediyne and epoxydiynes have found use as anticarcinogenic compounds. (9) this area of study is slowed, however, by complicated access into the skeletal systems required. (9) literature procedures that find use in epoxy alkyne formation such as halohydrin cylization,(12)m - cpba,(13) or oxone(14) have been shown to be successful, but limited examples are known with these systems at present . Under the barbier coupling conditions described in this paper, formation of compound 2 offers an efficient and controlled method for the formation of either an enediyne or epoxydiyne skeletal structure under benign conditions by allowing easy conversion to either the cis - epoxydiyne (from syn-2, scheme 3) or the cis - enediyne (from anti-2, scheme 4). Facile synthesis of epoxydiyne and enediyne functional groups under the reported conditions would allow for their use as template structures in organic synthesis, opening access to more sophisticated units for further exploration . In the following pages, we describe the transformation of 1 into either an epoxydiyne (5) or enediyne (6) skeletal structure in two steps with high regio- and stereocontrol using nmf as a new solvent to form 2 under barbier conditions, followed by a one - step transformation to yield either 5 or 6 . As shown in scheme 3, syn-2 can adopt the correct conformation to form a cis - oxirane upon reaction with either me3obf4 or tloet . Conversion of the hydroxy sulfide starting material (2) to the epoxyalkyne product (5) was quite successful as shown in table 2 . In all cases, this fact is worth mentioning as barbier reactions with propargyl halides can lead to rearrangement of the organometallic intermediate into an allenyl product . Reagent 4 has been previously shown to offer one of the few examples of a barbier reaction with a propargyl halide where rearrangement does not occur. (5b) use of tloet(17) gave yields similar to that of me3obf4(18) in all cases of oxirane formation, with stereoselectivity preserved under both sets of conditions . Use of the anti-2 allows formation of the (e)-alkene (the alkyne functional groups are cis to each other) functional group by the pathway shown in scheme 4 . Formation of the enediyne product was possible under slightly acidic conditions . A catalytic amount of p - tsoh is added to anti-2 in a mixture of water and thf (10% water) with stirring for 37 h. the reaction was also conducted under basic conditions after transformation of the oh group to a tosylate . Yields were quite low under basic conditions, presumably due to the competing acidity of the alkynyl proton, leading to decomposition and side product formation . Due to low yields, results of the acid - catalyzed conversion of anti-2 to (e)-6 are shown in table 3 . A slight amount of isomerization was witnessed under acidic conditions (about 3% of the product mixture), but overall, good stereocontrol was realized in these conversions, allowing for easy entry into the cis - enediyne ((e)-alkene) skeletal structure in a two - step sequence . The assignment of e- and z - isomers was made by comparison of the -vinyl proton shift . Alignment of the -vinyl proton and the heteroatom cis to each other gives rise to a lower field proton resonance than trans alignment of these two groups (figure 5). (19) the assignments for the enediyne products are compared correctly with the stereochemistry of compounds 2 and 3 within this study . Coupled with the original indium - promoted barbier reaction, the transformation of starting materials 1 and 4 into enediyne or epoxydiyne compounds proceeded in two steps in an overall 6080% overall yield, offering an efficient, benign, and good - yielding formation of these important skeletal structures . The use of n - methylformamide is shown to be a nice alternative to aqueous conditions in indium promoted coupling reactions between propargyl aldehydes (1) and -chloropropargylphenyl sulfide (4). This paper introduces the use of nmf as s viable solvent in a barbier coupling reaction, revealing increased rates and better selectivity when compared to reactions conducted in water and other organic solvents . The increased efficiency of the barbier coupling reaction as described in this paper allows for facile formation of epoxy - and enediyne skeletal structures in an overall two - step procedure . The introduction of nmf as an alternative solvent in barbier reactions will open new pathways to organic synthetic chemists . Further studies to test other halides and aldehyde functional groups under barbier coupling reaction conditions in nmf are currently underway and will be reported in due time . Tetrahydrofuran was purified by distillation under an argon atmosphere over sodium and benzophenone prior to use . Radial chromatography was performed using a chromatatron . To a solution of 30 mmol of alkyne in 80 ml of thf that had been cooled to 60 c under nitrogen was added 15 ml of n - buli (2.0 m in thf) with stirring . To this solution was added n, n - dimethylformamide (4.66 ml, 60 mmol) dropwise over 10 min . After 20 min of further stirring, the solution was added to a mixture of 75 ml of tert - butyl methyl ether and 160 ml of 10% kh2po4 solution which had been previously cooled to 0 c . The entire solution was stirred at 0 c for 30 min and then allowed to warm to room temperature . The organic layer was dried over mgso4, and the solvent removed under vacuum to give a clear oil . All propargyl aldehydes were used without further purification and could be stored at 0 c for 23 weeks . Yields for this reaction ranged from 85 to 95% . A magnetically stirred solution of aldehyde (1) (2.0 mmol) in deionized water (22 ml) was treated with -chloropropargylphenyl sulfide (702 mg, 3.0 mmol) and indium powder (228 mg, 2.0 mmol). The solution was allowed to proceed at room temperature until no 1 was seen (tlc analysis). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 10 ml). The combined organic layers were dried over mgso4 and evaporated to leave a dark yellow to brown oil . Purification was accomplished by flash chromatography or radial chromatography on silica gel (hexanesethyl acetate) to give a mixture of hydroxy sulfides (2)(11) as a light yellow oil . Purification was accomplished using radial chromatography on silica gel (40:1 hexanesethyl acetate) to give a mixture of diastereomeric hydroxy sulfides . Stereoselectivity was determined upon transformation to the epoxide product: yield = 471 mg (1.76 mmol) = 83%; h nmr (300 mhz, cdcl3) 0.91.5 (m, 7h), 1.82 (d, j = 1.7 hz, 0.2h), 1.87 (d, j = 1.6 hz, 0.8h), 2.13 (t, j = 6.5 hz, 2h), 4.09 (m, 1h), 4.86 (m, 1h), 7.17.8 (m, 5h), the oh proton was not detected; c nmr (75 mhz, cdcl3) 15.5, 18.2, 21.3, 34.2, (41.5, 42.9), (65.0, 66.1), 71.3, 78.5, 80.2, 84.7, 125.4, 126.1, 126.3, 127.6, 128.0, 136.4; ms m / z (m) calcd 258.1078, obsd 258.0998 . Calcd for c16h18os: c, 74.38; h, 7.02 . Found: c, 74.61; h, 6.99 . Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio: total yield = 489 mg (1.78 mmol) = 80% . Anti isomer: yield = 391 mg (1.41 mmol); h nmr (300 mhz, cdcl3) 1.94 (d, j = 1.8 hz, 1h), 4.10 (dd, j = 1.8, 4.7 hz, 1h), 5.11 (d, j = 4.7 hz, 1h), 7.27.7 (m, 10h), the oh proton was not detected; c nmr (75 mhz, cdcl3) 43.8, 66.0, 68.0, 81.3, 87.2, 93.6, 121.7, 125.8 (2), 127.3 (3), 128.1, 128.3(2), 129.3, 129.5, 136.1 . H nmr (300 mhz, cdcl3) 1.83 (d, j = 1.8 hz, 1h), 3.95 (dd, j = 1.8, 8.1 hz, 1h), 4.95 (d, j = 8.1 hz, 1h), 7.27.7 (m, 10h), the oh proton was not detected; c nmr (75 mhz, cdcl3) 44.1, 65.8, 68.0, 81.3, 87.2, 93.6, 121.7, 125.8 (2), 127.3 (3), 128.1, 128.3(2), 129.3, 129.5, 136.1 . Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) allowing isolation of two hydroxyl sulfide diastereomers in a 50:50 syn / anti ratio: total yield = 418 mg (1.54 mmol) = 70%; anti isomer: yield = 209 mg (7.7 mmol); h nmr (300 mhz, cdcl3) 0.24 (s, 9h), 1.84 (d, j = 1.6 hz, 1h), 4.23 (dd, j = 1.6, 5.3 hz, 1h), 4.80 (d, j = 5.3 hz, 1h), 7.17.4 (m, 5h), the oh proton was not detected; c nmr (75 mhz, cdcl3) 0.28(3), 44.4, 66.5, 67.5, 74.1, 84.3, 86.4, 125.0, 125.3, 126.5 (2), 127.3, 136.1; ms m / z (m) calcd 274.0848, obsd 274.0819 . Calcd for c15h18ossi: c, 65.64; h, 6.61 . Found: c, 65.17; h, 6.55 . Syn isomer: yield = 209 mg (7.7 mmol) = 70%; h nmr (300 mhz, cdcl3) 0.24 (s, 9h), 1.79 (d, j = 1.7 hz, 1h), 4.15 (dd, j = 1.7, 7.8 hz, 1h), 4.69 (d, j = 7.8 hz, 1h), 7.17.4 (m, 5h), the oh proton was not detected; c nmr (75 mhz, cdcl3) 0.28(3), 44.1, 67.9, 68.1, 75.0, 83.2, 86.9, 125.0, 125.3, 126.5 (2), 127.3, 136.1 . Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) to give a mixture of diastereomeric hydroxy sulfides: yield = 633 mg (1.69 mmol) = 77%; h nmr (300 mhz, cdcl3) 0.25 (s, 6h), 0.97 (s, 9h), 1.25 (m, 2h), 1.802.00 (m, 3h), 3.83 (t, j = 6.5, 2h), 4.11 (m, 1h), 4.99 (m, 1h), 7.17.4 (m, 5h), the oh proton was not detected; c nmr (75 mhz, cdcl3) 0.28 (2), 14.5, 15.1, 21.8 (2), 21.9 33.4, 47.0, 64.6, 65.1, 67.9, 75.2, 81.9, 87.3, 125.4, 126.6 (2), 127.1, 128.4, 135.9; ms m / z (m+) calcd 374.1736, obsd 365.1633 (loss of h2o). The general procedure was followed as described in section i.a with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used . Separation was accomplished using radial chromatography on silica gel (40:1 hexanesethyl acetate) to give a mixture of diastereomeric hydroxy sulfides . Stereoselectivity was determined upon transformation to the epoxide product: yield = 437 mg (1.63 mmol) = 77% . Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) allowing isolation of two hydroxyl sulfide diastereomers in a 75:25 syn / anti ratio: yield = total = 458 mg (1.65 mmol) = 75% . Anti isomer: yield = 115 mg (0.41 mmol); relevant h nmr shifts (300 mhz, cdcl3) 1.94 (d, j = 1.8 hz, 1h), 4.12 (dd, j = 1,8, 4.5 hz, syn isomer: yield = 343 mg (1.24 mmol); relevant h nmr shifts (300 mhz, cdcl3) 1.83 (d, j = 1.8 hz, 1h), 3.92 (dd, j = 1.8, 8.0 hz, 1h), 4.94 (d, j = 8.1 hz, 1h) processing and analysis of this reaction proceeded as described in section i.b . Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) allowing isolation of two hydroxyl sulfide diastereomers in a 60:40 syn / anti ratio: yield = total = 434 mg (1.58 mmol) = 72% . Anti isomer: yield = 174 mg (0.63 mmol); relevant h nmr shifts (300 mhz, cdcl3) 4.25 (dd, j = 1.7, 5.5 hz, 1h), 4.77 (d, j = 5.5 hz, 1h). Syn isomer: yield = 260 mg (0.95 mmol); relevant h nmr shifts (300 mhz, cdcl3) 4.15 (dd, j = 1.5, 7.8 hz, 1h), 4.71 (d, j = 7.8 hz, 1h). Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) to give a mixture of diastereomeric hydroxy sulfides . Stereoselectivity was determined upon transformation to the epoxide product: yield 583 mg (1.55 mmol) = 71% . Analysis as shown in section i.a.4 . A magnetically stirred solution of aldehyde 1 (2.0 mmol) in nmf (22 ml) was treated with -chloropropargylphenyl sulfide (702 mg, 3.0 mmol) and indium powder (228 mg, 2.0 mmol). The solution was allowed to proceed at room temperature until no 1 was seen (tlc analysis). The layers were separated, and the aqueous phase was extracted with dichloromethane (2 10 ml). The combined organic layers were washed with 1 n hcl (3 20 ml), dried over mgso4, and evaporated to leave a dark yellow to brown oil . Purification was accomplished by flash chromatography or radial chromatography on silica gel (hexanesethyl acetate) to give a mixture of hydroxy sulfides (2)(11) as a light yellow oil . Separation was accomplished using radial chromatography on silica gel (40:1 hexanesethyl acetate) to give a mixture of diastereomeric hydroxy sulfides . Stereoselectivity was determined upon transformation to the epoxide product: yield = 522 mg (2.02 mmol) = 92% . Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio: yield = total = 556 mg (2.00 mmol) = 91% . Anti isomer: yield = 445 mg (1.60 mmol); relevant h nmr shifts (300 mhz, cdcl3) 4.10 (dd, j = 1.8, 4.7 hz, 1h), 5.11 (d, j = 4.7 hz, 1h). Syn isomer: yield = 111 mg (0.40 mmol); relevant h nmr shifts (300 mhz, cdcl3) 3.95 (dd, j = 1.8, 8.1 hz, 1h), 4.95 (d, j = 8.1 hz, 1h). Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) allowing isolation of two hydroxyl sulfide diastereomers in a 40:60 syn / anti ratio: yield = total = 566 mg (2.06 mmol) = 94% . Anti isomer: yield = 340 mg (1.24 mmol); relevant h nmr shifts (300 mhz, cdcl3) 4.25 (dd, j = 1.7, 5.5 hz, 1h), 4.77 (d, j = 5.5 hz, 1h). Syn isomer: yield = 226 mg (0.82 mmol); relevant h nmr shifts (300 mhz, cdcl3) 4.15 (dd, j = 1.5, 7.8 hz, 1h), 4.71 (d, j = 7.8 hz, 1h). Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) to give a mixture of diastereomeric hydroxy sulfides . Stereoselectivity was determined upon transformation to the epoxide product: yield = 717 mg (1.91 mmol) = 87% . The general procedure was followed as described in section i.c with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used . Separation was accomplished using radial chromatography on silica gel (40:1 hexanesethyl acetate) to give a mixture of diastereomeric hydroxy sulfides . Stereoselectivity was determined upon transformation to the epoxide product: yield = 527 mg (2.05 mmol) = 93% . Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) allowing isolation of two hydroxyl sulfide diastereomers in a 85:15 syn / anti ratio: yield = total = 568 mg (2.05 mmol) = 93% . Anti isomer: yield = 85 mg (0.31 mmol); relevant h nmr shifts (300 mhz, cdcl3) 4.13 (dd, j = 1.8, 4.7 hz, 1h), 5.08 (d, j = 4.7 hz, 1h). Syn isomer: yield = 483 mg (1.74 mmol); relevant h nmr shifts (300 mhz, cdcl3) 3.92 (dd, j = 1.8, 8.1 hz, 1h), 4.97 (d, j = 8.1 hz, 1h). Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) allowing isolation of two hydroxyl sulfide diastereomers in a 70:30 syn / anti ratio: yield = total = 548 mg (2.00 mmol) = 91% . Anti isomer:: yield = 164 mg (0.60 mmol); relevant h nmr shifts (300 mhz, cdcl3) 4.23 (dd, j = 1.7, 5.5 hz, 1h), 4.80 (d, j = 5.5 hz, 1h). Syn isomer: yield = 384 mg (1.40 mmol); relevant h nmr shifts (300 mhz, cdcl3) 4.15 (dd, j = 1.5, 7.8 hz, 1h), 4.68 (d, j = 7.8 hz, 1h). Separation was accomplished using radial chromatography on silica gel (35:1 hexanesethyl acetate) to give a mixture of diastereomeric hydroxy sulfides . Stereoselectivity was determined upon transformation to the epoxide product: yield = 732 mg (1.96 mmol) = 89% . A solution of the hydroxy sulfide mixture (85:15 syn / anti) (100 mg, 0.39 mmol) in dichloromethane (10 ml) was treated with trimethyloxonium tetrafluoroborate (90 mg (0.60 mmol). The solution was stirred at room temperature for 8 h and diluted with 7% sodium hydroxide solution (aqueous, 8 ml). Purification was accomplished by radial chromatography on silica gel (elution with 80:1 hexanesethyl acetate) to give a mixture of diastereomers as a colorless oil in an 85:15 syn / anti ratio: yield = 52 mg (0.35 mmol) = 90%; h nmr (300 mhz, cdcl3) 0.9 1.5 (m, 7h), 1.82 (d, j = 1.8 hz, 0.15h), 1.86 (d, j = 1.9 hz, 0.85h), 2.17 (m, 2h), 3.25 (d, j = 4.6 hz, 0.85h), 3.40 (d, j = 2.3 hz, 0.15h), 3.50 (dd, j = 1.8, 2.3 hz, 0.15h), 3.61 (dd, j = 1.9, 4.6 hz, 0.85h); c nmr (75 mhz, cdcl3) (14.0, 14.1), (17.2, 17.4), 21.6, (31.7, 31.8), 44.5, (48.2, 48.3), 66.7, (77.5, 77.9), (79.5, 79.7), (85.2, 85.5) calcd for c10h12o: c, 81.04; h, 8.16 . Found: c, 80.99; h, 8.19 . A solution of the hydroxy sulfide mixture (85:15 syn / anti) (200 mg 0.72 mmol) in dichloromethane (20 ml) was treated with trimethyloxonium tetrafluoroborate (180 mg (1.20 mmol). The solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution (aqueous, 15 ml). Purification was accomplished by radial chromatography on silica gel (elution with 100:1 hexanesethyl acetate) allowing isolation of two diastereomers as colorless oils in an 85:15 syn / anti ratio: yield = total = 110 mg (0.64 mmol) = 89% . Anti isomer: yield = 12 mg (0.07 mmol); h nmr (300 mhz, cdcl3) 2.44 (d, j = 1.8 hz, 1h), 3.51 (dd, j = 1.8, 2.4 hz, 1h), 3.97 (d, j = 2.4 hz, 1h), 7.157.25 (m, 5h); c nmr (75 mhz, cdcl3) 48.2, 53.1, 66.2, 80.3, 87.3, 91.7, 123.5, 128.4 (2), 129.0, 133.1 (2). Calcd for c12h8o = c, 85.69 h, 4.79 . Found: c, 86.00 h, 4.71 . Syn isomer: yield = 98 mg (0.57 mmol); h nmr (300 mhz, cdcl3) 2.43 (d, j = 1.8 hz, 1h), 3.46 (dd, j = 1.8, 4.7 hz, 1h), 4.15 (d, j = 4.7 hz, 1h), 7.157.23 (m, 5h); c nmr (75 mhz, cdcl3) 48.7, 53.1, 66.5, 80.1, 87.3, 92.0, 123.0, 128.3 (2), 128.7, 132.3(2). A solution of the hydroxy sulfide mixture (70:30 syn / anti) (200 mg 0.73 mmol) in dichloromethane (20 ml) was treated with trimethyloxonium tetrafluoroborate (180 mg (1.20 mmol). The solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution (aqueous, 15 ml). Purification was accomplished by radial chromatography on silica gel (elution with 100:1 hexanesethyl acetate) allowing isolation of two diastereomers as colorless oils in an 70:30 syn / anti ratio: yield = total = 108 mg (0.66 mmol) = 90%; anti isomer: yield = 32 mg (0.30 mmol); h nmr (300 mhz, cdcl3) 0.13 (s, 9h); 2.21 (d, j = 2.0 hz, 1h), 3.25 (dd, j = 2.0, 2.3 hz, 1h), 3.55 (d, j = 2.3 hz, 1h); c nmr (75 mhz, cdcl3) 0.10 (3), 44.9, 49.9, 69.9, 81.7, 86.0, 102.0; ms m / z (m) calcd = 164.0657, obsd = 164.0656 . Syn isomer: yield = 76 mg (0.46 mmol); h nmr (300 mhz, cdcl3) 0.13 (s, 9h), 2.19 (d, j = 2.0 hz, 1h), 3.32 (dd, j = 2.0, 4.9 hz, 1h), 3.63 (d, j = 4.9 hz, 1h); c nmr (75 mhz, cdcl3) 0.10 (3), 45.1, 49.3, 70.1, 81.5, 85.6, 101.2 . A solution of the hydroxy sulfide mixture (90:10 syn / anti) (250 mg 0.67 mmol) in dichloromethane (20 ml) was treated with trimethyloxonium tetrafluoroborate (150 mg (1.00 mmol). The solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution (aqueous, 12 ml). Purification was accomplished by radial chromatography on silica gel (elution with 100:1 hexanesethyl acetate) to give a mixture of two diastereomers as a colorless oil in a 90:10 syn / anti ratio: yield = 148 mg (0.56 mmol) = 83%; h nmr (300 mhz, cdcl3) 0.22 (s, 6h); 0.97 (s, 9h), 1.27 (m, 2h), 1.82.0 (m, 3h), 3.23 (d, j = 4.4 hz, 0.9h), 3.31 (d, j = 2.5 hz, 0.1h), 3.53 (dd, j = 1.9, 2.5 hz, 0.1 hz), 3.69 (dd, j = 1.9, 4.4 hz, 0.9h), 3.83 (m, 2h); c nmr (75 mhz, cdcl3) 0.28 (2), 14.5, 14.7, 21.8 (2), (22.0, 22.3), (33.9, 34.2), (43.8, 44.0), (47.1, 47.9), (64.7, 65.0), 67.3, (78.7, 79.1), 81.8, (85.4, 85.8); ms m / z (m) calcd = 264.1546, obsd = 264.1545 calcd for c15h24osi: c, 68.13; h, 9.15 . Found: c, 67.79; h, 9.4 . A solution of the hydroxy sulfide mixture (85:15 syn / anti) (200 mg 0.72 mmol) in chloroform (20 ml) was treated with tloet (234 mg, 0.93 mmol). After 20 min of stirring, the insolubles were removed by filtration through a short celite pad . The resulting organic solution was washed with a saturated nahco3 solution (aq) and a saturated nacl solution (aq). The organic layer was dried and concentrated . Purification and analysis of products proceeded in the manner previously described (section ii.a). Subsequent hydroxy sulfides were treated in a fashion similar to that in ii.b.1 and analyzed in the manner previously described in section ii.a . A solution of the hydroxy sulfide mixture (25:75 syn / anti) (100 mg, 0.39 mmol) in a 9:1 thf / h2o mixture (10 ml) was treated with a catalytic amount of p - toluenesulfonic acid . The solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution (aqueous, 10 ml). After 5 min of stirring, 20 ml of dichloromethane was added to the solution with stirring for 10 min . The organic layers were combined, washed with 3 10 ml of water, dried over mgso4, and concentrated . Purification was accomplished by radial chromatography on silica gel (elution with 25:1 hexanesethyl acetate) to give a mixture of diastereomers as a colorless oil in a 73:27 cis / trans ratio: yield = 82 mg (0.34 mmol) = 87%; h nmr (300 mhz, cdcl3) 0.901.51 (m, 7h), 2.07 (m, 2h), 2.63 (s, 0.27h), 2.71 (s, 0.73h), 5.90 (s, 0.73h), 6.21 (s, 0.27h) 7.107.25 (m, 5h); c nmr (75 mhz, cdcl3) (13.9, 14.1), 17.1, 22.9, 31.6, 77.2, (79.5, 80.3), (83.1, 83.7), (90.5, 91.7), (113.3, 113.9), 125.1, 128.4(2), 129.3(2), 134.0, (145.9, 146.2); ms m / z (m) calcd 240.0973, obsd 240.0973 . Calcd for c16h16s: c, 79.95; h, 6.71 . Found: c, 80.09; h, 6.60 . A solution of the hydroxy sulfide (200 mg 0.72 mmol) in a 9:1 thf / h2o mixture (20 ml) was treated with a catalytic amount of p - toluenesulfonic acid . The solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution (aqueous, 20 ml). After 5 min of stirring, 30 ml of dichloromethane was added to the solution with stirring for 10 min . The organic layers were combined, washed with 3 10 ml of water, dried over mgso4, and concentrated . Purification was accomplished by radial chromatography on silica gel (elution with 25:1 hexanesethyl acetate) to give the product as a colorless oil in a 97:3 e / z ratio (the minor isomer was detected by gcms; the minor isomer was not seen by nmr): ield = 156 mg (0.60 mmol) = 83%; h nmr (300 mhz, cdcl3) 0.2.95 (s, 1h), 6.01 (s, 1h), 7.077.30 (m, 10h): c nmr (75 mhz, cdcl3) 81.7, 82.9, 89.3, 92.6, 114.0, 122.0, 125.7, 128.1 (2), 128.3, 128.8 (2), 129.5 (2), 133.6 (2), 134.0, 144.6 . A solution of the hydroxy sulfide mixture (20:80 syn / anti) (200 mg, 0.72 mmol) in a 9:1 thf / h2o mixture (20 ml) was treated with a catalytic amount of p - toluenesulfonic acid . The solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution (aqueous, 20 ml). After 5 min of stirring, 30 ml of dichloromethane was added to the solution with stirring for 10 min . The organic layers were combined, washed with 3 10 ml of water, dried over mgso4, and concentrated . Purification was accomplished by radial chromatography on silica gel (elution with 25:1 hexanesethyl acetate) to give a mixture of diastereomers as a colorless oil in a 77:23 e / z ratio: yield = 160 mg (0.61 mmol) = 85%; h nmr (300 mhz, cdcl3) 0.2.95 (s, 0.77h), 3.01 (s, 0.23h), 6.04 (s, 0.77h), 6.35 (s, 0.27h), 7.077.30 (m, 10h): c nmr (75 mhz, cdcl3) (81.5, 81.7), 83.0, 89.3, (92.6, 93.0), (114.0, 119.2), 122.0, 125.7, 128.1 (2), (128.3, 128.5), 128.8 (2), 129.5 (2), 133.6 (2), (134.0, 134.5), (144.6, 145.1). A solution of the hydroxy sulfide (200 mg 0.73 mmol) in a 9:1 thf / h2o mixture (20 ml) was treated with a catalytic amount of p - toluenesulfonic acid . The solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution (aqueous, 20 ml). After 5 min of stirring, 30 ml of dichloromethane was added to the solution with stirring for 10 min . The organic layers were combined, washed with 3 10 ml of water, dried over mgso4, and concentrated . Purification was accomplished by radial chromatography on silica gel (elution with 15:1 hexanesethyl acetate) to give the product as a colorless oil in a 97:3 e / z ratio (the minor isomer was detected by gcms; the minor isomer was not seen by nmr): yield = 123 mg (0.0.48 mmol) = 65%; h nmr (300 mhz, cdcl3) 0.0.11 (s, 9h), 2.87 (s, 1h), 6.20 (s, 1h), 7.107.29 (m, 5h); c nmr (75 mhz, cdcl3) 0.09 (3), 78.7, 81.2, 95.3, 100.9, 112.5, 124.8, 127.2(2), 128.7(2), 131.8, 142.1 . Calcd for c15h16ssi: c, 70.25; h, 6.29 . Found: c, 69.99; h, 6.43 . A solution of the hydroxy sulfide mixture (40:60 syn / anti) (200 mg 0.73 mmol) in a 9:1 thf / h2o mixture (20 ml) was treated with a catalytic amount of p - toluenesulfonic acid . The solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution (aqueous, 20 ml). After 5 min of stirring, 30 ml of dichloromethane was added to the solution with stirring for 10 min . The organic layers were combined, washed with 3 10 ml of water, dried over mgso4, and concentrated . Purification was accomplished by radial chromatography on silica gel (elution with 15:1 hexanesethyl acetate) to give a mixture of diastereomers as a colorless oil in a 50:50 e / z ratio: yield = 120 mg (0.48 mmol) = 65%; h nmr (300 mhz, cdcl3) 0.09 (s, 4.5h) 0.10 (s, 4.5h) 2.81 (s, 0.5h), 2.87 (s, 0.5h), 5.77 (s, 0.5h), 6.20 (s, 0.5h), 7.107.29 (m, 5h); c nmr (75 mhz, cdcl3) 0.09 (3), (78.7, 79.1), (81.2, 81.8), (95.3, 95.9), 100.9, (112.5, 116.1), 124.8, 127.2(2), 128.7(2), (131.8, 132.9), (142.1, 143.5). A solution of the hydroxy sulfide mixture (15:85 syn / anti) (250 mg 0.67 mmol) in a 9:1 thf / h2o mixture (10 ml) was treated with a catalytic amount of p - toluenesulfonic acid . The solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution (aqueous, 10 ml). After 5 min of stirring, 20 ml of dichloromethane was added to the solution with stirring for 10 min . The organic layers were combined, washed with 3 10 ml of water, dried over mgso4, and concentrated . Purification was accomplished by radial chromatography on silica gel (elution with 10:1 hexanesethyl acetate) to give a mixture of diastereomers as a colorless oil in an 82:18 cis / trans ratio: yield = 192 mg (0.54 mmol) = 80%; h nmr (300 mhz, cdcl3) 0.15 (s, 6h), 0.93 (s, 9h), 1.27 (m, 2h), 2.11(m, 2h), 2.71 (s, 0.82h), 2.80 (s, 0.18h), 3.75 (m, 2h), 5.93 (s, 0.82h), 6.16 (s, 0.18h), 7.077.26 (m, 5h); c nmr (75 mhz, cdcl3) 0.13(2), (13.9, 14.1), (15.0, 15.1), 21.3(3), 33.9, (64.9, 65.3), (77.3, 78.1), (79.7, 80.1), (83.2, 84.1), (93.1, 93.7), (114.1, 115.0), 125.4, 127.7(2), 129.1, 129.3, (133.7, 134.0), (144.9, 145.4); ms m / z (m) calcd 356.1630, obsd 355.9891 calcd for c21h28ossi: c, 70.73; h, 7.91 . Found: c, 70.81; h, 8.01.
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Sepsis is the systemic inflammation caused by infection with bacteria, viruses, fungi, parasites, or toxic products, and has become one of the leading causes of mortality in children . In developed countries, the mortality rate of pediatric sepsis is about 210% and is as high as 50% in developing countries . Pediatric sepsis usually leads to tachycardia, tachypnea, peripheral vasodilation, fever or hypothermia, and even multiple organ dysfunction syndrome (mods). Frequently - used applications in pediatric sepsis treatment concentrate on maintaining metabolic, circulatory, and respiratory stabilities, and antimicrobial agents, as well as surgical and adjunctive therapies such as nitric oxide and corticosteroids, which show great benefits in the outcome of pediatric sepsis treatment . Acute kidney injury (aki) is a severe consequence of sepsis and mods, and about 1527% of aki in children is generated from pediatric sepsis . The pathophysiology of septic aki is related to the altered global renal blood flow, as well as the comprehensive effects of inflammation, microvasculature blood flow, and bioenergetics factors . For example, glomerular endothelial injury during aki progression in a lipopolysaccharide - induced mouse sepsis model is closely related to tumor necrosis factor and receptor 1 . Due to the increased risk of mortality caused by septic aki, it is imperative to explore effective therapies for controlling aki in pediatric sepsis . Baicalin is one of the flavonoid constituents in scutellaria baicalensis georgi, a kind of perennial herb with protective effects against diseases like hydrogen peroxide - induced oxidative stress and cerebral ischemia injury [911]. Specifically, baicalin has been revealed to protect against oxidants, anxiety, acute hepatic injury, experimental periodontitis, and sepsis . It also alleviates reperfusion - induced damage to the myocardium, the mechanism of which may be associated with its anti - apoptotic roles . Moreover, a recent study found that baicalin can inhibit inflammation and cell apoptosis, which allows it to protect against ischemia - reperfusion injuries in the kidney . However, few studies have investigated the role of baicalin in septic aki or pediatric sepsis . This study aimed to uncover the effects of baicalin in treating aki in pediatric sepsis patients . The baicalin adjunctive therapy was tested in pediatric sepsis patients, after which the effect was compared based on the renal function assessment from bun and serum creatinine (cr). We also performed experiments in the cecal ligation and puncture (clp)-induced mouse sepsis model to investigate the effects and potential mechanisms of baicalin in septic aki . This study lays the foundation for future application of baicalin in adjunctive therapies for aki in pediatric sepsis . A total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 . All 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005, and with aki at the same time according to the criteria of aki proposed by acute kidney injury net (akin). The following conditions were excluded: patients with kidney injuries caused by prerenal or postrenal factors, and patients with a history of renal diseases such as acute or chronic renal failure . The 50 patients were randomly divided into 2 groups: control and baicalin, each group containing 25 patients . No significant difference existed in the age between the 2 groups (2.580.17 and 2.460.20). Patients in the control group received basic treatment, and those in the baicalin group received basic treatment plus oral baicalin (inner chengzi pharmaceutical, chifeng, china) according to the manufacturer s instructions . The blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital . These procedures were approved by our local ethics committee and were performed according to the instructions of the hospital . Clp was performed on male c57bl/6 mice (spf, age 810 weeks, weight 2226 g) (vital river laboratories, beijing, china) to induce a septic aki model based on the method in a previous report . Thirty individuals were randomly divided into 3 groups: sham, clp, and clp + baicalin . The cecum was ligated at 1 cm to the distal end, and double punctures were made to extrude feces into the abdominal cavity . Then the cecum was relocated to the abdominal cavity, and the abdomen was closed . The mice were resuscitated by intraperitoneal injection of 0.9% saline (24 ml / kg). Mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd) (pureone biotechnology, shanghai, china) for 6 days, after which all the mice were anesthetized and killed for blood and renal tissue sampling . Cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling (tunel) method with the in situ cell death detection kit, pod (roche, basel, switzerland) according to the manufacturer s instructions . Briefly, the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices . Fresh tunel mix (50 l) was added to the slices for 1-h incubation at 37c in the dark . Then, 50 l of converter - pod was added for incubation of 30 min at 37c in the dark, after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min . The slices were washed in phosphate - buffered saline (pbs) 3 times between steps, each time lasting 5 min . Slices were then dehydrated, mounted, and observed with an optical microscope (olympus, tokyo, japan). Total rna samples of the mouse renal tissue were extracted by trizol (invitrogen, carlsbad, ca) according to the manufacturer s instructions . Dna contamination was removed by dnase i (invitrogen) and then the quality and quantity of rna samples were examined by nanodrop 2000 (thermo scientific, carlsbad, ca). For each mouse, 1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase (invitrogen). Then the synthesized complementary dna (20 ng) was used in qrt - pcr on the quantstudio 6 flex realtime pcr system (applied biosystems, carlsbad, ca) with the specific primers for mouse bax (fw: 5-tgttt gctga tggca acttc-3 and rv: 5-gatgg ttctg atcag ctcgg-3) and bcl2 (fw: 5-gtaga agagg gtgtg acagc-3 and rv: 5-agcac tgact ctggg atcgc-3). Data were analyzed by the 2 method with gapdh (fw: 5-tcaac agcaa ctccc actct tcca-3 and rv: 5-accct gttgc tgtag ccgta ttca-3) as an internal control . Protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent (thermo scientific) and quantified by bio - rad protein assay (bio - rad, hercules, ca) according to the manufacturers instructions . The protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes . The blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 (ab32503 and ab32124, abcam, cambridge, uk) overnight at 4c . After washing in pbs 5 times, the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody (ab6721) for 1 h at room temperature . Ecl plus western blotting substrate (thermo scientific) was used to develop positive signals . Comparison between groups was performed with one - way analysis of variance and t test in spss 20 (ibm, new york, ny). A total of 50 pediatric sepsis patients ages 14 years were selected from the patients hospitalized from november 2013 to october 2015 . All 50 patients were diagnosed with pediatric sepsis based on the diagnostic standard proposed in 2005, and with aki at the same time according to the criteria of aki proposed by acute kidney injury net (akin). The following conditions were excluded: patients with kidney injuries caused by prerenal or postrenal factors, and patients with a history of renal diseases such as acute or chronic renal failure . The 50 patients were randomly divided into 2 groups: control and baicalin, each group containing 25 patients . No significant difference existed in the age between the 2 groups (2.580.17 and 2.460.20). Patients in the control group received basic treatment, and those in the baicalin group received basic treatment plus oral baicalin (inner chengzi pharmaceutical, chifeng, china) according to the manufacturer s instructions . The blood samples were collected 1 day before treatment and 15 days after treatment for bun and cr detection by the hospital . These procedures were approved by our local ethics committee and were performed according to the instructions of the hospital . Clp was performed on male c57bl/6 mice (spf, age 810 weeks, weight 2226 g) (vital river laboratories, beijing, china) to induce a septic aki model based on the method in a previous report . Thirty individuals were randomly divided into 3 groups: sham, clp, and clp + baicalin . The cecum was ligated at 1 cm to the distal end, and double punctures were made to extrude feces into the abdominal cavity . Then the cecum was relocated to the abdominal cavity, and the abdomen was closed . The mice were resuscitated by intraperitoneal injection of 0.9% saline (24 ml / kg). Mice in the clp + baicalin group were intragastrically administrated with baicalin 200 mg/(kgd) (pureone biotechnology, shanghai, china) for 6 days, after which all the mice were anesthetized and killed for blood and renal tissue sampling . Cell apoptosis in the mouse renal tissue was detected by tdt - mediated dutp nick - end labeling (tunel) method with the in situ cell death detection kit, pod (roche, basel, switzerland) according to the manufacturer s instructions . Briefly, the renal tissue of each mouse was embedded in paraffin and cut into 5-m slices . Fresh tunel mix (50 l) was added to the slices for 1-h incubation at 37c in the dark . Then, 50 l of converter - pod was added for incubation of 30 min at 37c in the dark, after which 50 l of diaminobenzidine was added to develop positive signals in the dark for 10 min . The slices were washed in phosphate - buffered saline (pbs) 3 times between steps, each time lasting 5 min . Slices were then dehydrated, mounted, and observed with an optical microscope (olympus, tokyo, japan). Total rna samples of the mouse renal tissue were extracted by trizol (invitrogen, carlsbad, ca) according to the manufacturer s instructions . Dna contamination was removed by dnase i (invitrogen) and then the quality and quantity of rna samples were examined by nanodrop 2000 (thermo scientific, carlsbad, ca). For each mouse, 1 g of total rna was used for reverse transcription catalyzed by superscript ii reverse transcriptase (invitrogen). Then the synthesized complementary dna (20 ng) was used in qrt - pcr on the quantstudio 6 flex realtime pcr system (applied biosystems, carlsbad, ca) with the specific primers for mouse bax (fw: 5-tgttt gctga tggca acttc-3 and rv: 5-gatgg ttctg atcag ctcgg-3) and bcl2 (fw: 5-gtaga agagg gtgtg acagc-3 and rv: 5-agcac tgact ctggg atcgc-3). Data were analyzed by the 2 method with gapdh (fw: 5-tcaac agcaa ctccc actct tcca-3 and rv: 5-accct gttgc tgtag ccgta ttca-3) as an internal control . Protein samples of mouse renal tissue were extracted by m - per mammalian protein extraction reagent (thermo scientific) and quantified by bio - rad protein assay (bio - rad, hercules, ca) according to the manufacturers instructions . The protein samples were separated on sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes . The blots were blocked in 5% skim milk for 2 h at room temperature and then incubated in rabbit monoclonal primary antibodies for bax and bcl2 (ab32503 and ab32124, abcam, cambridge, uk) overnight at 4c . Gapdh (ab181602) was used as an internal control . After washing in pbs 5 times, the blots were incubated in goat anti - rabbit horse - radish peroxidase - conjugated secondary antibody (ab6721) for 1 h at room temperature . Ecl plus western blotting substrate (thermo scientific) was used to develop positive signals . Comparison between groups was performed with one - way analysis of variance and t test in spss 20 (ibm, new york, ny). In the treatment of 50 pediatric sepsis patients, 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies . Results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies (p=0.2842 and 0.2135, figure 1a, 1b), while those of the baicalin group were significantly decreased after baicalin adjunctive therapy (p=0.0191 and 0.0077). It could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved, possibly due to the baicalin adjunctive therapy . We next performed experiments in the mouse model to analyze the function of baicalin in septic aki . Mice in the clp group showed significantly elevated bun and cr levels (p=0.0001 and 0.0013, figure 2a, 2b), suggesting that the kidney injury occurred after clp . Both bun and cr levels were significantly decreased in the clp + baicalin group (p=0.0181 and 0.0225), indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model . The possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis . Tunel assay showed more apoptotic cells in the clp group compared to the sham group, and baicalin treatment reduced apoptotic cell numbers (figure 3a), with significant differences between groups (p=0.0087 and 0.0359, figure 3b). Thus, baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model . Bax and bcl2, 2 factors indicating cell apoptosis, were detected at both mrna and protein levels to verify the changes in renal cell apoptosis . Qrt - pcr results showed that bax mrna level was significantly elevated in the clp group (p=0.0108) and then was down - regulated by baicalin treatment (p=0.0256, figure 4a). Bcl2 mrna level showed the opposite changing pattern to bax, with significant differences between groups (p=0.0004 and 0.0069, figure 4b). Protein level changes of the 2 factors were consistent with their mrnas, as shown by western blot analysis (figure 4c). Since up - regulated bax and down - regulated bcl2 are usually correlated with promoted cell apoptosis, these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model . In the treatment of 50 pediatric sepsis patients, 25 received adjunctive therapy of oral baicalin for 15 days and the other 25 patients received only basic therapies . Results showed that both bun and cr levels in the serum of the control group did not changed greatly after basic therapies (p=0.2842 and 0.2135, figure 1a, 1b), while those of the baicalin group were significantly decreased after baicalin adjunctive therapy (p=0.0191 and 0.0077). It could be inferred based on the 2 indexes that the renal functions of the baicalin group were improved, possibly due to the baicalin adjunctive therapy . We next performed experiments in the mouse model to analyze the function of baicalin in septic aki . Mice in the clp group showed significantly elevated bun and cr levels (p=0.0001 and 0.0013, figure 2a, 2b), suggesting that the kidney injury occurred after clp . Both bun and cr levels were significantly decreased in the clp + baicalin group (p=0.0181 and 0.0225), indicating that baicalin treatment effectively alleviated kidney injury in the clp - induced mouse sepsis model . The possible mechanism of baicalin in protecting against septic kidney injury of the mouse model was investigated from changes in cell apoptosis . Tunel assay showed more apoptotic cells in the clp group compared to the sham group, and baicalin treatment reduced apoptotic cell numbers (figure 3a), with significant differences between groups (p=0.0087 and 0.0359, figure 3b). Thus, baicalin had suppressive effects on renal cell apoptosis in the clp - induced mouse model . Bax and bcl2, 2 factors indicating cell apoptosis, were detected at both mrna and protein levels to verify the changes in renal cell apoptosis . Qrt - pcr results showed that bax mrna level was significantly elevated in the clp group (p=0.0108) and then was down - regulated by baicalin treatment (p=0.0256, figure 4a). Bcl2 mrna level showed the opposite changing pattern to bax, with significant differences between groups (p=0.0004 and 0.0069, figure 4b). Protein level changes of the 2 factors were consistent with their mrnas, as shown by western blot analysis (figure 4c). Since up - regulated bax and down - regulated bcl2 are usually correlated with promoted cell apoptosis, these results further confirmed that clp induced renal cell apoptosis and that baicalin suppressed apoptosis in the mouse sepsis model . This study performed investigations in pediatric sepsis patients and in a clp - induced mouse sepsis model to reveal the role of baicalin in aki of pediatric sepsis . Baicalin adjunctive therapy in the patients and treatment in the mouse model decreased bun and cr levels . Further histological and expression analyses suggested the suppressive function of baicalin in renal cell apoptosis . The 50 pediatric patients participating in this study were chosen from pediatric sepsis patients diagnosed with aki, and the mouse model induced by clp was detected with elevated bun and cr levels, which suggested potential kidney injury . Previous studies using mouse sepsis models used various baicalin treatment methods, including topical application on the mouse skin to analyze the effect of baicalin on epidermis, intraperitoneal injection to investigate the role of baicalin in mammary glands, and baicalin - containing diets to study liver injury, inflammation in the kidney, and lung carcinoma [22,2729]. In these studies, the dose of baicalin was usually 100200 mg/(kgd), and the duration ranged from 3 to 50 days . Thus, in the present study we chose to perform baicalin treatment in the model mouse via intragastric administration of 200 mg/(kgd) for 6 days, and the significantly elevated bun and cr levels in the clp group compared to the sham group indicated that this treatment method effectively induced kidney injury in the mouse sepsis model . Clinical trials with baicalin adjunctive therapy have focused on its protective roles in ulcerative colitis, hepatic fibrosis, and diabetes mellitus [3032], and several studies have investigated the potential of baicalin in treating patients with early diabetic nephropathy, in which baicalin inhibits aldose reductase activity and reduces urinary albumin excretion rate and the blood 2-microglobulin . The present study further discovered the protective role of baicalin against aki in pediatric sepsis patients, which was reflected in the reduced bun and cr levels . Moreover, the experiments in the mouse model confirmed that baicalin treatment in a sepsis animal model could improve renal functions . These findings show the potential of using baicalin to attenuate aki in pediatric sepsis patients, but the proper dosage and possible complications need to be assessed in future research . Tunel results showed that baicalin treatment in the mouse model significantly suppressed renal cell apoptosis, and western blot and qrt - pcr analyses indicated consistent changes in the expression of bax and bcl2 . A similar mechanism has been reported in mouse mammary glands, where baicalin inhibits cell apoptosis via regulating bax and bcl2 . In addition, baicalin inhibits hepatocyte apoptosis in mouse kidney injury induced by concanavalin a; therefore, suppressing renal cell apoptosis is likely to be one of the mechanisms by which baicalin attenuates septic kidney injury . However, the anti - proliferative role of baicalin has also been reported in studies on lung cancer and mouse embryonic stem cells, and it is converted to baicalin by intestinal gut flora to act as a pro - apoptotic and anti - proliferative substance . Baicalin is capable of promoting neuronal differentiation, and thus may benefit therapies for parkinson and alzheimer diseases . We speculate that there are multiple potential mechanisms behind the effects of baicalin in various diseases; therefore, it is necessary to uncover the detailed mechanisms in order to optimize use of baicalin to control aki in pediatric sepsis patients . The investigations of baicalin in pediatric sepsis patients and the clp - induced mouse sepsis model reveal the protective role of baicalin against aki in pediatric sepsis . Baicalin can inhibit renal cell apoptosis, which may be one of its functional mechanisms . This study provides a potential adjunctive therapy for treating aki in pediatric sepsis, and future research will concentrate on more detailed mechanisms of baicalin.
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The main cause of treatment failure and death in cancer patients is metastasis the formation of secondary tumors in organs distant from the original neoplastic cell tissue . Adjuvant therapy of proven efficacy is not currently available for cancer patients; therefore, the search for new targets of therapeutic reagents is required to prevent both proliferation and metastases . During metastasis, it is known that the mode of invasion is one of the markers of the malignancy and prognosis of cancer . The receptor for advanced glycation end - products (rage), a multiligand member of the immunoglobulin superfamily of cell surface molecules, interacts with distinct molecules implicated in homeostasis, development, and inflammation . Rage binding by ligands such as advanced glycation end - products (ages) triggers the activation of key cell signaling pathways, thereby reprogramming cellular properties . In addition, several reports have suggested that rage is associated with cancer malignancy [4, 5]. The advanced stage of the glycation process (one of the posttranslational modifications of proteins) leads to the formation of ages and plays an important role in the pathogenesis of angiopathy in diabetic patients, aging, and neurodegenerative diseases [69]. A growing body of evidence suggests that the interaction of glyceraldehyde - derived ages (glycer - ages), but not glucose - derived ages (glc - ages), with rage elicits oxidative stress generation in numerous types of cells (vascular wall cells, mesangial cells, schwann cells, and cortical neurons), all of which could contribute to the pathological changes seen in diabetic vascular complications of alzheimer's disease [1013]. We have recently found that glycer - ages stimulated the growth and migration of cultured human melanoma cells and that anti - rage antibodies inhibited the tumor formation and lung metastasis of melanoma cell xenografts and subsequently improved survival in athymic mice . However, the effects of glycer - ages on other cancer cells remains poorly understood, and the molecular mechanisms behind their effects have not been clarified . In the present study, we examined the effects of glycer - ages on cultured human lung adenocarcinoma a549 cells and showed that glycer - ages enhanced their malignancy rather than their proliferation . N - acetyl - l - cysteine (nac) was purchased from sigma - aldrich (st . Louis, mo, usa). Briefly, 25 mg / ml of bsa (a0281, sigma - aldrich) were incubated at 37c for 7 days under sterile conditions with 0.1 m glyceraldehyde (ga; nakalai tesque, kyoto, japan) and 5 mm diethylenetriamine - pentaacetic acid (dojindo, kumamoto, japan) in 0.2 m phosphate buffer (ph 7.4). The modified albumin was then purified by pd-10 column (ge healthcare, buckinghamshire, england) chromatography and dialysis against phosphate - buffered saline (pbs). Control unglycated bsa was incubated under the same conditions except for the absence of glyceraldehyde as a negative control . Protein concentrations were determined with the dc protein assay reagent (bio - rad, richmond, ca, usa) using bsa as a standard . The preparations were tested for endotoxin using the pyrotell - t test (seikagaku bio - business, tokyo, japan), but no endotoxin was detected . Human lung adenocarcinoma a549 and hepatocellular carcinoma hep3b cells were grown in dulbecco's modified eagle's medium (dmem; sigma - aldrich) supplemented with 10% fetal bovine serum (fbs; equitech - bio, kerrville, tx, usa) under standard cell culture conditions (humidified atmosphere, 5% co2, 37c). Yamagishi, and its mock vector - transfected hep3b cells were maintained in 10% fbs / dmem in the presence of 700 g / ml g418 (roche, mannheim, germany). The cells were washed with ice - cold ca and mg free pbs (pbs (-)) and subjected to lysis buffer (1% tritonx-100/nonidet p-40, 10 mm sodium fluoride, 1 mm sodium orthovanadate, 5 mm sodium pyrophosphate, 2 mm egta, 5 mm edta, and 1 protease inhibitor cocktail (complete, mini; roche)). Subsequently, the cell lysates were incubated on ice for 5 min and centrifuged at 10,000 g for 10 min at 4c . Their protein concentrations were then measured using the bradford assay (bio - rad). Cell lysates (30 g of proteins / lane) dissolved in sds sample buffer (62.5 mm tris - hcl (ph 6.8), 2% sds, 10% glycerol, and 0.01% bromophenol blue) containing 5% 2-mercaptoethanol (2-me) were boiled for 3 min at 95c, separated by sds - page, and then electrotransferred onto pvdf membranes (millipore, billerica, ma, usa). Biotinylated markers (cell signaling, beverly, ma, usa) were used as molecular weight markers . The membranes were blocked for 1 h using 5% skimmed milk in pbs containing 0.05% polyoxyethylene sorbitan monolaurate (pbs - t). After being washed twice with pbs - t, the membranes were incubated with goat anti - rage antibody (n-16) or mouse anti--actin antibody (santa cruz, santa cruz, ca, usa) for 1.5 h. subsequently, the membranes were washed twice with pbs - t and incubated with anti - goat igg antibody (santa cruz) or anti - mouse ig's antibody (biosource, camarillo, ca, usa) and anti - biotin antibody (cell signaling) for 1 h. after being washed a further two times with pbs - t, the immunoreactive proteins were detected with ecl plus western blotting detection reagents (ge healthcare) using a luminescent image analyzer (las-1000uvmini; fujifilm, tokyo, japan). The density of the bands was analyzed using a multi gauge version 3.0 (fujifilm). The cells (710 cells / cm) were seeded in various plates or culture dishes (bd biosciences, franklin lakes, nj, usa) and incubated for 24 h. the control unglycated bsa and glycer - ages treatments were carried out in 0.1% fbs / dmem for 48 and 72 h, and cell viability was determined by the wst-1 assay . After removing the medium, 100 l / well of 10% fbs / dmem and 10 l / well of wst-1 solution (5 mm wst-1, 0.2 mm 1-methoxy-5-methylphenazinium, and 20 mm hepes buffer (ph 7.4)) (dojindo) were added, and the cells were incubated for 2 h. absorbance was measured at 450 nm and 650 nm using a microplate reader (labsystems multiskan ascent, model no . 354; thermo fisher scientific, kanagawa, japan), and the net difference (a450 a650) was used to express cell viability . The migration and invasion capacity were evaluated in 24-well transwell chambers and biocoat matrigel invasion chambers (bd biosciences), respectively . In both assays, the upper and lower culture compartments were separated by polyethylene terephthalate filters with a pore size of 8 m . Ten percent fbs was used as a chemoattractant in the lower chamber compartments . Before starting the invasion assay, the matrigel matrix of the chambers was reconstituted by adding serum - free dmem for 2 h. in the migration assay, 1 10 cells in serum - free dmem with control unglycated bsa or glycer - ages (100 g / ml) were added into each of the upper chambers for 20 h. in the invasion assay, 2 10 cells in serum - free dmem with control unglycated bsa or glycer - ages were added to each of the upper chambers for 48 h. then, the nonmigrating or noninvading cells on the upper side of the chamber membranes were removed using cotton swabs . The cells that migrated to or invaded the opposite side of the chamber were counted . The active form of rac1 was detected with the ez - detect rac1 activation kit (pierce, rockford, il, usa). This assay uses the ability of a glutathione s - transferase (gst) fusion protein corresponding to the p21-binding domain (pbd) of human p21 activated protein kinase 1 (pak1) to specifically bind to the active gtp - bound and not the inactive gdp - bound forms of rac1 and cdc42 . Cell lysates were incubated with gst - pak1-pbd and swellgel immobilized glutathione discs for 1 h at 4c, leaving an aliquot for measuring the levels of total rac1 . The bound proteins were eluted in 2 sds sample buffer containing 5% 2-me for 5 min at 95c and characterized by western blotting using monoclonal anti - rac1 antibody . Rac1 activity was determined by densitometric quantification of the pulled - down rac1-gtp level and normalizing it to the level of total rac1 detected in the sample lysates . Total rna was isolated from the cells with isogen (nippon gene, tokyo, japan), and 50 ng of rna were reverse transcribed into cdna with primescript rt reagent kit (takara, shiga, japan) using a geneamp pcr system 9700 (perkin - elmer applied biosystems, foster city, ca, usa). Real - time pcr was performed with sybr premix ex taq using a smart cycler ii system (takara). The reaction mixture (25 l) contained 1 sybr premix ex taq, 0.2 m pcr forward primers, 0.2 m pcr reverse primers, and 10 ng of cdna as a template . The primers used were as follows: tgf-: 5- gcg tgc taa tgg tgg aaa cc -3 and 5- cgg agc tct gat gtg ttg aag a -3, mmp-2: 5-agt ctg aag agc gtg aag-3 and 5- cca ggt agg agt gag aat g -3, and -actin: 5- tcc acc tcc agc aga tgt gg -3and 5- gca ttt gcg gtg gac gat -3. all processes were performed according to the manufacturer's instructions . -actin was used as an endogenous control gene in order to normalize target gene expression values . The cells were seeded and incubated for 24 h. the control unglycated bsa and glycer - ages treatments were carried out in serum - free dmem for 48 h. the conditioned medium was collected and used with a mmp-2 biotrak activity assay system (ge healthcare). All experiments were performed in duplicate and repeated at least two to three times, with each experiment yielding essentially identical results . The significance of differences between group means was determined by one - way analysis of variance . To investigate whether rage proteins are present in human lung adenocarcinoma a549 cells, we carried out western blot analysis using anti - rage antibody (n-16). Rage proteins of different molecular weights were detected in a549 cells (figure 1). In full length rage cdna - transfected human hepatocellular carcinoma hep3b cells, the major band (57 kda) (indicated by an arrow in figure 1) represents the full - length rage protein . Likewise, the full - length rage protein was also detected in a549 and mock transfected hep3b cells . No bands were detected in a neutralization experiment using blocking peptide (data not shown) so the other bands may represent deglycosylated rage proteins . We examined the effect of glycer - ages on the cell viability of a549 cells . Cell viability was determined after 48 h incubation with control unglycated bsa or glycer - ages . Twenty, 50, and 100 g / ml of glycer - ages significantly decreased cell viability to 83, 64, and 54%, respectively, (figure 2(a)). In contrast, control unglycated bsa had no effect . Furthermore, glycer - ages significantly attenuated cell proliferation (figure 2(b)); however, no cell death was induced by glycer - ages according to cell death detection elisa (data not shown). We examined the effect of glycer - ages on cell migration and invasion . In the migration assay, the glycer - ages - treated cells showed significantly (2.7 times higher) greater migration than the control unglycated bsa - treated cells (figure 3(a)), and in the invasion assay, the glycer - ages - treated cells showed significantly (3.3 times higher) greater invasion than the control unglycated bsa - treated cells (figure 3(b)). When glycer - ages were added to the lower chamber, there was no effect on the results of the invasion assay (data not shown). These results suggest that glycer - ages - rage interaction is necessary for cell migration and invasion . Rho family small gtpases are widely accepted to be key regulators of actin reorganization and motility . Interestingly, rage signaling has been shown to result in the activation of rac1 [1720], and the activation of rage by ages has been shown to induce the generation of reactive oxygen species (ros). The glycer - ages - induced rac1 activity was about 2 times higher than that of the control unglycated bsa, and it was suppressed by pretreatment with n - acetyl - l - cysteine (nac), an ros scavenger (figure 4(a)). Likewise, the glycer - ages - enhanced migration capacity was also significantly suppressed by pre - treatment with nac (figure 4(b)). Thus, glycer - ages - rage interaction enhances migration capacity by activating rac1 via ros generation . Members of the matrix metalloproteinase (mmp) family are widely accepted to be key regulators of tumor invasion [2224]. In particular, mmp-2 and -9, which are called gelatinases, are key enzymes for degrading type iv collagen and are thought to play critical roles in tumor invasion and metastasis . We examined the expression of transforming growth factor- (tgf-) and mmp-2 mrna and assessed mmp-2 activity with the mmp-2 biotrak activity assay system . Neither the mrna expression of tgf- or mmp-2 was significantly increased by the addition of glycer - ages at 24 h (figures 5(a) and 5(b)). Pro - mmp-2 activity was also not significantly altered by the addition of glycer - ages at 48 h (figure 5(c)). However, glycer - ages only increased mmp-2 (the activated form) activity to 120% (figure 5(d)). Rage, which is a multiligand receptor, affects diseases such as cancer and diabetes through its ligands . Several reports have suggested that rage and amphoterin are closely associated with invasion and metastasis in cancer cells [26, 27]. Ages have been implicated in the development and progression of diabetic angiopathies, including skin dermopathy . However, it is still unclear which ages subtypes play a pathogenetic role and which ages receptors mediate the effects of ages on cells . We have provided direct immunochemical evidence for the existence of six distinct ages structures within the ages - modified proteins and peptides that circulate in the sera of diabetic patients . Recently, we found that glycer - ages perform a diverse range of biological activities on vascular wall cells, mesangial cells, schwann cells, and cortical neurons [1013]. We also found that glycer - ages, but not glucose - derived ages (glc - ages), significantly stimulated the growth and migration of human melanoma cells . Furthermore, the tumor formation of melanoma cells xenografts in athymic mice was prevented by treatment with anti - rage antibody . In tumor bearing - mice, survival rates were prolonged, and spontaneous pulmonary metastases were inhibited by treatment using anti - rage antibody . In addition, glycer - ages were present in the beds of human melanoma tumors, whereas they were hardly detected in normal skin . These results suggest that glycer - ages are involved in the growth and invasion of malignant melanoma through their interactions with rage . Recently, we found that glycer - ages have the strongest binding affinity for rage [28, 29] and subsequently elicit oxidative stress generation and evoke vascular inflammation, thereby implicating them in accelerated atherosclerosis in diabetes [10, 11]. Taken together, glycer - ages - rage interactions play an important role in the progression of melanoma cells to malignancy . However, the effects of glycer - ages on other cancer cells remain poorly understood, and the molecular mechanisms behind these effects have not been clarified . In the present study rage includes full - length, c - truncated, and n - truncated rage . The rage antibody (n-16) used in this study, which recognizes the n - terminus of rage, detects full - length and c - truncated rage . Indeed, full - length rage (57 kda) was detected in a549 cells . The other bands may have represented the de - glycosylated form or c - truncated rage . Glycer - ages are associated with cell cytotoxicity, but glc - ages are not [21, 31, 32]. In mesangial cells, intracellular ros produced by glycer - ages were found to induce apoptotic cell death . However, no a549 cell death induced by glycer - ages was observed in this study . It is thought that the cytotoxicity of glycer - ages depends on the sensitivity of their target cells to ros . Furthermore, glycer - ages enhanced the migration and invasion activity of the a549 cells, both of which are prominent features of cancer malignancy . Glycer - ages induced the invasion of a549 cells across matrigel, indicating that matrix degradation and migration mechanisms had been stimulated in these cells . At the molecular level, glycer - ages - induced phenomena resemble the effects of long - term oxidative stress or tgf-. Both oxidative stress and tgf- are key regulators of the malignancy rather than the proliferation of cancer cells [3335]. Tgf- strongly induces mmp-2 expression in a549 cells; however, mrna expression of tgf- was not induced by glycer - ages, and the mrna levels of mmp-2, which is produced by tgf-, did not change . In long - term oxidative stress, mori et al . Showed that intracellularly produced ros activated rac1 and enhanced the invasion capacity of tumor cells by activating mmp [33, 3739]. A recent report showed that sustained exposure of cells to h2o2, but not one time exposure to h2o2, increased pro - mmp-2 activation through the induction of membrane type 1-mmp (mt1-mmp) expression . Indeed, our results also showed that glycer - ages enhanced the migration capacity of a549 cells by activating rac1 via ros and increased their invasion capacity by activating mmp-2 . However, glycer - ages only increased mmp-2 (the activated form) activity to 120% . It is suggested that the activation of other mmp such as mmp-13 may also participate in the above - mentioned changes, and future studies are necessary to clarify this matter . In conclusion, we demonstrated that glycer - ages enhanced the migration and invasion of a549 cells rather than their proliferation . These results suggest that glycer - ages play a critical role in cancer malignancy and are potential targets for therapeutic intervention.
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Malaligned implants often complicate the clinical laboratory procedures employed for fabrication of superstructures . Due to improper load distribution, a stent is an appliance used for radiographic evaluation of height and width of the available bone during treatment planning for implant placement or during surgical procedures to provide site for optimum implant placement . The variable type of stents includes vaccuform or acrylic resin templates adapted over duplicated casts of diagnostic wax - up . These include plastic or metal tubes and channels in acrylic resin that dictate the position of implants . Stents should be made of transparent material, stable and rigid when in position, should cover enough teeth to stabilize its position, and when no teeth are present, stents should extend onto unreflected soft tissue regions . With the use of computed tomography (ct) and computer added designing (cad)/ computer added machining (cam) technology, it is now possible to construct a surgical implant stent that would allow the clinician to predetermine implant locations virtually and surgically place them without reflecting a tissue flap. [58] but the surgical stents prepared with ct and cad / cam technology are very much more cost effective than a conventional surgical stent and require enough time and special center and/or laboratories for their fabrication and later on their correction, if required . In this study, we used the conventional surgical stents to evaluate their efficacy in determining the position and diameter of implants . The aim of the study was to demonstrate the accuracy and clinical precision of conventional surgical stent protocol for optimum installation of dental implants in terms of position and diameter . The aim of the study was to demonstrate the accuracy and clinical precision of conventional surgical stent protocol for optimum installation of dental implants in terms of position and diameter . The study was conducted in the out - patient department of prosthodontics and dental material sciences in collaboration with department of oral and maxillofacial surgery, faculty of dental sciences, csmmu, lucknow . Patients were treated in this study with the conventional surgical stents; all the implants were placed to the desired depth as planned in virtual implant planning . All the patients (45 patients for whom 89 implants were placed at different sites) [figures 1, 2] visiting the department of prosthodontics and oral and maxillofacial surgery for implant supported prosthesis placement and who followed the standard inclusion criteria for placement of implants were included in the study . First of all, diagnostic cast of the patients was made and acrylic teeth / tooth were waxed - up [figure 3]. Then, the impression of that waxed - up cast was taken and poured to reproduce another cast with the missing teeth / tooth replaced by dental stone . Then, mesiodistal and buccolingual markings were drawn over these teeth / tooth to find a center point on the occlusal surface [figure 4]. Later on, after applying the separating media over these regions and filling the undercuts with wax, transparent self - curing material was applied at the teeth to be replaced along with some adjacent area antero - posteriorly, which confers the stability to the stent during implant placement [figure 5]. The holes were drilled in the stent so formed at the center point for the assessment of the diameter and position of implant and filled with gutta percha [figure 6]. This stent was placed in the mouth and radiograph was taken to evaluate the position of gutta percha . The efficacy of stents was then evaluated at the time of surgical exposure for implant placement and after the surgery, with the help of radiographs [figures 7, 8]. Intraoral view of the patient radiograph of the patients waxed - up missing teeth missing teeth replaced by dental stone in the cast with markings stent made of transparent material stent in place with gutta percha filled in holes post - operative radiograph philip j. described a technique to construct a surgical guide on mounted diagnostic casts . He also used these mounted casts to determine whether sufficient space exists for a fixed cantilevered implant prosthesis . Murat c cehreli suggested the fabrication of a bilaminar dual - purpose stent that facilitates implant placement with improved verification of implant positioning . The outer lamina is designed for use in the computed tomography (ct) evaluation using radiopaque markers . Yen - chen ku presented a simple method of fabricating a vacuum - formed matrix filled with clear acrylic resin and a gutta - percha marker . The matrix can be used as not only a radiopaque marker for evaluation but also a surgical guide during the surgical stage for single implant therapy . Roger a. solow showed that a radiographic - surgical template can facilitate consultation with a surgeon and patient, when implant - supported restorations are planned . A template that provides radiographic evaluation of the implant site and precise or modified surgical placement kivanc akea described a modified surgical stent that serves as a guide to proper mesiodistal paralleling of dental implants . He also used these mounted casts to determine whether sufficient space exists for a fixed cantilevered implant prosthesis . Murat c cehreli suggested the fabrication of a bilaminar dual - purpose stent that facilitates implant placement with improved verification of implant positioning . The outer lamina is designed for use in the computed tomography (ct) evaluation using radiopaque markers . Yen - chen ku presented a simple method of fabricating a vacuum - formed matrix filled with clear acrylic resin and a gutta - percha marker . The matrix can be used as not only a radiopaque marker for evaluation but also a surgical guide during the surgical stage for single implant therapy . Roger a. solow showed that a radiographic - surgical template can facilitate consultation with a surgeon and patient, when implant - supported restorations are planned . A template that provides radiographic evaluation of the implant site and precise or modified surgical placement kivanc akea described a modified surgical stent that serves as a guide to proper mesiodistal paralleling of dental implants . The foremost advantage of the stent used in this study is its surgical ease, simplicity, precise accuracy and low cost . It can be fabricated with minimum laboratory procedures which are used in routine dental practice . On the other hand, the fixed type screw retained implant stents which are fabricated with the help of ct and cad / cam technology are very costly and require more time and laboratory procedures . The stents fabricated with the radiopaque markers provide radiographic as well as clinical ease for optimum implant installation . Along with all these advantages the drill holes can directly be made through the stent, so the implant surgery may become less traumatic (with the preservation of soft tissue including the gingival margins of adjacent teeth and the interdental papilla) with decreased operative time which results in accelerated post - surgical healing, fewer post - operative complications and increased comfort and satisfaction for the patient . The possibility of displacement of stent during implant surgery was minimized by extension of the stent antero - posteriorly to cover enough area over the teeth / tooth present adjacent to edentulous area . In cases where no teeth are present, stabilization of stent was achieved by extending it over the unreflected areas like retromolar regions . This study shows the extreme accuracy of this conventional surgical stent . If each step of this protocol is followed precisely, it is possible to deliver an optimum implant installation in terms of position and diameter and later on function at a very much reduced rate and time, as taken in ct and cad / cam technology which are the most accepted methods
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Type 1 diabetes (t1d) is characterized by autoimmune destruction of pancreatic -cells, primarily mediated by t lymphocytes, resulting in insulin deficiency and lifelong exogenous insulin dependence (1). Latent autoimmune diabetes in adults (lada) is a subtype of autoimmune diabetes, but the progression of autoimmune -cell destruction is slow; therefore, patients with lada are not insulin dependent at the initial phase (at least 6 months) of disease onset . Clinical manifestation of lada shares the features of classical t1d and type 2 diabetes (t2d) (2). T1d, lada, and t2d present a spectrum of diabetes . They range from patients with classic childhood t1d characterized by autoimmune - mediated destruction of -cells with the lowest c - peptide levels to age - related exacerbation of glucose tolerance with the highest bmi seen in t2d (3). Furthermore, patients with lada have susceptibility genes for both t1d and t2d (4), suggesting that lada lies between t1d and t2d . B cells infiltrate the islets of young nod mice, the commonly used mouse model for human t1d, and play a role in the initiation of -cell destruction by the autoimmune response (5). Studies have shown that marginal zone b (mzb) cells and follicular b (fob) cells can effectively activate cd4 t cells and facilitate their proliferation by serving as important antigen - presenting cells, especially as antigen - specific antigen - presenting cells (6,7). B - cell targeted therapy has been shown to delay the fall of c - peptide levels in patients with recent - onset t1d (8). Clinical trials have identified b cells as a possible therapeutic target for the prevention and reversal of t1d . B - cell targeted therapy also has been successfully used in treating other autoimmune diseases, including rheumatoid arthritis (9), systemic lupus erythematosus (10), and multiple sclerosis (11). Moreover, studies have shown that b cells are important in regulating both innate and adaptive immunity (12). A subset of b cells expressing cd19cd5cd1d, which have a regulatory function in infection, inflammation, and autoimmune disease, recently has been identified . These regulatory b cells can regulate t - cell responses and inhibit inflammation in an interleukin-10 (il-10)dependent manner (1316). The maturation and expansion of b10 cells require il-21 and cd40 signals both in vivo and ex vivo (17,18). Considerable evidence shows that patients with autoimmune diabetes have altered frequencies of circulating immune cells, including t cells (19), dendritic cells (20), natural killer cells (21,22), and neutrophils (23). Although studies have reported no significant change in total b cells in peripheral blood of patients with t1d (21,24), little is known about b - cell subpopulations with distinct immune functions that may play a role in the diabetes spectrum of t1d, lada, and t2d . We hypothesized that an altered phenotype of b - cell subsets is associated with autoimmune diabetes . This study investigated a change of peripheral b - cell subsets in patients with t1d, lada, and t2d compared with healthy control subjects . The results show altered frequencies of various b - cell subsets associated with autoimmune diabetes . Two hundred fifty - eight patients with t1d (n = 81, mean diabetes duration 3.1 3.5 years), lada (n = 82, 6.1 5.9 years), or t2d (n = 95, 3.0 3.7 years) and 218 control subjects with normal glucose tolerance (ngt) (table 1) were enrolled in the current study from the second xiangya hospital of central south university (changsha, hunan, china). Diabetes was diagnosed according to world health organization (who) criteria (25). Diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy; at least one positive islet autoantibody (gad antibody [gada], insulinoma - associated protein-2 antibody [ia-2a], or zinc transporter 8 antibody [znt8a]); or impaired c - peptide secretion . Lada inclusion criteria were 1) any positive islet autoantibody (gada, ia-2a, znt8a), 2) age 30 years at onset of diabetes, 3) insulin independence for at least 6 months after diagnosis, and 4) no ketosis or ketoacidosis (2,26). Diagnostic criteria of t2d were 1) typical history of hyperglycemia according to who criteria, 2) negative for islet autoantibodies, and 3) not requiring immediate insulin treatment . Healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia (fasting plasma glucose [fpg] <6.1 mmol / l, 2-h plasma glucose <7.8 exclusion criteria for control subjects were secondary diabetes; inflammation, infectious disease, or other autoimmune disease; history of immunosuppressive medication or steroids for> 7 days; pregnancy; and malignant disease . This study was approved by the ethical committee of the second xiangya hospital of central south university, and all subjects provided written informed consent for the study protocol . Anthropometric and metabolic data of participants data are% (n), mean sd, and median (25th75th percentile). Compared after log transformation . * p <0.05 compared with ngt . * * p <0.01 compared with ngt . * * * p <0.001 compared with ngt . P <0.05 compared with t2d . P <0.01 compared with t2d . P <0.001 compared with t2d . P <0.001 compared with t1d . Study physicians recorded body height and weight, waist circumference, hip circumference, and blood pressure . Fasting venous blood samples were tested for triglycerides (tgs), total cholesterol (tc), hdl cholesterol (hdl - c), ldl cholesterol (ldl - c), fpg, hemoglobin a1c (hba1c), and fasting c - peptide (fcp). Hba1c was measured by automated liquid chromatography (variant ii hemoglobin testing system; bio - rad laboratories, hercules, ca). Fcp was measured by a chemiluminescence method (advia centaur; siemens, munich, germany). Gada, ia-2a, and znt8a were measured by radioligand assays as previously described (27,28). The cutoff indices of positivity for gada, ia-2a, and znt8a were 18 units / ml (who units), 3.3 units / ml, and 0.011 (znt8a index), respectively, which were determined as the 99th percentile of 405 healthy control subjects . The sensitivity of the current gada, ia-2a, and znt8a assays was 78%, 74%, and 70%, respectively, and the specificity was 96.7%, 96.7%, and 98.9%, respectively, in the islet autoantibody standardization program (iasp2012). Fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies (allophycocyanin - cy7-cd19 [hib19], fluorescein isothiocyanate - cd5 [ucht2], allophycocyanin - cd1d [51.1], peridinin chlorophyll protein - cy5.5-cd23 [ebvcs-5], and phycoerythrin - cy7-cd21 [lt21]; biolegend, san diego, ca) to determine the percentage of b - cell subsets according to the manufacturer s protocol . The stained cells were analyzed with a bd facscanto ii system, which was calibrated daily with appropriate single fluorochrome - stained samples . Fifty thousand lymphocytes were collected in a forward scatter / side scatter (fsc - a / ssc - a) lymphocyte gate and analyzed with flowjo version 7.6 software (tree star, inc ., dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining . 1 . The following antibodies were used to identify b - cell subsets: cd19 (pan b marker), cd19cd23cd21 (mzb cells), cd19cd23cd21 (fob cells), cd19cd23cd21 (transitional 2-marginal zone precursor [t2-mzp] b cells) (29), and cd19cd5cd1d (b10 cells). The intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15%, respectively . The initial cd19 gate was derived from a lymphocyte gate (defined on fsc and ssc) followed by single - cell discrimination . B: representative dot plot showing the gating strategy for mzb, fob, and t2-mzp b cells gated on cd19 b cells . C: representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells . Anova was performed to compare groups after adjustment for potentially confounding variables, including age, sex, and bmi . All the significant differences between groups are the results of adjusted analyses unless stated otherwise . Associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations . For the statistical analysis, we used spss version 19.0 (ibm corporation, chicago, il) and graphpad prism 5 (graphpad software, san diego, ca) software . Two hundred fifty - eight patients with t1d (n = 81, mean diabetes duration 3.1 3.5 years), lada (n = 82, 6.1 5.9 years), or t2d (n = 95, 3.0 3.7 years) and 218 control subjects with normal glucose tolerance (ngt) (table 1) were enrolled in the current study from the second xiangya hospital of central south university (changsha, hunan, china). Diabetes was diagnosed according to world health organization (who) criteria (25). Diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy; at least one positive islet autoantibody (gad antibody [gada], insulinoma - associated protein-2 antibody [ia-2a], or zinc transporter 8 antibody [znt8a]); or impaired c - peptide secretion . Lada inclusion criteria were 1) any positive islet autoantibody (gada, ia-2a, znt8a), 2) age 30 years at onset of diabetes, 3) insulin independence for at least 6 months after diagnosis, and 4) no ketosis or ketoacidosis (2,26). Diagnostic criteria of t2d were 1) typical history of hyperglycemia according to who criteria, 2) negative for islet autoantibodies, and 3) not requiring immediate insulin treatment . Healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia (fasting plasma glucose [fpg] <6.1 mmol / l, 2-h plasma glucose <7.8 exclusion criteria for control subjects were secondary diabetes; inflammation, infectious disease, or other autoimmune disease; history of immunosuppressive medication or steroids for> 7 days; pregnancy; and malignant disease . This study was approved by the ethical committee of the second xiangya hospital of central south university, and all subjects provided written informed consent for the study protocol . Anthropometric and metabolic data of participants data are% (n), mean sd, and median (25th75th percentile). Compared after log transformation . * p <0.05 compared with ngt . * * p <0.01 compared with ngt . * * * p <0.001 compared with ngt . P <0.05 compared with t2d . P <0.01 compared with t2d . P <0.001 compared with t2d . P <0.001 compared with t1d . Study physicians recorded body height and weight, waist circumference, hip circumference, and blood pressure . Fasting venous blood samples were tested for triglycerides (tgs), total cholesterol (tc), hdl cholesterol (hdl - c), ldl cholesterol (ldl - c), fpg, hemoglobin a1c (hba1c), and fasting c - peptide (fcp). Hba1c was measured by automated liquid chromatography (variant ii hemoglobin testing system; bio - rad laboratories, hercules, ca). Fcp was measured by a chemiluminescence method (advia centaur; siemens, munich, germany). Gada, ia-2a, and znt8a were measured by radioligand assays as previously described (27,28). The cutoff indices of positivity for gada, ia-2a, and znt8a were 18 units / ml (who units), 3.3 units / ml, and 0.011 (znt8a index), respectively, which were determined as the 99th percentile of 405 healthy control subjects . The positive samples were tested twice for confirmation . The sensitivity of the current gada, ia-2a, and znt8a assays was 78%, 74%, and 70%, respectively, and the specificity was 96.7%, 96.7%, and 98.9%, respectively, in the islet autoantibody standardization program (iasp2012). Fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies (allophycocyanin - cy7-cd19 [hib19], fluorescein isothiocyanate - cd5 [ucht2], allophycocyanin - cd1d [51.1], peridinin chlorophyll protein - cy5.5-cd23 [ebvcs-5], and phycoerythrin - cy7-cd21 [lt21]; biolegend, san diego, ca) to determine the percentage of b - cell subsets according to the manufacturer s protocol . The stained cells were analyzed with a bd facscanto ii system, which was calibrated daily with appropriate single fluorochrome - stained samples . Fifty thousand lymphocytes were collected in a forward scatter / side scatter (fsc - a / ssc - a) lymphocyte gate and analyzed with flowjo version 7.6 software (tree star, inc ., dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining . 1 . The following antibodies were used to identify b - cell subsets: cd19 (pan b marker), cd19cd23cd21 (mzb cells), cd19cd23cd21 (fob cells), cd19cd23cd21 (transitional 2-marginal zone precursor [t2-mzp] b cells) (29), and cd19cd5cd1d (b10 cells). The intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15%, respectively . The initial cd19 gate was derived from a lymphocyte gate (defined on fsc and ssc) followed by single - cell discrimination . B: representative dot plot showing the gating strategy for mzb, fob, and t2-mzp b cells gated on cd19 b cells . C: representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells . Anova was performed to compare groups after adjustment for potentially confounding variables, including age, sex, and bmi . All the significant differences between groups are the results of adjusted analyses unless stated otherwise . Associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations . For the statistical analysis, we used spss version 19.0 (ibm corporation, chicago, il) and graphpad prism 5 (graphpad software, san diego, ca) software . The control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d . The percentage of males in the lada group was higher than in the ngt group . The bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group . Patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . Patients with t2d had distinctive tg, tc, ldl - c, and hdl - c levels compared with the other groups . The fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt . There was a significant decrease of fcp in the lada and t1d (lowest) groups compared with the t2d and ngt groups . We did not find any significant changes in the frequency of cd19 b cells among the patient groups, regardless of age, sex, and bmi, compared with the ngt group (p> 0.05) (supplementary fig . The frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups (t1d vs. ngt, t1d vs. t2d, lada vs. t2d p <0.001; lada vs. ngt p <0.01) (fig . We found that mzb cells were more frequent in the t1d group than in the lada group, but the differences were diminished after adjustment for age, sex, and bmi . Fob cells were less frequent in the t1d group compared with the other three groups; however, the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age, sex, and bmi compared with the ngt and t2d groups (t1d vs. ngt, p <0.001; t1d vs. t2d, lada vs. ngt, p <0.01; lada vs. t2d, p <0.05) (fig . 2b). We also assessed the percentages of t2-mzp b cells, which were reported to have a regulatory function (29), and found these to be increased in the lada group compared with the ngt and t1d groups (lada vs. ngt p <0.001; t1d vs. lada p <0.01) (fig . 2c), whereas they were more frequent in the t2d group than in the ngt group (t2d vs. ngt p <0.001) (fig . The frequency of mzb (a), fob (b), t2-mzp b (c), and b10 (d) cells gated on cd19 b cells . P values refer to comparison of data after adjustment for age, sex, and bmi . * p <0.05, * * p <0.01, * * * p <0.001 . Of note, regardless of the adjustment for age, sex, and bmi, the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt, but these were lowest in the t1d group (t1d vs. ngt p <0.001; t2d vs. ngt, lada vs. ngt p <0.05) (fig . B10 cells were higher in the t2d and lada groups than in the t1d group (p <0.001 for both) (fig . After matching for age and sex, these findings remained the same for the percentages of cd19, mzb, b10, and t2-mzp b cells after adjustment for bmi among groups (supplementary table 1 and supplementary fig . However, patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d . We analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1) mzb and fob cells were correlated with age, 2) t2-mzp b cells were associated with sex, and 3) b10 and mzb cells were correlated with bmi (table 2), regardless of the type of diabetes . Of note, fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells . However, fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . In terms of lipid profile, we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level . Correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies (after log transformation) and anthropometric and metabolic variables were performed using pearson test . In addition to the associations in all participants, we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . In patients with t1d, the proportion of cd19 b cells associated negatively with fcp (r = 0.248, p <0.05), and the mzb cells exhibited a negative correlation with age (r = 0.498, p <0.001), bmi (r = 0.364, p <0.01), and hba1c (r = 0.324, p <0.01). In patients with lada, cd19 b cells negatively correlated with bmi (r = 0.498, p <0.001) and fcp (r = 0.248, p <0.05), whereas fob cells showed a positive correlation with age (r = 0.236, p <0.05). Finally, in patients with t2d, the percentage of t2-mzp b cells was positively associated with fcp (r = 0.364, p <0.05). The control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d . The percentage of males in the lada group was higher than in the ngt group . The bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group . Patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . Patients with t2d had distinctive tg, tc, ldl - c, and hdl - c levels compared with the other groups . The fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt . There was a significant decrease of fcp in the lada and t1d (lowest) groups compared with the t2d and ngt groups . We did not find any significant changes in the frequency of cd19 b cells among the patient groups, regardless of age, sex, and bmi, compared with the ngt group (p> 0.05) (supplementary fig . The frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups (t1d vs. ngt, t1d vs. t2d, lada vs. t2d p <0.001; lada vs. ngt p <0.01) (fig . We found that mzb cells were more frequent in the t1d group than in the lada group, but the differences were diminished after adjustment for age, sex, and bmi . Fob cells were less frequent in the t1d group compared with the other three groups; however, the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age, sex, and bmi compared with the ngt and t2d groups (t1d vs. ngt, p <0.001; t1d vs. t2d, lada vs. ngt, p <0.01; lada vs. t2d, p <0.05) (fig . 2b). We also assessed the percentages of t2-mzp b cells, which were reported to have a regulatory function (29), and found these to be increased in the lada group compared with the ngt and t1d groups (lada vs. ngt p <0.001; t1d vs. lada p <0.01) (fig . 2c), whereas they were more frequent in the t2d group than in the ngt group (t2d vs. ngt p <0.001) (fig . The frequency of mzb (a), fob (b), t2-mzp b (c), and b10 (d) cells gated on cd19 b cells . P values refer to comparison of data after adjustment for age, sex, and bmi . * p <0.05, * * p <0.01, * * * p <0.001 . Of note, regardless of the adjustment for age, sex, and bmi, the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt, but these were lowest in the t1d group (t1d vs. ngt p <0.001; t2d vs. ngt, lada vs. ngt p <0.05) (fig . B10 cells were higher in the t2d and lada groups than in the t1d group (p <0.001 for both) (fig . After matching for age and sex, these findings remained the same for the percentages of cd19, mzb, b10, and t2-mzp b cells after adjustment for bmi among groups (supplementary table 1 and supplementary fig . However, patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d . We analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1) mzb and fob cells were correlated with age, 2) t2-mzp b cells were associated with sex, and 3) b10 and mzb cells were correlated with bmi (table 2), regardless of the type of diabetes . Of note, fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells . However, fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . In terms of lipid profile, we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level . Correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies (after log transformation) and anthropometric and metabolic variables were performed using pearson test . In addition to the associations in all participants, we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . In patients with t1d, the proportion of cd19 b cells associated negatively with fcp (r = 0.248, p <0.05), and the mzb cells exhibited a negative correlation with age (r = 0.498, p <0.001), bmi (r = 0.364, p <0.01), and hba1c (r = 0.324, p <0.01). In patients with lada, cd19 b cells negatively correlated with bmi (r = 0.498, p <0.001) and fcp (r = 0.248, p <0.05), whereas fob cells showed a positive correlation with age (r = 0.236, p <0.05). Finally, in patients with t2d, the percentage of t2-mzp b cells was positively associated with fcp (r = 0.364, p <0.05). Increasing evidence suggests that b cells are as important as t cells in the immunopathogenesis of autoimmune diabetes . Most, if not all, diabetogenic t cells are known to be t - helper 1 subsets, but little is known about b - cell subsets in autoimmune diabetes . We investigated the phenotype and frequency of various b - cell subsets in the peripheral blood across the clinical spectrum of diabetes . The major finding of this study was a distinct alteration of b - cell subsets across the diabetes spectrum and their association with clinical features . We found no significant difference in the frequency of cd19 b cells, which is in accordance with the published studies (21,30); however, we did not find a negative correlation between total b cells and age, as previously reported (24,31). The current study went further than the examination of the frequency of total b cells, focusing on b - cell subsets with distinct immunological functions . Of note, we found that patients with t1d or lada had an increased frequency of mzb cells but decreased frequency of fob cells compared with control subjects with ngt and patients with t2d . In all groups, we found that fcp was negatively associated with mzb cells but positively associated with fob cells . Attanavanich and kearney (6) reported that mzb cells were more effective at activating naive cd4 t cells than fob cells . Mzb cells in nod mice were also shown to be important antigen - presenting cells for the activation of t cells in pancreatic - draining lymph nodes (32). Given the negative correlation between mzb cells and fcp, it is possible that mzb cells may promote -cell destruction in patients with t1d or lada . Decreased frequency of fob cells in the circulation of patients with t1d and lada suggests that some fob cells migrated from the blood to the pancreas or the draining lymph nodes as a result of the local inflammatory response . Along with the disease progression, more immune cells are known to infiltrate to the islets of both nod mice and patients with t1d (5,33), but the patients with t1d in the current study have a relatively long history of diabetes (3.1 years). Thus, fob cells appear to be more important in initial rather than established pancreatic -cell autoimmunity (34). We showed that patients with t2d and lada more frequently had t2-mzp b cells than control subjects with ngt, which associates with parameters of blood pressure, lipidemia, and glycemia but not with hba1c or fcp . These observations are consistent with a previous study showing that glycemia and lipidemia are associated with immune cells (35). Another interesting finding was the decrease of b10 cell frequency in all types of diabetes, being the lowest in t1d . Whether this phenotype is mediated through a common mechanism or different ones is not clear . What is clear is that immune mechanisms are involved and that more studies, especially functional studies, are required . Although b cells are generally acknowledged for their function in humoral immune response by producing antibodies, they also have been demonstrated to play an immunoregulatory role in the prevention of autoimmune disease by producing il-10 and downregulating t - cell responses (13,14,36). The observed b10 cell reduction could contribute to the breakdown of self - tolerance that leads to -cell destruction in patients with t1d (24) or lada . Antigen - activated il-10producing b cells may selectively inhibit autoreactive t - cell responses to islet - specific antigen to maintain self - tolerance, and hyperglycemic nod mice and patients with t1d lack this population of regulatory b cells (37). In the clinical trial where b cells in patients with new - onset t1d were depleted, the patients showed a transient remission (8). However, blanket targeting of all b cells, as used in the clinical trial and in treating other autoimmune disorders, needs to be improved by selectively deleting pathogenic b cells while preserving regulatory b cells to promote tolerance . Our preclinical study showed that more regulatory b cells were regenerated after b - cell deletion in an animal model (38). Furthermore, studies have shown that patients with long - standing t2d could have a secondary autoimmune response toward islet -cells (39). The reduction of b10 regulatory cells might contribute to the dysregulation of immune response in t2d . A functional study on il-10producing b cells and further phenotyping will be the goal of future work . In the current study, although we currently cannot identify a specific subset that could be used for diagnostic purposes, the results strongly imply that the change of b - cell subtypes is related to the pathogenesis of autoimmune diabetes . Therefore, it is important to reconsider the approach of b - cell targeted therapy in patients with t1d and aim to selectively remove pathogenic b cells but promote regulatory b cells . In summary, the current data demonstrate distinct differences in the frequencies of peripheral b - cell subsets in t1d and lada, which are associated with islet function and glycemia . The findings support the notion that these immunological alterations are involved in loss of self - tolerance and -cell destruction . The findings also suggest that, similar to regulatory t - cell therapy, transfusion of ex vivo expanded autologous b10 cells might open a new and effective therapeutic strategy in treating autoimmune diabetes.
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Human papillomavirus (hpv), a virus from the papillomavirus family, is capable of infecting humans . About 200 different strains of hpv identified, based on dna homology, have been found to be etiologically linked to cervical, vaginal, vulvar, penile, anal, oral, and plantar infectious lesions and cancers, as well [1, 2]. The hpv genome, a double - stranded dna molecule, consists of 8 kilobase pairs (kbp) of nucleotides, which comprises 3 regions: 6 early open reading frames (orfs)-e1, e2, e4, e5, e6, and e7; 2 late orfs - l1 and l2; and an upstream regulatory region . A considerable volume of hpv specific information pertaining to its genome, proteome, structure, and disease association is available scattered on the web with trivial annotations; however, it is not customized to explore for data analysis, host - pathogen interaction, strain - disease association, drug designing, and sequence analysis, etc . Therefore, we proposed to develop a comprehensive reserve on hpv with maximum possible inputs and outputs for the end users . Amongst the existing 200 strains of hpv, 150 have been sequenced as of now, and their data available at the national center for biotechnology information (ncbi). Genome and proteome information of those viral strains was retrieved from ncbi . Besides pubmed, various other online resources and published literature were also explored for validating genomic, proteomic, as well as strain and disease - associated information on hpv strains . Hpv strain - specific information, such as strain name, sequencing status, sequencing centre, ncbi accession i d, associated disease information with references, genome statistics (gc%, at%, a, t, g, c count, genes, and proteins), etc ., were curated from various online resources, and protein parameters (length, molecular weight, isoelectric point) were calculated using expasy protparam . Modeller9v10 and the swiss - model server were used for protein structure prediction . The stereochemistry of each protein was evaluated through procheck analysis, available at the rcsb validation server (http://deposit.rcsb.org/validate/), and validated using prosa - web (http://prosa.services.came.sbg.ac.at/prosa.php). Human papillomavirus proteome database (hpvpdb), the relational reserve, was developed using microsoft sql server 2005 as the back end . Html, javascript, and cgi - perl - based web interfaces were employed to execute sql queries . The application layer, the web interface, and the backend relational tables were integrated using cgi - perl . Home, about, tools, search, and advanced search interfaces can be explored to obtain strain- and protein - specific information . Reserve comprises the strain - specific detailed informationon its name, sequencing status, submission details, date of submission, ncbi ids, disease types and subtypes, type of dna, genome length, molecular weight, nucleotide composition (a, t, g, c, at, gc content), number of genes and proteins, and protein list . A genome map of each strain obtained by geneious 5.4.4 software (available from http://www.geneious.com/) is also integrated in this page . Users, through an advanced search option, can precisely access the genome and proteome information separately by selecting hpv genome or hpv proteome . Each protein entry comprises protein overview (name, locus, function, etc . ), protein sequence information (amino acid sequences with ncbi accession number with provision for direct protein blast against ncbi nr database), protein parameters (length, molecular weight, theoretical isoelectric point [pi], amino acid composition, etc . ), protein structure (predicted 3d structure by homology modeling viewed by jmol (available from http://www.jmol.org/) with the java platform, ramachandran plot obtained by procheck and z - score and energy plot obtained by prosa - web . Hpvpdb platform also provides a phylogeny analysis tool to perform multiple sequence alignment and phylogenetic tree construction of selected hpv proteins using the phylogeny . The original human papillomaviruses database was developed and hosted by the los alamos national laboratory (lanl) between 1994 and 1999 with funding from the national institute of allergy and infectious diseases (niaid).' Human papillomaviruses: a compilation and analysis of nucleic acid and amino acid sequences' contains four annual data books of papillomavirus information published in both paper and electronic form (1994, 1995, 1996, and 1997) but has not been updated since 1997 . This contains nucleotide sequences of few hpv strains and other papillomaviruses, amino acid and nucleotide sequence alignments, analysis, related host sequences, and database communication . We did not find any structural information in that database . In hpvpdb, along with updated protein sequence information, genome and protein structure information is also provided . Amongst the existing 200 strains of hpv, 150 have been sequenced as of now, and their data available at the national center for biotechnology information (ncbi). Genome and proteome information of those viral strains was retrieved from ncbi . Besides pubmed, various other online resources and published literature were also explored for validating genomic, proteomic, as well as strain and disease - associated information on hpv strains . Hpv strain - specific information, such as strain name, sequencing status, sequencing centre, ncbi accession i d, associated disease information with references, genome statistics (gc%, at%, a, t, g, c count, genes, and proteins), etc ., were curated from various online resources, and protein parameters (length, molecular weight, isoelectric point) were calculated using expasy protparam . Modeller9v10 and the swiss - model server were used for protein structure prediction . The stereochemistry of each protein was evaluated through procheck analysis, available at the rcsb validation server (http://deposit.rcsb.org/validate/), and validated using prosa - web (http://prosa.services.came.sbg.ac.at/prosa.php). Human papillomavirus proteome database (hpvpdb), the relational reserve, was developed using microsoft sql server 2005 as the back end . Html, javascript, and cgi - perl - based web interfaces were employed to execute sql queries . The application layer, the web interface, and the backend relational tables were integrated using cgi - perl . Home, about, tools, search, and advanced search interfaces can be explored to obtain strain- and protein - specific information . Reserve comprises the strain - specific detailed informationon its name, sequencing status, submission details, date of submission, ncbi ids, disease types and subtypes, type of dna, genome length, molecular weight, nucleotide composition (a, t, g, c, at, gc content), number of genes and proteins, and protein list . A genome map of each strain obtained by geneious 5.4.4 software (available from http://www.geneious.com/) is also integrated in this page . Users, through an advanced search option, can precisely access the genome and proteome information separately by selecting hpv genome or hpv proteome . Each protein entry comprises protein overview (name, locus, function, etc . ), protein sequence information (amino acid sequences with ncbi accession number with provision for direct protein blast against ncbi nr database), protein parameters (length, molecular weight, theoretical isoelectric point [pi], amino acid composition, etc . ), protein structure (predicted 3d structure by homology modeling viewed by jmol (available from http://www.jmol.org/) with the java platform, ramachandran plot obtained by procheck and z - score and energy plot obtained by prosa - web . Hpvpdb platform also provides a phylogeny analysis tool to perform multiple sequence alignment and phylogenetic tree construction of selected hpv proteins using the phylogeny . The original human papillomaviruses database was developed and hosted by the los alamos national laboratory (lanl) between 1994 and 1999 with funding from the national institute of allergy and infectious diseases (niaid).' Human papillomaviruses: a compilation and analysis of nucleic acid and amino acid sequences' contains four annual data books of papillomavirus information published in both paper and electronic form (1994, 1995, 1996, and 1997) but has not been updated since 1997 . This contains nucleotide sequences of few hpv strains and other papillomaviruses, amino acid and nucleotide sequence alignments, analysis, related host sequences, and database communication . We did not find any structural information in that database . In hpvpdb, along with updated protein sequence information, genome and protein structure information is also provided . Hpvpdb brings together comprehensive information on a total of 1,036 protein sequences and 743 predicted structures . The outcome of this study might provide a platform for simultaneous structural comparative analysis of these proteins and help in finding out variations in their structures to explore why different strains of hpv have causative associations with different types of cancers . Further, this might also help in designing specific drugs or vaccines against specific strains of hpv . Currently the hpvpdb is updated manually through online resources and available scientific publication review; however, to sustain the quality, these data are analyzed and checked before incorporation into this reserve . Meanwhile, to provide regular updates, our team is committed to searching for newly sequenced hpv strains, updating this reserve, and serving the users.
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According to numerous estimations, 15% to 29% of patients with cataract have 1.5 diopters (d) of refractive astigmatism.1,2 cataract surgeons usually prefer to treat cataract and correct refractive (spherical and astigmatic) disorders at the time of the surgery . Corneal incision (astigmatic keratotomy) and peripheral corneal relaxing incisions (pcris) were performed to treat these disorders.3,4 the main drawbacks of these approaches are that the outcome depends on multiple factors as the patient s age, the depth and length of the incision, complications related to wound healing, epithelial defects, or induction of dry - eye symptoms . These parameters affect the visual outcome in a unpredictable way so the corneal incisions are not considered a reliable method for astigmatism correction . Toric intraocular lens (iols) implantation was introduced in the 1990 s as an option for astigmatism correction in cataract patients . Initially they presented the disadvantage of postoperative rotation that decreased the visual outcome.58 new toric iol designs (acrysof toric iol; alcon, fort worth, tx), approved by the us food and drug administration (fda) at the end of 2005, have been found to be more stable and appear to be the preferred iol for correcting preexisting astigmatism in conjunction with cataract surgery.9,10 the acrysof iols are available in three options: t3, t4, and t5 of astigmatic correction 1.5, 2.25, 3.00 d, respectively (at the iol plane). The aim of this study is to report the clinical results of acrysof toric iols implantation for preexisting astigmatism correction and compare the postoperative rotation of the iols one and six months postoperatively . This prospective study included eyes that had cataract surgery at the papageorgiou general hospital, thessaloniki, greece, between may 2008 and december 2008 . The study was conducted according to the tenets of the declaration of helsinki and patients gave informed consent after the nature and intent of the study had been fully explained to them . Inclusion criteria were: cataract, age 70 years or younger, and preoperative regular corneal astigmatism greater than 1.00 d. exclusion criteria were: glaucoma, corneal disease, previous corneal or intraocular surgery, macular degeneration or retinopathy, and history of ocular inflammation . Each patient had a complete ophthalmologic examination, including visual acuity (va), slit - lamp examination, intraocular pressure (iop) measurement, and dilated fundus examination . Topography (magellan mapper; nidek technologies, padova, italy), automated refractometry (rk600; reichert, inc ., depew, ny), and ultrasonic immersion biometry (ocuscan rxp; alcon) were performed to determine the appropriate iol spherical power . Cylindrical power and axis placement to achieve emmetropia were calculated using an online toric iol calculator program (available from http://www.acrysoforiccalculator.com/). The 0180 axis was marked with the patient in a sitting position to avoid cyclotorsion using the nuijts / lane preoperative toric reference corneal marker (ae-2791tbl; asico, westmont, il) (figure 1). Intraoperatively, the desired implantation axis was marked using an intra - op toric axis marker ii (ae-2794; asico) (figure 2). A foldable iol was implanted in the capsular bag through a 2.75 mm limbal incision on 110. the toric iol was injected with a monarch - ii injector (alcon) and placed around 1015 off axis before the ophthalmic viscosurgical device (sodium hyaluronate 1%) was removed . After ophthalmic viscosurgical device removal, the iol was rotated to its final position by exactly aligning the toric reference marks with the limbal implantation axis marks . All surgeries were performed by the same experienced surgeon (it) using topical anesthesia and a standard divide - and - conquer phacoemulsification technique . Because of the intraoperative marker design and the pen mark fading during the operation in several cases, it was difficult to assess the proper alignment of the iol after its placement . Because of this difficulty, an image was captured from the real - time streaming recording of the surgery and was assessed blindly by a second operator using a commercially available software (screen protractor; iconico, ny, usa) (unpublished data). To overcome this difficulty we consider that the use of the marker set consisting of beveled degree gauge (ae-1590; asico) and maloney astigmatism axis marker (ae-2741; asico) is more appropriate for the whole procedure because it allows maximum visibility . The on - screen assessment of the axis remains a useful tool for postoperative evaluation of the iol location (figure 3). Measurements of visual acuity (using the snellen opto - type at a six - meter distance), iop, and comprehensive slit lamp examination were performed at one - day and one- and six - month postoperative visits . At the one- and six - month follow - up, a digital photograph and corneal topography (only at the one - month visit) were obtained to estimate the postoperative rotation of the iol using the software tools mentioned above (figure 1). Outcomes of interest included uncorrected va, cylindrical astigmatism power before and after iol implantation, and the possible rotation of the iol one and six months after the operation (when the initial desired place was at 0). Absolute values of the rotation used for the analysis after detecting rotation was not under investigation . Completed data forms were analyzed with microsoft excel 2007 (microsoft corporation, redmond, wa) and spss software (version 16.0; spss inc ., chicago, il). Twenty - nine eyes of 19 patients (mean age 63.03 5.42 years) were enrolled in this study . Uncorrected va was found to be 5/10 or more in 26 of 29 eyes (89.7%) and 8/10 or more in 19 of 29 patients (65.5%). Preoperative and postoperative corneal topography showed significant reduction of refractive astigmatism in all eyes after the surgery . Mean power of the astigmatism was 2.38 0.91 d (range 1.55 d) preoperatively and 0.64 0.61 d (range 02.5 d) postoperatively (figure 4). One month postoperation, the mean toric iol axis rotation was 2.2 1.5 (range 0.67.8). Six months postoperation, the mean toric iol axis rotation was 2.7 1.5 (range 0.98.4). The later rotation occurred between one and six months, and was found to be more than one degree (1.1) only in one eye (3%) and in all other cases was less than this value (figure 5). It is known that one degree of deviation causes 3.3% reduction of the iol cylindrical power . Calculated reduction of the desired correction (in diopters) because of the observed rotation (mean value, one and two standard deviations) for the three iol models is shown in table 1 . No eye had secondary surgery to reposition the iol axis within the six - month postoperative period . The results of our study corroborate previous studies,11 which demonstrates that proper selection and preoperative examination of patients followed by uncomplicated iol implantation of one - piece hydrophobic acrylic toric iols results in acceptable stability and visual outcome . In particular, the postoperative rotation of toric iols appears to occur in the early postoperative period (1 month) and remains constant later than six months . The lens rotates until a fibronectin and collagen adhesion develops between the iol and the posterior capsule, which prevents any further rotary motion.12 the amount of cylindrical correction reduction due to postoperative rotation was not large enough13 to affect the expected end result in final uncorrected distance va . Acrysof one - piece hydrophobic acrylic toric iols implantation shows satisfactory stability, acceptable clinical results, and is an exceptional option for correction of refractive astigmatism.
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Song is a complex, learned motor skill that involves the precise coordination of vocal and respiratory musculature in order to produce highly stereotyped renditions of a memorized song model . Two pathways contributed to this behavior are identified in the songbird forebrain: the motor pathway that is required throughout life for normal song production, and the anterior forebrain pathway (afp) which is necessary for song learning and plasticity [13]. Hvc (used as the proper name) is the beginning nucleus of both pathways and projects to the robust nucleus of the arcopallium (ra) and afp . The motor pathway is from hvc to ra, and then ra projects to brainstem nuclei controlling the vocal and respiratory muscles . During song, ra neurons in adult birds generate a highly stereotyped sequence of bursts [5, 6] and also receive input from lateral magnocellular nucleus of the anterior nidopallium (lman), the output nucleus of afp . Notably, recordings from lman neurons projecting to ra revealed highly variable spiking activity across song renditions, suggesting that lman may act as a source of variability . These results indicate that synaptic activity in lman - ra synaptic transmission is crucial to song plasticity [8, 9], but the detailed mechanism is unclear . It has been observed that ra received excitatory glutamatergic inputs from hvc and lman . The former is mediated by a mixture of n - methyl - d - aspartate (nmda) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid- (ampa-) type receptors [4, 10, 11], and the latter is mediated almost exclusively by nmda - type receptors . Lman - ra synaptic transmission is necessary for the variability of song in adult zebra finch, suggesting that the activity of nmda receptor on the lman - ra synapses might induce the activity pattern change of ra neurons and change the bursts that control the vocal and respiratory muscles . The control of gain, which refers to modulation of a neuron's responsiveness to input, is critically important for normal sensory, cognitive, and motor functions [1215]. Gain control is achieved through a divisive process and is observed as a change in the slope of the input - output relationship . Such gain changes are frequently observed in the responses of cortical neurons and are thought to play an important role in neural computations . Modulations of gain have been observed in the enhancement of neural responses by attention [12, 16] and can also be induced pharmacologically . In a neuron model, nmda receptors caused such membrane potential fluctuations between a hyperpolarized down - state and a depolarized up - state, which may gate the information and gain modulation . Here, we test the gain change of ra projection neurons after exogenous nmda was applied to selectively activate nmda receptors, using whole - cell current clamp recording in adult male zebra finch slices . Brain slices were prepared from adult male zebra finches (taeniopygia guttata) (> 90 days old) obtained from commercial suppliers as previously described [19, 20]. Brains were dissected into ice - cold, oxygenated (95% o2 and 5% co2) slice solution . Slice solution consists of (in mm) sucrose 248, kcl 5, nahco3 28, glucose 10, mgso47h2o 1.3, and nah2po4h2o 1.26 [21, 22]. Coronal brain slices (250 m thick) containing ra were cut with a vibrating microtome (ma752, usa) and collected in artificial cerebrospinal fluid (acsf) that had been warmed to 37c and subsequently allowed to cool to room temperature . Slices were allowed to recover in the holding chamber for at least 1.5 h and were equilibrated to room temperature before recordings were made . Standard acsf consists of (in mm) nacl 125, nahco3 25, nah2po4h2o 1.27, kcl 2.5, mgso47h2o 1.2, cacl2 2.0, and glucose 25 and was adjusted with sucrose to a final osmolarity of 350 mos . For recording, a slice was transferred to a submerge - type chamber where it was continuously exposed to acsf, saturated with 95% o2 and 5% co2 at room temperature (2226c) at the rate of 2.0 ml / min . Ra and the surrounding tissues were distinguishable under bx51wi microscope connected with a dic - ir video camera (olympus, japan). At high magnification (40x), ra neurons were visualized and the recordings were made from healthy cells . Recording pipettes were fabricated from borosilicate glass (sutter instruments, usa) using a flaming - brown puller (model p-97, sutter instruments, usa) and were filled with the pipette solution containing (in mm): k meso4 120, nacl 5, hepes 10, egta 2, mg - atp 2, and na3-gtp 0.3 (ph 7.3 - 7.4, and 340 mos). The recording pipettes, which had resistances ranging from 3 to 5 m, were positioned using an integrated motorized control system (sutter instruments, usa). The potential changes after application of nmda were corrected during the input - output stimulation protocols . The series resistance (rs), between 15 m and 30 m, was compensated using the bridge balance . All agents were applied by changing the bath perfusate from standard acsf to modified acsf in which various drugs were simply added . Unless indicated otherwise, the clampfit 9.2 (axon instruments, usa), mini 6.0 (synaptosoft inc ., fort lee, nj, usa), and originpro 8.0 (originlab, nothampton, ma, usa) were used for analysis . Events with peak amplitude of 50 mv or higher and a rise time of about 0.5 ms were detected automatically, and the results were analyzed with origin pro 8.0 . The steady - state spike rate was estimated by counting the number of spikes in 1 second, and the result was plotted versus the intensity of the injected current (f - i relationship). The slope of the f - i relationship (referred to as gain) was estimated by linear fitting . Slope parameters were estimated separately for individual neurons and that figures 4 and 5 contain mean slope values averaged for the whole groups of neurons . The amplitude of ahp was quantified as the difference between spike threshold and the lowest point of ahp . Throughout means were compared using paired two - tailed student's t - test or one - way anova followed by scheffe's multiple comparison . Stable whole - cell recordings were obtained in 160 ra projection neurons from 51 adult male zebra finches . A neuron with the resting membrane potential <50 mv and an overshooting action potential was considered viable . Ra projection neuron was identified by a larger soma, time - dependent inward rectifier induced by hyperpolarizing current and spontaneous regular spike firing or by depolarizing current pulses . The input - output relationship was examined by injecting current pulses from 0 to 90 pa with an increment of 10 pa and an interval of 15 s, and they were lasted for 1 s. traces of action potentials in response to 1 s current steps of 10, 50, and 90 pa were shown in figure 1(a). The slopes of the f - i relationships were 246.92 28.00 hz / na before nmda application and increased to 325.49 73.34 furthermore, the input resistances were decreased by 20 m nmda in ra projection neurons (p <0.05, n = 6) (figure 2). The afterhyperpolarization (ahp) of action potential underlies the modulation of firing frequency . To test the effect of nmda on spike ahp comparison of spike shape is shown in figure 3(a) with 90 pa current injection before and after nmda application . In average, there was no significant change in the amplitude of ahp after nmda application (p> 0.05, n = 5) (figure 3(b)). Then we used 50 m dl - apv, a nmda receptor antagonist, to check the selectivity of nmda receptor, and we found that the slope of f - i was not changed by dl - apv alone (p> 0.05). The slope was 255.78 66.69 hz / na and 269.34 56.96 hz / na (p> 0.05) before and after application of 50 m dl - apv (figure 4), respectively . Nmda increased the f - i slope 1.32 times (p <0.05); however, the gain modulation induced by nmda was completely abolished by dl - apv (figure 4). Previous studies reported that ra projection neurons received glutamatergic and gabaergic inputs [23, 24], which might increase background synaptic activities and modulate the gain of neurons [25, 26]. To determine the effect of ampa receptor and gaba receptor on gain modulation by nmda, we examined the effects of nmda in the presence of cnqx, an antagonist for ionotropic glutamate ampa receptors, or / and bicuculline methiodide (bic), a competitive antagonist of gabaa receptors, respectively . Bic (10 m) accelerated the depolarization by nmda application, but bic alone had little effect on the f - i slope . In the presence of bic, the mean initial slopes were 276.46 59.4 and 332.6 40.02 hz / na before and during application of nmda . The f - i slope was increased from 1.32 to 1.39 in the presence of nmda and bic (p> 0.05); that is, there is an increase of 105.30% over the nmda treatment (figure 5; p> 0.05, unpaired t - test). Cnqx (10 m) decreased the membrane potential and abolished the spontaneous spikes of ra projection neurons sometimes but did not affect the slope of f - i relationship . In the presence of cnqx, nmda application hardly changed the f - i slope of ra projection neurons no matter whether bic was present or not (figure 5; p> 0.05). These results indicated that the ampa receptor - dependent excitability played a crucial role in the gain modulation by nmda . The slope was 0.97 or 1.11 in the presence of cnqx or cnqx and bic, respectively . The gain induced by nmda was significantly decreased by cnqx (p <0.05). In this study, we showed that nmda increased the gain of ra projection neurons in adult zebra finch . Nmda - induced gain increase was limited to firing frequencies less than 40 hz . The effect of nmda on gain was mediated through nmda receptors and might involve synaptic activities . In songbirds, we found that nmda increased the slope of f - i relationship in ra projection neurons, which was completely blocked by dl - apv, a nmda receptor selective antagonist, suggesting that nmda increased the sensitivity of ra projection neurons . The pharmacology of nmda receptors involved in gain modulation has been examined in mammals . In cat visual cortex, nmda increased the gain of the contrast - response (c - r) curve of the neuron in layers ii / iii and v / vi . In mouse, the thalamocortical neurons in the dorsal lateral geniculate nucleus play a key role in the generation of firing patterns through nmda receptors . The neural activity in the afp of songbirds can direct moment - by - moment changes in the primary motor areas responsible for generating song . Singing - related activity in lman is more variable in pattern across repeated renditions during undirected song than directed song [28, 29]. Reported that the fundamental frequency of the syllable was shifted by stimulating the lman - ra pathway, which is mediated by nmda receptors . Our results indicated that the activity of nmda receptor changed the sensitivity of ra projection neuron . Ra, as a premotor nucleus, occupies an important position in the song system, integrating information from both pathways . Ra projection neurons are responsible for transmitting song information to midbrain and brainstem vocal and respiratory structures . Temporal pattern of spike bursts in ra projection neurons is associated with the timing of acoustic features of birdsong . Precise timing of individual spikes has a close relationship with stereotypic behavior, which suggests that the song is represented in ra by a temporal code [5, 31]. Thus, the modulation of nmda receptors in the activities of ra projection neurons could be very important for the plasticity of birdsong . Previous studies in slices demonstrated that nmda receptor achieves the nonlinear, multiplicative behavior seen in gain modulation by several mechanisms . Mel presented results from compartmental modeling studies showing that dendrites containing nmda - type synaptic conductances and voltage - dependent na, k, and ca channels can impose nonlinear interactions between nearby synapses that give rise to approximate multiplications [32, 33]. In thalamocortical cell of the rat and cat dlgn, nmda receptors contribute to the burst firing generated by the low - threshold ca potential and depend on the membrane potential range over which this type of firing occurs . In substantia nigra dopamine neurons, nmda receptor - mediated synaptic conductance generates transient high - frequency activity by rapidly but transiently overwhelming the conductance's underlying action potential ahp and/or engaging postsynaptic voltage - dependent ion channels in a manner that overcomes the limiting effects of ahp . But our results showed that there was no significant change in spike ahp after nmda application . So the gain induced by nmda in ra projection neurons may not be mainly modulated by the change of spike shape, but by synaptic activities . The change in membrane potential has been shown to play an important role in the firing pattern regulation by nmda receptor, supporting the idea that depolarization acutely alters firing frequency . It has been shown that simple excitability or inhibition alone can induce a multiplicative gain change in the cortical neurons . In this model, one set of synaptic inputs provided purely excitatory drive to a target neuron, and another set of combined excitatory / inhibitory inputs (or balanced inputs) provided the modulation . In this setup, changing the firing rates of the balanced inputs produced an approximately multiplicative gain modulation of the response to the purely excitatory drive . Here, blockade of the excitatory or inhibitory transmission alone had no effect on the f - i slope of ra projection neuron, implying that simple excitability or inhibition alone cannot induce a multiplicative gain change . However, our results did not exclude a role of spontaneous synaptic activity on gain modulation in vitro, which, due to the loss of synapses in slice, was not sufficient to produce a significant change in gain . Ampa receptors are required for the gain modulation in our study, because blocking excitatory transmission mediated by ampa receptors almost abolished the effect of nmda on gain modulation . Two reasons might be involved: one is the two states of nmda - base mechanism due to the special anatomical structure of ra . Ra projection neurons receive inputs from two areas, hvc and lman . Both are excitatory glutamatergic but have distinct postsynaptic properties . Hvc - ra is largely mediated by ampa receptor, whereas lman - ra is almost completely mediated by nmda receptor . Ampa / nmda receptor composition of different neuronal pathways is the foundation of gating and gain modulation . Nmda receptor - rich pathway can gate ampa - rich input and increase the gain of a neuron responding to the input of ampa - rich pathway . The second is related to the intrinsic properties of nmda receptors, which are uniquely voltage dependent . Except binding with neurotransmitter blockade of main excitatory inputs mediated by ampa receptor with cnqx hyperpolarized the membrane potential, which caused nmda receptors not activated . Therefore, the ampa receptor - mediated excitatory transmission is essential for the gain modulation induced by nmda . It is well known that the nmda receptor plays a critical role in many physiological and pathological processes, such as learning, memory, neurodegeneration, epilepsy, and ischemia . The nmda receptor - mediated synaptic transmission satisfies the associative property of hebbian learning and in fact plays a critical role in its cellular model, long - term synaptic plasticity . Nevertheless, pharmacological agents which block nmda receptors impair a variety of brain processes, suggesting that nmda receptor transmission also plays an important role beyond long - term memory and participates in shaping the dynamic activity of neural networks . In song system, the expression pattern of nmda receptor is related to the development of song learning [21, 3740]. Recent results show that the nmda receptor on the lman - ra synapses generates variability across different song renditions, thereby facilitating reinforced learning of songs . How nmda accomplishes this role is not clear . Our results showed that nmda application increased the gain of ra projection neurons, in which ampa receptor was necessary . The ampa receptor - mediated excitability is derived only from hvc [10, 11]. Therefore, nmda receptor in lman - ra synapse might regulate hvc - ra synaptic transmission, which implicated that nmda receptor is more effective to modulate the syllable online . These findings suggest a mechanism by which nmda receptor can selectively modulate behaviorally relevant excitatory inputs.
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First described by veterinarians, pythiosis is a serious life- and limb - threatening infection endemic to thailand, but rarely seen in the western hemisphere . Known to infect immunocompetent horses, dogs, and other large animals,2, 3 pythium infection was first reported in humans in 1989 in association with thalassemic hemoglobinopathy.4, 5 human pythiosis is known to present in one of four clinical entities: subcutaneous, ocular, vascular, and disseminated . Here, we present a unique case of vascular pythiosis managed with limb - sparing vascular bypass grafts complicated by pseudoaneurysm formation . The patient is a 17 year - old jamaican male with a history of paroxysmal nocturnal hemoglobinuria and severe aplastic anemia refractory to horse anti - thymoglobulin treatment, which was transfusion - dependent . One month prior to admission at our institution, he went fishing in a fresh water pond in jamaica and his left lower leg was caught in a bush . On the following day, he noticed a small bullae with a black center and later developed severely painful left inguinal swelling . He was admitted to a local hospital for the next several weeks, where all diagnostic testing performed was unremarkable . His pain persisted despite antibiotic and antifungal therapy, and he was later transferred to our institution for consideration of bone marrow transplant . On admission, he was a febrile with a white blood cell count of 6.2 (k/l) and platelet count of 18 (k/l) and was complaining of severe left leg pain . He had a 9 8 cm soft tissue defect with overlying escar on the posterior aspect of his left leg (fig . 1a), and his exam was also notable for an exquisitely tender and erythematous left inguinal mass . Motor and sensory exam were intact in both lower extremities without pain upon passive hip flexion . Several small, dark macules were noted along the lateral aspect of his left foot (fig . Mri showed a t2 bright left groin mass with edema along the fascial planes, and lower extremity angiogram demonstrated occlusion of the left common femoral artery with distal profunda femoris and mid - superficial femoral artery reconstitution (fig . The patient was not a candidate for percutaneous intervention, and was taken to the operating room for exploration . Visual inspection of the common femoral artery after exposure was significant for several patches of necrotic vessel wall . Initial attempt with thrombectomy was unsuccessful, as the fogarty catheter actually perforated the anterior wall at the areas of necrosis . The patient ultimately received an extra - anatomic left external iliac to superficial femoral artery bypass using a nonreversed greater saphenous vein graft, as the external iliac was the closest available inflow that did not visually appear to be involved with the infection . Intraoperative wound cultures were positive for pythium insidiosum and the patient was started on a combination of voriconazole, terbinafine, and amphotericin b. given his critical condition, an emergency investigational new drug request was drafted for permission to administer a p. insidiosum antigen vaccine available for compassionate use from the pan american veterinary laboratories . The vaccine was given once daily in one - week intervals in conjunction with subcutaneously injected interferon gamma (ifn-), for a total of four weeks . Three weeks after his initial surgery, the patient developed new sites of pain along the graft site and repeat imaging showed a 2.5 cm pseudoaneurysm at the proximal anastomosis to the external iliac artery with contrast extravasation (fig . He was taken to the operating room for an extra - anatomic left common iliac artery to the previous femoral popliteal bypass graft using a reverse saphenous vein graft . The patient later developed a polymicrobial superinfection of his wounds, and his vascular repair continued to break down leading to increased ischemic pain . After several multidisciplinary discussions with the family that all medical interventions were exhausted, and that even highly aggressive surgical measures including hip disarticulation or hemipelvectomy could not guarantee source control, conservative measures were initiated and ultimately, the patient expired . This patient presented a major diagnostic challenge, and his disease process and subsequent management were previously unknown to our hospital . In retrospect, he presented with a classic case of vascular pythiosis, with an antecedent history of exposure to surface water and exam findings of vascular insufficiency, including pain out of proportion to exam, distal emboli, and an ischemic ulcer . The correct infectious diagnosis was not made until 1 month after exposure, after a vascular bypass was already performed . Indeed, in a large multi - institutional study from thailand with a total of 102 cases of pythiosis, 60 patients received a diagnosis of vascular pythiosis, and only the patients who had undergone amputation survived . Further review of the literature for reports of vascular pythiosis demonstrated that the majority of patients were treated with amputation without any signs of recurrence, and were followed for 5 to 35 months post - operatively.4, 6, 8, 9 in our case, complete source control was not achieved during the first operation since the common femoral artery and the proximal portion of the superficial femoral artery were oversewn and left in place, standard practice for the treatment of peripheral arterial disease . Incomplete source control eventually led to development of a pseudoaneurysm at the proximal anastomosis and additional surgery . While the second repair was cited in previously uninvolved tissue planes, it did not protect against further infection, likely because p. insidiosum is known to invade through proteinase secretion . The authors could find no reported cases of confirmed vascular pythiosis treated with limb - sparing procedures.2, 7, 11, 12 medical therapy in the treatment of vascular pythiosis involves antifungal agents, immunotherapy, and iron chelation therapy . Much of the difficulty in eradicating this organism stems from its complicated phylogenesis;2, 6, 14 it shares features with true fungi at the same time it is phylogenetically more closely related to algae and diatomeae . P. insidiosum develops mycelium like fungi, but its cell walls do not contain chitin and its cytoplasmic membrane lacks ergosterol, both of which are important targets of traditional antifungal drugs . Despite this, drugs that affect cellular permeability, including amphotericin b, terbinafine, and the azoles, have been used with some clinical success.2, 15 more recently, immunotherapy has been used as an adjunct to surgery and antimycotics . Immunotherapy was first successfully used in humans in 1998 in a patient with a vascular p. insidiosum infection refractory to surgery and antifungals.2, 16 a p. insidiosum vaccine delivered twice with a two - week interval resulted in a cure 1 year later . Data collected from clinical trials and other reports estimate vaccine efficacy to be 5560%.2, 17 the proposed mechanism of action of the p. insidiosum vaccine is to shift host adaptive immune response from a predominantly t helper 2 (th2) response to a t helper 1 (th1) response.2, 17 pythium exoantigens are typically processed by antigen presenting cells (apcs) and presented to nave cd4 helper t cells (th0). This induces th0 cells to adopt a th2 phenotype secreting il-4 and il-5, which trigger many downstream effects, including mast cell and eosinophil migration to the site of infection with subsequent degranulation, releasing inflammatory mediators and causing tissue damage . Loss of tissue architecture then creates a more favorable environment for these pathogenic hyphae to take hold . Conversion of a th2 to th1 response would favor secretion of ifn- and il-2, leading to increased cytotoxic t lymphocyte recruitment and therefore, direct killing of p. insidiosum . In this case, the purpose of injecting ifn- subcutaneously was to augment a th1 response, and co - administration of gm - csf was to increase the efficiency of exoantigen presentation to apcs . Another treatment modality used to control this infection is iron chelation therapy . Given the association between pythium infection in patients with hemoglobinopathies with a long - standing history of blood transfusions4, 7 and the discovery of a ferrochelatase gene in p. insidiosum, other research efforts have focused on elucidating the role of iron overload in p. insidiosum pathogenesis . There is evidence that iron overload from transfusions contributes to derangements in host immunity by impairing lymphocyte function and altering phagocytosis, leading to a more susceptible state for infection6, 19 and is in fact, a key virulence factor for the survival of p. insidiosum in the host . Correcting iron overload has been cited as a treatment in infections common in thalassemia, and has also been used in pythiosis . In our patient, in conclusion, this patient's vascular p. insidiosum infection was refractory to optimized medical treatment, immunotherapy with cytokine injections, and surgery . Attempted source control with limb salvage necessitating vascular bypass was unsuccessful in treating this case of vascular pythiosis . Written informed consent was obtained from the patient's guardian for publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request.key learning pointsclinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy, exposure to flat water, and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation . Clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy, exposure to flat water, and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation . Clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy, exposure to flat water, and exam findings of vascular insufficiency . The most important part in managing vascular pythiosis limb infection is adequate source control with amputation . Clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy, exposure to flat water, and exam findings of vascular insufficiency.the most important part in managing vascular pythiosis limb infection is adequate source control with amputation . Clinicians should maintain a high level of suspicion for vascular pythiosis when presented with a patient with an underlying hemoglobinopathy, exposure to flat water, and exam findings of vascular insufficiency . The most important part in managing vascular pythiosis limb infection is adequate source control with amputation.
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Tissue culture methods and irradiation procedure have been described in . The three dimensional tissue culture model (3d epioral) and media were purchased from mattek corporation (ashland, ma). The 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . In order to induce differentiation and stratification of the keratinocytes of the 3d cultures, then the tissues were irradiated (dose of irradiation was 0, 2, or 12 gy) at the city of hope (duarte, ca) facility . Subsequently the tissues were incubated for 6 h at 37 c with 5% co2 after irradiation . Then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna . The rneasy plus mini kit (qiagen, germantown, md) was used to extract total rna of at least two identically treated tissues . The integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer (agilent technologies, palo alto, ca). Only rna samples with absorbance ratio, a260/280> 2.0, and rin> the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase; the transcription reaction included aminoallyl utp (aa - dutp) and the subsequent product was conjugated to cy5 nhs ester (ge healthcare life sciences, pittsburg, pa). The human whole genome onearray v4.3 (phalanx biotech, san diego, ca) was used to perform the dna microarray analysis . For this purpose, a quantity of 0.025 mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42 c using the hybbag mixing system with 1 onearray hybridization buffer (phalanx biotech) and 0.01 mg / ml sheared salmon sperm dna (promega, madison, wi). After the hybridization procedure the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format . The data from all the arrays of each experimental set were analyzed using rosetta resolver system (rosetta biosoftware, usa). Testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver . The error - weighted approach (which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy) was used to calculate the average expression values . In order to decide whether intensity data was significantly above background, p - values were calculated to test the null hypothesis that expression is absent (called p - value detected). A p - value detected <0.05 indicated that the transcript specific to a given probe was truly present or expressed . Lastly, we also calculated p - values for determining whether genes were differentially expressed . Rather than focusing solely on fold changes, rosetta resolver uses error - model - based hypothesis tests, which account for both fold change and expression level . Three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . Using the r function boxplot, raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes . This analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . For each sample the scattered plots of raw and normalized log2 data were compared using the r function pairs . Pearson correlation tables were used to define the linear relationship of the two variables, raw and normalized data, in the scatter plots . For each technical repeat, we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of <0.01 . Scatter plots showed that technical replicates had high repeatability and all the correlation values were> 0.953 . Our article,, emphasizes the differentially expressed genes of tissues that received different amounts of radiation (i.e. 0, 2, or 12 gy), while herein we show the profiling of all the transcriptomic data resulting from these treatments . We only used genes with | fold change |> 1.5 and p - value <0.05 . We input gene symbols into david bioinformatics, while throughout we used the default settings . The threshold for significance was a benjamini - adjusted p - value <0.05 . Based on our hypothesis, irradiated samples (compared to non - irradiated control samples) would display patterns of gene expression consistent with the physiological effects of irradiation . Table 2 includes selected enriched categories from our qc enrichment analysis (table s1 contains all enrichment analysis results). That is, up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death, cell proliferation, oncogenesis, and ubiquitin conjugation . Also, down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division . Overall, these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in . The following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics . Up- and down - regulated genes were analyzed separately for each condition, so there are two separate worksheets listing enrichment analysis results . Goterm cc = gene ontology cellular component, mf = molecular function, bp = biological process, and up_seq_feature = uniprot sequence feature . Other enriched categories are from the interpro, sp pir, smart, and kegg databases . Count = the number differentially expressed genes annotated with a given enriched term . Adj p - val = benjamini - adjusted p - values . More information on the output from david bioinformatics can be found here: http://david.ncifcrf.gov/content.jsp?file = function_annotation.html . Up- and down - regulated genes were analyzed separately for each condition, so there are two separate worksheets listing enrichment analysis results . Goterm cc = gene ontology cellular component, mf = molecular function, bp = biological process, and up_seq_feature = uniprot sequence feature . Other enriched categories are from the interpro, sp pir, smart, and kegg databases . Count = the number differentially expressed genes annotated with a given enriched term . More information on the output from david bioinformatics can be found here: http://david.ncifcrf.gov/content.jsp?file = function_annotation.html . Tissue culture methods and irradiation procedure have been described in . The three dimensional tissue culture model (3d epioral) and media were purchased from mattek corporation (ashland, ma). The 3d epioral tissue is a co - culture organotypic model and consisted of human fibroblasts on the bottom and human oral keratinocytes grown on top of the fibroblasts . In order to induce differentiation and stratification of the keratinocytes of the 3d cultures, then the tissues were irradiated (dose of irradiation was 0, 2, or 12 gy) at the city of hope (duarte, ca) facility . Subsequently the tissues were incubated for 6 h at 37 c with 5% co2 after irradiation . Then one of the tissues was placed in 10% formalin for histopathological studies and the others were used for the extraction of total rna . The rneasy plus mini kit (qiagen, germantown, md) was used to extract total rna of at least two identically treated tissues . The integrity and quality of rna was determined by evaluating the a260/280 absorbance ratio using an agilent 2100 bioanalyzer (agilent technologies, palo alto, ca). Only rna samples with absorbance ratio, a260/280> 2.0, and the rna was converted to double - stranded cdna and amplified using in vitro transcription with t7 polymerase; the transcription reaction included aminoallyl utp (aa - dutp) and the subsequent product was conjugated to cy5 nhs ester (ge healthcare life sciences, pittsburg, pa). The human whole genome onearray v4.3 (phalanx biotech, san diego, ca) was used to perform the dna microarray analysis . For this purpose, a quantity of 0.025 mg / ml fragmented cy5-labeled cdna was hybridized overnight at 42 c using the hybbag mixing system with 1 onearray hybridization buffer (phalanx biotech) and 0.01 mg / ml sheared salmon sperm dna (promega, madison, wi). After the hybridization procedure, the raw cy5 intensity signals produced by each of the microarrays were captured using a molecular devices axon 4100a scanner and measured using genepix pro software and stored in gpr format . The data from all the arrays of each experimental set were analyzed using rosetta resolver system (rosetta biosoftware, usa). Testing was done in triplicate by combining technical replicates while the statistical analyses were done using the proprietary modeling techniques of rosetta resolver . The error - weighted approach (which is specifically geared towards combining replicated hybridizations to improve measurement precision and accuracy) was used to calculate the average expression values . In order to decide whether intensity data was significantly above background, p - values were calculated to test the null hypothesis that expression is absent (called p - value detected). A p - value detected <0.05 indicated that the transcript specific to a given probe was truly present or expressed . Lastly, we also calculated p - values for determining whether genes were differentially expressed . Rather than focusing solely on fold changes, rosetta resolver uses error - model - based hypothesis tests, which account for both fold change and expression level . Three technical replicate hybridizations were averaged while ensuring that the technical replicates were of high repeatability . Using the r function boxplot, raw and normalized log2 data from each sample were plotted but we did not include control and flagged probes . This analysis not only helps finding hybridization with aberrant intensity distributions but also helps to confirm that the normalization of each replicate microarray has an appropriately centered distribution . For each sample the scattered plots of raw and normalized log2 data were compared using the r function pairs . Pearson correlation tables were used to define the linear relationship of the two variables, raw and normalized data, in the scatter plots . For each technical repeat, we calculated correlation values for raw and normalized log2 intensities and we included in the calculation only probes with p - value of <0.01 . Scatter plots showed that technical replicates had high repeatability and all the correlation values were> 0.953 . Our article,, emphasizes the differentially expressed genes of tissues that received different amounts of radiation (i.e. 0, 2, or 12 gy), while herein we show the profiling of all the transcriptomic data resulting from these treatments . We only used genes with | fold change |> 1.5 and p - value <0.05 . We input gene symbols into david bioinformatics, while throughout we used the default settings . The threshold for significance was a benjamini - adjusted p - value <0.05 . Based on our hypothesis, irradiated samples (compared to non - irradiated control samples) would display patterns of gene expression consistent with the physiological effects of irradiation . Table 2 includes selected enriched categories from our qc enrichment analysis (table s1 contains all enrichment analysis results). That is, up - regulated genes in irradiated samples were strongly enriched for functional categories involved in cell death, cell proliferation, oncogenesis, and ubiquitin conjugation . Also, down - regulated genes in irradiated samples were strongly enriched for functional categories involved in rna processing and cell division . Overall, these results confirmed the quality of the microarray data and facilitated further interpretation of the data presented in . The following are the supplementary data related to this article.table s1enrichment analysis results using david bioinformatics . Up- and down - regulated genes were analyzed separately for each condition, so there are two separate worksheets listing enrichment analysis results . Goterm cc = gene ontology cellular component, mf = molecular function, bp = biological process, and up_seq_feature = uniprot sequence feature . Other enriched categories are from the interpro, sp pir, smart, and kegg databases . Count = the number differentially expressed genes annotated with a given enriched term . More information on the output from david bioinformatics can be found here: http://david.ncifcrf.gov/content.jsp?file = function_annotation.html . Up- and down - regulated genes were analyzed separately for each condition, so there are two separate worksheets listing enrichment analysis results . Goterm cc = gene ontology cellular component, mf = molecular function, bp = biological process, and up_seq_feature = uniprot sequence feature . Other enriched categories are from the interpro, sp pir, smart, and kegg databases . Count = the number differentially expressed genes annotated with a given enriched term . More information on the output from david bioinformatics can be found here: http://david.ncifcrf.gov/content.jsp?file = function_annotation.html.
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Adolescents in sub - saharan africa face high exposure to human immunodeficiency virus (hiv) infection . Despite all efforts to prevent the disease, prevalence estimates in sub - saharan unaids (i.e., the joint united nations programme on hiv and aids) priority countries range from 1.3 to 15.6% among young women and 0.56.5% among young men aged 1524 years (unaids, 2011). Higher prevalence estimates are associated with certain regions (e.g., eastern and southern africa) and subpopulations (e.g., adolescent orphaned and/or married girls; birdthistle et al ., 2008; operario, underhill, chuong, & cluver, 2011; pascoe et al ., 2010). In addition, united nations children s fund (unicef) estimated there were 2 million adolescents aged 1019 years living with hiv globally in 2009 (unicef, 2011). Kenya and zimbabwe were among countries with the highest numbers of infected adolescents with an estimated 136,000 (prevalence estimate 1.5%) 1 and 104,000 (prevalence estimate 3.1%) adolescents living with hiv, respectively (unicef, 2011). The true measure of effectiveness for such interventions is reduction in hiv and other sexually transmitted infections (stis). However, very few adolescent prevention studies have used biomarker data as outcomes (michielsen et al ., 2010) because of concerns about the difficulties in collecting these data in resource poor settings (jukes, simmons, & bundy, 2008), even when power analyses suggest that moderate intervention effects could be detected . Behavioral prevention scientists have instead relied on self - reported sexual behavior, which is subject to social desirability bias and likely to differ between study arms (cowan & pettifor, 2009; kirby, obasi, & laris, 2006). Widespread access to testing and treatment facilitated by the president s emergency plan for aids relief (pepfar; http://www.usaid.gov/what-we-do/global-health/hiv-and-aids) have greatly enhanced opportunities to successfully incorporate biomarker procedures into adolescent hiv prevention research designs, since infected individuals identified through testing offered by studies may access free or subsidized hiv treatment and care (stringer et al ., 2006; wools - kaloustian et al ., 2006, 2009) moreover, including biomarker outcomes can greatly improve the credibility and legitimacy of hiv prevention research . To address the issue of feasibility, we present two case studies of prevention trials with biomarker data collection among adolescents in kenya and zimbabwe . Together these studies provide a valuable opportunity to examine the ethical and practical challenges of biomarker procedures within hiv prevention intervention clinical trials in developing countries . Specifically, we describe the challenges faced by investigators in the two cases in developing and implementing ethical procedures for informed consent, biomarker testing, and disclosure of test results . We also present the practical challenges of collecting biological samples in resource - limited countries and compare sample collection by blood spots relative to venipuncture . In addition, we examine difficulties with analyzing biological samples when the performance of assays in certain populations is unclear . We also provide specific suggestions for further research and development in collecting and analyzing biomarker data for adolescent prevention research . The primary goal of hiv prevention among adolescents (ages 1019 years) is to reduce new infections and related sexual risk factors [world health organization (who), 2006). Although the hiv prevalence of adolescents ages 15 and older is monitored through demographic and health surveys (dhs)2 in most sub - saharan africa countries, little is known about the prevalence of hiv in adolescents under 15 years . Moreover, these adolescents are rarely tested for hiv in clinical settings unless they present in poor health or with recurrent infections (ferrand et al ., 2009). Although a small number of recent randomized controlled trials have used biomarkers (baird, mcintosh, & ozler, 2010; cowan et al . ; pronyk et al ., 2006; ross et al ., 2007), most adolescent hiv prevention studies have relied on changes in self - reported sexual behavior to assess intervention effectiveness (see reviews: cowan & pettifor, 2009; michielsen et al ., 2010), with audio computer - assisted self - interviews intended to improve veracity (mensch, erulkar, & hewett, 2003). However, biomarker comparisons have suggested that self - reported measures have low validity (cowan et al ., 2002; gavin et al ., 2006; mensch, hewett, gregory, & helleringer, 2008; palen et al ., 2008; plummer et al ., 2004). For example, in one study, only one of 16 youth with hiv admitted to ever having sex, and four of the 16 also had either chlamydia or gonorrhea (cowan et al ., 2002). Inclusion of a highly prevalent and reliable biomarker of sexual activity in conjunction with self - reported sexual behavior can help to improve reliability of this outcome measure . Hiv infection is not the most useful biomarker of adolescent sexual activity by itself because of the relatively low prevalence among younger adolescents and the potential for transmission through perinatal and non - sexual blood - borne routes (amornkul et al ., 2009; ferrand et al ., 2009; stover, walker, grassly, & marston, 2006). Herpes simplex virus type 2 (hsv-2), the primary cause of genital herpes, is a commonly used biomarker of sexual activity because the presence of hsv-2 antibodies is highly associated with past sexual behavior (tobian et al ., 2009; van de laar et al ., 1998). In contrast to other stis such as chlamydia, gonorrhea, syphilis and trichomoniasis, infection with hsv-2 is life - long, and, once established, there is currently no treatment to eliminate it (geretti, 2006; gupta, warren, & wald, 2008). Hsv-2 prevalence in the adult general population in sub - saharan africa is high, ranging from 30 to 80% in women and from 10 to 50% in men, as assessed by detection of hsv-2 antibodies [amornkul et al ., 2009;, 2005; munjoma et al ., 2010; national aids & sti control programme (nascop), republic of kenya, 2009; tobian et al ., 2009; weiss, 2004]. Prevalence estimates for adolescents are only available through community - based studies and are lower . One study in zimbabwe among adolescent females aged 1519 years reported that 12% of participants tested positive for hsv-2 (birdthistle et al . A study in western kenya found hsv-2 prevalence to be 9% for females and 4% for males aged 1314, and 28% for females and 17% for males aged 1519 years (amornkul et al ., 2009). Another study conducted in zimbabwe among adolescents whose mean age was 15 years reported a prevalence estimate of 0.2%, suggesting a later stage of sexual debut (cowan et al . 2012) evaluated the use of hsv-2 as a biomarker of sexual debut among young people aged 1024 years . Following a comprehensive review of the literature, the authors concluded that the use of hsv-2 as a biomarker for sexual debut is limited because of its low transmissibility and the fact that not all potential sexual partners are infected with the virus . Building on these findings, our study describes the challenges of collecting hiv and hsv-2 biological data as biomarkers for sexual activity among sub - saharan african youth in prevention trials . We present two clinical trial case studies to examine important issues related to adolescent biomarker data collection in two clinical trials in kenya and zimbabwe (hallfors et al ., both studies were school - based, cluster randomized trials testing school support (including payment of school tuition and school uniforms) as a structural hiv prevention intervention for adolescent orphans . In both studies, we collected blood samples from participants for hiv and hsv-2 testing, but they differed as follows: we collected samples by venipuncture with results disclosed to participants and their guardians in kenya, whereas in zimbabwe, we collected samples by finger pricks for dried blood spots (dbs) and results were not disclosed . The kenya sample (n = 837) comprised young adolescent male (52%) and female (48%) orphans in grades 7 (61%) and 8 (39%) enrolled in school in siaya district, nyanza province . Their mean age was 14.9 (sd 1.5; range 1121 years); 22% reported ever having had sex . Among girls, hiv and hsv-2 prevalence was 1% (n = 10) and 3% (n = 28), respectively . Among those with positive hiv or hsv-2 test results, 70% (n = 7) and 64% (n = 18), respectively, reported never having had sex . The original sample for the zimbabwe study was orphan 6th grade girls (n = 328) in manicaland province primary schools; surveys were administered annually during 20072010 (hallfors et al ., 2011). After securing a project renewal grant, we collected biomarker specimens and a final survey in march through june 2012, when most participants were 1617 years old . From the original sample, we located 287 (88%) for the 2012 survey . Twenty - three percent reported ever having sex; 18% had ever been married and/or pregnant . Hiv and hsv-2 prevalence was 4% (n = 12) and 6% (n = 16), respectively . Among those with positive hiv or hsv-2 test results, 50% (n = 6) and 44% (n = 7), respectively, reported never having had sex . Although the protection of adult participants in hiv prevention research is well developed (national institute of mental health collaborative hiv / std prevention trial group, 2007), we found that clear guidance regarding hiv and other sti testing and disclosure is lacking for children and adolescents . As an example, our two clinical trials were peer - reviewed by two different nih scientific review panels; one set of reviewers thought that it was an ethical imperative to disclose hsv-2 as well as hiv results to adolescents, whereas the other was concerned that disclosure of biomarker results might lead to stigma and abuse . These divergent opinions led to discrepant protocols for disclosure, with one trial disclosing and the other not disclosing test results to participants . We used the national hiv testing and counseling (htc) guidelines [central statistical office (cso) & macro international inc ., 2007; nascop, republic of kenya, 2008] for hiv in both countries . Given the lack of national guidelines for adolescent sti testing in kenya [ministry of health (moh), republic of kenya, 2006), we developed a protocol in conjunction with the siaya district aids and std control office that closely followed hiv guidelines . Study protocols were reviewed and approved by irbs in the us, kenya and zimbabwe . The kenya study was approved by the ministry of education and siaya district education office; school head teachers identified orphans and organized meetings in which moi university research collaborators and a district education officer explained the study to students and their caregivers . Caregivers and students we invited a total of 923 orphans in grades 7 and 8 in 2011 to participate; 849 (92%) consented and 837 (91%) completed both the student survey and biomarker testing . Following national guidelines, we required kenya study participants under age 18 to have an adult guardian accompany them for hiv rapid testing (nascop, republic of kenya, 2008). In some instances, however, the guardian was not available because of work, other commitments, or illness . In such cases we permitted a relative, teacher or neighbor to stand - in for the guardian if they had a note from the guardian or if research staff were able to speak with the guardian by phone . Given the sensitive nature of hiv testing, the senior counselor interviewed all unclear or undocumented cases to determine whether or not to proceed with specimen collection and htc . We visited clinics several times to give participants the opportunity to return for testing with their guardian or authorized proxy . Although not required, all participants ages 18 and older were accompanied by an adult for support in case of a positive hiv test result . In accordance with national guidelines, we gave referrals to publicly funded hiv care and treatment centers to all adolescents with positive hiv test results, while hiv negative participants were counseled on risk reduction (nascop, republic of kenya, 2008). We disclosed results to both guardians and participants if the latter were under age 18 and to the participants alone if they were 18 years old according to the study protocol . In zimbabwe, with permission from the provincial education officer, the consortium principal investigator and a research associate held meetings at study schools to explain the continuing study to parents / guardians, participants, and key school staff . We obtained written parent / guardian consent and participant assent for continued voluntary participation in the study, including separate checkbox consent for survey and blood sample collection . Guardians raised few concerns, although they did ask why we were not disclosing test results . Participants who were married or 18 years or older provided their own consent . In deference to local cultural norms and to prevent potential spousal abuse, we prepared a second consent form for husbands to provide consent for their wives participation . Most of the wives gave their own consent for the survey and did not ask their husbands for consent . In four cases, the husband signed a consent form and in five additional cases, the husband refused and the wife did not take the survey . Although asking husbands for permission for their wives study participation is not consistent with us culture, it appears to be a useful protocol accommodation for some young wives in rural zimbabwe . We consistently relied on local researchers and key informants to make such culturally sensitive decisions . After 4 months, we found 88% of the original sample who consented to the survey; 85% consented to blood specimen collection . Of those not surveyed, two participants had died in childbirth, 2% refused, and approximately 10% of the sample could not be found . Of those not consenting to blood specimen collection (n = 8), six were married and refused for religious reasons or husband s objection . Two were consented for the survey by school officials who refused to provide additional consent for the blood collection, and no guardian was available to give permission . Hiv testing is technically far more advanced for widespread use in sub - saharan africa than hsv-2 testing . Rapid hiv antibody tests are now widely used and accepted (moh, republic of kenya, 2006), and sequential algorithms for retesting have been adopted in kenya and zimbabwe for rapid testing (cso & macro international inc ., 2007; nascop, republic of kenya, 2008; who, 2004). Rapid hiv tests meet high performance standards, sample collection procedures are simple, tests are easy to perform, and interpretation of test results is reasonably clear . In contrast, hsv-2 serologic testing presents some important challenges . Although a number of hsv-2 type - specific serologic tests are available, only a limited number have been tested among sub - saharan african populations . These include the herpeselect hsv-2 enzyme - linked immunosorbent assay (elisa; focus diagnostics, cypress, ca) and kalon hsv-2 elisa (kalon biological ltd ., guildford, uk; biraro, mayaud, morrow, grosskurth, & weiss, 2011). These tests require skilled laboratory staff, specialized equipment, and a constant source of electricity . They are appropriate for use with large batches of samples as in sentinel surveys and are inexpensive, costing about $3 per test . Although the literature reports variable performance of these tests with respect to sensitivity and specificity in sub - saharan populations, kalon is often found to be superior to focus herpeselect (see biraro et al ., 2011 for a review of the performance of hsv-2 tests in sub - saharan africa; 2008; ngayo, friedrich, holmes, bukusi, & morrow, 2010; smith et al ., 2009; van dyck et al ., 2004). When using the manufacturer s instructions (index cutoff value = 1.1 for both tests), kalon tends to have lower sensitivity but higher specificity than focus herpeselect (delany - moretlwe et al ., 2010; gamiel et al ., 2008; ngayo et al ., 2010; smith et al ., 2009; van dyck et al ., the low specificity of focus herpeselect is particularly problematic when disclosing results to research participants because of an unacceptably high rate of false positives when prevalence is low (gamiel et al ., 2008; mark et al ., 2008; ngayo et al, 2010; van dyck et al ., 2004). Increasing the assay cut - off values is one strategy for improving the performance of both the kalon and focus herpeselect tests . A number of studies have shown that increasing the cut - off value for focus herpeselect to 3.5 increases specificity, although sensitivity is necessarily reduced (delany - moretlwe et al ., 2010; smith et al ., other studies similarly improved performance at cut - off values of 3.2 and 3.3 (delany - moretlwe et al ., 2010; gamiel et al ., 2008; ngayo et al ., although findings are mixed, increasing the cut - off value for the kalon test to 1.5 has also been shown to increase specificity, with an attendant reduction in sensitivity (gamiel et al ., 2008; ngayo et al . In our studies, the kenya laboratory used the kalon assay, and the zimbabwe laboratory used the focus test . At the time of our studies, neither assay had been validated for dbs by the manufacturer in african populations . The efficiency of dbs for hsv-2 testing with each assay must be evaluated in specific populations prior to use (hogrefe, ernst, & su, 2002). Because the kalon test had not been validated for hsv-2 testing with dbs samples, specimens in the kenya study were obtained by venipuncture . The zimbabwe laboratory had validated the focus test with dbs (mudzori & mutsogoro, 2006). In both studies, we used a modified algorithm to interpret results: samples with initial index values <0.9 were reported as negative and no further testing was conducted . Samples with initial index values> 2.5 were defined as high positive and were reported as positive without additional testing . Samples with initial index values from 1.5 to 2.5 were considered low positive, and those with initial index values between 0.9 and 1.5 were considered equivocal; all low positives and equivocals were retested in duplicate . If both retest results had index values> 1.5, the final result was reported as positive . If both retest results were <0.9, the final result was negative . If retest results were equivocal (0.91.5) or discordant, the final result was indeterminate . Although test performance was improved by increasing the cut - off and expanding the range of results for repeat testing, the performance characteristics of tests to detect antibodies to hsv-2 are imperfect . Based on a recent systematic review of hsv-2 antibody test performance in sub - saharan africa (biraro et al ., 2011), sensitivity for the kalon test used in kenya was estimated at 94% and specificity at 92% with the higher cut - off value of 1.5 . The predictive values of all diagnostic tests are strongly influenced by the prevalence of infection in the population, and in low prevalence situations, even a very good test will have a poor positive predictive value (ppv). Using the sensitivity and specificity values for the kalon test under the testing conditions employed in our kenya study, the ppv was estimated at 26% with a 3% hsv-2 prevalence . That is, we can be confident that about one in four participants with positive test results truly had hsv-2 antibodies present in their blood sample; however, as many as three in four could have had a false positive test result . In kenya, we conducted sample collection and htc primarily in local public health facilities near the schools . Eighteen facilities participated . In two sites, with permission from the school headmasters and district education officer, we collected blood samples and conducted htc at the school . Venipuncture was conducted by six skilled phlebotomists trained in htc . To avoid obtaining two separate blood specimens (finger prick for rapid hiv testing and venipuncture for hsv-2 testing) the process, including counseling, blood draw, hiv testing, disclosure of results took on average 20 min . The process was longer for participants with discordant results who needed a tie - breaker test and for participants with hiv positive results . We offered all participants and their guardians a snack either before or after specimen collection . We stored blood samples for hsv-2 testing in labeled vacutainer tubes in a cooler box without ice and transported them to a local laboratory within 24 h for serum processing . Sera were stored at the local laboratory in a freezer at 20 c until transport on ice weekly to the hsv-2 testing laboratory . The logistics of specimen collection, processing and transport for this study were challenging but feasible . In comparison, we did not require guardians to be present because hiv results were not provided to participants . Sampling and dbs preparation were much less obvious to others nearby, helping to safeguard participant privacy and reduce concerns about witchcraft or other conspiracy theories regarding hiv / aids and blood, which circulate in sub - saharan african communities (tenkorang, gyimah, maticka - tyndale, & adjei, 2011). Blood from the finger prick was dropped on five circles on a dbs filter card labeled with the participant s study i d . Some participants may have been dehydrated or not very well nourished, making it difficult to fill all circles, but adequate samples were collected from all participants . Specimens were dried, stored with a desiccant, and transported to the testing lab at ambient temperatures . Dbs samples are easy to store and transport and require no processing or refrigeration (see table 1 for a summary of the study procedures).table 1comparison of biomarker testing procedures in two research studieskenyazimbabweethical considerationswritten guardian consent if <18/adolescent assentwritten guardian consent / adolescent assentresults disclosed to guardian and adolescentno results disclosedreferrals for carenot applicablecollecting and analyzing biological samples methodvenipuncturedried blood spot hiv testrapid testrapid test hsv testkalon elisafocus elisafeasibility of collection methods time20 min3 min temperature for specimen transportcooler with no iceambienttesting costsreference labreference lab bench fee, supplies, storage, labor us$5,784bench fee, supplies, labor us$8,000field site lab sample processing, labor, supplies, storage us$3,611additional fee us$480 other supplies us$100 transportation us$375 total us$9,919total us$8,480 elisa enzyme - linked immunosorbent assay comparison of biomarker testing procedures in two research studies elisa enzyme - linked immunosorbent assay given the experience we have described, we conclude that it is feasible to obtain hiv and hsv-2 biomarker data for adolescent hiv prevention intervention studies . In particular the us pepfar program has accomplished considerable staff training in sub - saharan africa; it has also stimulated the development of well - equipped laboratories, as well as useful tools for testing and protocols for improving test sensitivity, specificity, and cost (stringer et al ., 2006). In addition, it has stimulated the development of national guidelines (particularly for adults) for the disclosure of results . For hiv incorporation of hsv-2 serology results poses considerable challenges, particularly if results are disclosed to participants . Based on our experience, there are two reasons why we do not recommend disclosing hsv-2 serology results to adolescent human subjects in sub - saharan africa . First, in populations with low or moderate prevalence of infection, as might be expected for adolescents, the potential for false positive test results is substantial in combination with relatively minor imperfections in test specificity . Hsv-2 infection is often a silent disease, insofar as only 1025% of people with antibodies for the condition are aware that they have genital herpes (fleming et al ., 1997; leone, fleming, gilsenan, li, & justus, 2004; sizemore, lakeman, whitley, hughes, & hook, 2006). Adolescents without symptoms may deny ever having had sexual intercourse3, further complicating the clinical picture and raising concerns about false positives with the potential for psychological and social harm related to participation in the study . Second, local departments of health and sti authorities in sub - saharan african countries were much less familiar with hsv-2 pathology and treatment than with hiv and some other stis . Even in cases where subjects acknowledge sexual exposure or genital herpes symptoms, local health clinics may not be prepared to provide appropriate treatment or care . Given these vexing problems, the wisdom of disclosing hsv-2 results to adolescents after testing within a clinical prevention trial is questionable . In particular, decisions about whether to inform adolescent participants in prevention trials about hsv-2 test results should be made only after prevalence data are available and the predictive values of the test can be accurately assessed . In light of the feedback from the nih study section that raised concerns about possible stigma and abuse of hiv - positive participants in zimbabwe, as well as our own concerns, we recommend that more research be conducted to study the consequences of disclosure to adolescents in sub - saharan africa . A review of the literature indicates that sub - saharan adolescent prevention studies with biomarker outcomes have taken a variety of approaches to disclosure . In some studies, counselors disclosed participants hiv results immediately after rapid testing (baird, garfein, mcintosh, & ozler, 2012; dalal et al ., 2012; medley et al ., 2012). In other studies, participants could choose to receive their results after testing, either immediately or a few weeks later (amornkul et al ., 2009; jewkes et al ., 2006; ross et al ., 2007). In still other studies, the research did not provide test results but voluntary counseling and testing (vct) was made freely available for study participants (birdthistle et al . None of these studies, however, examined whether adolescents experienced any untoward outcomes with testing and disclosure, whether hiv positive youth enrolled in care or took steps to prevent transmission, whether hiv negative youth benefited from prevention counseling and engaged in subsequent testing, or if adolescents and their guardians felt it was appropriate to learn their status in the context of a research study . From our kenya study, the problem of stigma did not appear to be a major obstacle to adolescent testing and disclosure in our study area . Perhaps this is because hiv prevalence is among the highest in the country, and few families have been unaffected by hiv / aids . Moreover, high hiv prevalence has drawn research, education, and hiv counseling and testing campaigns to the area . For example, in our kenya clinical trial, more than 50% of participants (all in grade 7 and 8) reported that they had previously been tested for hiv, mostly through a home - based testing initiative of the kenya medical research institute (kemri) and us centers for disease control and prevention (cdc). Although our study provided hiv test results to youth, we relied on phlebotomist / nurse counselors to refer youth and their guardians for care, following national guidelines . The onus of making contact with the clinic was left up to the individual and was not tracked by the system of care; and follow up to make sure that hiv positive youth accessed care was well beyond the scope of our study . Since it is unknown whether youth experience significant mental trauma and social prejudice, and whether they actually engage in the system of care, we recommend that further research be conducted to examine the perceptions and behaviors of youth and particularly newly diagnosed youth after disclosure of their test results to ascertain the consequences of this aspect of research participation and whether it can be improved . This is particularly important for adolescents, since results are disclosed in the presence of their guardian . Regarding procedures for collecting biomarkers, we conclude that finger - sticks and dbs are overwhelmingly superior to venipuncture in sample collection efficiency and reduced burden on participants and community institutions . Manufacturer validations of dbs for hsv-2 are urgently needed, however, for this procedure to be more widely accessible to researchers . Because blood collection for dbs is minimally invasive, this procedure has excellent potential for widespread acceptability and consequent high participation in school and community research sites . In addition, rapid hiv testing in sub - saharan africa is typically conducted by finger prick, making this the preferred way for trained african health workers and counselors to participate in research data collection (who, 2004). Given the complex decisions required for biomarkers, we recommend that behavioral prevention scientists interested in using biomarker data collaborate from the early planning stages with biomedical scientists who have expert knowledge in hsv-2, as well as hiv, laboratory tests and testing procedures . We found that team members with such expertise provide valuable assistance in selecting qualified in - country laboratories, developing appropriate budgets, protocols, quality assurance and algorithms for interpreting results, and helping research staff to bridge communication with in - country laboratory staff . Further, we recommend that hsv-2 test kit manufacturers determine optimal cut - off standards for sub - saharan populations, and that researchers who conduct hsv-2 testing explicitly define the testing cutoffs they use in publications . There is now a substantial literature documenting validity problems when using manufacturers cutoffs with african populations potentially inflating prevalence findings (gamiel et al ., 2008; ngayo et al ., 2010; van dyck et al ., 2004 this suggests the need for action to improve assay validation practices on the part of manufacturers as well as methodological details from researchers documenting their findings . When conducting collaborative international research, it is important to recognize that alternative ethical systems exist . Thus, for international research we recommend inclusion of team members who are knowledgeable about local contexts . Indeed, successful collaborative research partnerships respect local cultures, values and practices; negotiate effectively within these systems; and incorporate into study designs ethical practices that are appropriate and sensitive to local cultural contexts (e.g., in our case, providing for husband consent for married participants; christakis, 1992; emanuel, wendler, killen, & grady, 2004). Behavioral prevention scientists traditionally have relied on self - reported sexual behavior survey item measures to evaluate adolescent hiv prevention interventions . Our experiences conducting research with orphan adolescents in kenya and zimbabwe suggest that collection of hiv and sti test results even in rural, resource - poor settings in sub - saharan africa is a feasible addition to the behavioral research toolkit . The use of sti biomarkers can greatly improve the validity of findings from adolescent behavioral intervention trials . Research is urgently needed to examine the risks and benefits of hiv testing and disclosure of test results in the context of a research study for adolescents . Further development and implementation of sti biomarker assessment techniques particularly pertaining to using dbs is needed to advance hiv prevention science.
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Stroke is rare in children, however, a common cause of neurological disease, and it is a major cause of death ranks in the top ten in pediatric period11,15). Documented incidence has been reported as 2.5 - 8/100000 children / year12,13). Medical treatment includes maintaining cerebral perfusion pressure with hyperventilation and osmotherapy, barbiturate coma and in case of thrombotic ischemia, thrombolytic procedure, and anticoagulanttherapy . However, if all medical treatment is fail to drop the intracranial pressure (icp) and deterioration of patient progressed, alternative treatments, such as surgical decompression could be mandatory . The common rationale of decompressive craniectomy with or without duroplasty is to let the volume expansion of the swelling brain to extracranial space via removed skull flap and prevent cerebral herniation and secondary damage of brain parenchyme . In case of traumatic brain injury, however, there has not been a definite evidence or standard guideline for decompressive craniectomy for pediatric patient with non - traumatic acute stroke with uncontrolled refractory high intracranial pressure . We will introduce our series about the functional and clinical outcome after decompressive craniectomy to control refractory high icp due to non - traumatic acute stroke in pediatric patient . Between march 2004 and december 2006, decompressive - hemicraniectomy and duroplasty was performed in 5 toddlers and preschool children with non - traumatic, malignant refractory high icp . Any patient have no trauma history, and refractory high icp was diagnosed as clinical evidence of acute stroke, massive infarction or hemorrhage with midline shift, compression of basal cisterns in conventional radiologic examination such as computed tomography (ct) and magnetic resonance image (mri) and, neurological deterioration consisting of decreased level of consciousness or increasing levels of sedation, or somnolence or stupor compared with baseline status at admission17). And all operations were performed by one pediatric neurosurgeon and all patients received treatment in pediatric intensive care unit of one medical center . In all patients, ct or mri blood sugar, body temperature, ventilation / oxygenation, blood volume and trans - cutaneous oxygen saturation, arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit . Clinical and neurologic status was evaluated with the glasgow coma scale (gcs). To reduce icp, hyperosmolar therapy craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal, parietal, and temporal bone . A large skin flap was lifted from the skull with meticulous blood control, multipleburr holes were made, large bone flap removed . In the temporalregion, the craniectomies were extended toward the floor of the middlefossa to maximize decompression . Multiple openings in the dura were made; a dural patchwas placed and sutured . In every case, intra - cerebral pressure monitor probe was positioned epidural space of bone margin . The bone flaps were stored in a bone bank and then, following improvement of clinical and neurologic status, cranioplasty was performed 3 to 6 months later . Glasgow outcome scale (gos)5), and pediatric cerebral performance category scale (pcpcs)4) (table 2) calculated every 6 months after discharge . In all patients, ct or mri was obtained immediately, after stabilization of ventilation and hemodynamicsat first . Blood sugar, body temperature, ventilation / oxygenation, blood volume and trans - cutaneous oxygen saturation, arterial blood pressure was continuously monitored and controlled in pediatric intensive care unit . Clinical and neurologic status was evaluated with the glasgow coma scale (gcs). To reduce icp, hyperosmolar therapy craniectomy involved the removal of a largebone flap ipsilateral to the involved hemisphere includingfrontal, parietal, and temporal bone . A large skin flap was lifted from the skull with meticulous blood control, multipleburr holes were made, large bone flap removed . In the temporalregion, the craniectomies were extended toward the floor of the middlefossa to maximize decompression . Multiple openings in the dura were made; a dural patchwas placed and sutured . In every case, intra - cerebral pressure monitor probe was positioned epidural space of bone margin . The bone flaps were stored in a bone bank and then, following improvement of clinical and neurologic status, cranioplasty was performed 3 to 6 months later . Glasgow outcome scale (gos)5), and pediatric cerebral performance category scale (pcpcs)4) (table 2) calculated every 6 months after discharge . Five pediatric patients were treated with decompressive hemicraniectomy with duroplasty for non - traumatic and refractory intracranial hypertension after unilateral hemispheric stroke . Four patients were toddler and one patient was pre - school girl . Three were boy and two were girl . Four cases were caused by ischemic stroke, and another one case was hemorrhagic stroke . One case of ischemic stroke, patient had atrial septal defect (asd). And including this patient three cases of ischemic stroke patients were taken angiography; however there was no abnormal findings in angiography . In all patients, conventional laboratory result and special serum anti - body test, including anti - phospholipid antibody were within normal rage . At the time of operation, all patients had a gcs score <8 (median 7, range 6 - 8) and all patients hadunilateral mydriasis . Surgical decompressive hemicraniectomies were performed at a mean of 12 hours (range 4 - 19 hours). In all cases we had performed supratentorial hemicraniectomies . The mean peak icp was 28.6 mm hg (range 25 - 30 mm hg). And the icp of all patients were dropped to normal range within one and half days after operation . There were no surgical complications, such as cerebrospinal fluid leak or wound infection, even after cranioplasty . One of ischemic stroke patient had got a ventriculoperitoneal shunt operation because of post - stroke hydrocephalus . The mean follow - up period was 47.6 months (36 - 68 months).gos and pcpcs for 5 patients had been measured to evaluate postoperative neurological outcome . Based on the regular follow up gos scores, 5 patients had shown satisfied recoveries: 4 had good recoveries (gos score of 5), and 1 had moderate disabilities (gos score of 4). The pcpcs scores also had revealed contend results: 4 patients received scores of 2, and only one patient scored of 3 . A 17-month - old girl was transferred to our emergency department with decreased mentality . Hemiplegia, hyper - reflexia, and babinski's sign were observed on the left side . In general physical examination, she had an ejection systolic murmur . By pediatriccardiologist consultation, patient was diagnosis with asd . Initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . And emergency mri sturdy was performed with mr angiography and diffusion - weighted image . But there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift . After surgery, she was treated in pediatric intensive care unit with be kept intubated for 7 days . A brain ct at the time of post - operative 7 days revealed sustained brain swelling, but midline shift did not noted anymore . The icp dropped to normal gradually within one day after surgery . At the time of postoperative 3-month, she returned for an elective cranioplasty . The bone flap was secured without any complications . At the end of follow - up, 41 months after surgery, this patient maintained a pcpcs of 2, and gos of 5, and she had showed mild left side hemiparesis, mild behavioral disorder and strabismus . A 17-month - old girl was transferred to our emergency department with decreased mentality . Neurological examination revealed a stuporous child without occasional spontaneous eye opening . Hemiplegia, hyper - reflexia, and babinski's sign were observed on the left side . In general physical examination, she had an ejection systolic murmur . By pediatriccardiologist consultation, patient was diagnosis with asd . Initial brain ct scan was suggestive of acute infarction and hemorrhagic transformation atright temporal area . And but there was appropriate for acute cerebral infarction with hemorrhagic transformation with significant mid - line shift . After surgery, she was treated in pediatric intensive care unit with be kept intubated for 7 days . A brain ct at the time of post - operative 7 days revealed sustained brain swelling, but midline shift did not noted anymore . The icp dropped to normal gradually within one day after surgery . At the time of postoperative 3-month, she returned for an elective cranioplasty . The bone flap was secured without any complications . At the end of follow - up, 41 months after surgery, this patient maintained a pcpcs of 2, and gos of 5, and she had showed mild left side hemiparesis, mild behavioral disorder and strabismus . In stroke patient, most common cause of death is due to uncontrolled icp associated with large hemispheric infarctions10,12). When it is present, it is associated with a dramatic increase in mortality . In addition, early icp elevation in patients with large hemispheric infarctions is highly concluded with high mortality16). It means that the one of most important goal of acute phase stroke treatment is control the icp and prevent a secondary damage due to brain swelling . In spite of many clinical and experimental trials on medical therapeutic method, there has been a widely accepted definite guideline for oxygen saturation, temperature, serum glucose level, blood pressure for adult stroke patient1). However, for pediatric stroke patient, even in many of mostly basic and elementary medical guideline has not been established yet14). To prevent and control icp, thrombolytic therapy such as clot lysis has an accepted role in acute stroke treatment . In adult stroke patient group, there are antithrombotic management of adult ischemic stroke protocol and guideline published by the american heart association1), such as intravenous tissue plasminogen activator, intra - arterial thrombolysis and oral administration of aspirin . But, for pediatric stroke patient, there is no guideline of this useful treatment method either14). To the best of our knowledge, the first report about the benefits of decompressive hemicraniectomy in pediatric patient was described by carter et al.3) at 1997 . However, from this time, only a few report deals with decompressive craniectomy for pediatric patient were published . In adult acute stroke patient group, the effect of decompressive craniectomy also has been established . Vahedi et al.21) reported that in a meta - analysis combining the data of 93 subjects from three small, randomized, controlled trials, mortality for conservative management in adult ischemic stroke patients with early brain edema was 71% as compared with 22% for decompressive surgical intervention . Nowadays, some case reports and review has been published favorable outcome after decompressive surgery in childhood acute stroke2,17). It is not often however in some article, decompressive craniotomy have been regarded as emerging procedure can save life, for pediatric stroke patient6,11), even guideline for craniectomy was introduced11). In point of fact widely accepted proper operation timing is immediately after signs of herniation and a midline shift which was defined as a more than equals 5-mm contralateral shift of the midline structures on the preoperative cranial ct scan on septum pellucidum level, compressed basal cisterns, or uncal herniation--had to be present on cranial ct had been noted . Recent studies in adult stroke group have shown that this procedure not only reduces mortality but also improves neurological functional outcome . This procedure may be life - saving if done early in cases of impending herniation, is associated with good outcomes in pediatric stroke patients9,11). Maybe if operation was performed earlier, it may minimize the vicious circle of brain swelling, increased icp, ischemia, and infarction12). In our series, four toddlers and one pre - school girl with refractory high icp due to non - traumatic, ischemic and hemorrhagic stroke four patient received pcpcs score of 2 (mild disability) and only one received that of 3 (moderate disability) after 3 years later post operation . These results showed that decompressive craniectomy cannot only play a rescuer therapeutic role but also guaranty acceptable clinical outcome in the treatment regimen in non - traumatic, refractory high icp in pediatric field . In some articles, decompressive aghakhani et al.2) report that decompressive craniectomy was performed in pediatric patient with malignant intracranial hypertension due to infectious encephalitis . In adult patient group, there had been not infrequently reported that malignant intracranial hypertension due to encephalitis treated with decompressive craniectomy . Now, even in case of pediatric patient with infectious disease in central nerve system, decompressive craniectomy could be considered as life - saving - treatment options . Gordon et al.7) report that pediatric stroke can affect not only patient itself, but also his or her whole family . Over half of stroke survivors have suffer from decrease in quality of life and affects the entire family, and relates to both neurological deficits and psychosocial factors11). Hence, if all medical treatment is fail to drop the icp in non - traumatic pediatric acute stroke patient, without hesitation, a decompressive craniectomy could be regarded as next step treatment option . In this study, we describes the young pediatric patients have undergone decompressivehemicraniectomy for a non - traumatic acute hemispheric stroke . It also shows that decompressive hemicraniectomy can be lifesaving and can besafely performed in toddler and pre - school children . And young children can get a survival and relatively independence and family satisfaction.decompressive hemicraniectomy should be considered as an alternative therapy for patients with life - threatening brains welling refractory to medical management.
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This was a double - blind, randomized, crossover trial in accordance with the declaration of helsinki and good clinical practice . The primary objective was to assess albuminuria during different treatments compared with that with placebo; secondary objectives were to assess effect on 24-h blood pressure, glomerular filtration rate (gfr), biomarkers, and raas components . After informed consent, patients attended a screening visit comprising laboratory tests and evaluations of inclusion / exclusion criteria . Slow - release furosemide in a fixed dose (mean dose 109 mg/24 h, range 60360 mg/24 h) was prescribed to prevent blood pressure elevation and fluid retention . Patients used an electronic blood pressure device (ua-779; a&d instruments, abingdon, u.k .) To measure home blood pressure throughout the study . Change in uaer (percentage) versus placebo during treatment with 300 mg aliskiren daily, 300 mg irbesartan daily, or the combination (p <0.001 vs. placebo for all treatments). From a list of prescreened candidates, 41 patients were screened for study participation (supplementary fig . 1, available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-0168/dc1). Nine of these were screen failures mainly due to albuminuria levels below the randomization requirement . Of the 10 randomly assigned patients who left the study before completion, 2 died, 1 was lost to follow - up, 4 had an adverse event that led to exclusion (diarrhea, severe hypertension, recurrent urinary tract infection, and dizziness), and 3 withdrew consent . Twenty - six patients had the primary end point, albuminuria assessed after randomization, and were included in the final analysis; the remaining six patients dropped out shortly after random assignment and were not included in the final analysis . After washout, patients attended a randomization visit before 2 months of treatment with placebo, 300 mg aliskiren once daily, 300 mg irbesartan daily, or the combination of the two, in random order . Patients with type 2 diabetes (world health organization criteria) aged 3080 years were eligible for randomization with baseline urinary albumin excretion rate (uaer)> 100 mg/24 h, hypertension (baseline office blood pressure> 135/85 mmhg), and baseline gfr> 40 ml / min per 1.73 m. exclusion criteria included major cardiovascular disease (within 6 months), heart failure (new york heart association class ii rather, we used active washout: during the first 14 days of all treatments, every patient received 150 mg aliskiren daily to avoid risk of hypotension or a drastic increase in blood pressure in the switch from placebo to combination treatment or vice versa . During the last 3 days of each treatment, patients collected three consecutive 24-h urine samples for assessment of geometric mean uaer . At the last day of each treatment period, patients attended our clinic for assessment of gfr and mounting of standard takeda 24-h blood pressure devices (tm2421, version 7; a&d medical, tokyo, japan). Measurements were performed every 15 min from 7 to 23 h (daytime) and every 30 min from 23 to 7 h (nighttime). Urinary albumin and creatinine concentrations were determined on a turbidimetric hitachi 912 system (roche diagnostics, mannheim, germany). Samples for prorenin, plasma renin activity (pra), high - sensitivity pra (hs - pra), immunoreactive plasma renin concentration (ir - prc), angiotensinogen, ang i, ace activity, ang ii, and aldosterone levels were determined after 30 min of supine rest, and the plasma was frozen after centrifugation (80c). Raas components and biomarkers were measured at baseline and at the end of treatment periods . Biomarkers of inflammation, endothelial dysfunction, and cardiovascular risk were measured: high - sensitivity c - reactive protein (hs - crp) (enzyme immunosorbent assay [eia]; dako, glostrup, denmark); serum soluble vascular adhesion molecule-1 and serum soluble intercellular adhesion molecule-1 (eia; diaclone, besanon, france); plasma plasminogen activator inhibitor-1 (hyphen biomed kit; andresy, france); serum nh2-terminal - probrain natriuretic peptide (eia kit; biomedica, wien, austria); fibrinogen (immunoturbidimetry); and plasma asymmetrical dimethyl arginine (high - performance liquid chromatography). Total renin concentration, prorenin concentration, plasma angiotensinogen, pra, and biomarkers of inflammation and endothelial dysfunction were measured using methodology described previously (4). Ir - prc was measured with an immunoradiometric kit (renin iii; cisbio, gif - sur - yvette, france). Hs - pra was measured by in - house radioimmunoassay of ang i formed during incubation of 25 l plasma and 50 pmol sheep angiotensinogen for 3 h at 37c in a total reaction volume of 100 l . The assay was double calibrated against ang i and the international reference preparation of renin, 68/356, from nibsc (hertfordshire, u.k . ). Plasma ang i and ang ii were measured using in - house radioimmunoassays and ethanol extraction of plasma samples . Ace activity was determined using a commercial radioenzymatic assay (ace direct; bhlmann - laboratories, schnenbuch, switzerland). Randomization was blinded to all investigators, and the study drugs were packed and labeled before delivery to the site . It was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect (uaer) if the true difference was 15% . This was based on the assumption that intrasubject coefficient of variation for the uaer was 13% . The log - transformed values of uaer were analyzed by a proc mixed model with sequence, treatment, and period as fixed factors and subject (nested in sequence) as a random factor . For 24-h blood pressure data, daytime average, nighttime average, and 24-h average values for systolic blood pressure and diastolic blood pressure were analyzed using a proc mixed model with sequence, treatment, and period as fixed factors and subject (nested in sequence) as a random factor . Extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . Gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data . A two - sided p value <0.05 was considered significant the correlation between changes in albuminuria and changes in hs - pra or ang ii were assessed by a nonparametric spearman correlation coefficient . Correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment (aliskiren, irbesartan, and aliskiren / irbesartan combination) and for all active treatments combined . Fractional clearance was calculated using urine samples collected during gfr measurements (urinary albumin excretion / serum albumin concentration gfr), and log - transformed levels were compared using a paired t test . Statistical analyses were performed using sas (version 8.2 or higher; sas institute, cary, nc) and spss (version 14.0; spss, chicago, il). It was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect (uaer) if the true difference was 15% . This was based on the assumption that intrasubject coefficient of variation for the uaer was 13% . The log - transformed values of uaer were analyzed by a proc mixed model with sequence, treatment, and period as fixed factors and subject (nested in sequence) as a random factor . For 24-h blood pressure data, daytime average, nighttime average, and 24-h average values for systolic blood pressure and diastolic blood pressure were analyzed using a proc mixed model with sequence, treatment, and period as fixed factors and subject (nested in sequence) as a random factor . Extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . Gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data . A two - sided p value <0.05 was considered significant . The correlation between changes in albuminuria and changes in hs - pra or ang ii correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment (aliskiren, irbesartan, and aliskiren / irbesartan combination) and for all active treatments combined . Fractional clearance was calculated using urine samples collected during gfr measurements (urinary albumin excretion / serum albumin concentration gfr), and log - transformed levels were compared using a paired t test . Statistical analyses were performed using sas (version 8.2 or higher; sas institute, cary, nc) and spss (version 14.0; spss, chicago, il). Baseline demographic data are shown in table 1 . Primary and secondary objectives were met . During placebo treatment geometric mean uaer was 258 (range 842,361) mg / day, mean sd 24-h blood pressure was 140/73 15/8 mmhg, daytime blood pressure was 144/76 17/9, and nighttime blood pressure was 130/68 17/9 . Gfr was 89 27 ml / min per 1.73 m and mean serum creatinine was 88 demographics of the 32 randomly assigned patients with type 2 diabetes, hypertension, and albuminuria and the 26 included in the final analysis data are means sd, n, or mean (range). Aliskiren treatment led to a significant reduction in albuminuria by 48% (95% ci 2762) compared with placebo (p <0.001) but not significantly different from irbesartan, lowering uaer by 58% (4270) (p <0.001 vs. placebo). Combination treatment reduced albuminuria by 71% (5979) (p <0.001) compared with placebo, significantly more than with either monotherapy (p <0.001 and p = 0.028). The relative difference between aliskiren and combination treatment was 31% . To adjust for treatment - induced changes in gfr and the potential influence on albuminuria reduction, we calculated fractional clearance, which was reduced by 46% versus placebo during aliskiren treatment (p = 0.021), by 56% versus placebo during irbesartan treatment (p = 0.002), and by 67% versus placebo during combination treatment (p = 0.001). Systolic / diastolic 24-h blood pressure was reduced 3/4 mmhg by aliskiren (ns / p = 0.009), 12/5 mmhg by irbesartan (p <0.001/p = 0.002), and 10/6 mmhg by the combination (p = 0.001/p <0.001) versus placebo . A correlation was found between change in albuminuria and change in 24-h diastolic blood pressure during all treatments (p = 0.039). Seated office systolic / diastolic blood pressure was reduced 7/4 mmhg by aliskiren, 6/4 mmhg by irbesartan, and 12/8 mmhg by the combination, all statistically significant compared with placebo, except for diastolic blood pressure during irbesartan treatment . Gfr was significantly reduced 4.6 (95% ci 8.80.3) ml / min per 1.73 m by aliskiren (p = 0.037), 8.0 (12.33.6) ml / min per 1.73 m by irbesartan (p <0.001), and 11.7 (15.97.4) ml / min per 1.73 m by the combination (p <0.001) compared with placebo . Aliskiren significantly reduced hs - pra, ang i, and ang ii by 87, 75, and 52%, respectively, compared with placebo; irbesartan had the opposite effect (table 2). When combined, the activating effect of irbesartan was counteracted by aliskiren, reducing hs - pra, ang i, and ang ii by 88, 78, and 56%, respectively, compared with irbesartan monotherapy . Whereas combination treatment caused a 1,068% increase in ir - prc versus a 279% increase during aliskiren monotherapy and a 178% increase during irbesartan monotherapy, hs - pra was reduced 47% compared with placebo after combination therapy . Pra measured by a conventional method was affected similarly to hs - pra, although the changes were smaller (table 2). The renin - specific activity (renin bioactivity / total renin mass) was 3 and 4% after aliskiren and combination therapy compared with placebo, respectively, thereby confirming that 9697% of renin was aliskiren bound . During irbesartan monotherapy, changes in raas components and cardiovascular biomarkers versus placebo all treatments were administered once daily . Adma, asymmetrical dimethyl arginine; icam-1, intracellular adhesion molecule-1; nt - probnp, nh2-terminal - probrain natriuretic peptide; pai-1, plasminogen activator inhibitor-1; vcam-1, vascular adhesion molecule-1; vwf, von willebrand factor . A significant correlation between reduction in albuminuria and increase in ir - prc was observed for all active treatments combined (r = 0.597, p = 0.0001). There was a significant correlation between changes in albuminuria and increase in ang ii during irbesartan treatment (correlation coefficient 0.486, p = 0.022); no significant correlations were observed in the other treatment groups . Hs - crp was reduced 35% from the placebo level with aliskiren (p = 0.047) and 35% with irbesartan (p = 0.043). Other statistically significant changes from placebo levels were a 6% reduction in soluble intracellular adhesion molecule-1 (p = 0.017) observed with the combination treatment and a 7% reduction in fibrinogen during aliskiren treatment (p = 0.037). No treatment led to significant changes from placebo levels in any of the other cardiovascular biomarkers (table 2). The most frequent adverse events were urinary tract infection (four patients, one male), pneumonia (three patients), and cough (three patients), occurring during different treatments . Anemia and hypomagnesemia were detected in two patients during the combination treatment . Compared with each monotherapy, combination treatment showed an increase in plasma potassium by 0.2 mmol / l (p = 0.036). One patient dropped out during the placebo period after several systolic blood pressure readings> 180 mmhg . Two patients died before the first measurement of albuminuria after the randomization and were not included in the final end point analysis . The first death was that of a 42-year - old obese man (bmi 42 kg / m) with a history of ischemic heart disease, myocardial infarction, and hypertension 4 years before study entry . The patient experienced sudden cardiac arrest, seemingly after a myocardial infarction during aliskiren treatment . The second death was that of a hypertensive, obese, 73-year - old man with diabetes duration of 16 years . The deaths were instantly reported to relevant authorities and were not suspected as being related to any of the drugs studied . Subsequently, home blood pressure measurement frequency was increased from twice weekly to twice daily, and patients were instructed to contact the investigator by direct phone (available around the clock), if any measurement was> 160/100 mmhg . Extra measurements of sodium, potassium, and creatinine were introduced 3 weeks into each treatment period . In this exploratory study, we demonstrated that treatment with 300 mg aliskiren once daily was as efficient in reducing albuminuria as standard therapy with 300 mg irbesartan once daily . When we combined the two treatments at the same doses, the reduction in albuminuria was enhanced . The added antiproteinuric effect with combination treatment compared with aliskiren alone was 31% . Given that the reductions in 24-h systolic blood pressure with aliskiren were unexpectedly small compared with those with placebo relative to 24-h diastolic blood pressure changes and were substantially lower than the office systolic blood pressure measurements in this study, we conducted a thorough review of potential flaws in data collection, storage, device calibration, reporting, and calculation . There was no evidence to indicate that the ambulatory data collection, storage, or reporting was flawed, and the unexpected results could be a chance occurrence . Our study suggested that the combination of aliskiren and irbesartan had an additional raas blocking effect compared with monotherapy because a synergistic increase in ir - prc was observed with the combination, which was related to the antiproteinuric effect, whereas hs - pra was reduced 50% compared with the reduction with placebo . As opposed to the aliskiren in the evaluation of proteinuria in diabetes (avoid) study (5), which showed an additional 20% albuminuria reduction after 24 weeks of treatment with aliskiren compared with placebo added to the maximal recommended dose of losartan and optimal antihypertensive therapy, this is the first study with a head - to - head comparison between aliskiren and irbesartan treatment . No other antihypertensive drugs except for furosemide were allowed in our study, thereby offering a clearer picture of the effect of the two compounds used, compared with the avoid study, in which aliskiren was combined with losartan and a mixture of other antihypertensive drugs . This study is different with regard to patient population, with a lower mean baseline uaer compared with that in the avoid study . In addition, we assessed the effect of renin inhibition on gfr, a measurement that is more precise than the estimated gfr used in the avoid study . Raas blockade is believed to reduce proteinuria through several different mechanisms: the mean transcapillary hydraulic pressure difference, the glomerular surface area, and the size and charge selectivity of the glomerular filter . In diabetic nephropathy several of these variables are abnormal, and raas blockade has been demonstrated to normalize directly measured or estimated glomerular hydraulic pressure (79), to reduce the shunt - like defects in the membrane, at least in part (10), and to restore the charge - selectivity properties of the glomerular membrane (11). Aliskiren is thought to reduce albuminuria by the same mechanism as during treatment with ace inhibitors or arbs . Recently, fisher et al . (12) have shown that aliskiren treatment increases renal plasma flow to a larger extent than the ace inhibitor captopril . The increase in renal plasma flow may be a response to angiotensin at1 receptor dependent reduction of the vascular tone in the efferent arteriole . Reduced vascular tone in the efferent glomerular arteriole could be responsible for the decrease in intraglomerular pressure, leading to the reduction in albuminuria and gfr as demonstrated in our study . Although the combination reduced gfr up to 12 ml / min (95% ci 15.97.4), we interpreted this as an reversible hemodynamic change and not as an indication of nephrotoxicity (13). In fact, it has been shown that an early hemodynamic reduction in gfr can translate into long - term renoprotection (13). When the albuminuria reduction was adjusted during combination treatment for changes in gfr (fractional clearance), it was 11% higher than during irbesartan treatment, a nonsignificant change that was possibly due to a small sample number . Signs of more effective raas blockade were evident from the synergistic effect of combination treatment on ir - prc . This conclusion is based on the fact that renin release into plasma is proportional to the interruption of the permanent negative feedback loop of ang ii on renin secretion (14). Combining aliskiren with irbesartan provided a 12-fold increase in ir - prc, but still with a 50% reduction in hs - pra compared with changes during the placebo period . This renin rise could reflect a high degree of intrarenal raas blockade during combination treatment as compared with that for the monotherapies, as has been suggested in nondiabetic patients (15). The reductions in albuminuria in our study were correlated with the rise in ir - prc, supporting the concept of increased intrarenal raas blockade underlying the additional effects observed during combination treatment . Compared with other studies of dual raas blockade, the rise in ir - prc is higher in dual raas blockade using aliskiren than in dual raas blockade with an ace inhibitor and an arb (5,16,17). Such marked increases in renin during aliskiren treatment have been noted before (18). Apart from reflecting more complete (intrarenal) raas blockade, they may also be due to the detection of prorenin as renin (19) or a change in the renin half - life after its binding to aliskiren (20). Pra was measured both by a conventional method and by a new high - sensitivity assay (hs - pra), which is independent of endogenous substrate variation (21). Because high pra levels confer the risk of cardiovascular disease (22), it will be interesting to evaluate long - term effects of direct renin inhibition in the ongoing aliskiren trial in type 2 diabetes using cardio - renal endpoints (altitude), providing data on hard cardiovascular and renal end points (23). The antihypertensive effect of aliskiren was smaller than that found in previous larger studies (16), although the office blood pressure reduction did not differ from that caused by irbesartan . The ongoing telmisartan alone and in combination with ramipril global endpoint trial (ontarget) investigators (24) concluded that in a cardiovascular risk population, dual raas blockade with the arb telmisartan and the ace inhibitor ramipril is equivalent in reducing cardiovascular events compared with either as monotherapy, although with more frequent adverse events, including renal adverse events . Almost 3,000 of the participating 25,260 patients had microalbuminuria at baseline, and substudies of albuminuria effects are expected . Several short - term studies using dual raas blockade in diabetic nephropathy have shown promising antiproteinuric effects, as reviewed by rossing (17), but the largest study so far (25) did not show additional benefits of a combination of ramipril and irbesartan, compared with ramipril monotherapy, in terms of albuminuria reduction after 20 weeks . The size was, however, sufficient to demonstrate the likely beneficial effect of combination therapy with aliskiren and irbesartan, although we evaluated a surrogate end point . In addition, the discrepancy between 24 h and office blood pressure readings complicates interpretation of the results . Studies evaluating mortality and morbidity are ongoing and will provide further information on dual raas blockade with aliskiren . In summary, we demonstrate an antiproteinuric effect of dual raas blockade with aliskiren and irbesartan in combination compared with either treatment alone in patients with type 2 diabetes, hypertension, and albuminuria . The synergistic effect on ir - prc illustrates a higher degree of intrarenal raas blockade during combination treatment.
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Nanobiomaterials are characterized by constituent particles and/or surface features less than 100 nm in at least one dimension . Starting with photolithography and dry etching in the 1980's to high - resolution electron beam lithography and other technologies in the 1990's, nanotechnology allows for making surface structures for cell engineering and has led to an increasing application in healthcare over the last decades . Nanolayers are used to enhance the surface biocompatibility of polymeric drug delivery systems, control the release of substances such as antibiotics or growth factors, act as gene - delivery vehicles, or serve as robust light emitters for cellular labeling and tracking [semiconductor nanocrystals, quantum dots (qds)]. Nanotechnology is also applied to modify and improve the surface structure in orthopaedic implants to promote their osseous integration . However, there are also side effects of nano- and microparticles in vivo . Micro- and nanoparticles released by friction of articulating partners from artificial joints are a major reason for aseptic implant loosening in orthopaedic surgery and may lead to severe peri - implant osteolysis (particle disease). In addition, nanoparticles can induce or promote allergic or inflammatory reactions or influence hemolysis and blood coagulation [57]. Although the cytocompatibility of a biomaterial is strongly influenced by its chemical composition, surface topography plays a crucial role for cell - surface interactions . Material surface properties have been studied intensively, but still lack from reliable data about cytocompatibility . Especially, the superordinate principles of cellular responses to surfaces with a defined topography are not well known and poorly understood . Because many variables influence cellular interactions to surface structures, it is difficult to draw conclusions and formulate general principles for nano- and microstructured surfaces . This review summarizes recent data of effects by nano- and microstructured biomaterials and particles in vitro designed for orthopaedic application to get a solid framework outlining the critical interactions that govern the cytocompatibility . Because biomaterials in orthopaedics are predominantly applied on bone, this review is focussed on the interactions of osteoblasts and bone - marrow - derived cells with structured biomaterials . Osteoblasts and osteoclasts are mainly responsible for the osteointegration of nanostructured biomaterials in orthopaedics . Osteoblasts derive from mesenchymal progenitor cells which are localized mainly in the bone marrow and periosteum . They are characterized by cuboidal and flat morphology (diameter about 20 m), present a large amount of rough endoplasmatic reticlum and a large golgi apparatus, and are potent to produce osteoid, a collagen i rich matrix . In addition, these mononuclear cells are also responsible for osteoid calcification (hydroxyapatite). Typical marker proteins for osteoblasts are cbfa1/runx2, osteocalcin, osteopontin, osteonectin, bone sialoprotein (bsp), osteoprotegerin (opg), collagen i, and alkaline phosphates (alp). Figure 1 gives a brief summary of the expression of several markers during osteoblast differentiation . Osteocytes act in a paracrine and mechanosensory manner, and can activate osetoblasts and osteoclasts . The latter cell type derived from the hematopoietic line, has multiple nuclei and is responsible for bone resorption . Its ruffled border is flanked by a sealing zone which facilitates local acidification and removal of bony matrix such as ca, h3po4, and h2co3 by endocytosis . Osteoclasts express high levels of tartrate - resistant acid phosphatase (trap) and cathepsin k. the interaction between osteoblasts and osteoclasts is complex . During differentiation, the ostoblast progenitors express receptor activator of nuclear factor ligand (rankl) and macrophage colony - stimulating factor (m - csf) which are strong stimuli for osteoclastogenesis . In contrast, osteoprotegerin (opg) is a potent inhibitor of osteoclasts . Moreover, the interactions between osteoblasts and osteoclasts in vivo are regulated by several hormones and cytokines, including parathyroid hormone (pth), calcitonin, and il-6 . It is generally accepted that the three - dimensional surface topography (size, shape, surface texture) is one of the most important parameters that influence cellular reactions [2, 1119]. Although many studies have investigated cellular reaction to different surface pattern, the significance of macro structure studies on bone cell behavior is questionable since in vivo adhesion structures (e.g., cell membranes, basement membranes) are comprised of much smaller nanometer scale features [20, 21]. The immature bone is characterized by an average inorganic grain size of 1050 nm whereas mature bone has an average inorganic grain size of 2050 nm (25 nm in diameter). Considering these parameters, modern implants for bone application have been designed with a smooth surface at the nanometer level . It was surprising that some of these have induced the formation of peri - implant fibrous tissue and implant loosening in vivo, while other implants with a higher degree of roughness showed significant better osteoconductive properties [2325]. There are various methods to modify the degree of roughness as well as surface energy and topography in orthopaedic implants . Typically applied techniques to enhance the degree of roughness and promote the osteointegrative properties of biometals (e.g., ti, cocrmo, ss) are chemical etching or anodization and also sand - blasting, sputter - coating, and machine - tooling . The lack of knowledge in cellular reaction to nanostructered biomaterials is based to a great extent on the difficulty in varying surface chemistry and topography independently . Moreover, the use of different cell lineages and culture conditions makes it difficult to compare results from different investigators [2631] (table 1). There is also a lack of consensus concerning the proper representation of implant surface topography . One major misunderstanding is the practice of defining a surface by its manufacturing process instead of concisely defining the topographic measurements [17, 33]. Considering these limitations for interpretation, the following review gives an overview of cellular reactions to surface structures of different orthopaedic biomaterials . The first step after exposure of any biomaterial to a biological environment results in the rapid adsorption of proteins to its surface . The composition, type, amount, and conformation of adsorbed proteins regulate the secondary phenomena such as cellular adherence and protein exchange [3537] and also following cellular reactions such as migration, proliferation, and differentiation . The potency for biomaterials to adsorb proteins is influenced by its physiochemical characteristics such as surface energy or hydrophobicity, and is also dependent on the local environment (ph, concentration of ions, composition and functional groups of proteins, strength of solution, temperature) (vroman effect) (figures 2, and 3). For inorganic nanocrystals and microstructured surfaces there are at least two approaches to change their hydrophobic surfaces: a ligand exchange reaction can replace the original hydrophobic surface with bifunctional coupling molecules or an inorganic coating such as silica (1) or an encapsulation of nanocrystals in an amphiphile organic coating (2). The first phase of protein adsorption onto a biomaterial's surface is characterized by the attachment of small rapidly diffusing proteins, followed by a progressive replacement by larger proteins with a high affinity to the substrate . Here, especially proteins with arg - gly - asp (rgd) containing sequences such as fibronectin or vitronectin act as cell receptors and have chemotactic or adhesive properties to bone cells . In addition, these rgd - peptides also have a strong effect on matrix maturation and biomineralization [4648]. After conditioning of a naked biomaterial by protein adsorption, cells attach rapidly on the protein - coated surface . Besides the influence of proteins, the cellular attachement to a nanostructed surface is also influenced by its physiochemical properties, especially by the outer functional groups [30, 50, 51]. Schweikl et al . Showed on self - assembly monolayers that the osteoblast proliferation on hydrocarbon chains, terminated by ch3, was as high as on amino groups (nh2) and hydrophilic oxidized surfaces, but significantly lower on fluorocarbon (cf3) groups . Mller et al . Showed that 3-aminopropyl triethoxysilane (apts) presents amine functional groups which allow for grafting rgd tripeptides and that the rgd - apts hybrid promotes cell adhesion, spreading, and cytoskeletal organization . Here, the zetal potential (differences in potentials between the surface of a tightly bounded layer and a diffuse layer) and the interfacial tension (wettability) of a surface is crucial [54, 55]. It was demonstrated for cpti surfaces that the contact angle (ca), parameter for wettability, increases linearly with the average roughness when the angles were higher than 45, but decreases linearly with roughness when the angle was less than 45 . Recent data examining osteoblast response to controlled surface chemistries indicate that hydrophilic surfaces (high number of polar components) improve cell attachment and matrix synthesis and also the osteogenic potency compared to hydrophobic surfaces [5759]. Compared ti alloys and cocr alloys towards protein absorptive properties and cell attachment with an osteoblast precursor cell line . They found no significant differences between ti alloys and cocr, but significantly greater cell adhesion rates for the ti implants and concluded that cell adhesion is a result of higher hydrophilicity of ti alloys . In contrast, other data showed that a low degree of wettability promotes protein adhesion and also cellular attachment to a biomaterial, and mller et al . Found no direct correlation between the wettability of the material surface and the osteoblast attachment and proliferation rate . Also qu et al . Found no significant differences of cell attachement on various titanium surfaces with different degrees of wettabilities (hydrophobic acid - etched, coarse - blasted large grit acid - etched, hydrophilic modified acid - etched, and modified coarse - blasted large grit, acid - etched) on mg68 cells . Heating (oxygen / atm) or peroxide treatment of biometals result in a thicker oxide layer and a more hydrophilic surface . . Showed that heat - treated titanium surfaces changed the wettability (more hydrophilic) but does not significantly affect the fibronectin and albumin adsorption as well as the initial osteoblast precursor cell attachment in vitro . Emphasized that the rate of protein correlates more with changes in chemical composition than with changes in wettability in metal surfaces . They showed that a preheating of ti6al4v specimen does not only lead to a thicker oxide layer but also results in an enrichment of v and al within the surface oxide . In contrast, post - treatment with butanol after preheating reduces the content of v, but not in al, and significantly increases the rate of fibronectin adsorption up to 2040% . Compared to the cellular attachment phase, the following adhesion phase lasts longer and involves various proteins and molecules (figure 2). As a link between cell and biomaterial, the interactions of a surface topography and serum proteins are crucial for the cytocompatibility of a biomaterial . Especially, the adsorption of adhesion proteins, such as fibronectin and vitronectin, from serum containing solutions and integrin - mediated signaling has been demonstrated to mediate cell adhesion and spreading . It has been shown that nanotube or nanoparticle surfaces created by anodization have promoted osteoblast adhesion up to three times compared to unanodized ti . These results were confirmed by the group of webster and other investigators [6871] who demonstrated that the initial attachment of osteoblasts onto the surface of biometals such as cpti, ti6al4v, and cocrmo is enhanced by submicron to nanometer consistent particles compared to metals composed of respective micron particles . One possible explanation of this phenomenon is the higher amount of particle binding sites for osteoblast adhesion at the surfaces of nanophase metals compared to micron particle size metals . The theory of enhanced protein and cell binding capacities by larger surface areas / roughness degrees was also confirmed for porous ha materials . Another example of the significance of surface structures for protein binding and osteoblast attachment is the helical rosette nanotubes (hrn) which can build self - assembly surface structures . It was demonstrated that a significant change of hrn coverage by heating correlated with the protein - binding and osteoblast adhesion potency in titanium surfaces [73, 74]. It is evident that not only the surface topography influences protein deposition and cell adherence but also proteins and cells modify the surface properties of a defined surface . Based on a surface analysis of the different biometal specimen before and after cell cultivation, we showed previously that a cell attachment and/or protein precipitation increase the roughness in polished biomaterials (steel, ti6al4v, and cocr). For porous coated cocr surfaces, we found only slight and no relevant changes in roughness whereas cell cultivation onto sandblasted ti6al4v lead to a strong decrease in specimen roughness . Both, the increase in roughness after cell culturing in the different biometals and the decrease in roughness of sandblasted ti6al4v could be explained by the dense cellular growth and accumulation of debris in depth of the structured surfaces and/or protein deposition as shown by other investigators [75, 76]. In addition, not only the amount but also the type of protein adsorption by a surface is crucial for cellular adherence and following reactions such as migration and differentiation . As an example, ti surfaces (ra: 0.370.01 m) adsorp fibronectin in higher concentrations compared to albumin, and fibronectin - coated ti surface promoted more osteoblast attachments in comparison to albumin - coated ti surfaces . These results correspond to the data of other authors who showed excellent osteoconductive properties after fibronectin adsorption onto a biomaterials' surface [7880]. Based on irm and tem analysis, the closest distance of cells to a surface (glass) was found to be approximately 10 nm [81, 82]. Historically, results from chicken fibroblasts have lead to a classification of three different types of separation . (1) focal contacts (fc): approximately 1015 nm separation from the substrate under the peripheral regions of the leading lamellae (appearing black in tem). Fc act as an interface between intra and extracellular components and occur linearly beneath the associated cytoplasmic stress fibres [83, 84]. They are tenacious adhesion sites that remain attached to the substratum even when cells are forcibly detached, indicating their function as anchorage structures . (2) close contacts: corresponding to approximately 30 nm separation (broader grey areas in tem). (3) greater separation: corresponding to approximately 100140 nm (white regions in tem). It is evident that not only fc appear soon after cellular attachment but also that (-catenin - positive) adherence junctions occur within 14 hours for grooved ti - based substrates . These observations underline the high significance of an early intercellular communication soon after adherence to a surface . The mechanisms of initial cellular adherence to a surface are different from long - term adherence as shown by a lack of statistical correlation between short - term adhesion (strength of cell attachment and early adhesion) and long - term adhesion (strength of cell - matrix interface) forces [14, 15, 86]. . Showed that the cultivation time has an influence on the long - term adhesion in biometal surfaces according to td (t) = atb, a being independent of b (td: time - dependend adhesion index, a: surface - dependent parameter, b: substrat - independent exponent, 0.5+/-0.03). For polylactides (plla), it was shown on oct-1 osteoblast - like cells that cell adhesion but not the proliferation could be enhanced by nanoscale and microscale roughness compared to smooth surfaces . In addition, there is evidence that fc show a dynamic behavior which allows for cellular migration and motility . Linear plla fibres with length scales of 0.52 m, constructed by electrospinning, have shown cellular contact guidance and enhanced osteoblastic differentiation . Here, cell morphology revealed that cells grown on fibres had smaller projected areas than those on planar surfaces . Also other polymers such as plga have been shown to be effective in enhancing osteoblast differentiation in vitro . Diener et al . Demonstrated on mg-63 osteoblastic cells that fc adhesion was smaller on ti and ss than on collagen - coated glass coverslips and that all fc showed a mobility of focal adhesions . However, anselme et al . Found higher adhesions on ti6al4v substrates than on noncollagen - covered glass samples, and emphasized that substrates with various surface compositions but with the same surface topography did not induce significant differences of adhesion . Based on the knowledge of protein adsorption and its effects on cellular attachment and adherence, a selective surface coating of nanostructured surfaces with rgd or collagen proteins offer a promising solution to improve the number of osteoblasts adhered on artificial surfaces [53, 95102]. Imprinting surfaces technology with deposition of specific protein - recognition sites can help to promote osteoblastic growth and differentiation [103106]. Protein - recognition can be based on a protein - ligand binding and/or electron donor - acceptor interactions or other types of binding forces . Integrin 51 and 5 v3 subunits competitively bind to rgd - sites of fibronectin [107, 108]. Dependent on the surface topography and chemistry of the biomaterial, fibronectin undergoes changes in structure including modulation in functional activity and shift in integrin binding capacity . Based on the data of self - assembled monolayers, it was shown that integrin subunits show selective binding capacities to different terminal groups . Integrin 51 shows a strong affinity to oh and nh2 surfaces, whereas 51 and 5v3 bind also to cooh but show poor binding capacities on ch3 surfaces [109113]. Furthermore, some data show that oh and nh2 surfaces can up - regulate osteoblast - specific gene expression but also matrix mineralization compared with cooh and ch3 functional groups [47, 112]. Cell migration and proliferation is the attachment following phase between the cell and the material surface . It is evident for designing nanostructured implants that cells use the nanotopography of a substrate for orientation and migration [117119]. Although it is known that bone cells align along defined substrate morphologies (contact guidance), the detailed relation between ordered nanotopography and cell behavior remains unknown in detail . For the first time, in 1964 it was shown that convex surfaces enhance cellular overlap, while grooves minimize cellular overlap . As pre - requisite to reach a defined cell colonization during directed tissue formation, structured nanophase surfaces lead to a predictable osteoblast orientation and migration on these surfaces [17, 121, 122]. Interaction between the ecm and associated changes in the orientation of the cytoskeleton are crucial for cell metabolism of cells and morphology due to actin - myosin tension structures . Anisotropic topographies (e.g., topographical grooves, chemically patterned stripes, or curved surfaces of a fibre) are potent to exert morphological as well as physiochemical features on cells at the same time, indicative for the complex environmental influence on cells . Focal contacts are important structures for cellular adherence onto a surface but may also delay migration and mobility of the cells . It was shown that bone - derived cells (mg63 cells) respond to a nanoscale roughness by a higher cell thickness and a delayed appearance of focal contacts . Especially, nanoporous ti - oxide surfaces promote cellular spreading and induce numerous filopods and osteoblastic differentiation [124, 125]. On electrochemically microstructured hexagonal pattern, mg63-cells go inside 30100 m but not in 10 m cavities . Most authors report a parallel orientation of cells cultured on polished (smooth) surfaces [57, 114, 126] (figure 4). Another method to not only enhance cellular adherence but also to promote osteoblastic differentiation and biomineralization of biometals is a surface anodization, for example, by -glycerophosphate sodium and calcium acetate [6671]. Cellular adhesion via fc may strengthen the linkage between cell and ecm and also impair the ability to dynamically remodelling the ecm and influence the migration rate . For collagen - coated coverslips, focal adhesion of mg-63 osteoblastic cells moved with a speed of 60 nm / min, whereas the speed was reduced in ti and more in ss surfaces . Another study on nb2o5-coated polished cpti samples showed that mc3t3-e1-osteoblast migration was fastest on smooth surfaces (ra = 7 nm), whereas adhesion strength, spreading area, and collagen - i synthesis were promoted by intermediate roughness (ra = 15 nm). However, it was surprising that higher degrees of roughness (ra = 40 nm) were rather peaked and reduced the speed of adhesion process in the same study . Besides the surface properties of a biomaterial, the cellular migration rate is dependent on the cell type and its differentiation stage . A higher migration rate is associated with a lower level of osteoblast differentiation . Cells with a low motility are characterized by a strong formation of fc while motile cells form less adhesive structures . It was found that mature osteoblasts spread out and form a greater number of fc when settled on smoother surfaces . Although cellular spreading is higher on smoother surfaces, some data indicate that the alp - expression is higher for rough isotropic surfaces (electro - erosion, acid - etching, sandblasted) compared to smoother substrates (machine tooling, polishing). Considering recent publications, there is no or only week statistical significance that there is a difference between the initial number of adherent cells and following proliferation of cells cultured onto a biometal or ceramic nano-/microscale surface in vitro . However, some authors emphasize that the influence of functional chemical groups for cellular migration and proliferation are stronger than general surface properties such as wettability . Especially a tio2-layer seems to promote cellular growth and proliferation on nanostructured biometals [128, 129]. Other examples for a promotion of cell - to - bone contact in vitro and also in vivo are machine - etched ti - surfaces (e.g., osteotite), defined sand - blasted implants [124, 125, 131], and hydroxyapatite (ha) coatings, for example, by plama - spray techniques [132134]. Recent studies investigating the response of adherent cells to nanography surfaces indicate that different cell phenotypes have different levels of sensitivities [117, 135137]. Here, osteoblasts react to features as low as to the 10 nm dimensions, which is comparable in size to a single collagen fibre . Moreover, the qualitative and quantitative kinetics in gene and protein expression is strongly influenced by topography and physiochemistry of a defined surface . Microporous ha surfaces seem to promote a high number of fc and increased levels of alp but short actin stress fibres compared to nonmicroporous ha surfaces [72, 139]. There is also evidence that ti and ha surfaces can activate early intracellular signalling pathways as shown by expression of relevant molecules such as - and 1-integrin, fak, erk followed by c - jun and c - fos genes for proliferation and alp for differentiation [139, 140]. However, hallgren et al . Found no significant histomorphometric and biomechanical differences between nanopatterned and control implants . . Showed that microfabricated discontinuous - edge surfaces (des), repeated open square boxes with a depth of 10 m, alter osteoblast adherence and migration but enhance cell multilayering, matrix deposition and mineralization when compared to smooth controls . In contrast to our data, anselme et al . Found higher proliferation rates on ss compared to ti6al4v . However, bigerelle et al . Demonstrated that neither material composition nor surface roughness amplitude influence cell proliferation, whereas they found a very significant influence on manufacturing process and surface topography for long - term adherence and proliferation in vitro . Our in vitro results confirm the well known osteogenic in vivo properties of ti implants, which may be based on surface factors observed on its outer tio2-layer [143146]. Mller et al . Demonstrated the ability of osteoblasts to grow into an open - porous ti implant (metal foam) and li et al . Also demonstrated that mc3t3-e1 cells attach to and are able to divide well in the inner surface of a highly porous trabecular ti6al4v implant . Some in vitro studies demonstrated an enhanced total protein and collagen production, as well as increased alp activity of osteoblasts cultured on nanoparticulate metals (cpti, ti6al4v, and cocrmo) indicating advantages for nanostructured surfaces for osteointegration [1, 149, 150]. Based on the data of redey et al ., it can be concluded that the low attachment and collagen production rates are related to a low wettability of a nanosurface . Nanotextured surfaces of ti surfaces prepared by chemical etching have upregulated the expression of bsp and op . As demonstrated by qu et al ., the expression of the bone - associated genes such as alp, oc, type - i - collagen, osteoprotegerin, and glyceraldehyde-3-phosphate - dehydrogenase is promoted by modsla ti surfaces . Some data also suggest that fluoride - modified ti surfaces can stimulate osteoblastic differentiation compared to unmodified titanium surfaces [151, 152]. . Showed in their in vitro experiments that nanophase biometals induce significantly greater calcium and phosphorus deposition by osteoblasts and also allow for calcium and phosphorous precipitation from culture media without osteoblasts in contrast to microphase ti6al4v and cocrmo . Furthermore, the authors found advantages in mineral precipitation without osteoblast for tial4v but no differences in dependency to the type of ti (wrought, microphase, or nanophase). It was evident that the increased calcium and phosphorus mineral content correlated to greater amounts of underlying aluminium content on ti6al4v surfaces . Although some data indicate that nanostructured ti alloys promote non - cell - mediated ca / po4-mineral deposition from culture media compared to cocrmo substrates, the greatest cell - dependend calcium and phosphorus mineral deposition occurred on nanophase cocrmo . It is evident that micropattern collagen films or scaffolds promote not only cellular adhesion but also allow for an osteoblastic differentiation and biocalcification in vitro [153155]. For ha- and dcpp - coated, ti surfaces the ca / p ratio influence the biomineralization rate in vitro . Besides the osteoblast - promoting effects of defined substrates and surface topographies, some data also allocate an inflammatory response induced by nano- or microstructured biomaterials . It was shown in many studies that cell - biomaterial interactions can activate macrophages which results in the synthesis of proinflammatory agents such as tnf, ifn, il-1 and -6, rankl and no [157159]. Some data have shown proinflammatory effects of different biomaterials which increase with the degree of surface roughness . Here, macrophage inflammatory protein-1, tnf, monocyte chemoattractant protein-1, and members of the interleukine and leukotriene family play a crucial role in biometal - induced inflammations [160164]. Most studies report about an enhanced expression of pro - inflammatory cytokines and chemokines by cells attached to rougher surfaces . Some data also indicate that anionic and neutral hydrophilic surfaces increase macrophage - monocyte apoptosis and reduce macrophage fusion to modulate inflammatory responses to implanted materials . However, adverse cellular effects seen with metallic implants may also be attributed to corrosion products or to the separation of metal ions (fe, cr, ni) which may have a major impact on cellular survival and differentiation [166168]. Those studies which suggest that a cell - mediated metal ion release by biometals that did not affect the cell viability or proliferation are characterized by short cultivation periods or other conditions which limit the reliability of data [169171]. Up to date, only few authors report about no significant influence of the cellular adherence and expression of osteoblast proteins by different biometals and surfaces such as alp expression [172, 173]. In contrast to the great opportunity enhancing biocompatibility and osteogenic potency of surfaces applied on bone by nanotechnology, micro- and nanoscaled particles released by friction of artificial joints can induce severe inflammation and may lead to osteolysis and implant failure [174, 175] (figure 5, table 2). There is a wide range in particles size and morphology produced by simulators for artificial joints . Particles released from metal - metal (crcomo alloys) are predominantly chromium oxide particles or cocrmo with varying ratios of co and cr . They show a round to oval morphology and also a substantial number of needle - shaped particles were found during the first circles . Emphasize the importance of particle size as a critical factor in osteoblasts proliferation and viability in vitro . Some data indicate that in contrast to ti - surfaces nano- and mircoparticles induce an inflammatory response although titanium is one of the biometals with the highest degree of cytocompatibility . As shown by miyanishi et al ., the release of vegf may play a crucial role in the pathogenesis of ti - induced osteolysis . Some data indicate that phagocytosis of ti particles is not a precondition for an inflammatory response such as a release of tnf or il-6 in cultured macrophages . It is evident that a binding of the macrophage cd11b / cd18 (macrophage mac-1 receptors / receptor of complement cr3bi, can also bind to icam-1 and icam-2) by integrin - specific antibodies also increased the release of tnf and il-6 in macrophages . This finding also suggests that the complement system plays a role in the pathogenesis of particle - induced inflammation, too . Especially, uhmwpe particles with a size range of 0.11.0 m have been shown to be most reactive for macrophage activation and cytokine secretion in bone marrow cells [179, 180]. However, not only the particle size but also the particle volume (number) is a critical factor for particle - mediated release of cytokines by macrophages . Green et al . Demonstrated for pe that the cell - particle ratios of 1: 100 (size 0.497.2 m) and 1: 10 (size: 0.494.2 m) induced significant stronger release of tnf and il-1 in macrophages . The authors conclude that especially particles in the phagocytosable size range of 0.310 m appear to be the most biologically active ones . The latter statement was also confirmed for silicon carbide (sic) particles and biometals such as cpti, ti6al4v and uhmwpe [184, 185]. Granchi et al . Investigated the in vitro effects of al2o3 and uhmwpe particles in an osteoblast - osteoclast co - culture system . Both particles did not affect either cell viability or tnf and gm - csf release, whereas il6 release was dependent on the particle concentration . Uhmwpe particles increased the release of rankl from osteoblasts and induced large amounts of multinucleated trap - positive giant cells in an osteoblast - osteoclast co - culture system . Also, carbon - based particles with low wear factors such as p25-cvd showed a high degree of cytocompatibility in vitro . Howling et al . Demonstrated on fibroblasts and monocytes that p25-cvd particles <100 nm were significantly less cytotoxic to both cell types than cocr metal wear particles . While the classical water - suspendable nanoc60 nanocrystal is apparently cytotoxic to various cell lines, the closely related fully hydroxylated, c60(oh)24, is nontoxic, thus producing no cellular response . Also, functionalized single - walled carbon nanotubes are nontoxic to cells in culture [198200]. There is evidence that not only particle size and chemical content but also the concentration strongly influence cellular reactions in vitro . Wilke et al . Showed a positive correlation between the release of proinflammatory cytokines (il-6, -1, and tnf) and amounts of ti6al4v - particles (10, 10, 10, and 10 particles / ml) by human bone marrow cells over 2 weeks . Some in vitro data also indicate that ti particles induce a stronger fibroblastic differentiation signal than uhmwpe in monocytes and other cells [182184]. . Showed that particles of high - density polyethylene (hdp) and ti6al4v induced significantly more proinflammatory mediators (il-1, il-6, tnf) and bone resorption compared to al2o3 and zro2 in vivo . Based on these data, it can be assumed that ceramics show a high degree of cytocompatibiltiy . For ha especially, particles with a size <53 m inhibit cellular proliferation, especially in osteoblasts and lead to a decrease in tgf1 and a significant increase in pge2 and ldh concentration, but did not influence the tnf or alp titer in vitro . It could be concluded that larger ha particles may be compatible with bone cells while smaller - sized ha particles can both activate the osteoclasts and decrease the cell population of the osteoblasts in vitro . Numerous variables influence the biocompatibility and osteogenic potency of nanostructured biomaterials in vitro and in vivo . Besides the locotypical environment in vivo or in vitro, the surface structure and the composition of a biomaterial affects cellular attachment, adherence, proliferation and migration, and also differentiation and survival of defined cell types . Here, information about typical parameters such as chemical composition, surface structure (topography, geometry, roughness, particle size), surface energy, hydrophobicity, and the degree of solubility in aqueous solutions of a biomaterial will help to value and grade a defined implant concerning its osteblast promoting potency . Considering recent publications, we could assume some general principles of cytocompatiblity and cell - surface interactions in nano- and microstructured surfaces . (1) wettability of a nanosurface influences significantly protein adsorption, which is a prerequisite of cellular adherence in serum containing solutions . (2) nanostructured surfaces enhance the surface area of biomaterials and promote cellular adherence . (3) the chemical outer functional groups of a nanosurface significantly influence cellular migration, proliferation, and differentiation but direct correlations between distinct parameters and cell functions are not entirely cleared . (4) the formation of fc underly a dynamic process and influence the motility and migration of cells . (5) a higher degree of differentiation is corresponding to a decreased cellular motility . (6) phagocytable particles with a size <10 m induce the strongest cellular response with regard to releasing inflammatory cytokines . (7) although ti has a high degree of cytocompatibility in vitro, phagocytable ti particles can induce a fibroblastic differentiation.
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Six a17 bac and one fosmid library were used to create mt3.5 (table s1). Most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries . Sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping . Separately, 25 gb of illumina sequence was generated using short (375 nt) inserts plus 2.1 gb from a 5 kb mate - pair library, then assembled using clcbio (www.clcbio.com) and soap (http://soap.genomics.org.cn/). Five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library (table s12). Three tissues were used for small rna analysis with ~3 million reads per illumina library (figures s17-s18, table s16, datafile s9). Six a17 bac and one fosmid library were used to create mt3.5 (table s1). Most were processed by sanger paired - end sequencing of 3 - 6 kb shotgun libraries . Sequences were downloaded in february / march 2009 with scaffolding performed by aligning all bac and fosmid ends against contigs and then anchored and ordered primarily by optical mapping . Separately, 25 gb of illumina sequence was generated using short (375 nt) inserts plus 2.1 gb from a 5 kb mate - pair library, then assembled using clcbio (www.clcbio.com) and soap (http://soap.genomics.org.cn/). Five tissues were used for rna - seq analysis with ~10 million illumina 36 bp reads per library (table s12). Three tissues were used for small rna analysis with ~3 million reads per illumina library (figures s17-s18, table s16, datafile s9).
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Surgical treatment of avascular necrosis (avn) of head of femur include core decompression, osteotomies, nonvascularized bone grafting, free vascularized fibular grafts, hip resurfacing, bipolar hip arthroplasty (bha) and total hip arthroplasty (tha).123 tha is indicated in the young individual in avn with acetabular involvement; however, its role is unclear in cases without acetabular involvement.4 bha was initially limited to be used in hip osteoarthritis, nonunions and acute neck femur fractures.56 bateman7 and giliberty8 were first to use bha in ficat stage 3 avn based on the hypothesis that acetabular floor retains a regenerative property, which regenerates bone in the subchondral region, if stimulation in the form of weight bearing is given through an accurately fitted cup and theorized that preferential motion at inner bearing will decrease the cartilage erosion . Papers have been published with both satisfactory91011121314 and unsatisfactory results using bha.5151617 the main limitations sighted are migration of the outer cup (secondary to the acetabular erosion) and unpredictable pain relief.3514 incidence of groin pain varied from 11.5% to 42%181920 and incidence of acetabular erosion and protrusio is reported to range from 0% to 45%.1819 the activity of the patient and the duration of followup determine the erosion of acetabulum and appearance of symptoms.21 however, the main issue seems to be motion between the outer cup and the acetabulum, a nonconcentric acetabulum and particulate wear.16222324 to decrease this motion between the outer cup and the acetabulum a gentle reaming of the acetabulum with insertion of tight fitting acetabular cup can be done as suggested above by bateman and giliberty.78 this is thought to decrease the incidence of groin pain and the acetabular erosion and also revision secondary to these issues.2526 in this retrospective series, we have assessed the midterm outcome of bha in young adults (age <60) with ficat stages 3 and 4 avn . A retrospective study with a prospective followup was planned . 80 consecutive patients below 60 years of age (96 hips) were operated by the senior author between 1995 and 2010 using bha with tight fitting cup technique . All patients were males with a mean age of 42 6.44 years (range 30 - 59 years). Out of a total of 96 hips, 76 were diagnosed as idiopathic avn, 8 were posttraumatic avn while 12 were post alcohol consumption avn . Ficat's classification system was used based on anteroposterior (ap) and lateral hip radiograph.26 clinically, the indication for surgery was pain with progressive inability to carry out daily activities . Patients with radiographs showing no or minimal acetabular involvement were included to undergo the procedure, while cases with advanced osteoarthritis with ostephytosis or protrusio were excluded . Surgeries were done in the lateral decubitus position using the posterolateral approach under epidural anesthesia . The hip joint capsule was excised if diseased . In our study, intraoperatively on gross examination, all the 96 hips (ficat stage 3 and 4) were found to have mild to moderate acetabular involvement as per steinberg et al.2 the cartilage was graded grossly as grade 0 (normal) if no abnormalities were observed; grade i (mild degeneration) if there was superficial fibrillation and slight irregularity of the surface; grade ii (moderate degeneration) if there was moderate fibrillation, alteration in color and consistency and thinning of cartilage without complete erosion to bone . Gentle superficial reaming of the acetabulum was done making the acetabulum concentric for a tight fitting bipolar cup [figure 1a]. The trial cups should fit snuggly enough so that when the hip is taken through a range of motion, no gross movement should be appreciated between the outer cup and the acetabulum . This assures that the prosthesis is tight fitting and movement between the outer cup and the acetabulum is avoided . During the process of making acetabulum concentric, sometimes subchondral bone was exposed . In these cases, pulse lavage of the prepared acetabulum was done to remove all debris before putting the bipolar cup . The size of the cup was equal to the size of the tight trial cup chosen . We did keep instrumentation for total hip replacement (thr) ready intraoperatively; however, we did not have to do thr in any of our selected cases . (a) peroperative photograph showing concentric acetabulum after superficial gentle reaming with the large hand reamer . (b and c) x - ray taken in neutral and abduction showing movement taking place mostly at inner bearing self - centering cup with 28 mm inner head and uncemented stems were used in 60 hips (zimmer, warsaw, usa, biomet, warsaw, usa). In the remaining 36 hips, normocentric hemispherical cups with 26 mm inner head and cemented stems were used (inor, mumbai, india and rch, mumbai, india). The patients were allowed ambulation with support of a walker or crutches on the 5 postoperative day and were advised to use the same for a period of 4 - 6 weeks . All patients were called for a final followup for clinical and radiological assessment . The clinical evaluation consisted of recording the details of thigh or groin pain, range of movements at the hip (patient was asked to sit cross legged on the bed), limb length discrepancy and harris hip score (hhs). The radiological evaluation comprised of looking for subsidence of the stem, recording of any evidence of stem loosening in the form of a radiolucent line surrounding the stem and superior or medial migration of the cup . Ap x - rays were taken with the patient lying supine with the hip in neutral and abduction position to check for movement in inner and outer bearing as shown [figure 1b and c]. The femoral component subsidence was evaluated by measuring the distance between the superior margin of the greater trochanter and the shoulder of the stem.3 medial migration of the bipolar cup was determined from the distance between a line perpendicular to kohler's line and center of the outer head, whereas superior migration was indicated by a change in distance between the inter teardrop line and the centre of outer head means centre of the circle that is calculated from the circumference of outer cup of bipolar prosthesis that is easily outlined on the radiograph.3 all radiographic measurements were done by either of two authors on ap radiograph of the hip and were compared to the immediate postoperative radiographs . A subgroup analysis was also done, by dividing the cohort according to ficat's class, to evaluate the effect of acetabular involvement on the outcome of the procedure . The patients were allowed ambulation with support of a walker or crutches on the 5 postoperative day and were advised to use the same for a period of 4 - 6 weeks . All patients were called for a final followup for clinical and radiological assessment . The clinical evaluation consisted of recording the details of thigh or groin pain, range of movements at the hip (patient was asked to sit cross legged on the bed), limb length discrepancy and harris hip score (hhs). The radiological evaluation comprised of looking for subsidence of the stem, recording of any evidence of stem loosening in the form of a radiolucent line surrounding the stem and superior or medial migration of the cup . Ap x - rays were taken with the patient lying supine with the hip in neutral and abduction position to check for movement in inner and outer bearing as shown [figure 1b and c]. The femoral component subsidence was evaluated by measuring the distance between the superior margin of the greater trochanter and the shoulder of the stem.3 medial migration of the bipolar cup was determined from the distance between a line perpendicular to kohler's line and center of the outer head, whereas superior migration was indicated by a change in distance between the inter teardrop line and the centre of outer head means centre of the circle that is calculated from the circumference of outer cup of bipolar prosthesis that is easily outlined on the radiograph.3 all radiographic measurements were done by either of two authors on ap radiograph of the hip and were compared to the immediate postoperative radiographs . A subgroup analysis was also done, by dividing the cohort according to ficat's class, to evaluate the effect of acetabular involvement on the outcome of the procedure . The mean followup was 7.52 1.80 years (range, 4 - 16). The hhs improved from a preoperative value of 39.33 6.11 (30 - 54) to 89.12 6.68 (74 - 96) (p = 0.0001, paired t - test) in the postoperative period . According to hhs grades, the final outcome was excellent in 52, good in 28 and fair in 16 hips . Hip and groin pain was reported in four hips (5%) but this did not limit activity in any of the patient [table 1]. 22 hips had flexion deformity of approximately 3 - 5 with a final range of flexion of 100.2 8.82 (85 - 116). Subsidence of the femoral component was seen in 8 hips (<5 mm); however, these hips were asymptomatic . Shortening of 1 cm was noted in 10 limbs, and 1.5 cm shortening was noted in 1 limb . There was no incidence of hip dislocation or dissociation of the bipolar cup and no case required revision . On ap neutral and abduction x - rays there was no appreciable movements noted between the outer cup and the acetabulum in any of the case . There was medial migration in 5 hips and proximal migration in 12 hips, but none more than 2 mm and all were asymptomatic . Comparison of results with various studies on subgroup analysis between the ficat stages 3 and 4 [table 2] it was noted that hips with ficat stage 3 were younger at the time of surgery and also had better final outcome in terms of range of motion . The final hhs score was however not statistically different in both groups indicating good results even in cases with acetabular involvement . None of the ficat stage 3 patients had any groin pain or femoral subsidence at the final followup while 4 cases of groin pain and 8 cases of femoral subsidence were seen in ficat stage 4 group . Figures 2 to 5 show case series with final result of four patients in our series . Comparison between the ficats 3 and 4 subgroups x - ray right hip joint anteroposterior view in a 35 year old male showing (a) preoperative radiograph with avascular necrosis right hip secondary to fracture neck femur . Also showing implant in situ (b) 4 years postoperative radiograph of the same patient treated with bipolar hip arthroplasty . (c) 16 years followup radiograph of the same patient without any loosening or migration and with subchondral sclerosis (a) x - ray pelvis with both hip joints anteroposterior view in a 40 year old male showing avascular necrosis ficat stage iii in right hip (post alcoholic) (b) x - ray of right hip joint anteroposterior view of same patient showing implant in position at 9 years followup without any sign of erosion or migration . (c) clinical photograph of same patient showing cross legged sitting (a) x - ray pelvis with both hip joints anteroposterior view in a 65 year old male showing posttraumatic avascular necrosis ficat stage iv (see non concentric acetabulum) and implant in situ . (b) x - ray pelvis with both hip joints anteroposterior view of same patient at 9.5 years followup showing well fitting large cup . (c and d) clinical photographs showing range of motion x - ray pelvis with both hip joints anteroposterior view in a 45 year old man with bilateral avascular necrosis of femoral head (a) 6 months followup showing right side bipolar arthroplasty left side ficat stage iv avascular necrosis . (b) at 5 years followup showing well fixed implant with large cup well placed in acetabulum surface replacement is an option in younger people with avn, but has limited indications and is a demanding procedure with high cost.27 the results published by for surface replacement are not uniform and a longterm followup is lacking.272829 tha is the method of choice for the treatment of advanced avn of the femoral head;53031 however, its overuse may lead to increased wear and need for early revision in young adults.14 various studies have shown that functional utility of tha reduces to 80% at 10 years, 33% at 16 years, subsequently requiring a revision surgery . Alternate bearing surfaces like metal on metal or ceramic on ceramic decrease the wear rate but have their own set of complications (metallosis, squeaking)3233343536373839 and long term results are still awaited.40 when compared with bha, conflicting results have been reported . Chan and shih30 have reported that there was no difference in the incidence of osteolysis, thigh or groin pain, dislocation rates and revision rates between bha and tha . They concluded that in young patients with ficat stage 3 avn, bha may be a useful alternative to tha . Furthermore, bha is less demanding, blood loss is comparatively less5 and revision is easier as compared with the revision of tha as the acetabulum is still intact.41 lee et al.3 have reported 23% outer cup migration rate, 15% gluteal pain and 20% groin pain in bha . Ito et al.15 have reported 42% radiological failures, 42% incidence of groin pain requiring a revision surgery in 25% hip undergoing bha . Similar results were shown by cabanela14 and lachiewicz and desman17 with groin pain and acetabular erosion as the two most important reasons for poor results in bha . Groin pain has been variably attributed to the preservation of diseased joint capsule, to irritation of the subchondral nerve endings of the acetabulum and to the acetabular erosion.1442 in our study, the diseased capsule was always excised to avoid capsular impingement . Furthermore, as nerve endings in the posterior capsule supply the acetabulum, excising it blocks the nerve supply to the acetabulum and thus helps in relieving pain . Poor fitting bipolar prostheses can lead to cartilage necrosis giving rise to groin pain and degeneration.3041 acetabular erosion has also been attributed to a fair amount of movement at the outer bearing due to a wide surface of the cup, which was greater than two third of a sphere.4243 reaming in bha technique creates a better fit that reduces movements between the outer cup and the acetabulum41 and ensures that on weight bearing, movements are transmitted to the inner bearing . In effect, this works like a low friction arthroplasty and results in reduced pain and damage to the acetabular bone stock.41 this also explains the low incidence of acetabular migration and low revision rate in our series . Dudani et al.25 too used the same technique and reported 80% good result at average follow of 7.2 years . Pellegrini et al.19 have reported a higher risk of revision with acetabular reaming while muraki et al.5 concluded that acetabular reaming increases the tendency of superomedial migration . The uniformly good results at the mid term seen in our series may be attributed to the technique and amount (depth) of reaming . Pellegrini et al.19 reamed the acetabulum till a depth when the osseous floor showed punctuate bleeding . We have used a more conservative reaming without breaching the integrity of the subchondral bone, and this is probably why we have low incidence and magnitude of medial migration . On the contrary [figure 2c] the subchondral bone shows sclerosis, asserting the bateman hypothesis regarding the regenerate potential of acetabulum.44 in our series, ten patients showed this sclerosis . Nagai et al.43 in 12 - 18 years old followup study of nonself centering bateman bipolar endoprosthesis for nontraumatic osteonecrosis of the femoral head concluded that the original bateman endoprosthesis was effective in delaying the need for tha for more than 10 years in ficat stage 3 avn of the femoral head . However, the sample size was very small, with 4 out of 12 patients having some groin pain and study limited to ficat stage iii avn only . In another study tsumura et al.45 found no difference in clinical results however incidence of migration in ficat 2 and 3 stage was less when compared with stage 4 . Based on this they recommended bha only for ficat stages 2 and 3 . In our study, we have used bha for both stages 3 and 4 stages . Although the final range of motion was better in ficat stage 3 group, the final hhs was similar in both the groups showing effectiveness of bha with tight fitting cup in both ficat 3 and 4 stages . Thus, even in cases with acetabular involvement this technique can be used with good clinical results and can defer the need for tha . Bipolar hip arthroplasty using tight fitting cup for avn hip has a low incidence of groin pain, acetabular erosion, and revision in midterm followup . This procedure can be used for treatment in young adults with ficat stages 3 and 4 avn of the femoral head to defer a definitive tha . Further large series with long term followups, multicentric randomized studies and reproducibility of results will be needed to establish this method.
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The success of valproic acid (vpa, 2-propylpentanoic acid), a short branched chain fatty acid, for the therapy of absence seizures, partial seizures, tonic - clonic seizures, bipolar disorder, schizoaffective disorder, social phobias, neuropathic pain and for the prophylaxis and treatment of migraine headaches has spurred investigation for its use in the treatment of other conditions . Treatments for encephalopathy secondary to hyperammonemia as a side - effect of vpa treatment remain empirical and restoration of possible hepatic carnitine deficiency remains controversial . While some authors have found a positive correlation between plasma ammonia level and vpa dose or concentration others have found none . Her elevated vpa levels were associated with elevated ammonia levels as expected of a positive correlation . In contrast, later on in the patient's clinical course her raised venous ammonia levels were found to be slowly down - trending in the presence of sub - therapeutic serum vpa . The present case report is about a 25-year - old hispanic female with a history of depression since age of 12 years, bipolar disorder, borderline personality disorder, purging type bulimia and history of suicide attempts at age 12 was admitted to the intensive care unit after attempted suicide by consuming an unknown number of vpa pills . This patient had recently started taking vpa (depakote) as a mood stabilizer at a dose of 250 mg at bed time about 3 days earlier, after a failed therapy . Patient was taking paroxetine 20 mg for depression; this was incremented to 40 mg at the time vpa was started . She reported that the change to vpa made her listless, lowered her mood, decreased her motivation, worsened her depression and caused suicidal thoughts . She overdoses herself on vpa which she took with alcohol . After ingesting the pills she notified her family who then brought her to the emergency room . On admission, her vitals were stable (blood pressure: 118/72 mm hg; temperature: 98.7f; pulse: 82 beats / min; oxygen saturation: 99% on room air). On initial examination she looked disheveled, although drowsy, was oriented to person, place and time . The complete blood count and comprehensive metabolic panel that was recorded initially and subsequently were unremarkable [table 1]. Subsequently, she was administered oral lactulose once and then 1400 mg of levocarnitine orally every 6 h for the next 2 days . Her vpa levels which peaked after admission continued to trend down over the next 4 days, the ammonia levels also peaked subsequent to admission then showed an initial dip followed by a very slow down - trend [figure 1a]. The normalized log - linear plot of vpa serum concentration over time showed a down - trending linear first - order kinetics with a calculated t = 15.08 h [figure 1b] (expected t for depakote: 9 - 16 h following oral dosing regimens of 250 - 1000 mg . The serum ammonia normalized log - linear plot in contrast showed two different decay kinetics: initial sharp down - trend for vpa levels greater than 90.9 g / ml but much slower downtrend for vpa levels less than 90.9 g / ml down into the subtherapeutic range of 8.5 g / ml . Patient continued to complain of lethargy for the next 4 days which subsequently alleviated . At 14 days laboratory values of the patient at the time of admission (a) valproic acid (vpa) and ammonia levels as a function of time (days). Vertical axes: straight line (left): vpa levels; dashed line (right): venous ammonia levels . (b) logarithm of normalized vpa and ammonia levels as a function of time in days . The normalization for each compound, for levels at each recorded time point (x) was to its recorded maximum level (xmax). The normalized log - linear plot of vpa showed linear first - order decay kinetics with a calculated t = 15.08 h (r = 0.994) recognition of symptoms of hyperammonemia remains a challenge especially in psychiatric patients . In a review of 11 case reports of symptomatic hyperammonemia in a psychiatric setting it was noted that acute hyperammonemic encephalopathy in psychiatric patients may present in various ways, sometimes with subtle clinical features . The previously reported spectrum of clinical features included: improvement in mood, worsening of psychiatric symptoms, mild complaints of fatigue and delirium . In our patient, hyperammonemia secondary to vpa overdose caused listlessness, lowering of mood, decreased motivation, worsening depression and suicidal ideation . We observed presence of vpa in serum caused increased venous ammonia levels, which eventually trended down to normal after its cessation . Increasing vpa levels in serum was associated with increasing venous ammonia levels initially [figure 1]. The peak concentration of ammonia noted was likely a decrement from the real peak based on the time to peak of depakote tablets being 4 h (the patients initial vpa levels were measured at 2 h after she allegedly consumed the vpa while the next blood draw was 9 h later). We also made the following observations: (1) raised venous ammonia levels may remain symptomatic for a significant period of time beyond discontinuation of vpa . (2) existence of more than one concurrent mechanism with different decay kinetics in the metabolism of ammonia in presence of high or low concentrations of serum vpa . Unmasking of an inherent urea cycle metabolic disorder could have amplified the differential decay rates of venous ammonia . The possibility of more than one mechanism causing hyperammonia secondary to vpa use is suggested by the different proposed mechanisms of vpa metabolites causing hyperammonemia, these include: (1) propionic acid inhibiting the urea cycle enzyme carbamoyl phosphate synthetase i; (2) valproyl - coa or a closely related compound as a proximate inhibitor of mitochondrial ureagenesis; (3) valproyl - carnitine ester causing a relative carnitine deficiency and (4) 2,4-dien - vpa inhibiting b - oxidation . Although, we cannot speculate on the mechanisms responsible for the different kinetics of decay of ammonia levels in the presence of down - trending vpa levels we can confidently state that non - elevated hepatic enzymes and hyperammonemia despite carnitine replacement indicated that hepatic damage and carnitine deficiency had very little role in causing the slowly down - trending elevated ammonia levels in the presence of subtherapeutic serum vpa levels . Hyperammonemia and psychiatric disturbance secondary to valproate use in our case showed a score of 9 by a naranjo algorithm, indicating strong causality with the drug and placing it in causality category of definite . Our assessment of a high adverse reaction probability scale was based on the fact that there are previous conclusive reports of hyperammonemia secondary to vpa use with similar psychiatric features as presented in this case report, hyperammonemia occurred only after vpa use and eventually trended down to normal levels with discontinuation of vpa, patients psychiatric disturbance correlated with hyperammonemia, the patient symptomatically improved with drug discontinuation, there are no alternative explanation of hyperammonemia and altered mood in this case, the serum levels of vpa were elevated with concentrations known to be toxic and that toxic effects observed were more severe with high dose and less severe when secondary hyperammonemia decreased . This case report suggests that hyperammonemia, which is known to occur in about 50% patients treated with vpa, may have more than one concurrent etiologic mechanism with different decay kinetics which are not related to hepatic damage or carnitine deficiency.
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Intramammary infection (mastitis) is the most common reason for the use of antimicrobials in dairy cows . Antimicrobials have been used to treat mastitis for more than fifty years, but consensus about the most efficient, safe, and economical treatment is still lacking . The concept of evidence - based medicine has been introduced to veterinary medicine and should apply also to treatment of mastitis . The impact on public health the aim of this article is to review current treatments of mastitis during lactation and seek for evidence - based, best practice treatment recommendations for bovine mastitis . Penetration of substances into milk when administered parenterally or absorption and distribution throughout the udder when infused intramammarily (imm) depends on their pharmacokinetic characteristics . These are lipid solubility, degree of ionization, extent of binding to serum and udder proteins, and the type of vehicle . Antimicrobial treatment of dairy cows creates residues into milk, and residue avoidance is an important aspect of mastitis treatment . The activity of macrolides, tetracyclines and trimethoprim - sulphonamides has been shown to be reduced in milk . Selecting a substance with a low minimum inhibitory concentraton (mic) value for the target pathogen the antimicrobial should have bactericidal rather than bacteriostatic action, because phagocytosis is impaired in the mammary gland . However, activity in vitro does not guarantee efficacy in vivo when treating bovine mastitis . Antimicrobial resistance amongst mastitis pathogens has not yet emerged as a clinically relevant issue, but geographical regions may differ in this respect . The biggest problem is the widespread resistance of staphylococci, particularly staphylococcus aureus, to penicillin g . Cure rates for mastitis caused by penicillin - resistant strains of s. aureus seem to be inferior to those of mastitis due to penicillin - susceptible strains . It is not known if this is due to pharmacologic problems of the drugs used, or virulence factors possibly linked to -lactamase gene of the resistant isolates . Using an in vitro -lactamase test for determining resistance to penicillin g of staphylococci before treatment coagulase - negative staphylococci tend to be more resistant than s. aureus and easily develop multiresistance . Mastitis causing streptococci have remained susceptible to penicillin g, but emerging resistance to macrolides and lincosamides has been detected . Antimicrobial susceptibility of coliform bacteria varies but normally is not a limiting factor for therapy . An important question regarding the treatment of mastitis is whether the antimicrobial should accumulate in the milk or in the udder tissue . The target site may depend on the causative agent: streptococci are known to remain in the milk compartment, but s. aureus penetrates udder tissue and causes deep infection (table 1). The advantages of this route are high concentrations of the substance achieved in the milk and low consumption of the antimicrobial as the drug is directly infused into the diseased quarter . For example, concentration of penicillin g in milk after imm administration is 100 - 1000 times as high as the concentration after systemic (parenteral) administration . A disadvantage of the imm administration is uneven distribution throughout the udder and the risk of infecting the quarter when infusing the product via the teat canal . Efficacy of imm treatment varies according to the causative pathogen, with the best therapeutic response being shown for mastitis caused by streptococci, coagulase - negative staphylococci, and corynebacterium spp .. where to target antimicrobial therapy in clinical mastitis due to different pathogens the systemic route of administration has been suggested to be more efficient than imm for the treatment of clinical mastitis as antimicrobials theoretically have better penetration of the udder tissue by this route . However, it is difficult to attain and maintain therapeutic concentrations in milk or udder tissue following systemic administration . Many commonly used broad - spectrum antimicrobials such as oxytetracycline, trimethoprim - sulphonamide and ceftiofur, it is difficult to produce and maintain therapeutic concentrations in the milk . Macrolides would have ideal pharmacokinetics, but clinical studies have failed to demonstrate efficacy when used for the systemic treatment of clinical mastitis . In streptococcal mastitis, spiramycin and tylosin have shown reasonable efficacy . One additional problem for the bovine practitioner is that the recommended dosage for many antibiotic preparations for adult cattle may be too low when pharmacological aspects are considered, but residue studies have been carried out using the approved dosages . Repeated intramuscular injections of large volumes of antibiotics can be irritating and cannot be recommended from the animal welfare point of view . One substance used for systemic treatment is penicillin g, which as a weak acid penetrates poorly into the mammary gland, however, due to the very low mic values of susceptible organisms, therapeutic concentrations can be achieved in milk . Penethamate is a more liphophilic penicillin g formulation and diffuses better than penicillin g procaine into milk . The efficacy of systemic treatment with penicillin g or penethamate has been shown in clinical trials . Combinations of penicillin and aminoglycosides should not be used, as there is no scientific evidence demonstrating a better efficacy for the combination and aminoglycosides are known to produce long - lasting residues . The only type of mastitis where systemic treatment would be clearly advantageous may be mastitis caused by s. aureus . In severe mastitis due to coliform bacteria, parenteral administration of antimicrobials has been suggested to combat bacteraemia . The general benefit of antimicrobial treatment in coliform mastitis has been questioned, but systemic antimicrobial treatment is recommended in cases of severe escherichia coli mastitis with heavy bacterial growth in the udder . Fluroquinolones and cefquinome have shown efficacy in experimental trials and ceftiofur in a clinical field trial . There is no evidence that administering bactericidal antimicrobials to cows with severe coliform mastitis causes the release of massive amounts of endotoxin . Finally, the antimicrobial used for systemic treatment of mastitis must be approved for dairy cattle . For example, penicillin g procaine or fluoroquinolones are not approved for dairy cattle in the united states . Treatment of mastitis should be targeted towards the causative bacteria whenever possible, but in acute situations, treatment is initiated based on herd data and personal experience . Rapid or on - farm bacteriological diagnosis would facilitate the selection of the most appropriate antimicrobial . Treatment protocols and drug selection for each farm should be made by veterinarians familiar with the farm . The use of on - farm written protocols for mastitis treatment can promote judicious use of antimicrobials . Therapeutic response of the cows can be monitored using individual somatic cell count data if available, or using the california mastitis test, and with bacteriological samples in herds with contagious mastitis . In general, the use of narrow - spectrum antimicrobials is preferable (table 2). First choice antimicrobials for treating mastitis caused by streptococci and penicillin - susceptible staphylococci are -lactam antimicrobials, particularly penicillin g. broad - spectrum antimicrobials such as third or fourth generation cephalosporins should not be used as first alternatives for mastitis, as they may increase emergence of broad - spectrum -lactam resistance . Systemic treatment is recommended in clinical mastitis due to s. aureus and in severe cases of coliform mastitis, preferably in combination with imm treatment . Too short a duration of standard treatment is probably an important reason for poor cure rates in mastitis therapy . A longer treatment improves cure rates, and duration of treatment should generally be extended in mastitis caused by s. aureus and streptococcus uberis . Clinical mastitis should be treated for at least three days; this recommended treatment duration is longer than label treatments in many countries . All mastitis treatment should be evidence based i.e., the efficacy of each product and treatment length should be demonstrated by scientific studies . Treating subclinical mastitis with antimicrobials is generally not economical during lactation because of high treatment costs and poor efficacy . In a study with a large number of subclinical mastitis cases, the overall bacteriological cure rate for antimicrobial treatment was 75% and that for no treatment 68% . The marginal benefit applied for streptococcal mastitis only; in mastitis due to s. aureus, antimicrobials were equal to no treatment . Treatment of subclinical mastitis will not affect the incidence of mastitis in the herd unless other preventive measures are taken . Studies on treating cows based on high somatic cell counts have generally shown that no effect on milk production has been achieved in herd problems caused by very contagious bacteria such as s. aureus or streptococcus agalactiae treatment of subclinical mastitis is advised.
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Approximately 40 million people are infected with the human immunodeficiency virus (hiv) worldwide (unaids 2006). Infections by this lentivirus from the family retroviridae are characterized by a long asymptomatic period after host immune defenses control the initial infection . During this subsequent chronic phase of infection, hiv slowly diminishes the host the virus replicates by hijacking the intracellular molecular machinery to transcribe viral rna, and eventually the productively infected cells die (fauci 1988). Over time, the density of virions in the blood stream increases and the immune system functioning becomes progressively compromised, leaving hiv - infected individuals increasingly susceptible to opportunistic infections . Being blood - borne, hiv is transmitted via contact with the blood of an infected individual: through transfusions, needle - sharing, sexual contact or from mother to child during childbirth or breast - feeding . Initially there were concerns that hiv might be vector - borne (e.g., transmitted via mosquitoes) but it has since become widely accepted that such transmission does not occur at any significant level (bockarie and paru 1996). This current belief stems both from epidemiological data and experimental studies that directly examine the potential for hiv transmission via arthropods (lawrence 1987; lifson 1988; bockarie and paru 1996). Why is hiv not vector - borne (throughout this article we use the term vector synonymously with arthropod)? The majority of medical scientists, when asked this question, will offer the following explanations (bockarie and paru 1996): (i) hiv concentrations in the blood are too low during human infection to permit vector transmission; (ii) hiv is unable to survive long enough outside of humans (or primates) for vector transmission; (iii) hiv is not able to replicate within arthropod vectors . Each of these explanations has empirical support (lifson 1988; bockarie and paru 1996) and thus all three are good explanations for the lack of vector transmission in hiv . If most arthropods pick up very little hiv when feeding on humans, and if the level of hiv in (or on) these vectors quickly decays, then no significant vector transmission is expected to occur . The above findings provide a satisfying proximate explanation for the lack of vector transmission in hiv . Given the current characteristics of hiv, these three features of the virus make it unlikely that vector transmission will occur . In this article, we approach this question from an evolutionary perspective . Therefore, rather than taking the characteristics of hiv as given, we are interested in understanding why hiv has evolved these particular features and not others instead . For example: (i) why has hiv not evolved a replication strategy that results in viral concentrations high enough to allow vector transmission? (ii) why has hiv not evolved to be more durable and thus to survive longer outside of humans? (iii) why has hiv not evolved the ability to replicate in arthropod vectors? Asking such questions might strike many readers as strange since much of evolutionary biology proceeds by asking why certain features of organisms are as we observe them rather than by asking why some features are absent . This is only natural, since it is difficult to decide which, among the infinite number of missing features, should be the focus of study . It is useful to view such questions as falling on a continuum, from questions about the lack of traits that are virtually non - existent in all taxa (e.g., why do dogs not have wheeled appendages) to questions about the lack of traits that are common in some populations or species but not others (e.g., why do german shepherds not have curly hair?). Our contention is that, by asking such questions about the absence of some traits, we can gain deeper insight into biology . To quote philosopher arthur eddington (1927), the contemplation in natural science of a wider domain than the actual leads to a far better understanding of the actual . . As in all science, however, not all questions are interesting, and this serves as our primary guide for focusing on some missing features and not others . In particular, we focus on the lack of vector transmission in hiv, because of the profound epidemiological significance of its absence . An arthropod - transmissible form of hiv would clearly exacerbate the already devastating impact of the disease, opening up routes of transmission to groups previously at low - risk (e.g., children). Therefore, it is worth asking why, from an evolutionary standpoint, this has not occurred (weiss 2001). More to the point, if there are conditions under which hiv could have evolved vector transmission we would do well to understand these, not only from the standpoint of scientific curiosity, but also to prevent such an outcome in the future . In this article, we address the question of why hiv lacks vector transmission, both through a consideration of available empirical data and through the construction of mathematical models . Although our results are necessarily speculative, we believe that they shed some light on the evolutionary biology of hiv, and on the evolutionary biology of blood - borne pathogens more generally . There are two broad reasons why a trait of interest (in this case vector - transmission) might not evolve . First, the necessary genetic variation for the trait might arise only very rarely (if at all). For instance, the evolution of rna viruses, such as hiv, could be strongly constrained by the size of their genome (holmes 2003). Second, the necessary form and strength of natural selection might not be present for the trait to evolve, at least over the timescale under consideration . Thus, from an evolutionary standpoint, hiv is not vector - borne because either the necessary genetic variation for such transmission has never arisen, or the necessary selective factors that would make such variants increase in frequency over the relevant timescale have not occurred . Our evolutionary explanation for why hiv is not vector - borne will be sought within these two possibilities . Before beginning to consider these explanations there are two different processes that might result in vector - transmission, and that are often lumped under this single heading . This occurs when the arthropod acts solely as a means of physical transport of viral particles between hosts (e.g., having viral particles in and around mouthparts). This occurs when the virus replicates within the arthropod vector during the time period between feeding events . How likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation? The most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population . Unfortunately, performing such an assay on all genotypes within the population would be next to impossible . Furthermore, if variants capable of vector transmission are selectively disadvantageous, then their frequency in the population might be extremely low . Nevertheless, there are some studies available that take this approach as far as is possible . A second approach to addressing this issue if some of hivs close relatives have evolved vector - transmission, then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened . Below we review both types of evidence, for both mechanical and biological transmission . Mechanical transmission: a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods . Such transmission is often difficult to assess under natural conditions, and therefore most studies have employed artificial experimental setups, using a variety of arthropods including mosquitoes, stable flies, and tabanids (e.g., horse flies and deer flies). For example, hiv was found to be viable for up to 10 days in african soft ticks, and perhaps even up to 14 days (humphrey - smith et al . 1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus . Most research on this topic has stressed the need for large bloodmeal size, however, because hiv tends not to have a very high viral titre during infection in humans (lifson 1988; webb et al . This has led to a focus on arthropods, like ticks, whose bloodmeal sizes are often 70 times larger than that of mosquitoes (humphrey - smith et al . 1993). It has also been suggested that squashing mosquitoes while they are feeding, and subsequently scratching the area (which might result in lacerations) could increase the likelihood of transmission as well (siemens 1987). Other studies of closely related viruses suggest that, in principle, there is no obvious barrier to mechanical transmission of hiv by arthropods . In fact, it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans (eigen et al . At least three other retroviruses can be transmitted mechanically, including bovine leukemia virus, friend murine leukemia virus, and equine infectious anemia virus (foil and issel 1991; humphrey - smith et al . The latter (equine infectious anemia virus) is believed to be a close relative of hiv (mcclure et al . 1988), and epidemiological evidence suggests that vector transmission might play a significant role in its transmission (foil and issel 1991). Interestingly, however, all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv (foil and issel 1991). It has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods (jupp et al . Even if this does occur, however, the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses (foil and issel 1991), again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus . Biological transmission: despite evidence that mechanical transmission of hiv by arthropods can occur, there is no evidence that biological transmission is possible . (1993) found that hiv can remain viable in ticks for up to 2 weeks, they failed to find any evidence that hiv can replicate in these vectors . Evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa (becker et al . 1986), but this has been controversial, and epidemiological evidence is not indicative of vector transmission (noireau et al . Furthermore, experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells (srinivasan et al . In fact, no evidence exists to date that any retrovirus is capable of biological transmission by arthropods (webb et al . 1989; foil and issel 1991; kuno 2004; kuno and chang 2005). It remains unclear exactly why such replication is not possible, but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation (van den heuvel et al . Alternatively, it remains possible that appropriate genetic variation can arise, but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses . The above empirical findings suggest that, in principle, hiv is capable of being transmitted mechanically . In practice, however, the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur (lifson 1988; webb et al . There is evidence of genetic variation for differences in viremia in hiv - infected patients (kanki et al . 1999) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous . Thus, we are forced to ask: why have genetic variants of hiv that induce higher viremia, and thus that transmit mechanically via arthropods, not increased in frequency? On the other hand, there is no evidence to suggest that biological transmission can occur, even in principle . This might be because the relevant genetic variation for such transmission has not appeared (or perhaps is not possible). Alternatively, perhaps such variation does occasionally arise, but that such strains are acted against by natural selection . In this section we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv . Mechanical transmission: to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . At present hiv infection is increasing in prevalence in the human population (unaids 2006). The simplest description of this is exponential growth in the number of infected individuals; where y(t) is the number of hiv - positive individuals at time t, and r is the per capita growth rate . Although the rate of increase of hiv appears to be slowing in recent years, exponential growth nevertheless provides a useful benchmark for its initial spread in humans . The per capita growth rate will depend on various properties of hiv, including its mode of transmission . To derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters, we first need to specify a model for the epidemiological dynamics . This model will allow for both sexual transmission of hiv as well as insect transmission, and therefore it will also track the number of insects carrying hiv . Using w(t) for the number of insects carrying hiv at time t, and y(a, t) as the number of hiv - positive people who were infected a years ago, we specify the dynamics as with boundary condition . In equations (2), v is the population size of insects free of hiv, a is the insect - biting rate, b1 is the probability of an insect picking up hiv, given it feeds on an infected human, and is the per capita loss rate of infected insects (which subsumes both insect mortality and the decay of hiv stores in or on the insect). Note that, although many insects display a characteristic time lag between feeding events, for simplicity we have ignored this . Also note that, for simplicity, equations (2) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages (i.e., it does not depend on a). The parameter b2 is the probability that an insect - carrying hiv infects a human when feeding, and x is the number of hiv - negative people . We assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population (unaids 2006). The parameter is the transmission rate through sexual contact of hiv, and is assumed to be independent of infection age . Our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection, and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase (kelley et al . 2007). Results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption . Lastly, (a) is a function describing the mortality rate of hiv - positive people as a function of infection age . In particular, we will suppose that (a) has the form where is the time during the infection at which aids develops . This model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase, r, is defined implicitly, as a function of various epidemiological parameters, by the equation: some of the parameters in equation (4) will depend on the level of viremia in humans, and therefore viremia will affect the per capita rate of spread of hiv . In particular, the amount of hiv picked up by an arthropod during a feeding, b1, is expected to increase with viremia . Similarly, evidence suggests that sexual transmission rate,, also increases with viremia (operskalski et al . 1997 lastly, evidence also shows that high levels of viremia lead to a more rapid development of aids, and thus a lower value of in untreated patients (mellors et al . With these specifications, our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms . Suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission (i.e., b1 0). Also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission . Assuming that multiple infections do not occur (an assumption that we relax below), the rate of change in frequency, p, of this mutant strain is (day and gandon 2007) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . Specifically, ra and rb are implicitly defined, from equation (4), as respectively . Equations (6) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate . More precisely, sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold . Empirical evidence in support of this functional form for hiv exists (quinn et al ., a comparison of equations (6a) and (6b) reveals that rb will be smaller than ra (i.e., the mutant strain will decrease in frequency) and hiv will be predominately sexually transmitted, when the following conditions hold: vector mortality, or the hiv decay rate, is high (i.e., large), vector population size is small (i.e., small v), vector biting rate is small (i.e., small a), or the hiv transfer rates to and from vectors, b1 and b2, are small . Indeed, rb is not only less than ra in such situations (as in fig . 1), but often negative as well, meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . Therefore, some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission . Conversely, if the opposite conditions hold, then the mutant will increase in frequency, and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology (fig . 1). Sexual transmission rate (solid line) and vector transmission rate (dashed line) as a function of viremia, . The resulting per capita growth rate based on equation (4) is also plotted (growth rate is negative where is falls below the horizontal axis, meaning that such strains can never increase in number). B denote the sexually transmitted and vector transmitted genotypes considered in model (5) of the text . Parameter values: 0 = 1/65, a = 1, v = 100, x = 5, = 15 . These considerations provide the conditions required for the spread of a mutant with mechanical vector transmission, but they do not tell us the time frame over which such spread occurs . For example, it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans . The calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv, in order for the relative frequency of the mutant, p/(1 p), to increase by a factor of k over a period of t years, is given by all parameters in equation (7) can be estimated except k, t, and b (appendix 2), yielding fig . The factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k, as a function of the amount of time over which evolution occurs (between 30 and 50 years for hiv in humans). Solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years . The parameter k has very little effect over several orders of magnitude, meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b . First, note that changes in k (the amount of increase that must occur for evolutionary change to be deemed, we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years . Also, as expected, the curves are decreasing, reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act . More interestingly we can see that, if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years, then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . On the other hand, if increased viremia results in the development of aids after only 1 year, then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred . Unfortunately, there are currently no estimates available of these parameters, but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . Therefore, the lack of vector transmission in hiv cannot immediately be attributed to an insufficient evolutionary history of hiv in humans . Biological transmission: although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv (or any other retrovirus; foil and issel 1991; kuno 2004; kuno and chang 2005; webb et al . 1989), it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved . Equation (5) can again be used in this context, with equation (4) again defining the per capita growth rate for different strains of hiv . Biological transmission need not require an increased viremia in humans, however, because the pathogen would replicate to transmissible levels once in the arthropod vector . As a result, strains that are capable of biological vector transmission need not result in the more rapid development of aids . Without some associated cost, however, biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve . Thus, if an absence of such transmission is to be explained in terms of selection (as opposed to an explanation based on a lack of genetic variation) then there must be some associated cost . There are at least two biologically plausible mechanisms through which such a cost might arise . First, effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . In this case, the cost stems from an evolutionary trade - off between these two transmission routes . The sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations (8) could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain . There is, however, no a priori reason why this would be expected rather than the reverse . Therefore, it does not provide a very compelling answer as to why biological transmission has not evolved in hiv, particularly given that it is absent in all other retroviruses as well . The second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts, and natural selection acting on the virus replicative capacity within a host . It is well documented in hiv (and other retroviruses) that extensive genetic variation arises within an infected host via mutation . If vector - transmissible strains suffer a cost in terms of their replicative ability within humans, then this within - host natural selection acting against vector transmission might be enough to prevent its spread . Modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants, some of which will be better at exploiting the human host . This will cause evolutionary change in the genetic composition of hiv within the infected individuals . At the same time, this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission . The assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host . The simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection . Specifically, we suppose that humans almost always harbor only a single strain, but occasionally new strains arise by mutation . When such a mutation occurs, the mutant then either takes over the host or dies out instantaneously, resulting in a single strain infection once again (levin and pimentel 1981; nowak and may 1994). In keeping with our earlier notation we will use b to denote the vector - transmissible form, and a to denote the form best able to compete within a host and thus to transmit sexually . Letting be the rate at which new mutations arise within an infected host, turning either an a pathogen into a b pathogen or vice versa, and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j, where i and j are either a or b, model (5) can be extended to yield (day and proulx 2004; day and gandon 2006) with ra and rb again given by equations (6). The hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form, and thus we take ab = 0 in equation (9). In this case, there are then two possible evolutionary outcomes . First, if rb ra> ba, then the frequency of vector transmission will ultimately evolve to the equilibrium value . On the other hand, if rb ra <ba, then vector transmission will never evolve . In other words, if the significance of within - host evolution is large relative to the benefit of vector transmission, then vector transmission will never evolve . This will be true whenever the mutation rate of the virus is high (i.e., large) and when the selective advantage of sexual transmission in terms of within - host competition is large . The first of these is certainly true of most retroviruses, although the second requirement is less well documented . Nevertheless, this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission . How likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation? The most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population . Unfortunately, performing such an assay on all genotypes within the population would be next to impossible . Furthermore, if variants capable of vector transmission are selectively disadvantageous, then their frequency in the population might be extremely low . Nevertheless, there are some studies available that take this approach as far as is possible . A second approach to addressing this issue if some of hivs close relatives have evolved vector - transmission, then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened . Below we review both types of evidence, for both mechanical and biological transmission . Mechanical transmission: a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods . Such transmission is often difficult to assess under natural conditions, and therefore most studies have employed artificial experimental setups, using a variety of arthropods including mosquitoes, stable flies, and tabanids (e.g., horse flies and deer flies). For example, hiv was found to be viable for up to 10 days in african soft ticks, and perhaps even up to 14 days (humphrey - smith et al . 1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus . Most research on this topic has stressed the need for large bloodmeal size, however, because hiv tends not to have a very high viral titre during infection in humans (lifson 1988; webb et al . This has led to a focus on arthropods, like ticks, whose bloodmeal sizes are often 70 times larger than that of mosquitoes (humphrey - smith et al . It has also been suggested that squashing mosquitoes while they are feeding, and subsequently scratching the area (which might result in lacerations) could increase the likelihood of transmission as well (siemens 1987). Other studies of closely related viruses suggest that, in principle, there is no obvious barrier to mechanical transmission of hiv by arthropods . In fact, it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans (eigen et al . At least three other retroviruses can be transmitted mechanically, including bovine leukemia virus, friend murine leukemia virus, and equine infectious anemia virus (foil and issel 1991; humphrey - smith et al . The latter (equine infectious anemia virus) is believed to be a close relative of hiv (mcclure et al . 1988), and epidemiological evidence suggests that vector transmission might play a significant role in its transmission (foil and issel 1991). Interestingly, however, all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv (foil and issel 1991). It has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods (jupp et al . 1983), although this has been controversial (kuno 2004). Even if this does occur, however, the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses (foil and issel 1991), again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus . Biological transmission: despite evidence that mechanical transmission of hiv by arthropods can occur, there is no evidence that biological transmission is possible . (1993) found that hiv can remain viable in ticks for up to 2 weeks, they failed to find any evidence that hiv can replicate in these vectors . Evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa (becker et al . 1986), but this has been controversial, and epidemiological evidence is not indicative of vector transmission (noireau et al . Furthermore, experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells (srinivasan et al . In fact, no evidence exists to date that any retrovirus is capable of biological transmission by arthropods (webb et al . 1989; foil and issel 1991; kuno 2004; kuno and chang 2005). It remains unclear exactly why such replication is not possible, but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation (van den heuvel et al . Alternatively, it remains possible that appropriate genetic variation can arise, but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses . The above empirical findings suggest that, in principle, hiv is capable of being transmitted mechanically . In practice, however, the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur (lifson 1988; webb et al . There is evidence of genetic variation for differences in viremia in hiv - infected patients (kanki et al . 1999) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous . Thus, we are forced to ask: why have genetic variants of hiv that induce higher viremia, and thus that transmit mechanically via arthropods, not increased in frequency? On the other hand, there is no evidence to suggest that biological transmission can occur, even in principle . This might be because the relevant genetic variation for such transmission has not appeared (or perhaps is not possible). Alternatively, perhaps such variation does occasionally arise, but that such strains are acted against by natural selection . In this section we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv . Mechanical transmission: to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . At present hiv infection is increasing in prevalence in the human population (unaids 2006). The simplest description of this is exponential growth in the number of infected individuals; where y(t) is the number of hiv - positive individuals at time t, and r is the per capita growth rate . Although the rate of increase of hiv appears to be slowing in recent years, exponential growth nevertheless provides a useful benchmark for its initial spread in humans . The per capita growth rate will depend on various properties of hiv, including its mode of transmission . To derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters, we first need to specify a model for the epidemiological dynamics . This model will allow for both sexual transmission of hiv as well as insect transmission, and therefore it will also track the number of insects carrying hiv . Using w(t) for the number of insects carrying hiv at time t, and y(a, t) as the number of hiv - positive people who were infected a years ago, we specify the dynamics as with boundary condition . In equations (2), v is the population size of insects free of hiv, a is the insect - biting rate, b1 is the probability of an insect picking up hiv, given it feeds on an infected human, and is the per capita loss rate of infected insects (which subsumes both insect mortality and the decay of hiv stores in or on the insect). Note that, although many insects display a characteristic time lag between feeding events, for simplicity we have ignored this . Also note that, for simplicity, equations (2) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages (i.e., it does not depend on a). The parameter b2 is the probability that an insect - carrying hiv infects a human when feeding, and x is the number of hiv - negative people . We assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population (unaids 2006). The parameter is the transmission rate through sexual contact of hiv, and is assumed to be independent of infection age . Our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection, and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase (kelley et al . Results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption . Lastly, (a) is a function describing the mortality rate of hiv - positive people as a function of infection age . In particular, we will suppose that (a) has the form where is the time during the infection at which aids develops . This model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase, r, is defined implicitly, as a function of various epidemiological parameters, by the equation: some of the parameters in equation (4) will depend on the level of viremia in humans, and therefore viremia will affect the per capita rate of spread of hiv . In particular, the amount of hiv picked up by an arthropod during a feeding, b1, is expected to increase with viremia . Similarly, evidence suggests that sexual transmission rate,, also increases with viremia (operskalski et al . Lastly, evidence also shows that high levels of viremia lead to a more rapid development of aids, and thus a lower value of in untreated patients (mellors et al . 1997; levy 1998; raffanti et al ., our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms . Suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission (i.e., b1 0). Also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission . Assuming that multiple infections do not occur (an assumption that we relax below), the rate of change in frequency, p, of this mutant strain is (day and gandon 2007) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . Specifically, ra and rb are implicitly defined, from equation (4), as respectively . Equations (6) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate . More precisely, sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold . Empirical evidence in support of this functional form for hiv exists (quinn et al ., a comparison of equations (6a) and (6b) reveals that rb will be smaller than ra (i.e., the mutant strain will decrease in frequency) and hiv will be predominately sexually transmitted, when the following conditions hold: vector mortality, or the hiv decay rate, is high (i.e., large), vector population size is small (i.e., small v), vector biting rate is small (i.e., small a), or the hiv transfer rates to and from vectors, b1 and b2, are small . Indeed, rb is not only less than ra in such situations (as in fig . 1), but often negative as well, meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . Therefore, some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission . Conversely, if the opposite conditions hold, then the mutant will increase in frequency, and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology (fig . Sexual transmission rate (solid line) and vector transmission rate (dashed line) as a function of viremia, . The resulting per capita growth rate based on equation (4) is also plotted (growth rate is negative where is falls below the horizontal axis, meaning that such strains can never increase in number). B denote the sexually transmitted and vector transmitted genotypes considered in model (5) of the text . Parameter values: 0 = 1/65, a = 1, v = 100, x = 5, = 15 . These considerations provide the conditions required for the spread of a mutant with mechanical vector transmission, but they do not tell us the time frame over which such spread occurs . For example, it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans . The calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv, in order for the relative frequency of the mutant, p/(1 p), to increase by a factor of k over a period of t years, is given by all parameters in equation (7) can be estimated except k, t, and b (appendix 2), yielding fig . The factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k, as a function of the amount of time over which evolution occurs (between 30 and 50 years for hiv in humans). Solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years . The parameter k has very little effect over several orders of magnitude, meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b . First, note that changes in k (the amount of increase that must occur for evolutionary change to be deemed, we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years . Also, as expected, the curves are decreasing, reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act . More interestingly we can see that, if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years, then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . On the other hand, if increased viremia results in the development of aids after only 1 year, then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred . Unfortunately, there are currently no estimates available of these parameters, but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . Therefore, the lack of vector transmission in hiv cannot immediately be attributed to an insufficient evolutionary history of hiv in humans . Biological transmission: although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv (or any other retrovirus; foil and issel 1991; kuno 2004; kuno and chang 2005; webb et al . 1989), it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved . Equation (5) can again be used in this context, with equation (4) again defining the per capita growth rate for different strains of hiv . Biological transmission need not require an increased viremia in humans, however, because the pathogen would replicate to transmissible levels once in the arthropod vector . As a result, strains that are capable of biological vector transmission need not result in the more rapid development of aids . Without some associated cost, however, biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve . Thus, if an absence of such transmission is to be explained in terms of selection (as opposed to an explanation based on a lack of genetic variation) then there must be some associated cost . There are at least two biologically plausible mechanisms through which such a cost might arise . First, effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . In this case, the cost stems from an evolutionary trade - off between these two transmission routes . The sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations (8) could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain . There is, however, no a priori reason why this would be expected rather than the reverse . Therefore, it does not provide a very compelling answer as to why biological transmission has not evolved in hiv, particularly given that it is absent in all other retroviruses as well . The second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts, and natural selection acting on the virus replicative capacity within a host . It is well documented in hiv (and other retroviruses) that extensive genetic variation arises within an infected host via mutation . If vector - transmissible strains suffer a cost in terms of their replicative ability within humans, then this within - host natural selection acting against vector transmission might be enough to prevent its spread . Modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants, some of which will be better at exploiting the human host . This will cause evolutionary change in the genetic composition of hiv within the infected individuals . At the same time, this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission . The assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host . The simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection . Specifically, we suppose that humans almost always harbor only a single strain, but occasionally new strains arise by mutation . When such a mutation occurs, the mutant then either takes over the host or dies out instantaneously, resulting in a single strain infection once again (levin and pimentel 1981; nowak and may 1994). In keeping with our earlier notation we will use b to denote the vector - transmissible form, and a to denote the form best able to compete within a host and thus to transmit sexually . Letting be the rate at which new mutations arise within an infected host, turning either an a pathogen into a b pathogen or vice versa, and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j, where i and j are either a or b, model (5) can be extended to yield (day and proulx 2004; day and gandon 2006) with ra and rb again given by equations (6). The hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form, and thus we take ab = 0 in equation (9). In this case, there are then two possible evolutionary outcomes . First, if rb ra> ba, then the frequency of vector transmission will ultimately evolve to the equilibrium value . On the other hand, in other words, if the significance of within - host evolution is large relative to the benefit of vector transmission, then vector transmission will never evolve . This will be true whenever the mutation rate of the virus is high (i.e., large) and when the selective advantage of sexual transmission in terms of within - host competition is large . The first of these is certainly true of most retroviruses, although the second requirement is less well documented . Nevertheless, this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission . Experiments and epidemiological data have unequivocally demonstrated, however, that such vector transmission does not occur at any significant level, and various aspects of hiv biology have been implicated as proximate reasons (bockarie and paru 1996). These reasons do not offer an explanation for why vector transmission has not evolved, however, and as weiss (2001) points out, we ought to seriously consider whether such evolution might occur in the future (for a summary, see table 1). Main conclusions of the study existing data suggest that the lack of mechanical vector transmission in hiv is not due to genetic constraints . While ecological constraints, such as number of vectors and biting rates, may limit vector transmission in certain areas, these constraints would likely not explain why hiv has not evolved this form of transmission in areas where vector - borne diseases (e.g. Malaria) are endemic . Rather, there must presumably be a reason why such transmission is selectively disadvantageous in hiv . Effective mechanical vector transmission can be brought about only through the evolution of higher levels of viremia, and this also results in a more rapid onset of aids . This reduces the duration over which such strains can be transmitted from an infected human, more than is made up for by the occurrence of vector transmission . It also remains possible that insufficient time has elapsed for the evolution of vector transmission to occur, but our calculations suggest that this is not a very compelling possibility . On the other hand, existing data is largely consistent with the hypothesis that biological vector transmission has not evolved in hiv because of genetic constraints . At the same time, it is not possible to rule out a selective explanation instead . In particular, if there is a genetic trade - off between efficient replication in humans and replication in arthropod vectors, then a conflict between selection favoring effective replication within humans, and selection favoring arthropod transmission between humans can readily prevent biological transmission from evolving . This is particularly likely when the mutation rate of the virus is high, and thus might provide an explanation for the lack of biological vector transmission in all retroviruses . Our analysis might also be extended to include other forms of transmission, for instance needle transmission (see bruneau et al . Several evolutionary consequences of this are possible depending both on the level of viremia required for such transmission to occur and the resulting transmission rate . For instance, if needle transmission can be achieved with a lower viremia than sexual transmission, and if this leads to a sufficiently high transmission rate, less virulent strains could be favored . Conversely, if needle transmission requires a high viremia and leads to a sufficiently high transmission rate, more virulent strains would be favored . The only situation in which enhanced needle use could lead to the evolution of vector - borne transmission would be if effective needle transmission requires a viremia close to that of vector - borne transmission, while leading to a much higher transmission rate than vector - borne transmission . This way, strains with high viremia could be maintained in the population through needle transmission, and vector - borne transmission would then occur largely as a byproduct . Our conclusions in this article are necessarily speculative, but such speculation is a necessary part of the initial stages of any research . One of our aims is to stimulate future research into the evolutionary biology of hiv transmission . From the results presented here, a number of different directions might be taken to ground these evolutionary ideas more firmly in empirical data . One possibility would be to examine more closely mechanical vector transmission in immunodeficiency viruses of other species . For example, more data on the epidemiological patterns of siv and its potential for alternative routes of transmission would be enormously useful . Since siv is believed to be at the evolutionary ancestor of hiv, it would be very interesting to know if the longer evolutionary history it has had with its host has resulted in different transmission patterns . To the best of our knowledge, there are no empirical studies testing the potential for vector transmission of siv . Another fruitful approach might be to conduct artificial selection experiments with hiv in arthropod tissue culture . Experiments have demonstrated that hiv cannot currently replicate significantly in arthropod cells, but no study to our knowledge has attempted to select for the evolution of hiv replication in such cells . One could even imagine doing such experiments with both mammalian and arthropod cell cultures to determine of the evolutionary trade - off postulated here actually occurs . Ultimately, it will require innovative experiments and empirical studies to push the boundaries of our knowledge of hiv, and the use of evolutionary biology as a powerful tool for designing sensible intervention strategies . These kinds of studies are beginning to appear for other aspects of hiv biology (e.g., see mller et al . 2006 for an interesting evolutionary analysis of hiv virulence) but more work on transmission biology would be useful . For example, if further empirical research validated the hypothesis presented here, that mechanical vector transmission has not evolved because of its associated mortality costs, this would then have important implications for how we attempt to stop the spread of hiv . Strategies such as condom use, while beneficial for reducing the extent of sexual transmission, could thereby enhance the relative benefit of vector transmission, potentially resulting in the evolution of this new route of transmission . The use of antibiotics against bacterial pathogens has clearly brought home the fact that pathogens can readily evolve the means to circumvent our control measures, and there is no reason to expect things to be any different for other control measures . The use of antiviral medication, on the other hand, not only reduces sexual transmission but also the level of viremia, and therefore would presumably not move the selective balance more towards vector transmission . It is only by asking these kinds of questions, however, that we will have a chance at preventing adverse future outcomes . Finally, the question of biological vector transmission addressed here is really a special case of the more general question of the evolution of a pathogen s host range . Why do some pathogens have a relatively broad taxonomic host range while others are much more conservative? This continues to be an interesting and important question in the evolutionary ecology of parasites (poulin 2007) and there are some theoretical results predicting when we might expect different outcomes (gandon 2004). From the standpoint of human diseases this is also clearly an important question since emerging diseases, such as pandemic influenza, are precisely instances in which a pathogen evolves a different host range . A better understanding of the evolutionary biology of parasite host ranges is an important goal for future research.
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In the normal kidney, immunohistochemical expression of contractile protein alpha - smooth muscle actin (asma) is limited to the vascular smooth muscle cells . In pathological conditions, the expression of asma the first experimental publication on this subject, by johnson et al, involved a series of clinical studies on the role of asma in diffuse renal diseases . In 1992 alpers and colleagues found higher expression of asma in proliferative types of glomerulonephritis gn . In iga nephropathy, asma expression in interstitium is related to damaged renal function and progression of disease . The expression of asma in interstitial myofibroblasts helps to differentiate between minimal change disease and the early phase of idiopathic membranous gn . In patients with iga nephropathy, higher mesangial expression of asma predicts a progressive decline in renal function . In focal segmental glomerulosclerosis,, interest in the asma is increasing due to the fact that the asma in cytoplasm of the podocytes is associated with nephrin and thus is indirectly involved in the glomerular filtration barrier . Asma is analyzed in various types of gn together with markers such as metalloproteinase and nestin in investigation of tissue remodulation and epithelial - mesenchymal transition . To our best knowledge, this is the first morphometrical study of the correlation of asma expression in gn and renal function at the time of biopsy . This retrospective study analyzed 142 patients 82 adults and 60 children who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics, university hospital, split, croatia in the period from 1994 to 2007 . Patients data were collected from the hospital records . Due to the small numbers of each type of gn in the sample, pathological diagnoses were classified into 2 categories proliferative glomerulonephritis (pg) in 98 cases and non - proliferative glomerulonephritis (npg) in 44 cases . In the pg category were: 29 iga nephropathy, 24 mesangioproliferative gn, 11 henoch - schenlein purpura, 9 focal segmental gn, 6 rapidly progressive gn, 6 endoproliferative gn and 6 lupus nephritis, 4 membranoproliferative gn and 1 hemolytic uremic syndrome, 1 alport syndrome and 1 churg - strauss syndrome . In the npg category were: 17 membranous gn, 15 focal segmental glomerulosclerosis, 5 minimal change disease, 2 fibrillar gn, 1 thin basement membrane disease, c1q nephropathy, igm nephropathy, amyloidosis and hereditary nephropathy . Absolute values of blood pressure (bp), serum creatinine (sc), creatinine clearance (ccr) and 24-hour urine protein, measured within 7 days before the biopsy, were collected from the hospital records . For adults, absolute values were categorized according to the common terminology criteria for adverse events (ctcae). For children, bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey . Serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories: normal, high and low . Ccr was determined from sc, the patient s height and proportionality constant using the schwartz method . The 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits . Asma expression was analyzed by indirect immunohistochemistry (envision / hrp system (dako, denmark) using mouse monoclonal anti - alpha smooth muscle antibody (asma / hrp dako, denmark). Paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient . Tissue sections were incubated with primary asma antibody (dilution 1:50) for 60 min and peroxidase - labeled secondary antibody for 20 min, followed by 10 min incubation with diaminobenzidine substrate - chromogen solution (dako, denmark). Hematoxylin counter - staining was done; slides were dehydrated in alcohol gradient, cleared in xylol and mounted with canada balsam . Positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer . Computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera (olympus 4.1 zoom) connected with olympus bx41 microscope (olympus, japan). A computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections, using analysis software (analysis soft imaging system, usa). All foci of asma expression in the interstitium (including atrophic tubules), as well as the perimeter of histological slide, were measured in m at 100 magnification; their ratio was calculated as a percentage of asma expression in interstitium . Area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli (figure 1a). Their ratio was calculated as a percentage of asma expression in glomeruli (figure 1b). In 5 case controls, renal cortex in the highest distance from the tumor the area measuring 0.14 mm was defined, and glomeruli and interstitium were analyzed in the same manner . In the control kidneys, nonparametric spearman s correlation, mann - whitney test, and analysis of variance (anova, kruskal - wallis) were made using graphpad prism statistical software (graphpad software, inc . This retrospective study analyzed 142 patients 82 adults and 60 children who underwent percutaneous renal biopsy in the department of internal medicine and department of pediatrics, university hospital, split, croatia in the period from 1994 to 2007 . Patients data were collected from the hospital records . Due to the small numbers of each type of gn in the sample, pathological diagnoses were classified into 2 categories proliferative glomerulonephritis (pg) in 98 cases and non - proliferative glomerulonephritis (npg) in 44 cases . In the pg category were: 29 iga nephropathy, 24 mesangioproliferative gn, 11 henoch - schenlein purpura, 9 focal segmental gn, 6 rapidly progressive gn, 6 endoproliferative gn and 6 lupus nephritis, 4 membranoproliferative gn and 1 hemolytic uremic syndrome, 1 alport syndrome and 1 churg - strauss syndrome . In the npg category were: 17 membranous gn, 15 focal segmental glomerulosclerosis, 5 minimal change disease, 2 fibrillar gn, 1 thin basement membrane disease, c1q nephropathy, igm nephropathy, amyloidosis and hereditary nephropathy . Absolute values of blood pressure (bp), serum creatinine (sc), creatinine clearance (ccr) and 24-hour urine protein, measured within 7 days before the biopsy, were collected from the hospital records . For adults, absolute values were categorized according to the common terminology criteria for adverse events (ctcae). For children, bp was determined by body size and age using center for disease control and prevention growth charts and data from the national health and nutrition examination survey . Serum creatinine was categorized according to the pediatric reference ranges and divided into 3 categories: normal, high and low . Ccr was determined from sc, the patient s height and proportionality constant using the schwartz method . The 24-hour urine protein was corrected for body surface area and categorized according to 95% confidence limits . Asma expression was analyzed by indirect immunohistochemistry (envision / hrp system (dako, denmark) using mouse monoclonal anti - alpha smooth muscle antibody (asma / hrp dako, denmark). Paraffin - embedded tissue sections of renal biopsies were deparaffinized in xylol and rehydrated in alcohol gradient . Tissue sections were incubated with primary asma antibody (dilution 1:50) for 60 min and peroxidase - labeled secondary antibody for 20 min, followed by 10 min incubation with diaminobenzidine substrate - chromogen solution (dako, denmark). Hematoxylin counter - staining was done; slides were dehydrated in alcohol gradient, cleared in xylol and mounted with canada balsam . Positive control samples were normal renal tissues of 5 adult patients who underwent nephrectomy for renal cancer . Computer - assisted morphometric image analysis was used to measure glomerular and interstitial asma expression using ibm computer and digital camera (olympus 4.1 zoom) connected with olympus bx41 microscope (olympus, japan). A computer mouse was used to trace the perimeter of the area of interest on a computer monitor in successive sections, using analysis software (analysis soft imaging system, usa). All foci of asma expression in the interstitium (including atrophic tubules), as well as the perimeter of histological slide, were measured in m at 100 magnification; their ratio was calculated as a percentage of asma expression in interstitium . Area of asma expression in each glomerulus was measured in m at 400 magnification and added together for all glomeruli (figure 1a). Their ratio was calculated as a percentage of asma expression in glomeruli (figure 1b). In 5 case controls, renal cortex in the highest distance from the tumor the area measuring 0.14 mm was defined, and glomeruli and interstitium were analyzed in the same manner . In the control kidneys, nonparametric spearman s correlation, mann - whitney test, and analysis of variance (anova, kruskal - wallis) were made using graphpad prism statistical software (graphpad software, inc . The expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults (table 1). The children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure (10.79.4% vs. 3.74.3%, p=0.014) (figure 2). The difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure (7.94.4% vs. 15.410.6%, p=0.051). In adults, expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure (10.714.9% vs. 9.912.6%, p=0.692) and (13.69.5% vs. 12.97.8%, p=1.00), respectively . In children, positive correlation was found between asma expression in interstitium and absolute value of sc (r=0.45, p=0.002) (figure 3a). In categorized sc, the negative correlation of sc and expression of asma in glomeruli was not significant (r=0.154, p=0.291). In adults, expression of asma in interstitium was correlated to sc (r=0.528, p<0.001) (figure 3b). The significance was confirmed in categorized sc, where grade iii had higher expression of asma in interstitium (p=0.0009). Significant negative correlation was found between expression of asma in glomeruli and sc (r=0.395, p=0.002) (figure 3c). In children, absolute values of ccr negatively correlated to expression of asma in interstitium (r=0.375, p=0.009) and positively to expression of asma in glomeruli (r=1.00, p<0.001) (figure 4a, b). In adults, there were no significant correlations between asma expression in glomeruli and ccr (r=0.058, p=0,643). Children and adults with ccr grade ii and iii had higher expression of asma in interstitium (p=0.0152 and p=0.0007, respectively) (figures 5a, b). No significant association between renal expression of asma and 24-hour urine protein was found, regardless of the patient s age . The expression of asma in glomeruli and interstitium was not significantly different between pg and npg categories of gn in both children and adults (table 1). The children with high blood pressure had greater expression of asma in interstitium compared to children with low blood pressure (10.79.4% vs. 3.74.3%, p=0.014) (figure 2). The difference in expression of asma in glomeruli was not statistically significant between children with high and low blood pressure (7.94.4% vs. 15.410.6%, p=0.051). In adults, expression of asma in interstitium and in glomeruli were not significantly different between patients with high and normal blood pressure (10.714.9% vs. 9.912.6%, p=0.692) and (13.69.5% vs. 12.97.8%, p=1.00), respectively . In children, positive correlation was found between asma expression in interstitium and absolute value of sc (r=0.45, p=0.002) (figure 3a). In categorized sc, the negative correlation of sc and expression of asma in glomeruli was not significant (r=0.154, p=0.291). In adults, expression of asma in interstitium was correlated to sc (r=0.528, p<0.001) (figure 3b). The significance was confirmed in categorized sc, where grade iii had higher expression of asma in interstitium (p=0.0009). Significant negative correlation was found between expression of asma in glomeruli and sc (r=0.395, p=0.002) (figure 3c). In children, absolute values of ccr negatively correlated to expression of asma in interstitium (r=0.375, p=0.009) and positively to expression of asma in glomeruli (r=1.00, p<0.001) (figure 4a, b). In adults, there were no significant correlations between asma expression in glomeruli and ccr (r=0.058, p=0,643). Children and adults with ccr grade ii and iii had higher expression of asma in interstitium (p=0.0152 and p=0.0007, respectively) (figures 5a, b). No significant association between renal expression of asma and 24-hour urine protein was found, regardless of the patient s age . Studies of asma expression in renal parenchymal diseases began more than 2 decades ago, when it was noticed that a) damaged glomerular mesangial cells change their immunophenotype expressing asma and b) asma - positive myofibroblasts start interstitial fibrosis . A number of papers were published about asma expression in different types of human gn, its connection to proliferation markers and prognostic impact . Most authors agree that asma expression in glomeruli was higher in proliferative gn and increases as the disease worsens, which makes asma expression a potential clinical prognostic factor . Some authors determined the relationship between iga nephropathy, lupus nephritis and other types of gn with expression of asma . Kim in 2001 found correlation between proliferation marker ki-67 and asma in different types of gn . In this study we focused on the connection of asma expression with impaired renal function in glomerulonephritis, measured at the time of biopsy . We did not find correlation with a special type of gn nor proliferation, possibly because of heterogeneity of the sample and the arbitrary method of categorization into proliferative and non - proliferative gn . Increased expression of asma in the interstitium can be found in different diseases such as proliferative gn, diabetic nephropathy and renal transplant rejection . The asma has prognostic value due to the association with interstitial fibrosis, urine protein and sc [8,2528]. Asma - positive myofibroblasts are responsible for the increased amount of extracellular matrix and renal fibrosis . This study also found higher expression of asma in interstitium of all patients with higher sc . Greater expression of interstitial asma in children and adults was associated with higher grades of ccr and lower absolute values of ccr . We confirm that elevated sc or lower values of ccr are associated with higher expression of asma in interstitium . Several studies have attempted to predict the development of progressive renal failure, measuring histomorphometric changes in the tubulointerstitial compartment . The best correlating parameters of interstitial fibrosis with renal function are the ratio of the accumulation of tgf - beta-1 and its antagonist decorin, interstitial expression of asma, and accumulation of interstitial collagen . According to jiang et al ., who analyzed asma production in peritoneal fibroblasts stimulated by tnf - beta-1, hepatocyte growth factor could be important in blocking postoperative peritoneal adhesion . Analyzed 133 biopsies of various human renal diseases, and found tubular epithelial cells with mesenchymal phenotype (vimentin and asma positive) whose numbers have been associated with the level of sc and degree of interstitial damage . Analyzed 27 patients with iga nephropathy who had normal ccr at the time of biopsy, and found that expression of asma in mesangium predicts a progressive decline in renal function . In our study, a negative connection of asma expression in glomeruli and sc at the time of biopsy was found, statistically significant in adults and non - significant in children, but with the same trend . It is well known that asma expression in the mesangial cell indicates its change to myofibroblastic immunophenotype . Our presumption is that asma - expressing mesangial cells are capable of higher contraction activity; this leads to glomerular hyperfiltration as an early adaptive mechanism to glomerular damage . In a recent experimental study of membranous glomerulonephritis in rats, osteopontin and asma are expressed together in myofibroblasts in the crescents of damaged glomeruli . In children, the absolute values of ccr a possible explanation that children have a better ccr is due to phenotypic modulation of human mesangial cells to more contractile cells, which makes the glomerular function transitory normal or even increased . We analyzed association of expression of asma and hypertension, because it is known that glomerular mesangial cells during hypertensive damage express myofibroblast phenotype and expression of asma increases . The only significant association that we found was between expression of asma in interstitium and increased blood pressure in children . We did not find significant correlation between expression of asma in glomeruli nor interstitium with 24-hour urine protein in our patients . This result confirms previous findings that urine protein is primary linked to the changes in permeability of glomerular filtration barrier . Nevertheless, according to some studies asma expression in interstitium is linked to the degree of proteinuria and sc, and higher interstitial asma expression is an indicator of poor prognosis . Asma expression in interstitium is associated with sc and ccr and consecutively with decrease of renal function . Asma expression in glomeruli is associated with lower values of sc in adults, as well as with normal or higher ccr in children . This correlation suggests that myofibroblastic phenotypic modulation of glomerular cells has a favorable impact on filtration . When calculated with precise computer - assisted quantitative morphometric technology, renal expression of asma the disadvantage of this study is its small sample size with different types of gn; the next survey should include a much narrower cohort of patients with a specific type of gn, as well as better phenotypic identification of asma - positive glomerular cells.
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Chondroblastoma accounts for <1% of all primary bone tumors . Usually occurring in young adolescents, however, unusual sites of presentation such as patella, talocalcaneal, and temporomandibular joint has been reported . Since last decade, fine needle aspiration cytology (fnac) has evolved as a simple, safe, and noninvasive preoperative tool in the diagnosis of bone tumors . It is necessary to differentiate chondroblastoma from other giant cell - rich lesions, as chondroblastoma exhibits predominantly benign behavior, though recurrences and metastasis are on record . We report cytomorphology in two cases of chondroblastoma confirmed by histopathology with a brief review of literature . A 16-year - old male presented with pain and diffuse swelling on the knee joint since 2 months . Swelling was noted in the upper end of tibia measuring 3 cm 3 cm, and was tender on palpation . X ray showed typically lytic, centrally placed, sharply demarcated lesion with sclerotic border in the epiphysis . A 10-year - old male presented with diffuse swelling on the shoulder joint of 1-month duration . Local examination revealed swelling at the upper end of humerus measuring 4 cm 4 cm . Fnac was performed by the nonaspiration technique in the first patient using a 24 gauge needle while for the second case, ultrasound - guided aspiration was done because the lesion was deep - seated . Smears were highly cellular, polyhedral cells arranged in sheets and small clusters, giving a pebble stone pattern appearance . The individual cells were monomorphic with a well - defined cell membrane, glassy cytoplasm at places showing microvacuolation . Additionally seen was cartilaginous matrix, plenty of scattered osteoclast - type multinucleated giant cells, and bluish granular calcification surrounding individual cells chicken wire calcification [figure 1a c]. (h and e, 40) (b) cytology smears showing polygonal cells with vacuolization in clusters, with calcification seen in the letilower corner . (h and e, 100) (c) cytology smears showing chicken wire calcification . (h and e, 100) (d) histopathology section showing cartilage, chicken wire calcification, and polygonal cells with vacuolization of cytoplasm . (h and e, 40) at surgery, curettage and bone grafting was performed . Grossly, the specimen consisted of multiple irregular grey - brown tissue bits with areas of hemorrhage . Histopathology in both the cases showed sheets of polygonal cells with thick cell membranes and fine pale vacuolated cytoplasm . A 16-year - old male presented with pain and diffuse swelling on the knee joint since 2 months . Swelling was noted in the upper end of tibia measuring 3 cm 3 cm, and was tender on palpation . X ray showed typically lytic, centrally placed, sharply demarcated lesion with sclerotic border in the epiphysis . A 10-year - old male presented with diffuse swelling on the shoulder joint of 1-month duration . Local examination revealed swelling at the upper end of humerus measuring 4 cm 4 cm . Fnac was performed by the nonaspiration technique in the first patient using a 24 gauge needle while for the second case, ultrasound - guided aspiration was done because the lesion was deep - seated . Smears were highly cellular, polyhedral cells arranged in sheets and small clusters, giving a pebble stone pattern appearance . The individual cells were monomorphic with a well - defined cell membrane, glassy cytoplasm at places showing microvacuolation . Additionally seen was cartilaginous matrix, plenty of scattered osteoclast - type multinucleated giant cells, and bluish granular calcification surrounding individual cells chicken wire calcification [figure 1a c]. (h and e, 40) (b) cytology smears showing polygonal cells with vacuolization in clusters, with calcification seen in the letilower corner . (h and e, 100) (c) cytology smears showing chicken wire calcification . (h and e, 100) (d) histopathology section showing cartilage, chicken wire calcification, and polygonal cells with vacuolization of cytoplasm . (h and e, 40) at surgery, curettage and bone grafting was performed . Grossly, the specimen consisted of multiple irregular grey - brown tissue bits with areas of hemorrhage . Histopathology in both the cases showed sheets of polygonal cells with thick cell membranes and fine pale vacuolated cytoplasm . Unusual sites of location include pelvis, patella, talus, calcaneum, and temporomandibular joint . Most of the chondroblastomas are benign in nature requiring simple curettage and bone grafting . However, recurrence is known to occur in 10 - 38% of the cases . Metastases are unusual but occur in the lungs at the time of recurrence . As in most of the cases of bone tumors, this, compounded with nonconforming radiological features, leads to the tragic delay in diagnosis . Though the age of the patient and location of the lesion in the given bone are key pieces of information, accurate and early diagnosis is a must for planning appropriate treatment . The past decade has witnessed the impressive use of fnac in the diagnosis of bone tumors . Despite this, we have a limited case series describing fnac findings in chondroblastoma . Fna aspirates of chondroblastoma show mononuclear cells arranged in a dispersed pattern like pebbles . These cells have well - defined cell margins, glassy cytoplasm, and nucleus with indentation and grooving . Apart from associated giant cells and chondroid matrix, the chicken wire calcification that has been described in histopathology sections can be readily appreciated in fnac smears and can be a clue to diagnosis . However, errors in the fnac diagnosis of this rare tumor are known to occur, with the major pitfalls being its association with aneurysmal bone cyst (abc) leading to a nonrepresentative aspirate and the occurrence of a plethora of giant cell - rich lesions in the differentials . In one large series of 12 cases of chondroblastoma, the authors described the cytomorphology of this tumor in detail . The diagnosis was missed on fnac, among which, one was associated with abc and in another case an erroneous diagnosis of a giant cell tumor was made . The cause of misdiagnosis in one case was due to its association with abc, leading to nonrepresentative aspirate . In the second case, due to the location of the tumor in preauricular region, cartilaginous stroma aspirated, in a study of 110 cases of bone lesions attributed the missed diagnosis in two cases of chondroblastoma at fnac to inadequate sampling and misinterpretation as giant cell tumor . In giant cell tumor, the mononuclear cells are spindle - shaped and occur in clusters . In chondromyxoid fibroma, the smears are sparsely cellular and lack the mononuclear cell component . Instead, stellate cells with pleomorphism are noted . Aspirates in abc are usually hemorrhagic, scantily cellular, composed of osteoclast giant cells, histiocytes, osteoblasts, and spindle cells . In addition to chondroblastoma, abc can occur secondarily in giant cell tumor and osteosarcoma . This report reiterates the cytomorphologic findings in chondroblastoma and its differential diagnosis from other giant cell containing lesions of bone . The collective application of the knowledge of the cytomorphological features of this rare bone tumor would aid in accurate preoperative diagnosis even in the absence of typical clinical and radiologic presentations.
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Suicide is a grave public health issue with a global mortality rate of 16 per 100,000 individuals.1 suicide is one of the top three leading causes of death, particularly among individuals aged 1544 years, which is a productive age group.1 suicide is a complex event that is influenced by biological, cultural, psychological, and socioeconomic factors . More than 90% of suicide victims were diagnosed with psychiatric problems at the time of death, and approximately two thirds had been diagnosed with depression.2,3 depressed patients tend to consult physicians other than psychiatrists, and a variety of medical professionals, including general practitioners, the staff of emergency departments, and non - psychiatric nurses, are key interveners for preventing suicide . Among medical staff members, negative attitudes toward suicide such as anger could affect nontherapeutic reactions to suicidal individuals.4 furthermore, a belief that suicide is a personal right was negatively related to suicide intervention skills.5 an accurate assessment of the attitudes toward suicide held by medical staff members is relevant . Previous studies have shown that difficulties with parental bonding during childhood could be a predisposing factor for the onset of many psychiatric conditions, such as anxiety, depressive states, and maladjusted behaviors.68 parental bonding and premorbid personality traits play an important role in shaping the developmental trajectory of an individual, including his / her ability to adjust to stressful events . Although few studies have assessed the relationship between parental bonding and mental conditions among medical staff members,9,10 no study has evaluated the association between parental bonding and attitudes toward suicide . The objective of this study was to investigate whether parental bonding is associated with attitudes toward suicide among medical college students in japan . Students in their fifth year of medical school at hirosaki university, hirosaki, japan, participated in the study . The surveys were distributed to 226 medical students . Of the distributed 226 surveys, 160 questionnaires (116 males and 44 females) the demographic data (age and sex) were obtained from self - questionnaires and interviews . The data collection for this study was approved by the ethics committee of the hirosaki university school of medicine, and the subjects provided written informed consent before participating in the project . The parental bonding instrument (pbi) was administered to each participant to measure the attitudes and behavior of the parents.11 the pbi is a 25 item self - rating instrument by which individuals over the age of 16 report their experiences of being parented to the age of 16 . A total of 12 items measure the dimension of care, and 13 items measure protection, both on a likert scale . The care dimension of the pbi reflects parental warmth and involvement in contrast to rejection and indifference . The overprotection dimension of the pbi reflects parental overprotection and control in contrast to the encouragement of autonomy . Higher scores on the care and protection dimensions reveal that participants perceive their parents to be more caring and/or protective . We employed the japanese version of the attitudes toward suicide questionnaire (atts) to assess the attitudes toward suicide held by the study participants.12 we employed a six factor model that was previously developed in studies of japanese attitudes, including common occurrence, suicidal expression as mere threat, unjustified behavior, impulsiveness.12,13 each item, with the exception of items 10 and 28, was scored on a five point scale from 1 (strongly agree) to 5 (strongly disagree). Items 10 and 28 were scored using a different scoring method that employed a five point scale from 1 (strongly disagree) to 5 (strongly agree). Multivariate regression analysis was applied to assess the relationship between parental bonding and attitudes toward suicide . The data were analyzed using the pasw statistics software program for windows, version 18.0 . Students in their fifth year of medical school at hirosaki university, hirosaki, japan, participated in the study . The surveys were distributed to 226 medical students . Of the distributed 226 surveys, 160 questionnaires (116 males and 44 females) the demographic data (age and sex) were obtained from self - questionnaires and interviews . The data collection for this study was approved by the ethics committee of the hirosaki university school of medicine, and the subjects provided written informed consent before participating in the project . The parental bonding instrument (pbi) was administered to each participant to measure the attitudes and behavior of the parents.11 the pbi is a 25 item self - rating instrument by which individuals over the age of 16 report their experiences of being parented to the age of 16 . A total of 12 items measure the dimension of care, and 13 items measure protection, both on a likert scale . The care dimension of the pbi reflects parental warmth and involvement in contrast to rejection and indifference . The overprotection dimension of the pbi reflects parental overprotection and control in contrast to the encouragement of autonomy . Higher scores on the care and protection dimensions reveal that participants perceive their parents to be more caring and/or protective . We employed the japanese version of the attitudes toward suicide questionnaire (atts) to assess the attitudes toward suicide held by the study participants.12 we employed a six factor model that was previously developed in studies of japanese attitudes, including common occurrence, suicidal expression as mere threat, unjustified behavior, impulsiveness.12,13 each item, with the exception of items 10 and 28, was scored on a five point scale from 1 (strongly agree) to 5 (strongly disagree). Items 10 and 28 were scored using a different scoring method that employed a five point scale from 1 (strongly disagree) to 5 (strongly agree). Multivariate regression analysis was applied to assess the relationship between parental bonding and attitudes toward suicide . The data were analyzed using the pasw statistics software program for windows, version 18.0 . Table 1 presents the items included in the six factor atts with their descriptive data . Paternal care was significantly associated with paternal protection, maternal care, maternal protection, and right to suicide . Maternal care was significantly associated with maternal protection, right to suicide, and common occurrence . Right to suicide was significantly associated with common occurrence, unjustified behavior, and preventability / readiness to help . Table 3 shows the multiple regression analysis of the atts sub - scales with the pbi scores . After adjusting for age and sex, maternal care approached a statistically significant association with the right to suicide . Under the same conditions, maternal care the present study assessed attitudes toward suicide and their associations with parental bonding among japanese medical college students . The majority of the participants in our study agreed that anyone could commit suicide (88.8%) and that suicide is preventable (86.3%). In addition, the multiple regression analysis revealed that participants who reported a higher level of maternal care thought that suicide was a common occurrence and tended to think that people do not have the right to commit suicide . Domino and takahashi used the suicide opinion questionnaire to investigate the differences in attitudes toward suicide between students in japan and those in the united states, and the results demonstrated that the scores for right to die and normality were higher in japanese students and that those for aggression were higher in american students.14 etzersdorfer et al compared the attitudes towards suicide held by medical students in madras with those in vienna, finding that medical students in madras rejected the right to commit suicide and assisted suicide to a greater degree than the students in vienna.15 in a swedish study to assess whether attitudes differ between students at the beginning and end of their studies, students in the final year of medical school more frequently considered suicide to be an expression of psychiatric disease and thought that people attempting to commit suicide were not responsible for their actions.16 in addition, a study from japan revealed that sympathetic comments increased with the amount of years in school among students while critical comments decreased.17 in japan, mentality on suicide might be more culturally visible and acceptable than other countries . In past years, suicide was regarded as an honorable solution to personal guilt and failure . Regarding occupational differences in attitudes toward suicide, onc et al found that medical students and general practitioners held the most permissive attitudes in turkey.18 in addition, they showed that the attitude scores of general practitioners regarding their preparedness to prevent suicide were lower compared to those of other groups . In japan, kodaka et al reported attitudes toward suicide among pharmacists using the atts.13 more than half of pharmacists reported that people understand the wishes of others to commit suicide, and 16.8% of the participants thought that people who mention suicide do not actually take their own lives . A 10 year follow - up study of norwegian physicians (n=631) revealed that a low level of maternal care predicted severe depressive symptoms and partially mediated low self - esteem.9 lung et al assessed the psychological effects on 127 health care workers who had cared for suspected severe acute respiratory syndrome (sars) patients . Early maternal attachment and neuroticism were found to heavily affect mental health during life - threatening stress.10 distinct patterns of parental bonding may contribute to the development of different dysfunctional schemas which may influence attitudes toward suicide.19 there are several limitations in this study . First, we used pbi to assess the child rearing behavior experienced by our participants . Although pbi has shown basic reliability for long periods,20 we could not completely rule out possible influences of the current mood state or recall bias in individuals because pbi is an instrument for measuring recalled parental behaviors . Second, certain parameters were not assessed in this study personality traits, a history of psychiatric disorders, suicidal behavior, and current mood state could affect the attitude towards suicidal behavior . Third, because the participants in our study are medical students, we cannot generalize these results to general practitioners . Our study indicated that the majority of participants seriously believe that anyone could commit suicide . Participants who have experienced a higher level of maternal care thought that suicide occurs commonly and tended to think that people do not have the right to commit suicide . Although parental bonding predisposes these attitudes toward suicide, educational programs could change caretaking practices.21 to achieve more positive attitudes towards suicidal behavior, promoting the best practices for suicide prevention is needed for medical students.
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Copd is characterized by persistent airway obstruction, usually of a progressive nature and associated with an enhanced chronic inflammatory response in airways to noxious particles or gases.1 copd is associated with increased mortality and reduced life expectancy.2 of the 3.8 million mortalities caused by chronic respiratory system diseases in 2010, 2.9 million were by copd.3 the first consensus report for the diagnosis, management, and prevention of copd was published in 2001 by the global initiative for chronic obstructive lung disease (gold) committee, and since then it has been updated several times . The major objectives of gold are to increase the awareness of copd and to help the millions of people who suffer from this disease and die prematurely from it or its complications by increasing the standards of care . The low adherence to guideline treatment recommendations has been reported in several studies.46 under or overprescription cause the inappropriate treatment of the disease.4,7 undertreatment of copd has also been reported in turkey.8 the bold study in adana, turkey showed that only 12.3% of the adults with copd used medication for their diseases in 2004.9 the chronic diseases and risk factors in turkey survey reported that 46.1% of the patients clinically diagnosed with copd used regular medication in 2011.10 to date, there has been no study on adherence to the gold guidelines for the treatment of copd among pulmonary physicians in turkey . At the time of the study, the patients with copd were still treated according to the gold 2010 guideline recommendations . Therefore, stages and treatments in this study are also classified in accordance with the gold 2010.1 the aim of this study is to investigate the adherence rates to gold 2010 recommendations for stable copd treatment by pulmonary physicians with a national survey in outpatient clinics of turkish pulmonary units from different regions of the country . This multi - center, cross - sectional study was conducted with the participation of the pulmonary outpatient clinics of eleven centers (university hospitals, research and training hospitals, state hospitals, and private hospitals) from eight cities (adana, ankara, diyarbakir, erzurum, istanbul, izmir, kocaeli, and samsun) across turkey . Sample size estimation has been calculated in order to have the representation of all gold stages, and sequential recruitment of 100 patients was anticipated per center . The study was approved by the institutional ethics committee of ankara university school of medicine (november 21, 2010; #24,596) prior to its initiation and conducted in accordance with the latest version of declaration of helsinki and international conference of harmonization / good clinical practice guideline . The inclusion criteria included doctor s diagnosis of copd, age 40 years, available pulmonary function test (pft) results from within the past 2 years and copd treatment data from any time within 6 months of the test, and formal written consent . Patients with a concomitant chronic disease that leads to obstructive (ie, asthma) or restrictive (ie, disseminated bronchiectasis) changes on pft, with pft results discordant to gold diagnostic criteria (symptoms of copd, and postbronchodilator spirometric evaluation confirming forced expiratory volume in 1 second (fev1)/forced vital capacity [fvc] <70%, exclusion of other obstructive lung diseases),1 and those with acute copd exacerbations were excluded from the study analysis . In nonsmoker patients, biomass exposure is considered as one of the major risk factors especially in rural areas, and they were not excluded from the study if they meet the gold spirometric diagnostic criteria . Patient recruitment has been performed according to the study inclusion and exclusion criteria in each study center . Patient data were collected from hospital records on standardized forms and following parameters were recorded: any chronic diseases accompanying copd, age at the time of copd diagnosis, spirometry results (fev1 and fev1/fvc) and copd treatment recommended within 6 months of this result, and demographic information such as sex, age, location of residence, occupation, level of education, and health insurance status . Overall, 1,125 patients have been recruited, and 719 patients meeting the gold diagnostic criteria (fev1/fvc <70%) were included in this analysis . Appropriateness of treatment was established in accordance with the 2010 gold guideline recommendations below: in stage i, add short - acting bronchodilator when needed.in stage ii, add regular treatment with one or more long - acting bronchodilator (long - acting 2-agonist [laba] or long - acting muscarinic antagonist [lama]).in stages iii and iv, add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid (ics) for those experiencing frequent exacerbations (gold 2010).1 in stage i, add short - acting bronchodilator when needed . In stage ii, add regular treatment with one or more long - acting bronchodilator (long - acting 2-agonist [laba] or long - acting muscarinic antagonist [lama]). In stages iii and iv, add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid (ics) for those experiencing frequent exacerbations (gold 2010).1 all other treatment approaches (under- or overtreatment) were evaluated as inappropriate treatment . Data are reported as mean and sd; median, minimum, and maximum; or frequency and percentage . Monte carlo simulation was used for multigroup comparisons where chi - square test assumptions were not fulfilled . Bonferroni - corrected mann whitney u - test was used for subgroup comparisons within numeric variables . Seven - hundred and nineteen patients (614 males, 105 females) with mean sd age of 62.99.7 years were included . Six - hundred and forty - two patients (89.3%) had smoking history, and health insurance coverage rate was 95.3% . Of all the patients, 2.2% (n=16) were in stage i, 33.1% (n=238) in stage ii, 48.1% (n=346) in stage iii, and 16.6% (n=119) in stage iv . Mean sd fev1 value by stage was 88.6%9.3% in stage i, 60.4%8.1% in stage ii, 39.4%5.6% in stage iii, and 23.9%4.2% in stage iv . There were significant differences between the disease stages in terms of age (p=0.004) and duration of disease (p<0.001). Of the 547 (76%) patients with available data on comorbidities, 412 (57.3%) had at least one comorbidity . The most common comorbidity was hypertension (60.9%), followed by cardiovascular diseases (43.9%), diabetes mellitus (22.6%) and depression / anxiety (10.4%). Overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group . Appropriate and inappropriate treatment approaches in different stages are represented in table 2 . In stage i, only one patient (6.25%) was treated with appropriate regimen, and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss . The two most common treatment options were long - acting muscarinic antagonist (lama) (37.5%) and laba - ics - lama (37.5%). In stage ii, adherence to gold recommendations was 14.7% (n=35), and this group was also overtreated by a combination of bronchodilators and icss . In this stage, frequently preferred treatment options were laba - ics - lama (47.5%), laba - ics - lama - theophylline (t) (13%), and laba - ics (12.6%). Of all the patients in stage ii, 81.5% (n=194) received treatment containing ics . In stage iii, adherence to guideline increased to 84.4% (n=292) because of the wide use of bronchodilator - ics combinations . The most preferred treatment combination was laba - ics - lama (45.1%), followed by laba - ics - lama - t (27.2%). Of all the patients in stage iii, 89.3% (n=309) received treatment containing ics . The undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use, 1.7% (n=6) of the patients were treated by ics alone without bronchodilator drug and 3.8% (n=9) were not on any medication for copd . In stage iv, the appropriate treatment rate was also high, as 83.2% (n=99) received dual, triple, and quadruple therapies with bronchodilators and icss . The most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations (both 31.1%). Of all the patients in stage iv, 87.3% (n=104) received treatment containing ics . The inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics, use of ics only (4.3%, n=5), and lack of treatment (5.9%, n=7). According to these findings, while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline, 40.5% was inappropriately treated . Inappropriate treatment rate were 93.8% in stage i, 85.3% in stage ii, 15.6% in stage iii, and 16.8% in stage iv (table 2). The cause of inappropriate therapies was due to over - treatment (100%) among stage i. in stage ii overtreatment (91.1%) was the main reason for guideline discordant therapies, followed by undertreatment (8.9%), and lack of treatment (3.8%). Only 14.7% of the stage ii patients were treated appropriately . In severe and very severe patients, guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . In stage iii, 13.3% of patients were undertreated, and 2.3% of very severe copd patients did not receive any regular treatment . While undertreatment rate was 10.9% among stage iv patients, 5.9% of stage iv patients had no treatment for copd . The most administered treatment regimen was laba - ics - lama combination (43.4%, n=312), followed by laba - ics - lama - t (22.5%, n=162) and laba - ics (10.9%, n=78). In patients treated with regular bronchodilators, the most frequently used drug was laba (n=613), followed by lama (n=575), t (n=197), short - acting 2-agonist (n=13) and short - acting muscarinic antagonistic drugs (n=13), respectively . The distribution of patients on bronchodilator alone, treatment containing ics, and no maintenance drug by copd stage is shown in table 3 . Of all the patients, 614 (89%) received treatment containing ics or ics alone . The distribution of patients who received ics was 43.8% in stage i, 81.5% in stage ii, 89.2% in stage iii, and 86.5% in stage iv . Of the 25 (3.5%) patients who were not on any medication, one patient was in stage i, nine in stage ii, eight in stage iii, and nine in stage iv . There were 327 (45.5%) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy . Of all the patients, 56 (7.8%) were on monotherapy, 125 (17.4%) on dual, 350 (48.7%) on triple, and 163 (22.6%) on quadruple therapy . Long - term oxygen use rate was 22.4% (n=112 patients) in stages iii and iv patients . Seven - hundred and nineteen patients (614 males, 105 females) with mean sd age of 62.99.7 years were included . Six - hundred and forty - two patients (89.3%) had smoking history, and health insurance coverage rate was 95.3% . Of all the patients, 2.2% (n=16) were in stage i, 33.1% (n=238) in stage ii, 48.1% (n=346) in stage iii, and 16.6% (n=119) in stage iv . Mean sd fev1 value by stage was 88.6%9.3% in stage i, 60.4%8.1% in stage ii, 39.4%5.6% in stage iii, and 23.9%4.2% in stage iv . There were significant differences between the disease stages in terms of age (p=0.004) and duration of disease (p<0.001). Of the 547 (76%) patients with available data on comorbidities, 412 (57.3%) had at least one comorbidity . The most common comorbidity was hypertension (60.9%), followed by cardiovascular diseases (43.9%), diabetes mellitus (22.6%) and depression / anxiety (10.4%). Overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group . Appropriate and inappropriate treatment approaches in different stages are represented in table 2 . In stage i, only one patient (6.25%) was treated with appropriate regimen, and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss . The two most common treatment options were long - acting muscarinic antagonist (lama) (37.5%) and laba - ics - lama (37.5%). In stage ii, adherence to gold recommendations was 14.7% (n=35), and this group was also overtreated by a combination of bronchodilators and icss . In this stage, frequently preferred treatment options were laba - ics - lama (47.5%), laba - ics - lama - theophylline (t) (13%), and laba - ics (12.6%). Of all the patients in stage ii, 81.5% (n=194) received treatment containing ics . In stage iii, adherence to guideline increased to 84.4% (n=292) because of the wide use of bronchodilator - ics combinations . The most preferred treatment combination was laba - ics - lama (45.1%), followed by laba - ics - lama - t (27.2%). Of all the patients in stage iii, 89.3% (n=309) received treatment containing ics . The undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use, 1.7% (n=6) of the patients were treated by ics alone without bronchodilator drug and 3.8% (n=9) were not on any medication for copd . In stage iv, the appropriate treatment rate was also high, as 83.2% (n=99) received dual, triple, and quadruple therapies with bronchodilators and icss . The most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations (both 31.1%). Of all the patients in stage iv, 87.3% (n=104) received treatment containing ics . The inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics, use of ics only (4.3%, n=5), and lack of treatment (5.9%, n=7). According to these findings, while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline, 40.5% was inappropriately treated . Inappropriate treatment rate were 93.8% in stage i, 85.3% in stage ii, 15.6% in stage iii, and 16.8% in stage iv (table 2). The cause of inappropriate therapies was due to over - treatment (100%) among stage i. in stage ii overtreatment (91.1%) was the main reason for guideline discordant therapies, followed by undertreatment (8.9%), and lack of treatment (3.8%). Only 14.7% of the stage ii patients were treated appropriately . In severe and very severe patients, guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . In stage iii, 13.3% of patients were undertreated, and 2.3% of very severe copd patients did not receive any regular treatment . While undertreatment rate was 10.9% among stage iv patients, 5.9% of stage iv patients had no treatment for copd . The most administered treatment regimen was laba - ics - lama combination (43.4%, n=312), followed by laba - ics - lama - t (22.5%, n=162) and laba - ics (10.9%, n=78). In patients treated with regular bronchodilators, the most frequently used drug was laba (n=613), followed by lama (n=575), t (n=197), short - acting 2-agonist (n=13) and short - acting muscarinic antagonistic drugs (n=13), respectively . The distribution of patients on bronchodilator alone, treatment containing ics, and no maintenance drug by copd stage is shown in table 3 . Of all the patients, 614 (89%) received treatment containing ics or ics alone . The distribution of patients who received ics was 43.8% in stage i, 81.5% in stage ii, 89.2% in stage iii, and 86.5% in stage iv . Of the 25 (3.5%) patients who were not on any medication, one patient was in stage i, nine in stage ii, eight in stage iii, and nine in stage iv . There were 327 (45.5%) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy . Of all the patients, 56 (7.8%) were on monotherapy, 125 (17.4%) on dual, 350 (48.7%) on triple, and 163 (22.6%) on quadruple therapy . Long - term oxygen use rate was 22.4% (n=112 patients) in stages iii and iv patients . This is the first large - scale real - life study in turkey that reflects the clinical practice of pulmonary specialists through an observational analysis of their adherence to treatment recommendations of gold 2010 guideline . The survey was carried out in eleven pulmonary outpatient clinics spread over the national territory . Our findings show that only 59.5% of overall patients were treated in accordance with the guideline recommendations . While inappropriate treatment regimens were more frequently prescribed for stages i (93.8%) and ii (85.3%), overtreatment in early stages and high rate of ics administration (89%) in all stages of disease resulted in improper therapeutic approach according to gold 2010 recommendations . In more severe disease stages, guideline - adherent therapeutic approaches were detected by wide use of ics addition to one or more long - acting bronchodilators . Most of the provider s guideline adherence studies have reported the current practices of general practitioners . A few recent studies have reported similar rates of adherence to guideline recommendations for copd patients (37.9%54.7%) between pulmonologists in other countries.4,5 stage i patients were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss, while guidelines recommend short - acting bronchodilators as initial therapy . In a study conducted among the pulmonary specialists in italy, corrado and rossi reported that 87.7% of the stage i patients received regular treatment containing long - acting bronchodilators, ics alone, or other treatment combinations of ics plus long - acting bronchodilators.4 in a retrospective database study in the united states, fitch et al reported that only 1% of the stage i patients received ics added to laba and lama, while 6% received ics plus single long - acting bronchodilators, and 76% did not receive any medication in stage i.11 shariff et al reported that 7.7% of the patients in stage i were overtreated and received inappropriate treatment according to the guidelines, in another study on guideline adherence among practitioners and pulmonary specialists.5 in our study, only 14.7% of the patients in stage ii received appropriate therapy, and this was mainly due to overtreatment which was combined long - acting bronchodilators and ics in 77.3% of the patients . Other reasons for nonadherent therapeutic approaches were single use of ics in 3.4% of the patients and lack of treatment in 3.8% of the patients . Other studies found 26%49.2% adherence to gold recommendations in this stage . Overtreatment was 67.3% in one study,4 and lack of copd treatment has been reported in 54% of the patients in another report for stage ii.11 severe and very severe copd patients were treated by the combined use of one or more long - acting bronchodilators and ics which was compliant to gold recommendations . This finding can be explained by the features of this group of patients who are more symptomatic and have more frequent exacerbations . Similar studies on guideline adherence in different countries also reported frequent use of dual and triple therapies in stages iii and iv.4,5,11 the ics was frequently used at all stages (43.8% in stage i, 81.5% in stage ii, 89.3% in stage iii, and 87.4% in stage iv) mostly as a part of combination therapy . This study demonstrates that the pulmonary specialists in turkey have a high tendency to use ics for the treatment of patients with copd . In a study by de miguel - diez et al in spain, 34.5% of the patients were prescribed ics, alone or in combination, with a rate of inappropriate use of 18.2%.12 asche et al reported that 33% of the patients received combination treatment containing laba, lama, and/or ics consistent with the current gold guidelines.13 pulmonary specialists in our study mostly preferred to prescribe combination treatments containing ics . This might be related to a number of factors: inadequate time with patients, which leads to opting for combination therapies that can provide a broad - spectrum treatment.concerns about symptom control, exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist.reimbursement of all copd drugs in turkey, making them easily accessible for patients with health insurance.preferring to prescribe drug groups that are effective in the treatment of both asthma and copd, where a differential diagnosis is not available.increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use . Inadequate time with patients, which leads to opting for combination therapies that can provide a broad - spectrum treatment . Concerns about symptom control, exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist . Reimbursement of all copd drugs in turkey, making them easily accessible for patients with health insurance . Preferring to prescribe drug groups that are effective in the treatment of both asthma and copd, where a differential diagnosis is not available . Increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use . In patients treated with regular bronchodilators (alone or a part of a combination therapy), the most frequently used drug was laba (n=613), followed by lama (n = 575), t (n=197). Among these results, t use was prescribed for 27.4% of stable copd patients which was remarkably higher than the finding of corrado and rossi that reported 2.2% of patients were using xanthines.4 gold remark on t is that it is effective in copd but, due to its potential toxicity, inhaled bronchodilators are preferred when available.1 in our study, this high rate of t prescription in addition to other inhaled bronchodilators may be explained by the choice of an oral bronchodilator drug even if it has potential toxicity and drug interactions in older patients . Another finding of this study was that the use of long - acting bronchodilators such as laba and lama alone remains considerably low, and they have been mostly preferred in stages i, ii, and iii . The most commonly used bronchodilator in monotherapy was lama (4.2%), and this might be due to its single - dose use and duration of action of more than 24 hours.14,15 this study has several limitations . Individual patient data were retrospectively collected through patient records: number of acute exacerbations in the previous year, complete information on comorbidities, adherence to nonpharmacologic interventions were lacking . Gold guidelines were updated in 2011 to incorporate clinical presentation, including dyspnea as measured by copd assessment test and the modified british medical research council . The updated version of the guidelines incorporates clinical presentation with spirometry findings (gold 2011).16 this study was undertaken between december 2010 and november 2011 . Study protocol had been designed to evaluate gold 2010 guideline adherence . Due to lack of knowledge on dyspnea level (medical research council scale) and exacerbation frequency during the last year even this new gold classification (updated in 2011) has been changed by considering sypmtoms and exacerbations in addition to spirometric staging, we have not enough data as to the exact superiorities over the old gold classification . A very recent analysis showed that gold 2011 shifted the overall copd severity distribution to more severe categories . There were nearly three times more copd patients in stage d than in former stage iv (p<0.05). The predictive capacity for survival up to 10 years was significant for both systems (p<0.01) and gold 2007 and 2011 did not differ significantly . In this study, the percent predicted fev1 thresholds of 85%, 55%, and 35% were found better to stage copd severity for mortality, which are similar to the ones used previously in the gold 2010 classification . Increasing intensity of treatment of patients with copd according to their gold 2011 reclassification is not known to improve health outcomes.17 the classification recommended by the gold committee since 2011 does not seem to be adopted universally throughout europe . Even if there is a real improvement over the previous classification due to taking into account clinical criteria, quality of life, and exacerbations, criticisms have arisen concerning the choice of certain pathways and therapeutic recommendations not based on prospective studies with a high level of evidence.18 at the national level, we do not have any published data on the adoption of the new gold 2011 classification . According to the previously mentioned obstacles of the new classification, and lack of the knowledge about the frequency of new gold 2011 classification use, instead of the gold 2010 . We believe this data on the guideline adherence among pulmonologists may still reflect the current attitude toward the routine approach of the diagnosis and therapies of copd in turkey, and also is concordant with the real life situation around the world . Turkish thoracic society has published an updated report of copd prevention, diagnosis and treatment in 2014,19 and included in the global alliance against chronic respiratory disease (gard) with the ministry of health.20 the improvement of adherence to national and international copd guidelines may be achieved by continuing medical education supported by national thoracic societies . Increased awareness of guidelines may influence the standardized care of copd patients, and will have prognostic influences . Promotion of the compliance to evidence - based medicine by health authorities may have also positive effects on the attitude change in the real - life applications . This study showed that the pulmonary specialists in turkey have low rates of adherence to gold guidelines for treatment of stable copd patients . Major improper approaches were overtreatment in early stages and excessive use of inhaler corticosteroids in all stages of disease . New strategies are needed to achieve a standardized approach for the treatment of copd, and thus to improve adherence to guideline recommendations, with particular focus on treatment indications and overtreatment in turkey.
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Amphotericin b (amb), an amphoteric polyene macrolide, has been considered one of the main options of antifungal therapy for serious and life - threatening mycotic infections for over 50 years despite the introduction of newer antifungal medications such as the azoles (e.g., voriconazole) and echinocandins (e.g., caspofungin), which have better safety profiles (1, 2). This may be due to the fact that azoles and echinocandins are costly, ineffective against certain pathogenic fungi (e.g., candida krusei and candida parapsilosis), and not readily available in developing countries such as iran (3). Many acute and chronic adverse reactions have been associated with amb, such as infusion - related reactions (e.g., fever, chills, and hypotension), normocytic normochromic anemia, cardiac toxicity (e.g., ventricular tachycardia and hypertension), hepatic toxicity (e.g., increase in liver enzymes and bilirubin), neurologic toxicity (e.g., confusion, delirium, tremor, and seizure), and nephrotoxicity (4, 5). Major features of amb - induced nephrotoxicity include an increased serum creatinine level, a decreased glomerular filtration rate (gfr), urinary potassium wasting, hypokalemia, urinary magnesium wasting, and hypomagnesemia (6). Although it is a common event in clinical settings, different aspects of amb nephrotoxicity have not been studied well or in detail in our population . The purpose of this investigation was to specifically assess the frequency, time onset, and possible associated factors of amb nephrotoxicity in hospitalized patients in hematology - oncology wards in the southwest of iran . A cross - sectional, observational study was performed over a period of 9 months from august 2015 to april 2016 in 2 hematology - oncology and 1 hematopoietic stem cell transplantation wards of namazi hospital, which is affiliated to shiraz university of medical sciences, shiraz, iran . The medical ethics committee of the hospital approved the study, and all patients signed and approved a written informed consent form . The inclusion criteria for recruiting patients were as follows: 1) age 15 years or older; 2) no documented history of acute kidney injury defined by an increase in serum creatinine 0.3 mg / dl within 48 hours, or an increase in serum creatinine by 1.5 times the baseline in the previous 7 days, or a urine volume <0.5 ml / kg / h for 6 hours (7); 3) no documented history of chronic kidney disease (clearance creatinine below 60 ml / min/1.73 m calculated by the simplified modification of diet in renal disease equation); 4) no documented history of peritoneal or hemodialysis for> 3 months (8); 5) no documented history of having received amb by any administration route in the previous 14 days; and 6) having received any formulation of amb intravenously for at least 1 week . They included age, sex, weight, amb dose, duration and indication, duration of amb infusion, type of co - administered medications that may exacerbate or attenuate amb nephrotoxicity, length of hospital stay, and mortality . Serum urea and creatinine were monitored daily during the course of the amb treatment . According to routine ward practice, serum potassium and magnesium were checked daily and once weekly for patients, respectively . Because most cases of amb nephrotoxicity occur during the first 2 weeks of treatment (9, 10), urine urea, creatinine, sodium, potassium, and magnesium levels were determined at days 0, 3, 5, 7, 10, and 14 of the amb treatment . Serum as well as urine urea, creatinine, and the aforementioned electrolytes were determined using an auto - analyzer (shanghai xunda medical instrument, shanghai, china). The creatinine levels in the serum and urine samples were established using the modified jaffe colorimetric reaction . Amb nephrotoxicity was defined as an estimated clcr 50% (calculated by the cockcroft - gault formula) or an increase in serum creatinine (double the baseline value) (11). Either the fractional excretion of sodium> 2% or the fractional excretion of urea> 50% (in cases of diuretic co - administration) was considered as acute tubular necrosis (atn) (12). Serum level potassium and magnesium below 3 meq / l and 1.2 meq / l were defined as hypokalemia and hypomagnesemia, respectively (13). A urine potassium - to - creatinine ratio above 13 meq / g was considered renal potassium wasting (14). The time onset of nephrotoxicity, hypokalemia, and hypomagnesemia after the initiation of amb was also determined . Daily dose reductions, every other day dosing, discontinuation, or performing dialysis as probable measures of managing amb nephrotoxicity were recorded . Continuous data were expressed as either mean standard deviation (sd) or mean standard error (se). Probable associations between the categorical variables were evaluated by the chi - squared or fisher s exact test . The fisher s exact test was applied if more than 25% of the categories had expected frequencies of less than 5 . The parametric and non - parametric continuous variables were examined by the independent t- and mann - whitney u tests, respectively . Using the stepwise method, the logistic regression analysis with the odds ratio (or) and 95% confidence interval (ci) were employed to determine the associated factors of amb nephrotoxicity . In the first step, the possible association of each independent variable, namely age, gender, amb cumulative dose, duration of amb infusion, baseline gfr value, mean daily oral / intravenous sodium supplementation, and the co - administration of aminoglycosides, calcineurin inhibitors, vancomycin, acyclovir, loop diuretics, corticosteroids, and sodium bicarbonate, with amb nephrotoxicity (as the dependent variable) was assessed separately by univariate analysis . Those with p values of less than 0.4 statistical significance in all analyses was defined by p values <0.05, except for the first step of the logistic regression analysis (p values <0.4). Continuous data were expressed as either mean standard deviation (sd) or mean standard error (se). Probable associations between the categorical variables were evaluated by the chi - squared or fisher s exact test . The fisher s exact test was applied if more than 25% of the categories had expected frequencies of less than 5 . The parametric and non - parametric continuous variables were examined by the independent t- and mann - whitney u tests, respectively . Using the stepwise method, the logistic regression analysis with the odds ratio (or) and 95% confidence interval (ci) were employed to determine the associated factors of amb nephrotoxicity . In the first step, the possible association of each independent variable, namely age, gender, amb cumulative dose, duration of amb infusion, baseline gfr value, mean daily oral / intravenous sodium supplementation, and the co - administration of aminoglycosides, calcineurin inhibitors, vancomycin, acyclovir, loop diuretics, corticosteroids, and sodium bicarbonate, with amb nephrotoxicity (as the dependent variable) was assessed separately by univariate analysis . Those with p values of less than 0.4 were then considered together for the multivariate logistic regression analysis . Statistical significance in all analyses was defined by p values <0.05, except for the first step of the logistic regression analysis (p values <0.4). During the 9-month study period, 90 patients who were scheduled to receive amb were screened . A flowchart detailing the reasons patients dropped out of the study is presented in figure 1 . The most common admission diagnosis of the patients was acute myeloid leukemia (60%) followed by acute lymphoid leukemia (20%), hodgkin disease (5%), and aplastic anemia (5%). Prophylaxis of aspergillosis (52.5%), treatment of aspergillosis (17.5%), and treatment of mucormycosis (12.5%) were the three most frequent indications of amb administration (table 1). The mean sd daily doses of conventional and liposomal amb were 28.79 10.84 mg and 212.78 99.33 mg, respectively . The mean sd cumulative dose of conventional amb was 223.79 125.53 mg and 2367.15 2137.89 mg for liposomal amb . All courses of either conventional or liposomal amb infusions were administered within 2 - 6 hours . Potential nephrotoxic medications, namely acyclovir, corticosteroids, calcineurin inhibitors (cyclosporine), vancomycin, loop diuretics (furosemide), and aminoglycosides (amikacin), were given to 38 (95%), 28 (70%), 21 (52.5%), 19 (47.5%), 5 (12.5%), and 1 (2.5%) patients, respectively . In contrast, spironolactone and sodium bicarbonate as potential nephroprotective agents were administered to 7 (17.5%) and 4 (10%) individuals, respectively . No patient received cisplatin, ifosfamide, cyclophosphamide, dopamine, and mannitol during the study period . The mean sd daily oral / intravenous sodium supplementation administered in conjunction with the amb treatment was 124.75 62.15 meq . During the study, 11 (27.5%) and 21 (52.5%) patients developed amb nephrotoxicity and atn, respectively . The mean sd onset of amb nephrotoxicity was 6.73 2.36 days . In accordance with the univariate analysis, amb indication (p = 0.259), vancomycin co - administration (p = 0.388), duration of liposomal amb infusion (p = 0.141), and amount of oral / intravenous sodium supplementation (p = 0.375) based on this model, none of these variables were significantly associated with amb nephrotoxicity (table 2). Amb administration was discontinued permanently in 2 individuals who developed nephrotoxicity, and they underwent emergent hemodialysis . There was no statistically significant difference in the duration of hospitalization between the patients with and without amb nephrotoxicity (27.72 4.81 and 30.33 13.57 days, respectively; p = 0.541). The mortality rate was also comparable between the patients with (54.54%) and without (48.27%) amb nephrotoxicity (p = 0.723). The mean sd onset of hypokalemia during amb administration was 5.06 3.35 days . Only one documented episode of hypomagnesemia was detected in the study population during the amb treatment . Considering the fact that serum magnesium levels were not measured routinely for most of the patients, determining the rate of renal magnesium wasting was not feasible . The co - administration of neither loop (p = 0.642) nor potassium - sparing diuretics (p = 0.211) was significantly associated with hypokalemia . The mean se daily amounts of intravenous potassium and magnesium administered during the course of the amb treatment were 52.43 12.13 and 3.51 2.04 meq, respectively . The incidence (27.5%) and time onset (5.06 3.35 days) of amb nephrotoxicity in the present study were within the ranges reported in the literature . Some degree of increase in serum creatinine has been detected within 2 weeks in up to 80% of patients who received amb (9, 10). In one of the most prominent studies in this regard, a 9-year retrospective analysis demonstrated that 138 (28%) of 494 adult in - patients experienced some type of nephrotoxicity during amb treatment (10). In a 1-year prospective observational study by tavakoli - ardakani et al . (15) in the hematology - oncology and stem cell transplantation wards at taleghani hospital in tehran, 9 (25.71%) out of 35 patients developed an increase in serum creatinine and bun during the course of amb treatment . This rate was reported to be 27.8% at another referral hematology - oncology and stem cell transplantation center in tehran (16). In the adult infectious diseases ward at imam khomeini hospital in tehran, khalili et al . (17) demonstrated that 10 (76.92%) of 13 individuals receiving amb alone developed acute kidney injury (aki); however, the incidence of aki among the patients given amb along with ceftriaxone and/or vancomycin was 86.68% . The time onset of amb nephrotoxicity was not generally reported in the aforementioned iranian studies . The wide variations in the incidence of amb nephrotoxicity noted above could be due to different study methodologies, clinical settings, relevant risk factors, and definitions . Notably, 62.5% of the individuals in our study were given liposomal amb, which has a considerably less nephrotoxic formulation . This could partially justify the relatively lower rate of amb nephrotoxicity in our cohort than in similar studies in iran . In the present study, amb nephrotoxicity in 45.45% of the affected patients resolved spontaneously without any intervention . Only 2 patients in our cohort required emergency hemodialysis due to the severity and persistency of amb nephrotoxicity . It has been reported that amb nephrotoxicity is predominantly reversible within a few months after discontinuation of treatment (18); however, about 15% of affected patients may require renal replacement therapy such as dialysis (19). In the shariati hematology - oncology and stem cell transplantation wards, hayatshahi et al . (16) showed that amb nephrotoxicity led to a dose reduction of this agent in 3.7% of cases . In terms of clinical outcomes, mortality and duration of hospitalization were comparable between patients with and without amb nephrotoxicity in the present population . In contrast, bates et al.s (20) study of a large population (707 adults) in the united states reported that acute renal failure due to amb increased the mean length of hospital stay by 8.2 days (p <0.0001) and increased the total cost of treatment by $29,823 . The mortality rate was also much higher (54% vs. 16%; p = 0.001). Differences in the confounding factors (e.g., the severity of the underlying disease), the type of amb formulation used (conventional vs. lipid - based), and sample size could partially account for these disparities . We considered p values greater than 0.05 (0.4) in the univariate analysis to select all possible variables linked to the dependent variable . Among the different demographic, clinical, and paraclinical features, amb indication, vancomycin co - administration, duration of liposomal amb infusion, and amount of oral / intravenous sodium supplementation nevertheless, none of these variables were significantly associated with amb nephrotoxicity in the multivariate logistic regression model . Several large - scale retrospective and prospective studies have identified males, an average daily dose of amb above 35 mg, a cumulative dose of amb greater than 2 - 5 g, dehydration, co - administered diuretics and nephrotoxic agents (e.g., aminoglycosides, cyclosporine, foscarnet, cisplatin, and ifosfamide) or corticosteroids, and baseline renal dysfunction as potential risk factors for amb nephrotoxicity (21 - 23). Inadequate statistical power resulting from the relatively small sample size and the relatively low incidence of amb nephrotoxicity due to the administration of liposomal amb appear to be the main reasons for our findings in this regard . It has been suggested that conventional amb should preferably not be administered to patients with two or more of the aforementioned risk factors of amb nephrotoxicity (10, 24). Notwithstanding, with the introduction of considerably less nephrotoxic formulations of amb into the market, this suggestion does not seem to make sense in clinical practice . During the course of the amb treatment in the current study, hypokalemia and renal potassium wasting developed in 45% and 27.5% of the patients, respectively . In contrast, hypomagnesemia was detected in only 1 patient (2.5%). The incidence of amb - induced hypokalemia in our cohort was lower than that reported in the literature (75% - 90%) (25). This may be due to the fact that 62.5% of the patients in the present study received liposomal amb . Several publications have estimated the frequency of amb - induced hypomagnesemia to be between 15% and 100% depending on the dose and formulation of amb (26). Apart from the type of amb formulation and the presence of relevant risk factors, such as the co - administration of loop and potassium - sparing diuretics, the low rate of hypomagnesemia in our population could have been because the patients serum magnesium levels were measured only once a week during the amb treatment in the studied wards . Severe hypokalemia and hypomagnesemia due to amb can cause metabolic complications, rhabdomyolysis, and life - threatening arrhythmias (25); however, these adverse events were not observed in our cohort at least during the course of the amb treatment . In conclusion, nearly one - third (27.5%) of our cohort developed nephrotoxicity within the first week of amb treatment . Amb nephrotoxicity resolved spontaneously in about half (45.45%) of the affected patients without any intervention . Mortality and the duration of hospitalization were comparable between patients with and without amb nephrotoxicity . No studied demographic, clinical, and paraclinical features of the study population were significantly associated with amb nephrotoxicity . Among the studied electrolyte abnormalities, hypokalemia and renal potassium wasting were the most notable, affecting about one - half and one - third of amb recipients, respectively . The co - administration of either loop or potassium - sparing diuretics did not significantly affect electrolyte abnormalities during the course of the amb treatment . Close monitoring of renal function indexes, including serum creatinine, bun, serum potassium, and magnesium, during amb treatment is highly recommended . Additionally, implementing approved prophylactic measures, such as saline loading (150 meq / day) before and/or during amb infusion, and exploiting lipid - based formulations of amb (especially liposomal), if available and affordable, should be considered to minimize the possibility of amb nephrotoxicity.
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O - glycans have been found to play an important role in protein stability and tertiary structure, stabilize protein conformation, modulate the activity of enzymes (e.g. By reversible attachment of o - linked glcnac to cytoplasmic and nuclear proteins) and signaling molecules, are essential for a number of recognition processes and are sorting determinants guiding the modified protein in the cell from the place of biosynthesis to its target location [13]. Unlike the n - glycans they may be linked to several different amino acid residues (very often serine or threonine, but also hydroxylysine, tyrosine or hydroxyproline) of the protein and their occurrence cannot be predicted easily by a common consensus sequence . Extended galnac residues linked to serine or threonine residues (mucin - type glycans) are the most frequent and best investigated o - glycans in higher eukaryotes . Eight core subtype formations showing cell - tissue specific patterns related to their function have been described (for a review see). Also glcnac--ser / thr without any elongation, a typical feature for nuclear and cytoskeletal proteins existing in a dynamic equilibrium with phosphorylation, and fuc--ser / thr as well as glc--ser, primarily found in epidermal growth factor domains, have been found in eukaryotes . A linkage via mannose has been found to be the main o - glycan type in yeasts, but also some mammalian tissues carry this structural feature, which seems to be relevant in some muscular dystrophies, while o - xylosylation is the starting point for the biosynthesis of chondroitin and heparan sulfates, which have roles in development and morphogenesis . The o - glycans of nematodes are rather complex with methylation as further modification [9, 10]. Xenopus eggs have o - glycans with up to 10 monosaccharide constituents, often highly fucosylated, with a linkage to the protein via galnac [11, 12]. Even parasites (helminths and t. cruzi) display small galnac linked o - glycans . In plants o - glycans occur as arabinogalactans linked to hydroxyproline or serine (gal--hyp / ser), or hydroxyproline is glycosylated with short arabinofuranosides (ara--hyp, ara--hyp). Here we present for the first time an overview on o - glycans of some species of the mollusk branch of the evolutionary tree . The glycosylation potential of snails is important in general, because some of them are intermediate hosts of pathogens (biomphalaria glabrata and lymnaea stagnalis) or a pest in agriculture . A broad knowledge of their glycosylation abilities may show targets for pest control in the near future . In the course of the years, n - glycan patterns of several snail species have been presented [1417]. Methylated mannose and galactose residues were found to be frequent constituents . In the present study we determine the main o - glycan structures in snails and characterize a common core - trisaccharide . Cepaea hortensis, planorbarius corneus, arion lusitanicus, limax maximus and helix pomatia were collected by the authors under the supervision of dr . Manfred pintar (department of integrative biology and biodiversity research, institute of zoology, university of natural resources and life sciences, vienna) in the vicinity of vienna . All chemicals purchased were of the highest quality available from sigma or fluka . While water living snails could be dissected right away, land living snails were extensively washed to remove the extraneous mucous components . Snails were dissected as previously described and proteins were purified according to with minor modifications: 1 g of wet tissue was homogenised in 10 ml of chaps - based lysis buffer (0.5% (w / v) chaps, 150 mm nacl, 20 mm tris / hcl, 2.5 mm sodium pyrophosphate, 1 mm ethylene - glycol - bis(2-aminoethylether)-n, n, n,n-tetraacetic acid and 1 mm edta, ph 7.5) and incubated for 72 h at 4 c . Snail proteins were then reduced by the addition of 500 l of 10 mm dithiothreitol and incubation at 56 c for 45 min . Then 500 l of 55 mm iodoacetamide were added and incubated for 30 min at room temperature in the dark . The aqueous samples were precipitated with a 4-fold amount of pre - chilled acetone at 20 c for at least 4 h and then centrifuged at 32000 g for 60 min . The pellets were resuspended in 0.1 m sodium citrate buffer, ph 4.6, and incubated with 2 units of -glucosidase from rice [ec 3.2.1.20] at 37 c for 16 h. sugars and polysaccharides were eluted from cation exchange chromatography (ag 50 w - x2, biorad, ca) with 2% of acetic acid, while proteins were eluted by 1.0 m ammonium acetate, ph 9.0 . The protein fraction was again precipitated by acetone followed by a similar precipitation by methanol . The dry sample was dissolved in 0.1 m naoh containing 0.8 m nabh3 and incubated at 37 c for 68 h. the sample was carefully neutralised by the addition of 2 m acetic acid, dried in a vacuum centrifuge and washed at least twice with 30% (v / v) methanol . The glycans were directly extracted by solid phase extraction on a porous graphitized carbon cartridge (supelclean envi - carb, 100 mg bed weight, 1 ml column volume or 500 mg bed weight, 6 ml column volume, sigma - aldrich, germany) according to by using ammonium formate buffer / acetonitrile for elution and then on a reversed - phase c18 cartridge (strata, c18-e, 55 m, 50 mg bed volume, 1 ml column volume, phenomenex, germany) using 5% (v / v) acetic acid as loading solvent and 25% (v / v) isopropanol in 5% (v / v) acetic acid for elution . Hplc separation was carried out on a porous graphitized carbon (pgc) column (hypersil - keystone, hypercarb 5 150 3 mm, thermo scientific, austria) at a flow rate of 0.6 ml / min using 0.3% (v / v) formic acid adjusted to ph 3.1 with ammonium as solvent a and 95% (v / v) acetonitrile in solvent a as solvent b. the elution protocol consisted of 2 min at 100% (v / v) of solvent a followed by a gradient of 28 min till 25% (v / v) of solvent b and a cleaning step of 4 min at 60% (v / v) of solvent b. each fraction was tested by esi - ms for its glycan content . Single glycan structures were pooled and lyophilised . For monosaccharide analysis alditol acetates and for linkage analysis gc analysis was carried out on a vf 5 ms capillary column (60 m, 0.25 mm inner diameter; 0.1 mm film thickness; varian, darmstadt, germany) and electron impact (ei) mass spectrometry was performed in the positive ion mode using a polaris q instrument (thermoquest - analytical systems). For analysis of small sample amounts with esi - ms an in - gel release method for glycans bound to coomassie reduced oligosaccharides were analyzed by pgc - lc - esi - ms on a hypercarb column (0.32 150 mm, thermo fisher scientific, austria) coupled to an ultimate 3000 (dionex) capillary hplc and a q - tof ultima ms (waters) as described previously [21, 26]. The interpretation of the ms data was performed with the help of the software tool glycoworkbench . Arion lusitanicus proteins were isolated and separated from contaminants through several purification steps in order to obtain sufficient amounts (12 g) of pure glycans for further gc - ms analysis (total monosaccharide and linkage analysis). The preparation of o - glycans was carried out via -elimination followed by further separation steps . As a final procedure the glycans were separated on a pgc (porous graphitized carbon) hplc - column by collecting 1-minute fractions which were screened by esi - ms to gain clean single structures . In the course of the -elimination releasing the glycan from the protein backbone, sodium borohydride was used . In contrast, for reduction of monosaccharides before gc - ms analysis sodium borodeuteride was employed, which allowed further the discrimination between the sugar released by -elimination and those linked to other sugars previously . In fig . 1 monosaccharide constituents of the core trisaccharide representing the main structure found in a. lusitanicus, with a pseudomolecular mass ([m+h]) of m / z 576.3 da, were determined by gc - ms of the corresponding alditol acetates and identified on the basis of retention times in gc and their characteristic electron impact (ei) mass spectra . The results revealed the presence of a galnac - residue which was h - reduced, while all the other sugars were h reduced . Therefore, galnac can be considered as the protein bound sugar . The high amount of 4-o - me - gal indicated that this residue is the one which is mainly involved in the elongation of the glycan . Minor compounds were identified as contaminations by n - glycans (glcnac and man) or storage glycans (glc and gal). The lc - esi - ms / ms fragmentation pattern confirmed the occurrence of two methylated hexoses linked to the amino sugar (fig . 1monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [h] reductive -elimination . Alditol acetates obtained after acid hydrolysis, reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms . Peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed (1: man, h - reduced, 2: glc, h - reduced, 3: gal, h - reduced, 4: glcnac, h - reduced)fig 2lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus . Registered ions represent proton adducts [m+h] monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [h] reductive -elimination . Alditol acetates obtained after acid hydrolysis, reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms . Peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed (1: man, h - reduced, 2: glc, h - reduced, 3: gal, h - reduced, 4: glcnac, h - reduced) lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus . Registered ions represent proton adducts [m+h] moreover, gc - ms analysis of partially methylated alditol acetates was carried out for further confirmation of the compounds and also for linkage determination . Methylated sugar standards purified by preparative gc were used to elucidate the type of methylated hexose (man or gal) based on retention time . The selected ion chromatogram of the monosaccharide derivatives obtained from the o - glycan core structure identified 2,3,4,6-tetra - o - methyl - galacitol at 20.66 min and 1,4,5-tri - o - methyl galnac - ol at 33.33 min (fig . 3a) which could be verified by ei - ms data (figs . 3b and c). To determine the position of the naturally occurring methyl group on the galactose this allows in the ei - ms fragmentation pattern the discrimination between the naturally occurring and introduced methylgroups in the fragments by a mass difference of 3 da . That way the occurrence of terminal 4-o - methylated galactose 4). Combining these data, the main o - glycan structure of a. lusitanicus was elucidated as a trisaccharide containing two terminal 4-o - methylated galactoses which are 3- and 6-linked to an inner galnac residue (fig . 5).fig 3linkage analysis of the core trisaccharide - alditol from a. lusitanicus (a) selected ion chromatogram (m / z 145 and m / z 318) of partially methylated alditol acetates obtained after permethylation with [h] methyliodide, hydrolysis, reduction with sodium borohydride and peracetylation; (b) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and (c) 2,3,4,6-tetra - o - methyl - galacitol . Characteristic primary selected secondary fragment ions are assignedfig 4ei - ms spectra and fragmentation patterns of (a) 1,4,5-tri - o - deuteromethyl - galnac - ol and (b) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [h] methyliodide . The structure plot is generated in the notation of the consortium for functional glycomics (http://www.functionalglycomics.org) using the visual editor of glycoworkbench . This software application is developed and available as part of the eurocarbdb project (http://www.eurocarb.db.org/applications/ms-tools). Square = galnac, circles = gal, me = methyl group linkage analysis of the core trisaccharide - alditol from a. lusitanicus (a) selected ion chromatogram (m / z 145 and m / z 318) of partially methylated alditol acetates obtained after permethylation with [h] methyliodide, hydrolysis, reduction with sodium borohydride and peracetylation; (b) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and (c) 2,3,4,6-tetra - o - methyl - galacitol . Characteristic primary selected secondary fragment ions are assigned ei - ms spectra and fragmentation patterns of (a) 1,4,5-tri - o - deuteromethyl - galnac - ol and (b) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [h] methyliodide . The structure plot is generated in the notation of the consortium for functional glycomics (http://www.functionalglycomics.org) using the visual editor of glycoworkbench . This software application is developed and available as part of the eurocarbdb project (http://www.eurocarb.db.org/applications/ms-tools). Square = galnac, circles = gal, me = methyl group a small amount of an isoform with the same molecular weight, but slightly later retention time in the pgc (porous graphitized carbon) selected ion chromatogram (fig . 6c), was identified analogously by gc - ms and ei - ms as trisaccharide containing a galnac residue substituted by one 4-o - methylated galactose and one 3-o - methylated mannose (data not shown).fig 6selected ion chromatograms of partially methylated structures obtained by lc - esi - ms . A core trisaccharide carrying in addition one methylated and one unmethylated hexose (m / z 914.4 [m+h]); b core trisaccharide with one additional hexose (m / z 738.3 [m+h]); c core trisaccharide and its isoform (m / z 576.3 [m+h]). Square = amino sugar, circle = hexose, me = methyl group selected ion chromatograms of partially methylated structures obtained by lc - esi - ms . A core trisaccharide carrying in addition one methylated and one unmethylated hexose (m / z 914.4 [m+h]); b core trisaccharide with one additional hexose (m / z 738.3 [m+h]); c core trisaccharide and its isoform (m / z 576.3 [m+h]). Square = amino sugar, circle = hexose, me = methyl group besides the trisaccharide core several other glycan species were found during purification on pgc - column . A glycan consisting of the methylated core trisaccharide and one further hexose (m / z 738.3 [m+h]) was found . This additional hexose was an unmethylated gal residue linked alternatively to one of the two arms of the trisaccharide explaining the two isoforms detected by pgc selected ion chromatogram (fig . The fourth structure (m / z 914.4 [m+h]) which was obtained in sufficient amounts for gc - ms consisted of the core trisaccharide elongated by an unmethylated hexose on one arm and a methylated hexose on the other one (fig . The presence of significant amounts of man and 3-o - me - man indicated that these two residues were responsible for the elongation . Gc - ms analysis using different derivatisation protocols requested a high amount of material and high quality of purification . Especially for snail tissues this was a challenge and therefore this method could not be carried out for a large number of samples in due time . An in - gel -elimination after sds - page followed by a short clean - up (pgc - cartridge) procedure was sufficient . The shortened protocol also ensured that no weakly bound modifications, such as sulfate or sialic acids for example, got lost . Combining our data on snail monosaccharide analysis, data from previous gc - ms methylation analysis and the fragmentation results of lc - esi - ms / ms analysis of selected snails we could determine the main structures of snail o - glycans and classify them according to their modification of the established core structure into six groups: (i) glycans smaller than the core, missing one or both methyl groups or missing one hexose; (ii) the trisaccharide core containing galnac and two 4-o - methylated hexose residues; (iii) the core with additional methylated hexoses; (iv) the core with one or two additional unmethylated hexoses and sometimes one more methylated hexose; (v) glycans containing fucose; (vi) glycans containing one hexnac and up to six unmethylated hexoses . For an overview on the structures see table 1.table 1overview of o - glycan distribution in selected snail species given in [%] of total o - glycans . Compositions are given in terms of hexose (hex), methylated hexose (mehex), n - acetylhexosamine (hexnac) and deoxyhexose (fucose; dhex)glycan compositiongroupobserved mass [m+h]calculated mass [m+h]arion lusitanicusachatina fulicacepaea hortensisplanorbarius corenusbiomphalaria glabratahelix pomatialimax maximusclea helenahexhexnac(i) - glycans smaller than the core386.2386.1707000000mehexhexnac400.2400.1850310200hex2hexnac548.2548.22traces000traces000hexmehexhexnac562.3562.2390012182600(mehex)2hexnac(ii) - core576.3576.2554692612292910089(mehex)3hexnac(iii) - core with additional methyl hexoses752.3752.325212302700(mehex)4hexnac928.4928.39traces0000000(mehex)5hexnac1104.41104.46traces0000000hex(mehex)2hexnac(iv) core with additional hexoses and methyl hexoses738.3738.30141617164512011hex2(mehex)2hexnac900.4900.36203720100hex(mehex)3hexnac914.4914.3725505400(mehex)2hexnacdhex(v) - fucosylated structures722.3722.31220traces1traces00(mehex)3hexnacdhex898.4898.3860000000(mehex)4hexnacdhex1074.41074.44traces0000000hex3hexnac(vi) hexnac and hexoses710.3710.2700061traces00hex4hexnac872.3872.32000460000hex5hexnac1034.31034.3800030000hex6hexnac1197.01196.43000traces0000 overview of o - glycan distribution in selected snail species given in [%] of total o - glycans . Compositions are given in terms of hexose (hex), methylated hexose (mehex), n - acetylhexosamine (hexnac) and deoxyhexose (fucose; dhex) analyzing snails in detail, it was obvious that the o - glycan trisaccharide core is an important decoration of snail proteins in all investigated species . In five of the analyzed snails (a. lusitanicus, a. fulica, h. pomatia, l. maximus, c. helena) this glycan was the most abundant type of o - glycan . B. glabrata and c. hortensis displayed as their main glycan the trisaccharide elongated by one or two unmethylated hexoses, respectively . Only p. corneus was an exception possessing a large amount of hex4hexnac glycan . Whereas the addition of one hexose to the core was relatively frequent, larger structures were minor compounds or were even not present in several species . The only detected amino sugar was galnac which provided in all cases the connection to the protein . Gal and man were present in unmethylated or mono - methylated (3-o - me - gal, 4-o - me - gal, 3-o - me - man) versions . Fucose, which had been identified as such in previous works [23, 28], occurred only in low amounts and was detected linked to galnac as well as to terminal sugar residues by ms / ms fragmentation . During the last decades our knowledge on glycosylation processes - structure of the glycans and the corresponding biochemical pathways including the responsible enzymes - increased enormously, especially for glycans of mammalian origin . The glycosylation capabilities of other species were only under investigation if their glycans were for any reason connected with human life (e.g. Some recognition processes of pathogens or allergy on food or plant glycans) or if they were potent candidates for cell culture systems for the expression of therapeutics (some insect, yeast and plant cells). In the meantime more and more invertebrate systems are investigated because of their potential usefulness for biotechnological reasons or to get some deeper insights into the processing pathways of unusual glycan structures . Previous studies on various invertebrates showed that glycosylation has a lot more varieties than we expected as long as only mammals were investigated, but the data are scattered . Still the main focus is laid on n - glycans but the interest in o - glycans is growing . A number of difficulties arose during snail glycan analysis due to the tough tissue, which was not easy to homogenize and the rigid mucus, which had to be discarded diligently . The removal of storage carbohydrates, which might influence monosaccharide analysis and the separation of n- and o - glycans were other struggles to resolve . While the first was done by -glucosidase digestion from rice, the latter was an even more difficult task . N - glycosidases (pngase a and f) did not remove the n - glycans completely and even mild -elimination conditions seemed to be able to release, at least in part, residual n - glycans which resulted in n - glycan impurities in o - glycan fractions . Furthermore some n - glycans of snails and digested storage glycans are so small (45 sugar residues [16, 17]) that they may co - migrate on hplc with o - glycans . Therefore only a limited number of glycans could be prepared in terms of sufficient amount and purity for permethylation followed by maldi - tof - ms and gc - ms linkage analysis . However, in combination with lc - esi - ms of native glycans which is capable of a high throughput of samples the o - glycan pattern of 8 snail species (land snails with shell: achatina fulica, cepaea hortensis and helix pomatia; slugs: arion lusitanicus and limax maximus; water snails: biomphalaria glabrata, clea helena, and planorbarius corneus) could be identified . Four monosaccharide constituents galnac, gal, man and fuc are the building blocks of the structures . The only further modification is a methylation of the hexoses, resulting in 3-o - me - gal, 4-o - me - gal and 3-o - me - man, which has already been shown for snail n - glycans [1417, 23]. Each o - glycan contains only one amino sugar, which is the protein linked galnac . The linkage amino acid was not determined in detail, but due to the easy release by -elimination the common galnac - ser / thr motif is very likely . The connection to other amino acids, for example tyrosine, is usually more stable . In snail o - glycans the inner galnac is frequently elongated by two 4-o - me - gal residues in (13)- and (16)-linkage . Elongations of this trisaccharide core by one or two hexoses with or without methyl group appeared in most snail species, whereas further elongation or fucosylation are rare events . Insects, one neighbor of mollusks in the phylogenetic tree, display also internal galnac elongated by one gal in their o - glycan structures, but further elongations and/or branching are frequently done by another amino sugar (galnac or glcnac). The main o - glycans of toxocara canis are trisaccharides consisting of galnac, gal or 4-o - gal and 2-o - fuc . So far nothing is known about biological functions of snail glycans . For sure these glycans are similarly involved in recognition processes as they are in all other species, but especially the significance of the high degree of methylation of hexoses of n- as well as of o - glycans is waiting for elucidation . Here we enlarge the current knowledge on glycosylation abilities of lower animals by presenting the first o - glycan analysis of snails, representatives of the mollusk phylum.
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Adiponectin (arcp 30, adipoq, apm1 or gbp28), secreted by adipose tissue, is a 247-amino acid peptide which was discovered in 1995 (1, 2). The amounts of adiponectin account for 0.01% of total plasma protein . The concentration of adiponectin circulating in plasma is very high (2 to 20 g / ml) (3). Plasma levels of adiponectin in the japanese population is about 5 to 10 g / ml (4) and serum adiponectin is lower in indo - asians when compared with caucasians (median 3.3 vs. 4.9 g / ml) (5). Adiponectin has gained particular interest on account of its relation with insulin sensitivity, atherosclerosis, and inflammation (6). Current projections estimate that cardiovascular disease, especially atherosclerosis, will be the leading cause of all disease in 2020 (7). Recent findings suggest that high adiponectin concentration is an independent predictor of mortality in chronic heart failure patients, chronic kidney patients, and elderly patients, who are at high risk for cardiovascular events (8 - 12). Obesity has been proven to be an independent risk factor for cad in both genders, and is a growing health problem in most developed and some developing countries (13 - 15). Body weight, body mass index (bmi), waist circumference (wc) and whr are primary methods to determine obesity . While bmi reflects general obesity, waist circumference and waist hip ratio are related to central - type obesity, where body fat is primarily located in the abdomen . Prospective epidemiological studies have revealed that central obesity (determined by wc and whr) is more relevant in cad risk compared to general obesity (determined by bmi) (16 - 18). While whr is commonly used in reflecting central obesity, waist circumference is shown to have a better correlation with abdominal fat localization (19 - 22). To our best knowledge, this is the first study performed in kosovo focusing on this particular issue . The study population consisted of 82 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis at the university clinical center of kosovo . With the exception of acute coronary syndromes, the patients had to present in a stable clinical condition without major concomitant non - cardiovascular disease . The study protocol was approved by the ethical committee of the faculty of medicine, university of prishtina, and research was conducted in accordance with declaration of helsinki guidelines . Written informed consent cad was defined as more than 50% luminal stenosis identified in a minimum of two views, in the case of single - vessel involvement . Gensini score assesses the severity of coronary artery disease: it grades narrowing of the lumen of the coronary artery and scores it as 1 for 1 - 25% narrowing, 2 for 26 - 50% narrowing, 4 for 51 - 75%, 8 for 76 - 90%, 16 for 91 - 99% and 32 for a completely occluded artery . This score is then multiplied by a factor, according to the importance of the coronary artery . The multiplication factor for a left main stem (lms) lesion is 5, it is 2.5 for proximal left anterior descending artery (lad) and proximal circumflex artery (cx) lesions, 1.5 for a mid - lad lesion, and 1 for distal lad, mid / distal cx and right coronary artery lesions . Gensini score was expressed as the sum of the scores for all three coronary arteries to evaluate the entire extent of coronary artery disease (23). Were taken after the patients had removed their shoes and upper garments and had donned an examining gown . Waist height ratio (whtr) is measured by division of weight circumference by their height, always expressed in same units . Body mass index (bmi) was calculated as weight (kg) divided by the square of height (m). The wc was measured by tape in cm and was also categorized according to the world health organization (who) criteria (24). Blood pressure was measured by european society of hypertension - international protocol 2 (esh - ip2). With the whr, the waist is measured at the narrowest part of the waist, between the lowest rib and iliac crest, and the hip circumference is taken at the widest area of the hips at the greatest protuberance of the buttocks . The who defines the ratios of> 9.0 in men and> 8.5 in women as one of the decisive benchmarks for metabolic syndrome . The 12-h fasting blood samples were taken in the morning and the sera were stored at -70 c immediately after centrifugation until being assayed . The laboratory was monitored for the precision and accuracy of glucose and lipid measurements by the surveillance program . The primary predictor variable was serum adiponectin level as determined by immunoassay of thawed fasting serum samples . Each sample was assayed in duplicate, and adiponectin level was calculated as the average of the two measurements . The continuous variables were primarily analyzed using a t - test, while the binary data were primary analyzed using a chi - squared test . The continuous variables are presented in the tables with their means sd (standard deviation). Additionally, single (unadjusted) and multiple (adjusted) binary logistic regressions were carried out with cad group as the dependent variable, and the other significant variables as independent predictors . The cut p value of 0.1 was used as a criterion to identify the variables which need to be included in the binary logistic regression models . Adiponectin as the focusing variable of this study was included in the model regardless of its statistical significance . Other adjusting variables included are: diastolic blood pressure (dbp), whr, lvef, gender male, and smoking . The study population consisted of 82 consecutive patients who underwent coronary angiography for suspected or known coronary atherosclerosis at the university clinical center of kosovo . With the exception of acute coronary syndromes, the patients had to present in a stable clinical condition without major concomitant non - cardiovascular disease . The study protocol was approved by the ethical committee of the faculty of medicine, university of prishtina, and research was conducted in accordance with declaration of helsinki guidelines . Written informed consent cad was defined as more than 50% luminal stenosis identified in a minimum of two views, in the case of single - vessel involvement . Gensini score assesses the severity of coronary artery disease: it grades narrowing of the lumen of the coronary artery and scores it as 1 for 1 - 25% narrowing, 2 for 26 - 50% narrowing, 4 for 51 - 75%, 8 for 76 - 90%, 16 for 91 - 99% and 32 for a completely occluded artery . This score is then multiplied by a factor, according to the importance of the coronary artery . The multiplication factor for a left main stem (lms) lesion is 5, it is 2.5 for proximal left anterior descending artery (lad) and proximal circumflex artery (cx) lesions, 1.5 for a mid - lad lesion, and 1 for distal lad, mid / distal cx and right coronary artery lesions . Gensini score was expressed as the sum of the scores for all three coronary arteries to evaluate the entire extent of coronary artery disease (23). Were taken after the patients had removed their shoes and upper garments and had donned an examining gown . Waist height ratio (whtr) is measured by division of weight circumference by their height, always expressed in same units . Body mass index (bmi) was calculated as weight (kg) divided by the square of height (m). The wc was measured by tape in cm and was also categorized according to the world health organization (who) criteria (24). Blood pressure was measured by european society of hypertension - international protocol 2 (esh - ip2). With the whr, the waist is measured at the narrowest part of the waist, between the lowest rib and iliac crest, and the hip circumference is taken at the widest area of the hips at the greatest protuberance of the buttocks . The who defines the ratios of> 9.0 in men and> 8.5 in women as one of the decisive benchmarks for metabolic syndrome . The 12-h fasting blood samples were taken in the morning and the sera were stored at -70 c immediately after centrifugation until being assayed . The laboratory was monitored for the precision and accuracy of glucose and lipid measurements by the surveillance program . The primary predictor variable was serum adiponectin level as determined by immunoassay of thawed fasting serum samples . Each sample was assayed in duplicate, and adiponectin level was calculated as the average of the two measurements . The continuous variables were primarily analyzed using a t - test, while the binary data were primary analyzed using a chi - squared test . The continuous variables are presented in the tables with their means sd (standard deviation). Additionally, single (unadjusted) and multiple (adjusted) binary logistic regressions were carried out with cad group as the dependent variable, and the other significant variables as independent predictors . The cut p value of 0.1 was used as a criterion to identify the variables which need to be included in the binary logistic regression models . Adiponectin as the focusing variable of this study was included in the model regardless of its statistical significance . Other adjusting variables included are: diastolic blood pressure (dbp), whr, lvef, gender male, and smoking . Of the eighty - two study patients, 41 patients comprised the cad group (study subject) and 41 patients were classified as the non - cad group (control subject). Study group (mean age 66.76 + 9.12) were on the average, 2 years older than the controls (mean age 64.80 8.30). The baseline characteristics of the two groups are depicted in table 1 . According to table 1, in comparison with the controls, the cad group patients were older and were more likely to be male and smokers . The number of male patients in the study group was 29 (70.7%) and in the control group was 17 (41.5%), which is significantly higher (p=0.008), also smokers were 27 (65.9%) vs 13 (31.7%) (p=0.002) significantly more numerous in the study group than the controls . Bmi: basal metabolic index, wc, whtr, whr, systolic blood pressure (sbp), dbp . Fifteen patients (18.2%) had normal bmi (mean 23.221.4), 37 patients (45%) were overweight, 30 patients (43.4%) were obese . Bmi in case group (mean 28.17 3.32) and control (mean 28.764.68) and wc in case (96.978.63) vs control (95.1311.52) they were no significant . Whtr in case (1.76 7.56) vs control (0.570.08), and whr is significant between case and control (0.930.06) vs (0.880.07) (p - value: 0.0001) as seen in table 1 . Cad patients had higher mean gensini scores than non - cad patients (53.16 33.46 vs. 1.21 5.66, p<0.0001), but there was no statistically significant difference detected between cad and non cad for plasma adiponectin levels, presented in table 2 . Total cholesterol in the populations averaged (3.58 1.07 mmol / l) in cases and (3.59 1.19 mmol / l) in the control g biochemical characteristics of the two groups . Cad, ast: aspartate aminotransferase, crp: c reactive protein, hdl - c: high density lipoprotein cholesterol, ldl - c: low density lipoprotein cholesterol, lvef . Roups . Baseline adiponectin concentrations correlated significantly in terms of the lipid parameters, positively with hdl cholesterol concentrations (r= 0.365, p=0.001) and serum triglyceride concentrations were correlated negatively (r=-0.336, p=0.001). Total cholesterol concentration and ldl cholesterol concentrations sixty - two patients (75%) had hypertension, 29 patients (35%) had diabetes, 23 patients (28%) dyslipidemia . Forty - two patients (51%) had family history of coronary heart disease and sudden non - accidental death or a combination of these . Correlations of adiponectin with other variables of all patients 14.6% had triple - vessel disease, 18.3% had double - vessel disease and 17% single - vessel disease . Adiponectin levels show a non - significantly progressive decline as the angiographic severity of coronary artery stenosis increases (value=0.335) as shown in table 4 cag: coronary angiography, svd: single vessel disease, dvd: double vessel disease, tvd: triple vessel disease . In control group 37 patients (45%) have (gensini score = 0), 15 patients (18%) had mild disease (gensini score <32), 14 patients (17%) had moderate disease (gensini score= 32 - 58), 16 patients (19.5%) had severe disease (gensini score 58). Bmi did not correlate with gensini score by severity of coronary arteries, as seen in table 5 . Distribution of gensini in different bmi categories significant differences were seen in binary logistic regression models for cad analysis for adiponectin, whr, lvef, dbp, male gender and for smoker . Whr (or 14; 95% ci 0.94 - 207.60; p=0.06), lvef (or 4.61; 95% ci 1.49 - 14.21; p=0.01), dbp (or 2.75; 95% 2.75 (1.12 - 6.87; p=0.03), smoking (or 4.15; 95% ci 1.65 - 10.44; p=0.001), male patients (or 3.41, 95% ci 1.37 - 8.52; p=0.01), all presented in table 6 . Binary logistic regression models for cad (unadjusted and adjusted), dbp, whr, lvef . The present study found that there was a strong association between adiponectin and coronary artery disease . These findings provide further evidence for an adiponectin paradox in which higher levels of adiponectin may be secreted as a protective or a compensatory response to worsening cardiovascular disease . In our study, we found medium significant positive correlation between gensini and adiponectin (r= 0.272, p=0.01). This is in compliance with the saphir study, which suggests that the role of adiponectin differs between early atherosclerosis and advanced stage vascular disease (25). Adiponectin protects against the development of disease, once the disease is established, adiponectin concentrations are elevated as a counter - regulatory response to protect against further inflammation and atherosclerosis (26). Findings of positive correlation between adiponectin and culprit lesion necrotic core (nc) content is consistent with the hypothesis that in advanced cad, serum adiponectin levels are elevated in a counter - regulatory response (27). High serum adiponectin levels, instead of being protective, have been shown to be associated with increased mortality and adverse event rates . This paradoxical relation is further supported by study that included patients undergoing pci for symptomatic cad (28). Despite the potential association between heart failure and high plasma adiponectin shown by several clinical studies, other experimental studies have demonstrated a beneficial, protective effect of adiponectin on myocardium . Elevated plasma levels of adiponectin among patients with heart failure furthermore, several statins are also effective for elevating plasma adiponectin (29, 30). There were no significant differences in plasma concentrations of adiponectin between patients with stable angina pectoris (sap) and control patients (11.3 v 12.8 g / ml) (31). Our study findings were similar to this study, where plasma concentrations of adiponectin between patients with sap and control group (3.45 v 3.49 g / ml) had no significant differences . In the present study, patients in our sap group had already been receiving some treatment with antianginal and antiatherosclerotic drugs . As certain medical treatments may confound interpretation of plasma concentrations of adiponectin measurements and there may not be significant differences in plasma concentrations of adiponectin between patients with sap and control patients . Another important clinical finding showed that plasma concentrations of adiponectin were considerably increased in older and female patients with cad (31). As a consequence of their findings they suggested that unstable plaque may stabilize as the patient grows older or that accumulation of adiponectin in atherosclerotic vascular walls may suppress its elimination half - life from plasma, resulting in an increase in plasma concentrations of adiponectin in older patients with cad . Although a significant relation between serum adiponectin levels and age was not found in our study, it is known that age has an effect on serum adiponectin levels and that there is a positive relationship between age and serum adiponectin level (32). Including both individuals with and without cardiovascular disease, higher adiponectin has protecting role in those without cardiovascular disease and predictive of worse outcomes in those with existing disease (33). Moreover, it has been suggested that normal and even higher serum adiponectin levels may prevent the development of cardiovascular diseases and complications in healthy individuals (34). Male patients with hypoadiponectinemia (<4.0 g / ml) had a two - fold increase in cad prevalence, independent of well - known cad risk factors (35). Similar to our study in male gender with hypoadiponectinemia (<2.0 g / ml) increase the odds of having cad (table 6). Our current study did not show a significant correlation between bmi and cad (p - value 0.115), despite the presence of significant association between whr and cad (p - value= 0,0001). This may be explained by the fact that bmi quantifies general adiposity, although person who is overweight or obese is likely to have excess fat but bmi does not give an indication as to how this fat is distributed in the body . However, fat distribution is an important determinant of cad independent of bmi and other classic risk factors of cad (36). Welborn and dhaliwal (37) and srikanthan, seeman, and karlamangla (38) confirmed, and cited several other investigations that show whr being the superior clinical measurement for predicting all cause and cardiovascular disease mortality . Welborn and dhaliwal added that the hip circumference indicated a lower risk for body fat accumulation, and thus including it into the waist - to - hip equation enhances the accuracy of this measurement technique . Relatively small sample of patients that were selected randomly from the general population, may have limited the ability to detect significant relationship between adiponectin and coronary artery disease, thus making it difficult to indicate the results to the general population . There is a significant positive correlation between waist - hip ratio and severity of coronary artery disease . Also, there is a significant positive correlation between adiponectin and gensini score among kosovar patients.
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Neurofibromatosis (nf) is an autosomal dominant disease, discovered in 1882, by the german pathologist friedrich daniel von recklinghausen, characterized by disordered growth of ectodermal tissues, and is part of a group of disorders called phakomatoses (neurocutaneous syndrome). Neurofibromatosis type - i (nf-1), also known as von recklinghausen syndrome, is caused by the mutation of a gene on the long arm of chromosome 17, which encodes a protein known as neurofibromin . It is characterized by spots of increased skin pigmentation (caf au lait spots), combined with peripheral nerve tumors and a variety of other dysplastic abnormalities of the skin, nervous system, bones, endocrine organs, and blood vessels . The localized form of neurofibromatosis type - i, first described by gammel in 1931, is very rare . Crowe et al . Proposed the term sectorial neurofibromatosis for this localized form of neurofibromatosis, and miller and sparkes modified the nomenclature to segmental neurofibromatosis (sn) the current term for neurofibromas of segmental distribution . The commonly affected sites for sn are the thorax and abdomen (55%), upper extremities (20%), and lower limb and face (10% each). Only few cases (less than 10) of segmental neurofibromatosis over the face have been described so far . A 26-year - old female patient reported to the dental department complaining about the unesthetic appearance of her face, since eight years [figure 1]. History revealed that eight years back, the patient underwent surgery in the neck region for a cyst and one to two days after surgery the patient got a swelling on the face . The swelling was of the same size since then and there was no pain associated with the swelling . The unesthetic appearance of the face on examination, the face of the patient appeared asymmetrical, bilaterally, with a growth on the right side of the face . The skin over the right side of her face was thickened and hyperpigmented, with a soft and loose hanging overgrowth . There were numerous small and large, sessile and pedunculated nodular growths localized on the right side of her neck, with dark - colored underlying skin . The skin over the right cheek and chin area showed coffee - colored pigmentation (caf - au - lait spots) [figure 2]. Nodular growths localized on the right side of the neck with dark colored underlying skin there was a soft, compressible, nontender growth on the lower lip . The right ear showed disfigurement and hypertrophy, with thickened cartilage and overlying hyperpigmented skin [figure 3]. Disfigurement of the ear and hypertrophy with thickened cartilage and overlying hyperpigmented skin intraorally there were numerous soft tissue growths with a sessile base seen on the right buccal mucosa [figure 4] and tongue [figure 5]. Soft tissue growths with sessile bases on right buccal mucosa soft tissue growths with sessile bases on the tongue laboratory findings revealed a raised erythrocyte sedimentation rate (esr) (70 mm in the first hour), a decreased hemoglobin level (7.5 gm%), and the enzyme - linked immunosorbent assay (elisa) was found to be nonreactive . A panoramic radiograph disclosed asymmetric enlargement of the right side of the mandible, with loss of cortication of the lower border in the midline area, expansion of the body of the mandible causing displacement of the teeth, seen on right side, expansion of the right inferior alveolar nerve canal, enlarged mental foramen, resorption of the roots of the mandibular right teeth, and loss of the right antegonial notch . A deficiency was seen on the right side of the maxilla, with crowding of the posterior teeth [figure 6]. A panoramic radiograph the right lateral oblique radiograph revealed a deep sigmoid notch, loss of the antegonial notch, an enlarged and rounded coronoid process, with resorption of the mandibular right teeth [figure 7]. A right lateral oblique radiograph subsequently, biopsy of one of the nodules was taken and sent for histopathological examination . The histopathological slide revealed a non - encapsulated tumor of the dermis with a normal overlying epidermis . The tumor consisted of loosely spaced spindle cells and wavy collagenous strands in a clear matrix [figure 8]. A non - encapsulated tumor of the dermis, with a normal overlying epidermis these tumors are composed of schwann cells, fibroblasts, mast cells, and vascular components . The caf au lait spots are irregularly - shaped, evenly pigmented, brown macules . Most individuals with neurofibromatosis have six or more spots that are 1.5 cm or greater in diameter . In young children, five or more caf au lait macules, greater than 0.5 cm in diameter, are suggestive of neurofibromatosis . Less than 1% of the healthy children have three or more such spots, although one or two caf au lait macules are commonly encountered in healthy individuals without disease . Bone involvement includes pseudoarthrosis of the tibia, bowing of the long bones, and orbital defects . Treatment of neurofibromatosis is predominantly surgical . When neurofibromas increase in size or cause pain, malignant transformation must be suspected, and excision or biopsy must be performed . Acoustic neuromas and tumors that cause tinnitus and vertigo must be excised with great caution . Plastic surgeons may be included in the correction of deformities, especially those of the face . Considering the autosomal dominant inheritance pattern of neurofibromatosis, genetic counseling
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The zygomaticomaxillary complex is an important functional and aesthetic landmark of the midface, and it provides prominent cheek structure . However, unfortunately it is very vulnerable to injury because of its intrinsically prominent convexity . There are four bony attachments between zygoma and other facial bones: a superior attachment to the frontal bone (frontozygomatic suture line), a medial attachment to the maxilla (zygomaticomaxillary suture line), a lateral attachment to the temporal bone (zygomaticotemporal suture line), and a deep attachment to the greater wing of the sphenoid bone (zygomaticosphenoidal suture line). When blunt trauma to the zygomatic complex results in fractures of all four suture lines, it is referred to as a tetrapod fracture . Closed reduction without surgical incisions has virtually been abandoned because judgment on the adequate reduction of fracture could not been made and it often resulted in disappointing cosmetic results . Open reduction with surgical incisions has been accomplished through keen's approach, gillies' approach, bicoronal scalp flap approach or the more popular dingman's approach (1). This procedure has advantages in that it leaves no facial scars and is simple to perform . However, the fracture site is not directly visualized and access to the orbit is not possible, which may result in incomplete surgical reduction . However, it necessitates a lengthy scalp incision and provides inadequate exposure to the inferior, medial aspect of orbit, and may leave a conspicuous long scalp scar . Also, because of its extensive dissection, the temporal branch of the facial nerve may be damaged . The dingman's approach has become the most popular method in repairing zygomatic complex fractures . This procedure utilizes a lateral eyebrow incision line and a second incision across the entire lower eyelid in the subciliary line . This technique provides good exposure and direct visualization of the frontozygomatic area, inferior orbital rim and orbital floor . But, it provides limited exposure of the lateral orbital rim because it consists of two separate facial incisions, and it leaves external cutaneous scars . Also, there is some risk of lower eyelid ectropion due to cicatrical contracture . Since its first description for the removal of herniated orbital fat, there have been subsequent reports demonstrating the efficacy of transconjunctival approach not only for the lower eyelid blepharoplasty but also for the repair of orbital fractures (2 - 5). Mccord and moses (6) described the addition of the lateral canthotomy incision to gain access to the lateral orbital wall and inferior orbital rim, and nunery (1) reported a lateral superior cantholysis for the repair of trimalar fractures . Since then, a few authors (7 - 9) have described the advantages of this type of incision, but there has been no report that describe the long term results of two - point fixation through the single transconjunctival incision for the treatment of zygoma fracture . Between march 2002 and january 2006, a total of 53 patients with zygomatic complex fractures, which were not severely comminuted, were treated with this surgical method . Included were 48 men and five women, aged 15 to 88 yr (mean age 33.2 yr). Twenty eight patients (52.8%) had associated blow out fracture, two patients (3.7%) had associated nasal bone and mandible fracture, and one patient (1.9%) had associated nasal bone fracture (table 1). Upon knight and north classification, four patients (7.5%) belonged to group i, twenty one patients (39.6%) belonged to group ii, eight patients (15%) belonged to group iv, eleven patients (20.7%) belonged to group v, and nine patients (16.9%) belonged to group vi . All patients were treated with two - point fixation through a single transconjunctival approach with a lateral canthal extension . If the reduction of zygoma was not complete, an additional small gingivobuccal incision (less than 2 cm) was made and subperiosteal dissection was performed to expose zygomaticomaxillary buttress and posterior surface of zygoma . Through the small gingivobuccal incision, the follow - up period ranged from 8 to 46 months (average 24.8 months). Preoperative and postoperative clinical examinations were performed with special attention paid to features such as symmetry of zygoma and canthal ligaments, enophthalmos, diplopia, limited ocular function, scarring and ectropion . Follow - up radiographs (waters' view and zygomatic arch view) were routinely used to evaluate the adequacy of reduction . Under general endotracheal anesthesia, a lateral canthal incision, about 1.0 - 2.0 cm, then, the conjunctiva is incised 2 - 3 mm below the tarsus extending from the commissure to a point just lateral to the punctum, and the transconjunctival incision is connected to the lateral canthal incision (fig . The lower eyelid flap is pulled down by cutting the superficial lateral canthal tendon and inferior limb of deep lateral canthal tendon with fine scissors . Dissection proceeds anteriorly and inferiorly between the orbicularis oculi muscle and orbital septum until the periosteum of the inferior orbital rim is exposed . Through the extended lateral canthal incision, the lateral aspect of the upper lid is retracted superiorly, and the periosteum of lateral orbital rim is exposed . The periosteum is incised and a subperiosteal dissection is done to expose the orbital floor, the inferior orbital rim, and the lateral orbital rim including the zygomaticofrontal suture area (fig ., the dingman's elevator is placed posteriorly to the body of the zygoma along the zygomatic arch and a reduction of zygoma is done . If the reduction of zygoma is not complete or unsatisfactory, an additional small gingivobuccal incision (less than 2 cm) is made and a limited subperiosteal dissection is performed to expose the zygomaticomaxillary buttress and posterior surface of zygoma . Dingman's elevator is inserted through the small gingivobuccal incision and an accurate reduction can be accomplished . In all cases, complete reduction was confirmed by the accurate alignment of the lateral and inferior orbital wall . Then, the two - points (frontozygomatic suture and inferior orbital rim) are fixated with plates and screws (fig . Resorbable 2.0-mm miniplates systems, macrosorb (macropore, san diego, ca, u.s.a .) Were used in 35 patients (87.5%) and titanium miniplates (osteomed, dallas, tx, u.s.a .) Were used in 5 patients (12.5%). If there is an associated impure blow - out fracture, orbital floor reconstruction with porous polyethylene implant (medpor) is performed . The periosteum is closed with a 4 - 0 absorbable suture, and the conjunctiva is closed by a 7 - 0 absorbable suture . In case of old patient with lid laxity, the canthopexy is accomplished by a 5 - 0 absorbable suture fixation in the direction of posterior - superiorly to the periosteum of lateral orbital rim to prevent the postoperative ectropion . In other cases, finally, the skin and subcutaneous layer at the lateral canthal incision site is closed with a 6 - 0 suture . Between march 2002 and january 2006, a total of 53 patients with zygomatic complex fractures, which were not severely comminuted, were treated with this surgical method . Included were 48 men and five women, aged 15 to 88 yr (mean age 33.2 yr). Twenty eight patients (52.8%) had associated blow out fracture, two patients (3.7%) had associated nasal bone and mandible fracture, and one patient (1.9%) had associated nasal bone fracture (table 1). Upon knight and north classification, four patients (7.5%) belonged to group i, twenty one patients (39.6%) belonged to group ii, eight patients (15%) belonged to group iv, eleven patients (20.7%) belonged to group v, and nine patients (16.9%) belonged to group vi . All patients were treated with two - point fixation through a single transconjunctival approach with a lateral canthal extension . If the reduction of zygoma was not complete, an additional small gingivobuccal incision (less than 2 cm) was made and subperiosteal dissection was performed to expose zygomaticomaxillary buttress and posterior surface of zygoma . Through the small gingivobuccal incision, the follow - up period ranged from 8 to 46 months (average 24.8 months). Preoperative and postoperative clinical examinations were performed with special attention paid to features such as symmetry of zygoma and canthal ligaments, enophthalmos, diplopia, limited ocular function, scarring and ectropion . Follow - up radiographs (waters' view and zygomatic arch view) were routinely used to evaluate the adequacy of reduction . Under general endotracheal anesthesia, a lateral canthal incision, about 1.0 - 2.0 cm, is made down to the periosteum of lateral orbital rim . Then, the conjunctiva is incised 2 - 3 mm below the tarsus extending from the commissure to a point just lateral to the punctum, and the transconjunctival incision is connected to the lateral canthal incision (fig . The lower eyelid flap is pulled down by cutting the superficial lateral canthal tendon and inferior limb of deep lateral canthal tendon with fine scissors . Dissection proceeds anteriorly and inferiorly between the orbicularis oculi muscle and orbital septum until the periosteum of the inferior orbital rim is exposed . Through the extended lateral canthal incision, the lateral aspect of the upper lid is retracted superiorly, and the periosteum of lateral orbital rim is exposed . The periosteum is incised and a subperiosteal dissection is done to expose the orbital floor, the inferior orbital rim, and the lateral orbital rim including the zygomaticofrontal suture area (fig ., the dingman's elevator is placed posteriorly to the body of the zygoma along the zygomatic arch and a reduction of zygoma is done . If the reduction of zygoma is not complete or unsatisfactory, an additional small gingivobuccal incision (less than 2 cm) is made and a limited subperiosteal dissection is performed to expose the zygomaticomaxillary buttress and posterior surface of zygoma . Dingman's elevator is inserted through the small gingivobuccal incision and an accurate reduction can be accomplished . In all cases, complete reduction was confirmed by the accurate alignment of the lateral and inferior orbital wall . Then, the two - points (frontozygomatic suture and inferior orbital rim) are fixated with plates and screws (fig . Resorbable 2.0-mm miniplates systems, macrosorb (macropore, san diego, ca, u.s.a .) Were used in 35 patients (87.5%) and titanium miniplates (osteomed, dallas, tx, u.s.a .) Were used in 5 patients (12.5%). If there is an associated impure blow - out fracture, orbital floor reconstruction with porous polyethylene implant (medpor) is performed . The periosteum is closed with a 4 - 0 absorbable suture, and the conjunctiva is closed by a 7 - 0 absorbable suture . In case of old patient with lid laxity, the canthopexy is accomplished by a 5 - 0 absorbable suture fixation in the direction of posterior - superiorly to the periosteum of lateral orbital rim to prevent the postoperative ectropion . In other cases, finally, the skin and subcutaneous layer at the lateral canthal incision site is closed with a 6 - 0 suture . During the average 24.8 months of follow up, most patients had functionally and esthetically satisfactory results . All patients showed satisfactory facial symmetry, no lateral canthal displacement, and no functional impairment . None of them had persistent diplopia, enophthalmos or alteration of visual acuity . Upon postoperative radiography (skull waters view and zygomatic arch view), one patient (1.9%) had persistent scleral show and was corrected three months after the repair of zygomatic complex fracture . This case was 78 yr - old patient who had lid laxity preoperatively and showed ectropion postoperatively, which may be due to scar contracture . And two patients (3.7%) had mild hyperpigmentation at the lateral canthal incision site even after six months postoperatively . But most of patients showed hardly visible scar in the lateral canthal area (fig . Although subciliary incision with lateral eyebrow incision (dingman's approach) is the most common approach to the zygomatic complex (7, 10), it can leave a visible scar and ectropion of the lower eyelid . Factors predisposing to an eyelid retraction and ectropion include hematoma, eyelid edema, adhesions of the orbital septum, scar contracture, horizontal laxity of the eyelid margin, weakening of the pretarsal muscle, and wide dissection of the anterior periosteum (11). To decrease the complication rates of the subciliary approach, many authors have tried to treat zygoma complex fracture repair using the transconjunctival approach (1, 6 - 9). They emphasized the advantage of this approach over the dingman's approach, in terms of minimal ectropion, hidden scars, and minimal lid lymphedema . In the transconjunctival approach, the dissection can be performed in one of two separate planes; preseptal approach and retroseptal approach . Some authors (12, 13) advocated that the retroseptal approach is preferred because it can be performed easily and the integrity between orbital septum and orbicularis oculi muscle is not disturbed . However, the retroseptal approach has increased risk of injury to the inferior oblique muscle and prolapse of orbital fat into the surgical field (10, 11). Hence, all our patients were treated with the transconjunctival preseptal approach . In the management of the lateral canthal extension site, manson et al . (9) advocated that the canthal reattachment is not required in an acute fracture treatment but is necessary in fractures when treatment is delayed, in patients with established canthal deformities, or in patients in whom a simultaneous coronal incision is employed . According to them, the anterior limb of the lateral canthal tendon which is also referred to as the superficial lateral canthal tendon by other authors (14), is continuous to the temporal fascia and galea aponeurotica and the diffuse connections of this limb facilitate proper positioning of the canthus after mobilization of the posterior limb (deep lateral canthal tendon). The current authors think that in addition to some indications mentioned above, patients of old age with lid laxity also needs lateral canthal reattachments or canthopexy . Its significance began to be considered following our case of a 78 yr - old patient who developed persistent ectropion when canthal reattachment had not been performed . The precise physical stability of the zygoma with respect to the number and location of miniplates applied is not known, because it depends on fracture anatomy, extent, and the amount of displacement . Some laboratory studies, using human cadaver heads, have been conducted to investigate the stability of fixated zygoma . In 1989, rinehart et al . (15) simulated noncomminuted zygoma fractures in eight human cadaver heads and the stability of fracture fixation was studied for various fixation patterns . In their study, double - miniplate fixation across the zygomaticofrontal and zygomaticomaxillary fracture lines is sufficient to withstand static and oscillating loading similar to physiologic masticatory forces . In 1990, davidson et al . (16) analyzed different methods of internal fixation of simple displaced fractures of the zygoma in an attempt to define the simplest method of achieving postreduction stability . In their report, the three - point fixation (frontozygomatic suture, inferior orbital rim, and zygomaticomaxillary buttress) using either miniplates alone or interfragmentary wiring conferred the greatest stability . In addition, they proposed that the two - point fixation using miniplate alone conferred a degree of stability comparable to most methods of three - point fixation regardless of the site in which the miniplates were applied . We concur with the above assertions and think that two - point miniplate fixation at the infraorbital rim and zygomaticofrontal suture would suffice, provided the comminution of the zygoma is not severe . Although not the focus of our attention in this study, in case of severely comminuted fracture, three - point fixation through the transconjunctival incision and gingivobuccal incision is preferably performed . To fixate the fracture site and provide stability, we used four hole resorbable 2.0-mm miniplates systems, macrosorb (macropore, san diego, ca, u.s.a .) In most patients, and titanium miniplates (osteomed, dalllas, tx, u.s.a .) Only when there was a segmental fracture at the infraorbital rim or when requested by the patient due to the lighter financial burden . In addition to the inconspicuous scarring, the main advantage of this method is a direct and full visualization of the lateral and inferior orbital wall to be served as a guidance for an accurate reduction, which makes the reduction more successful . The only drawback we experienced in our method is a little difficulty in reducing the fracture - displaced zygoma because of limited space and a slightly inadequate vector for the elevator . Usually a fractured zygoma is displaced and rotated posteriorly and medially, so an anterior - lateral pull is required . However, the lateral canthal incision is located medially to the zygoma body and lateral pulling of the zygoma body may be a little difficult . In conclusion, we believe that the proposed technique is very useful for the treatment of zygomatic complex fracture which is not severely comminuted, because it provides excellent exposure and postoperative stability of the zygoma with a lower incidence of complications, including visible scarring and ectropion.
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The gram - negative coccobacillus pasteurella multocida is normally found in the oral cavity of dogs and cats . It is a recognised cause of wound infection as a result of bites, licking or scratching . Bacteraemia and endocarditis are uncommon complications and, from literature reviews, are found to be associated with prior animal trauma, a second source of infection (e.g. Cellulitis) and high mortality rates . We herein report a case in which a pet owner developed p. multocida endocarditis and was subsequently treated via opat (outpatient parenteral antimicrobial therapy) service with an excellent clinical outcome . Species identification of p. multocida was performed using traditional microbiological methods and confirmed with 16s rrna sequencing and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry (maldi - tof). A 38-year - old man was admitted to our hospital with a 2 days history of fever, malaise and cellulitis around a mitrofanoff abdominal catheter . Multiple comorbidities included marfan syndrome (with metallic aortic valve inserted sixteen years before) and congenital urological abnormalities that led to the mitrofanoff appendicovesicostomy and a recent renal transplant, five years and six months before admission respectively . He was on steroids and tacrolimus as immunosuppressive therapy . On presentation, the patient was febrile, hypotensive and tachycardic, had a normal leukocyte count (5.0910/l) but raised creactive protein (crp) 45 mg / l with mild anaemia (hb of 10.1 g / dl from a baseline of 14.6 g / dl) and renal impairment (creatinine 163 umol / l and urea 8.1 mmol / l). The cellulitis around the mitrofanoff abdominal catheter was noted and a swab was taken for culture . His chest x - ray did not show any relevant consolidation and abdominal ultrasound noted two small abdominal collections that had already been noted post - transplant and had since decreased in size . The patient was documented to be penicillin allergic (previous skin rash), so was initiated on ertapenem iv 1 gr / od . The cellulitis improved and the patient was clinically well within 48 hours of commencing antibiotics . The blood culture set (aerobic and anaerobic bottles) taken on admission was positive with p. multocida but the abdominal swab grew only mixed anaerobes . Urine culture was negative and all subsequent blood cultures taken after starting ertapenem were negative after 5 days of incubation . Transesophageal (tee) echocardiography revealed the presence of a vegetation (0.57 0.2 cm) above the atrium of the right coronary artery . A previous transthoracic echocardiogram (tte), around one month prior to admission, was reported as negative . Therefore, the patient was treated following our local protocol for infective endocarditis: he was switched to ceftriaxone iv 2 gr / od, a picc line was inserted in the right arm and he was discharged under the opat (outpatient parenteral antimicrobial therapy) service to complete six weeks iv antibiotics as an outpatient . On follow - up, two repeat ttes showed clear aortic valve . We reviewed all the previous reported cases of p. multocida endocarditis in the literature through pubmed and cross - references search . To our knowledge this is the twenty - first reported case of endocarditis but the first to be reported in a penicillin allergic patient . As with previous cases, the diagnosis of infective endocarditis was made according to duke criteria: one major criterion (evidence of endocardial involvement) and three minor criteria (predisposing heart condition, fever and microbiological evidence with positive blood culture). Laboratory isolation and identification of p. multocida was performed using standard microbiological techniques: api-20ne, api-20e (biomerieux) and the phoenix automated system (bd). Interestingly, both api profiles reported a low confidence value for identification as p. multocida to the species level (cv 53%) and the phoenix assay only identified the isolate to genus level as pasteurella spp . Thus, confirmation of species identification was performed using 16s rrna sequencing and maldi - tof protein profiling: 16s rrna sequence was performed using pcr parameters detailed in maskell na et al . (2006) and we obtained a 584bp fragment sharing 99% sequence homology to that of p. multocida sub species septica . Maldi - tof profiling was performed using a bruker daltonics microflex platform with recommended -cyano-4-hydroxycinnamic acid matrix under pre - defined conditions as specified by bruker . The maldi - tof mass spectra for the clinical isolate was analysed against maldi - tof mass spectra for 3,438 species of bacteria located in the bruker daltonics flexanalysis 3.0 database . The clinical isolate was identified as p. multocida with a confidence value of 2.321 indicating highly probable species identification, with no differential bacterial species being reported . Sensitivities were as follow (eucast breakpoints): penicillin mic (mg / l) was 0.19, ciprofloxacin 0.016, doxycycline 0.5, co - trimoxazole 0.032, ertapenem 0.016 and ceftriaxone <0.016, all being susceptible . Of note the accuracy of previous reports in the literature has been questioned as their standard microbiological description was incomplete and pasteurella species may be misidentified as haemophilus spp . They both belong to the pasteurellaceae family and based on the genomic sequence it appears they diverged approximately 270 million years ago . Are gram - negative coccobacilli generally present in the oral cavity of cats and dogs . To date twenty - one species have been identified and the species p. multocida can be further divided into three different subspecies (subspecies multocida, septicum and gallicida). The spectrum of infections varies from skin and soft tissue to central nervous system infections . Ten out of twenty - one (47.6%) endocarditis cases described in the literature had close contact with animals detailed in the clinical history . Our patient did not have a clear history of bites or scratches but he did have two cats at home and close contact with dogs at his sister's house . In fact, of the cases of p. multocida endocarditis reviewed, two out of twenty - one (9.5%) had cirrhosis as underlying condition . Penicillin still remains the best antimicrobial agent for the treatment of virtually all forms of pasteurella infection . A fluoroquinolone, doxycycline, or co - trimoxazole should be considered for skin and soft tissue infections as an alternative for patients with intolerance to -lactams . There are no clear guidelines about the treatment of p. multocida endocarditis in penicillin - allergic patients . The patient was known to be penicillin allergic (previous rash) and ciprofloxacin and doxycycline were contraindicated as causing interaction with his anticoagulant treatment . We decided then to switch to ceftriaxone as this antibiotic is already vastly used in the treatment of endocarditis caused by other gram - negative organisms and is more cost effective . Aminoglycosides have moderate to poor in vitro activity against pasteurella and probably should not be used, particularly given the paucity of clinical experience . Finally, our patient improved within 48 hours of commencing antimicrobial therapy and surgical intervention was unnecessary . From the literature, only three other cases have been successfully treated with solely antibiotics and valve replacement was mandatory in seventeen out of twenty - one (81%) of cases described . Clinicians may face various challenges, from achieving the correct species identification to chosing treatment regimens in penicillin - allergic patients . In our case new diagnostic techniques such as 16s rrna sequencing and maldi - tof were demonstrated to be invaluable tools for species confirmation . We propose that maldi - tof is a rapid, accurate method for the identification of p. multocida from bacterial cultures and can significantly reduce the time to diagnosis of p. multocida bacteraemia . Furthermore we have shown that cephalosporins and carbapenems are probably good substitutes for penicillin in case of allergy.
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Patient aged 64 was admitted to the county hospital urology ward in september 2010 and diagnosed with tumor of the left kidney . Ultrasonographic exam revealed the structure with a diameter of 45 mm in the central field of the left kidney . Ct showed a solid focal lesion of the left kidney (30 x 40 x 32 mm) located below the renal stalk and covering the renal pelvis and the paranephric section of the left ureter, apart from that no changes were observed (figure 1). Radiological image of the chest did not reveal any pathological changes . In order to make the results of pre operational diagnosis sided ureterorenoscopy was conducted and a urine sample was taken for cytology to exclude epithelial nature of the tumor . After undergoing the above mentioned test, the patient was qualified for transperitoneal radical nephrectomy . With general anesthesia applied, the left kidney was removed through an incision below the left costal margin . During the operation, a significant peripheral infiltration was discovered which hindered access to the renal stalk of the left kidney . Operative course passed without complications and the patient was discharged in good general condition 8 days after the surgery . Left kidney tumor in ct exam . On pathological examination (samples no 12399241239928) in all samples after detailed hematological work up which did not show any other tumors, the patient was qualified for radiation treatment and currently remains under permanent oncological supervision . Medical records and literature mention several cases of malignant kidney lymphoma [1, 2]. Some authors claim that this entity accounts for 3% of all solid renal tumors among adults . Lymphomatous renal involvement can have three different causes: most commonly it appears along with the generalized disease and enlarged lymph nodes . Renal involvement develops without any symptoms then.it may be related to organ transplantation and infection with epstein such cases are rare and difficult to diagnose which may lead to mistakes in treatment . Such cases are rare and difficult to diagnose which may lead to mistakes in treatment . The case presented in the report illustrates diagnostic and therapeutic difficulties that a urologist might encounter during treatment of patients with possible renal tumor . Results provided by clinical imaging of focal lesions are inconsistent . What must always be taken into account is an atypical tumorous process, autoimmunological disorders as well as inflammatory condition . In this particular case control appointments in the department of hematology have been scheduled to take place every four months and the prognosis for the patient has been described as good . They develop from lymphocytes and, depending on the variety of b and t lymphocytes, those processes can have different clinical outcome . Some of them are located solely within the lymph nodes or tissues and internal organs, while others are accompanied by leukemia . It might seem that when it comes to lymphomas, the pathogens initiating the course of the disease could be both, the epstein barr viruses which attack the b lymphocytes, and by htlv1 viruses dwelling in t lymphocytes and causing a subacute form of leukemia among adults . There can also be some co factors of environmental character: chloro organic compounds, ionization energy, benzene like substances, states of immunosuppression and autoimmunological diseases . The malignant lymphoma morbidity rate can also be increased by extensive exposition to herbicides, and is even 35 times higher among patients with a kidney transplant . It usually dwells within the lymphoid tissue of waldeyer's ring, spleen and thymus . Extranodal lymphomas can most often be found in the gastrointestinal tract, other types can be located in skin, bones, eye kidney might be at the stage of involvement which spreads through lymphatic system from the nodes in retro peritoneum . If the renal involvement occurs at an advanced stage of the disease, as yet another location of it, it is relatively easy to diagnose . Once a patient is diagnosed with lymphoma of kidney, further examinations are required in order to rule out a systemic disease . The necessary test in such cases include: blood tests, bone marrow biopsy, radiological examination, pathomorphological evaluation and histo immunological evaluation of enlarged lymph nodes . The malignant types, however, build up rapidly, are resistant to treatment and show no signs of improvement within a short period of time . How long the patient will live depends on the histopathological diagnosis of the tumor and its stage of development . Non hodgkin lymphomas are treated with chemotherapy, radiation, and, in some cases, surgeries . Low malignant, originally extranodal, tumors are usually surgically removed and treated with radiotherapy . Cell lymphoma associated with chronic inflammation arising in a renal pseudocyst with barely noticeable clinical symptoms . Publications in english present several cases of spontaneous rupture of the ureter as primary symptom of malignant lymphoma . Risk of their occurrence increases from 1.2% in 5 years after the operation, to 6.8% in 20 years after the transplantation, which is related to epstein barr virus infections . Some authors also report on cases of co existence of inflammatory intestines disease, including the crohn disease, and malignant lymphomas but then changes can most frequently be located in the vicinity of intestines or other adjacent sections of the digestive tract . The presented case leads to a conclusion that each focal lesion of unclear etiology found on imagining studies should be treated as a possible malignant lymphoma and ought to be histopathologically verified as quickly as possible.
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Assessment of myocardial viability in patients with coronary artery disease is of great clinical importance, as dysfunctional but viable myocardium has the ability to regain contractility after coronary revascularization with subsequent improvements in cardiac function and prognosis . The perfusable tissue index (pti) can be used as a marker of myocardial viability, having been validated in patients with ischaemic heart disease [27]. However, pti has never been used in clinical practice, primarily due to the complex imaging protocol consisting of dynamic [o]h2o and [o]co scans, together with a transmission scan, and the lack of high - quality, clinically usable parametric images . Recently, however, a method was developed to derive the pti from a single [o]h2o pet / ct scan . Given the current potential of clinical [o]h2o - based perfusion imaging and the rapid growth in the availability of cardiac pet / ct systems, pti viability measurements could become incorporated into clinical practice [9, 10]. Parametric pti images derived from [o]h2o pet / ct scans, however, have not yet been validated . The purpose of this study was to compare these novel parametric pti images with late gadolinium - enhanced cardiovascular magnetic resonance (lge - cmr) imaging, an established method for quantifying scar size and a marker of viability . The study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period . All patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations . Patients with contraindications for pet / ct or cmr (e.g. Pacemaker, claustrophobia, atrial fibrillation) were excluded . The study was approved by the institutional medical ethics review committee, and all participants gave written informed consent prior to inclusion . [o]h2o scans were acquired using a gemini tf-64 (philips healthcare, best, the netherlands) pet / ct scanner . [o]h2o (370 mbq) was injected as a 5-ml bolus (0.8 ml s) followed by a 35-ml saline flush (2 ml s), simultaneously starting a 6-min list mode emission scan . This scan was followed immediately by a respiration - averaged slow low - dose (ld) ct scan (55 mas, rotation time 1.5 s, pitch 0.825, collimation 16 0.625, acquiring 20 cm in 37 s) during normal breathing . All scans were checked for misalignment between the ld ct scan and the [o]h2o scan; in none of the patients were corrections needed . Dynamic [o]h2o images were reconstructed into 22 frames (1 10, 8 5, 4 10, 2 15, 3 20, 2 30, 2 60 s) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner, normalization, dead time, decay, scatter, randoms and attenuation based on the corresponding ld ct scan . Using capp software (siemens / cti, knoxville, tn), regions of interest (rois) of 1 cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus . These rois were combined into one volume of interest (voi) for the ascending aorta . A second set of rois were placed over the right ventricle (rv) cavity in at least five transaxial planes, with roi boundaries at least 1 cm from the rv wall to avoid spill - in from myocardial activity . Both vois were then projected onto all dynamic [o]h2o images, thereby generating arterial (ca(t)) and rv (crv(t)) time activity curves . Parametric pti images were calculated as previously described . In brief, parametric images of myocardial blood flow (mbf), perfusable tissue fraction (ptf), and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [o]h2o [1114]. Parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images, resulting in parametric anatomic tissue fraction (atf) images . Finally, parametric pti images were calculated as the ratio of ptf and atf images (fig . 1). Finally, 16 myocardial vois were defined manually on parametric ptf images, according to the 16 segments model of the american heart association, after which this voi template was projected onto the parametric pti images.fig . (b) atf (g ml), (c) ptf (g ml), and (d) pti in a patient without coronary artery disease example of short axis images of (a) blood volume, (b) atf (g ml), (c) ptf (g ml), and (d) pti in a patient without coronary artery disease cmr studies were performed on a 1.5-t whole - body scanner (magnetom sonata or avanto; siemens, erlangen, germany), using a six - channel phased - array body coil . After survey scans, a retrotriggered, balanced steady - state free precession gradient - echo sequence was used for cine imaging . Image parameters included: slice thickness 5 mm, slice gap 5 mm, temporal resolution <50 ms, repetition time 3.2 ms, echo time 1.54 ms, flip angle 60, typical image resolution 1.3 1.6 5.0 mm . The cardiac cycle consisted of 20 phases . After obtaining four-, three- and two - chamber view cines, stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle (lv). Contrast images were acquired 10 to 15 min after administration of 0.2 mmol kg gadolinium - dtpa in the same views as in the cine images, using a two - dimensional segmented inversion - recovery prepared gradient echo sequence (te 4.4 ms, tr 9.8 ms, inversion time 250 to 300 ms, typical voxel size 1.3 1.6 5.0 mm). Images were analysed off - line using the software package mass (mr analytical software system; medis, leiden, the netherlands). The endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images . End - diastolic volume, end - systolic volume and ejection fraction were computed using these analyses . The amount of fibrosis was calculated using the full - width at half - maximum method, which defines fibrosis as myocardial tissue with a signal intensity 50% of the maximum hyperenhancement intensity . If no enhancement was found in a slice, the maximum signal of the nearest slice with enhancement was used . All areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area . Cmr images were analysed according to the same 16-segment model as used for the parametric pet images . Finally, myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50% lge per segment [15, 17]. Continuous variables are presented as means sd, and categorical data are summarized as frequencies and percentages . Multiple datasets were compared using analysis of variance (anova), and specific differences were identified using student s t - test with bonferroni inequality adjustment . Receiver operating characteristic curves were generated for ptf, pti and mbf for the prediction of myocardial viability assessed by lge cmr . The area under the curve (auc) was considered a measure of accuracy to discriminate between viable and nonviable myocardium . The study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period . All patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations . Patients with contraindications for pet / ct or cmr (e.g. Pacemaker, claustrophobia, atrial fibrillation) were excluded . The study was approved by the institutional medical ethics review committee, and all participants gave written informed consent prior to inclusion . [o]h2o scans were acquired using a gemini tf-64 (philips healthcare, best, the netherlands) pet / ct scanner . [o]h2o (370 mbq) was injected as a 5-ml bolus (0.8 ml s) followed by a 35-ml saline flush (2 ml s), simultaneously starting a 6-min list mode emission scan . This scan was followed immediately by a respiration - averaged slow low - dose (ld) ct scan (55 mas, rotation time 1.5 s, pitch 0.825, collimation 16 0.625, acquiring 20 cm in 37 s) during normal breathing . All scans were checked for misalignment between the ld ct scan and the [o]h2o scan; in none of the patients were corrections needed . Dynamic [o]h2o images were reconstructed into 22 frames (1 10, 8 5, 4 10, 2 15, 3 20, 2 30, 2 60 s) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner, normalization, dead time, decay, scatter, randoms and attenuation based on the corresponding ld ct scan . Using capp software (siemens / cti, knoxville, tn), regions of interest (rois) of 1 cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus . These rois were combined into one volume of interest (voi) for the ascending aorta . A second set of rois were placed over the right ventricle (rv) cavity in at least five transaxial planes, with roi boundaries at least 1 cm from the rv wall to avoid spill - in from myocardial activity . Both vois were then projected onto all dynamic [o]h2o images, thereby generating arterial (ca(t)) and rv (crv(t)) time activity curves . Parametric pti images were calculated as previously described . In brief, parametric images of myocardial blood flow (mbf), perfusable tissue fraction (ptf), and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [o]h2o [1114]. Parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images, resulting in parametric anatomic tissue fraction (atf) images . Finally, parametric pti images were calculated as the ratio of ptf and atf images (fig . 1). Finally, 16 myocardial vois were defined manually on parametric ptf images, according to the 16 segments model of the american heart association, after which this voi template was projected onto the parametric pti images.fig . (b) atf (g ml), (c) ptf (g ml), and (d) pti in a patient without coronary artery disease example of short axis images of (a) blood volume, (b) atf (g ml), (c) ptf (g ml), and (d) pti in a patient without coronary artery disease cmr studies were performed on a 1.5-t whole - body scanner (magnetom sonata or avanto; siemens, erlangen, germany), using a six - channel phased - array body coil . After survey scans, a retrotriggered, balanced steady - state free precession gradient - echo sequence was used for cine imaging . Image parameters included: slice thickness 5 mm, slice gap 5 mm, temporal resolution <50 ms, repetition time 3.2 ms, echo time 1.54 ms, flip angle 60, typical image resolution 1.3 1.6 5.0 mm . The cardiac cycle consisted of 20 phases . After obtaining four-, three- and two - chamber view cines, stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle (lv). Contrast images were acquired 10 to 15 min after administration of 0.2 mmol kg gadolinium - dtpa in the same views as in the cine images, using a two - dimensional segmented inversion - recovery prepared gradient echo sequence (te 4.4 ms, tr 9.8 ms, inversion time 250 to 300 ms, typical voxel size 1.3 1.6 5.0 mm). Images were analysed off - line using the software package mass (mr analytical software system; medis, leiden, the netherlands). The endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images . End - diastolic volume, end - systolic volume and ejection fraction were computed using these analyses . The amount of fibrosis was calculated using the full - width at half - maximum method, which defines fibrosis as myocardial tissue with a signal intensity 50% of the maximum hyperenhancement intensity . If no enhancement was found in a slice, the maximum signal of the nearest slice with enhancement was used . All areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area . Cmr images were analysed according to the same 16-segment model as used for the parametric pet images . Finally, myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50% lge per segment [15, 17]. Continuous variables are presented as means sd, and categorical data are summarized as frequencies and percentages . Multiple datasets were compared using analysis of variance (anova), and specific differences were identified using student s t - test with bonferroni inequality adjustment . Receiver operating characteristic curves were generated for ptf, pti and mbf for the prediction of myocardial viability assessed by lge cmr . The area under the curve (auc) was considered a measure of accuracy to discriminate between viable and nonviable myocardium . A p value of <0.05 was considered statistically significant . Lge was seen in the cmr images of 34 patients (74%). Of the 736 myocardial segments, 364 (49%) showed some degree of lge.table 1patient characteristics (n = 46)characteristicvalueage (years)65 10gender (male)36 (78%) previous myocardial infarction34 (74%) lv end - diastolic volume (ml)226 65lv end - systolic volume (ml)135 71lv ejection fraction (%) 42 16lv mass (g)132 39 patient characteristics (n = 46) pet / ct and cmr data are summarized in table 2 . There was a gradual decrease in ptf, pti and mbf values with increasing degree of lge on cmr images (p <0.001 by anova). Atf values remained relatively constant, except for a significant decrease in the (near) transmurally enhanced segments (p <0.001 by anova).table 2segmental pet / ct and lge dataextent of lge (%) control (n = 372)025 (n = 190)2550 (n = 83)5075 (n = 54)75 (n = 37)p value (anova)atf0.76 0.090.77 0.090.75 0.110.73 0.080.68 0.11**<0.001ptf (g ml)0.72 0.080.72 0.090.70 0.090.60 0.08**0.51 0.12***<0.001pti0.91 0.080.89 0.090.89 0.090.77 0.10**0.70 0.16***<0.001mbf (ml g min)1.02 0.300.91 0.26 * 0.85 0.30 * 0.83 0.24 * 0.67 0.30**<0.001*p <0.05 vs. control; * * p <0.05 vs. control, 025% lge and 2550% lge; * * * p <0.05 vs. control, 025% lge, 2550% lge and 5075% lge . Segmental pet / ct and lge data * p <0.05 vs. control; * * p <0.05 vs. control, 025% lge and 2550% lge; * * * p <0.05 vs. control, 025% lge, 2550% lge and 5075% lge . Figure 2 shows a concordant pattern between parametric ptf, pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig . 2long axis images (a d) and polar maps (e h) in a patient with an anterior myocardial infarction: a, e cmr with lge (arrow,% transmurality); b, f ptf (g ml); c, g pti; d, h mbf (ml g min)fig . 3long axis images in a patient with a nontransmural anterior myocardial infarction: a cmr with lge; b pti long axis images (a d) and polar maps (e h) in a patient with an anterior myocardial infarction: a, e cmr with lge (arrow,% transmurality); b, f ptf (g ml); c, g pti; d, h mbf (ml g min) long axis images in a patient with a nontransmural anterior myocardial infarction: a cmr with lge; b pti using cmr as a reference, 91 of 364 (25%) segments showing some degree of lge were judged to be nonviable (lge> 50%). As shown in fig . 4, the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable (auc 0.87, ci 0.830.90, and 0.86, ci 0.820.91, respectively, p = 0.541). Mbf was able to predict myocardial viability with less accurate (auc 0.69, ci 0.630.75, p <0.001). Optimal cut - off values of ptf, pti and mbf for predicting (near) transmural lge on cmr were 0.69 g ml, 0.80, and 0.78 ml min g with sensitivities of 69%, 91% and 72%, and specificities of 87%, 73% and 56%, respectively . Figure 3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig . 4receiver operator characteristics curves for the abilities of ptf, pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf, pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement lge was seen in the cmr images of 34 patients (74%). Of the 736 myocardial segments, 364 (49%) showed some degree of lge.table 1patient characteristics (n = 46)characteristicvalueage (years)65 10gender (male)36 (78%) previous myocardial infarction34 (74%) lv end - diastolic volume (ml)226 65lv end - systolic volume (ml)135 71lv ejection fraction (%) 42 16lv mass (g)132 39 patient characteristics (n = 46) pet / ct and cmr data are summarized in table 2 . There was a gradual decrease in ptf, pti and mbf values with increasing degree of lge on cmr images (p <0.001 by anova). Atf values remained relatively constant, except for a significant decrease in the (near) transmurally enhanced segments (p <0.001 by anova).table 2segmental pet / ct and lge dataextent of lge (%) control (n = 372)025 (n = 190)2550 (n = 83)5075 (n = 54)75 (n = 37)p value (anova)atf0.76 0.090.77 0.090.75 0.110.73 0.080.68 0.11**<0.001ptf (g ml)0.72 0.080.72 0.090.70 0.090.60 0.08**0.51 0.12***<0.001pti0.91 0.080.89 0.090.89 0.090.77 0.10**0.70 0.16***<0.001mbf (ml g min)1.02 0.300.91 0.26 * 0.85 0.30 * 0.83 0.24 * 0.67 0.30**<0.001*p <0.05 vs. control; * * p <0.05 vs. control, 025% lge and 2550% lge; * * * p <0.05 vs. control, 025% lge, 2550% lge and 5075% lge . Segmental pet / ct and lge data * p <0.05 vs. control; * * p <0.05 vs. control, 025% lge and 2550% lge; * * * p <0.05 vs. control, 025% lge, 2550% lge and 5075% lge . Figure 2 shows a concordant pattern between parametric ptf, pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig . 2long axis images (a d) and polar maps (e h) in a patient with an anterior myocardial infarction: a, e cmr with lge (arrow,% transmurality); b, f ptf (g ml); c, g pti; d, h mbf (ml g min)fig . 3long axis images in a patient with a nontransmural anterior myocardial infarction: a cmr with lge; b pti long axis images (a d) and polar maps (e h) in a patient with an anterior myocardial infarction: a, e cmr with lge (arrow,% transmurality); b, f ptf (g ml); c, g pti; d, h mbf (ml g min) long axis images in a patient with a nontransmural anterior myocardial infarction: a cmr with lge; b pti using cmr as a reference, 91 of 364 (25%) segments showing some degree of lge were judged to be nonviable (lge> 50%). As shown in fig . 4, the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable (auc 0.87, ci 0.830.90, and 0.86, ci 0.820.91, respectively, p = 0.541). Mbf was able to predict myocardial viability with less accurate (auc 0.69, ci 0.630.75, p <0.001). Optimal cut - off values of ptf, pti and mbf for predicting (near) transmural lge on cmr were 0.69 g ml, 0.80, and 0.78 ml min g with sensitivities of 69%, 91% and 72%, and specificities of 87%, 73% and 56%, respectively . Figure 3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig . 4receiver operator characteristics curves for the abilities of ptf, pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf, pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement the present study was conducted to validate the use of a parametric myocardial viability imaging technique using [o]h2o pet / ct in patients with ischaemic heart disease . Viability assessed using parametric ptf and pti imaging was in good agreement with that assessed using lge cmr . Furthermore, these images and myocardial perfusion imaging are obtained simultaneously allowing both myocardial viability and ischaemia to be evaluated in a single scanning session . [o]h2o is generally considered to be the gold standard for absolute quantification of myocardial perfusion in vivo [10, 18, 19]. Apart from mbf, [o]h2o also provides estimates of the extent of myocardium within a roi that is able to exchange water rapidly, i.e. Ptf . Subsequently, ptf can be corrected for partial volume effects by dividing it by its anatomical counterpart atf, resulting in pti . It has been shown that both ptf and pti can identify myocardial scarring and thus can act as markers of viability . Only recently, however, a method has been developed that enables generation parametric atf, ptf and pti images from a single [o]h2o pet / ct scan . In addition, the traditional (long) transmission scan can be replaced by a (rapid) low - dose ct scan, thereby shortening the total scanning time substantially . Now that the method has been shown to produce high - quality parametric images, its implementation into clinical practice needs investigation . Using the described parametric imaging approach, the present study demonstrated that atf was relatively constant independent of tissue characteristics of the myocardium . Only in (near) transmurally infarcted segments was atf significantly reduced, most likely due to wall thinning, as atf is prone to partial volume effects . In contrast, ptf and pti progressively decreased with increasing extent of scarring, as shown by lge cmr . These results are in line with those of previous studies in which reductions in both ptf and pti were observed in nonviable scarred myocardium . Pti in control segments appeared to be somewhat lower than the expected value of unity for normal segments (0.91 0.08). This phenomenon has previously been observed in cardiomyopathy in both animal experiments and human studies [6, 20]. Slight misalignment between pet and cmr segments may have occurred to additionally account for this reduction in pti . Furthermore, the presence of interstitial fibrosis in dilated cardiomyopathy may also explain this reduction in normal segments that remains undetected on lge cmr [21, 22]. Taking lge as a reference, the optimal cut - off values for discriminating between viable and nonviable myocardium although this would reduce the data processing time, it would not affect the scanning protocol, as for both parameters a dynamic [o]h2o pet scan in combination with a low - dose ct scan are required . In addition, the sensitivity of pti exceeded that of ptf (91% and 68%, respectively), whereas for specificity the opposite pattern was observed (73% and 78%, respectively). Although further study is need to determine the cause of this discrepancy, as a potential clinical marker of viability pti may be favoured over ptf to reduce false - negative findings in patients who might benefit from revascularization . It is of interest to note that, compared to ptf and pti, mbf performed relatively poorly in distinguishing viable from nonviable tissue, rendering it less suitable for viability imaging . Previous studies have indicated that the optimal cut - off value for pti is in the range 0.70.9 . Lge was used as a surrogate end - point of myocardial viability instead of functional recovery of dysfunctional myocardium after revascularization . Lge has been shown to be a good, but not perfect, marker of myocardial viability [15, 17]. Therefore, the results should be interpreted with caution, and more studies are warranted to establish the value of parametric pti as a viability marker . Ptf and pti, obtained from a single [o]h2o pet / ct scan, can be used as markers of myocardial viability in patients with coronary artery disease . Ptf and pti, obtained from a single [o]h2o pet / ct scan, can be used as markers of myocardial viability in patients with coronary artery disease . 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While advances in free trade and globalization increase the movement and accelerate the accumulation of invasive species (lockwood et al . 2005), it is still unclear how introduced populations can successfully establish . As elton (1958) pointed out, for every successful invasion that occurs, there are enormously more invasions that never happen, or fail quite soon or even after a good many years (page 109). This modern biological paradox cannot readily be reconciled, especially in the characteristic case where the founder population is small, as such populations are definitely in a precarious position (mayr 1965; page 42). Introductions of populations at low density and/or small size are often faced with inverse density dependent effects, attributed to demographic stochasticity or reduced cooperative interactions (courchamp et al . Allee (1931) first proposed that under these conditions, populations may suffer a decrease in the per - capita rate of increase, from here on referred to as the allee effect . Upon arrival in a novel environment, individuals need to overcome a series of challenges in order to reduce the population's risk of extinction . The time period in which this occurs is generally considered the initial establishment phase, and is thought to be a common feature and general pattern of invasion and the process of growth and expansion (shigesada and kawasaki 1997; sakai et al . The occurrence of a lag phase that precedes a noticeable increase in population growth and density can result from ecological and/or evolutionary phenomena (sakai et al . Small populations that undergo logistic growth slowly increase through the initial phase of the exponential curve, leading to the perception of a time lag . Where this time lag is more pronounced, populations may be recovering from inverse density dependent effects (i.e., allee effects). Individuals may suffer a reduction in fitness at low densities for many reasons (reviewed by courchamp et al . Even when the initial population is large, rapid dispersal required for expansion could be suicidal as the population density decreases, thereby enhancing inverse density dependent effects (lewis and kareiva 1993; drake et al . Component allee effect where the population size and density affects the mean overall fitness or some component of individual fitness, respectively (stephens et al . 1999; gascoigne et al . It is often difficult to decipher the exact mechanism that manifests allee effects (and it is not always the case that component allee effects lead to demographic allee effects). Nevertheless, we focus our attention on population level demographic allee effects with the underlying assumption that a component allee effect led to the demographic allee effect . Essentially, it is the case that introduced individuals may be maladapted to small population sizes where their survival and reproductive ability are significantly impacted, and these impacts on individual fitness combine to produce an overall decrease in abundance (i.e., demographic allee effects). As allee effects impact individual fitness, the underlying traits that influence these effects (i.e., component allee effects) may be subject to natural selection (courchamp et al . 2008). Whereas propagule pressure is an emerging explanation for the establishment of invasive species (lockwood et al . 2005), it relies on an obvious relationship between the number and size of introduction events and the probability of success, as safety in numbers helps combat allee effects and stochastic extinction . In the event that propagule size is not large enough to overcome inverse density dependent effects, a population traits that may be responsible for reproductive success and survival at small population densities and sizes include mate finding cues (pheromones and vocal / visual signals), dispersal / aggregation behavior, habitat preferences, mating synchronicity, and gamete morphology and performance (see courchamp et al . Direct evidence for the evolution of these traits as functional adaptations to allee effects is limited, but we can infer an adaptive evolutionary origin of these traits from studies of sexual selection (courchamp et al . 2008; gascoigne et al . An evolutionary response to intensive selection pressure imposed by density dependent survival and reproduction relies on genetic variants for adaptive evolution . According to neutral quantitative genetic theory, a loss of genetic variation is expected from population bottlenecks and founder effects (nei et al . However it is not neutral variation that matters, but rather evolvability depends on the variation relevant to selection . Maintenance or even increases of this (additive) genetic variation has been theoretically and empirically observed following a bottleneck or in small founder populations (bryant et al . 1986; goodnight 1988; willis and orr 1993; cheverud and routman 1996; briggs and goldman 2006; turelli and barton 2006). Additionally, many recent studies suggest that there is actually no significant reduction in genetic diversity in most successful invaders (lee 2002; allendorf and lundquist 2003; roman and darling 2007 and references therein), and that evolution can occur on contemporary timescales (reznick and ghalambor 2001; carroll et al . 2007; kinnison and hairston 2007). Our purpose here is to explore the feasibility of small populations that may adaptively respond to overcome allee effects in order to establish, given any amount of genetic variation . In this paper, we present evidence for the enhanced potential for growth and spread of a small introduced population of organisms faced with allee effects when adaptation occurs . We model the growth and spread of this population according to a reaction - diffusion equation, and allow evolution to influence inverse density dependent effects through a genetic subsystem that provides the opportunity for successful invasion when otherwise (under the current, ecological paradigm) the population would go extinct . The deterministic model that we explore in this paper broadly describes population dynamics with density - mediated growth (i.e., an allee effect) and diffusive dispersal . This type of demographic model has been used as a compact and tractable representation of invasion (e.g., skellam 1951; lewis and kareiva 1993). Specifically, it has been applied to systems such as introductions of nonindigenous freshwater and marine species through ballast water discharge (drake et al . 2005; drury et al . 2007). Using this approach, drake et al . (2005) report acceptable volumes of discharge for various organisms (with differing reproductive rates) for a range of invasion risk tolerances . Here, we consider populations that are introduced below the invasion risk threshold, but nonetheless succeed if evolutionary dynamics are considered in conjunction with the ecological system . The growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by (lewis and kareiva 1993): which determines the rate of change in the local population density relative to the carrying capacity [u which denotes u(x, t)] over time, at a point in space . This equation models the growth of the population (the first term on the right hand side of eqn . 1) at a spatial location that is subject to an allee effect in addition to migration (which depends on the second term on the rhs of eqn . 1). The diffusion coefficient (d) scales the rate of population spread, in this case across a one - dimensional habitat x. the reproductive rate is regulated by r, and a (which is a function of space and time, derived below) is the local critical density or allee threshold that determines if population growth is positive or negative (fig . Growth dynamics of the model population (a), and the solution of eqn . (1) without diffusion (b) with reproductive rate, r = 0.6, and allee threshold, a = 0.3 . Trajectories for population size (b) are given for initial densities from 0 to 1 in increments of 0.05 . There are many variations of single - species models of population dynamics that exhibit allee effects (see table 1 of boukal and berec 2002), however the growth function of eqn . The behavior of this verhulst (1838) logistic model modified to include a nonlinear cubic term (based on the fitzhugh - nagumo equations; fitzhugh 1960; nagumo et al . 1962), is bistable, with equilibria at u = 0 (extinction), u = a (unstable threshold), and u = 1 (carrying capacity). Figure 1a shows this behavior in terms of the growth of the population (change in population density with respect to time) versus the population density . At densities below the critical threshold (a) there is negative population growth declining to extinction (from here on the population is considered extinct below a cutoff density of 0.0001, as a declining population trajectory will only asymptotically approach zero in a deterministic model; gomulkiewicz and holt 1995); otherwise the population will reach carrying capacity . 1b with the graph of the solution of the growth function (population size versus time) at various initial densities . When diffusion is added to this model of population growth, there are two critical elements that emerge based on the solution to the partial differential equation (pde). The first is the wave speed, which is determined by the allee threshold (a). As we are considering a strong allee effect in this model (i.e., 0 <a <1, where the population below this threshold exhibits negative growth versus reduced positive growth from a weak allee effect), there exists a unique wave speed of the invasion front that is a result of being pushed from the inside out, as opposed to being pulled by the leading edge (lewis and kareiva 1993). This velocity can be derived through the solution of the pde (1): (lewis and kareiva 1993; murray 1993). This result suggests that in order for a wave to maintain a positive velocity of advance, the magnitude of the allee threshold (a) must be less than half of the maximum value of the population density relative to the carrying capacity . In addition to the velocity of the wave front, the region occupied by the invading population must exceed a certain critical size for positive growth to occur (skellam 1951; kierstead and slobodkin 1953). This phenomenon is clearly explained by okubo (1980) by noting that whereas reproduction takes place within a region or patch, diffusion takes place at the boundaries resulting in a loss of organisms, thus reducing the density within the patch . This tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth . This relationship gives a minimum region within which reproduction cannot compensate for loss due to diffusion, especially when allee effects influence population growth . Thus, lewis and kareiva (1993) derive a minimum size condition (i.e., the radius of the initial beachhead) based on the wave speed that is required for the population to establish and radially expand . We address this critical size threshold qualitatively, as the analytical solution (i.e.,; lewis and kareiva 1993) is for two - dimensional spread, while we work with a simpler one - dimensional model . The minimum critical radius is proportional to the ratio of diffusivity (i.e., diffusion coefficient, d) to the reproductive rate (controlled by r). The inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success . The diffusion process has been extensively analyzed in invasion processes (e.g., fisher 1937; skellam 1951; okubo 1980). In order to incorporate evolutionary factors that may influence invasion success, this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population's likelihood of survival . Specifically, we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait (e.g., a trait impacting the component allee effect). From here on, except in the absence of evolution, referring to the allee threshold implies that that value is the initial value, as it changes over time . This quantitative trait influences an organism's ability to survive and reproduce in a small population . The results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . Including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies . Therefore, it is important to investigate the possibility of evolution in the analysis of invasions . The framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast, ecological and slow, evolutionary timescales in order to make these rates comparable (kondrashov and khibnik 1996). In the coupled evolutionary ecology model, the reaction - diffusion eqn . (1) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance . As the population dynamics vary across space, the genetic subsystem describes the rate of change of the trait mean (i.e., the allee parameter) at each location x by: (pease et al . 1989; garca - ramos and kirkpatrick 1997; kirkpatrick and barton 1997; hare et al . The first term on the right hand side reflects the force of local directional selection, where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function (i.e., per - capita growth rate:) with respect to the trait, a (lande 1976; falconer 1989). Thus,, where we assume that individual fitness approaches the population mean fitness, as most individuals are close to the average phenotype (webb 2003). This suggests that the genetic variance () is small (and constant in this model). This small parameter for the genetic variance can be used to couple the fast ecological timescale, t, with the slow evolutionary timescale, = t (kondrashov and khibnik 1996; webb 2003). Combing these two components of genetic variance and selection, quantifies the effect of natural selection on the local mean value of the quantitative trait (the allee parameter; lande 1976; falconer 1989). In order to account for the influence of migration on the trait's local mean, the latter two terms in eqn . The middle term takes into account asymmetrical gene flow caused by the variation of density across space (pease et al . 1989; garca - ramos and kirkpatrick 1997; kirkpatrick and barton 1997; hare et al . This captures the influence of the mean trait value (i.e., genetic contribution) from more abundant populations to less abundant neighboring locations due to the spatial gradient, as more individuals migrate from areas with relatively high population densities . The last term mirrors the diffusion term from the ecological model, and describes the homogenizing effect of random dispersal . We solved the spatially explicit system numerically using matlab 7.0 (2004, the mathworks, natick, ma, usa) using a finite difference method to incorporate diffusion and gene flow (adapted from garvie 2007). By iterating eqns (1) and (2) forward in time, the population density and allee threshold at each location are updated with diffusion following growth and selection, respectively, while incorporating the spatial gradient . The simulated populations, with and without evolution, behaved as we expected from the model eqns (1) and (2), and adequately approximate / represent the critical conditions that govern this dynamical system . The growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by (lewis and kareiva 1993): which determines the rate of change in the local population density relative to the carrying capacity [u which denotes u(x, t)] over time, at a point in space . This equation models the growth of the population (the first term on the right hand side of eqn . 1) at a spatial location that is subject to an allee effect in addition to migration (which depends on the second term on the rhs of eqn . 1). The diffusion coefficient (d) scales the rate of population spread, in this case across a one - dimensional habitat x. the reproductive rate is regulated by r, and a (which is a function of space and time, derived below) is the local critical density or allee threshold that determines if population growth is positive or negative (fig . Growth dynamics of the model population (a), and the solution of eqn . (1) without diffusion (b) with reproductive rate, r = 0.6, and allee threshold, a = 0.3 . Trajectories for population size (b) are given for initial densities from 0 to 1 in increments of 0.05 . There are many variations of single - species models of population dynamics that exhibit allee effects (see table 1 of boukal and berec 2002), however the growth function of eqn . The behavior of this verhulst (1838) logistic model modified to include a nonlinear cubic term (based on the fitzhugh - nagumo equations; fitzhugh 1960; nagumo et al . 1962), is bistable, with equilibria at u = 0 (extinction), u = a (unstable threshold), and u = 1 (carrying capacity). Figure 1a shows this behavior in terms of the growth of the population (change in population density with respect to time) versus the population density . At densities below the critical threshold (a) there is negative population growth declining to extinction (from here on the population is considered extinct below a cutoff density of 0.0001, as a declining population trajectory will only asymptotically approach zero in a deterministic model; gomulkiewicz and holt 1995); otherwise the population will reach carrying capacity . 1b with the graph of the solution of the growth function (population size versus time) at various initial densities . When diffusion is added to this model of population growth, there are two critical elements that emerge based on the solution to the partial differential equation (pde). The first is the wave speed, which is determined by the allee threshold (a). As we are considering a strong allee effect in this model (i.e., 0 <a <1, where the population below this threshold exhibits negative growth versus reduced positive growth from a weak allee effect), there exists a unique wave speed of the invasion front that is a result of being pushed from the inside out, as opposed to being pulled by the leading edge (lewis and kareiva 1993). This velocity can be derived through the solution of the pde (1): (lewis and kareiva 1993; murray 1993). This result suggests that in order for a wave to maintain a positive velocity of advance, the magnitude of the allee threshold (a) must be less than half of the maximum value of the population density relative to the carrying capacity . In addition to the velocity of the wave front, the region occupied by the invading population must exceed a certain critical size for positive growth to occur (skellam 1951; kierstead and slobodkin 1953). This phenomenon is clearly explained by okubo (1980) by noting that whereas reproduction takes place within a region or patch, diffusion takes place at the boundaries resulting in a loss of organisms, thus reducing the density within the patch . This tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth . This relationship gives a minimum region within which reproduction cannot compensate for loss due to diffusion, especially when allee effects influence population growth . Thus, lewis and kareiva (1993) derive a minimum size condition (i.e., the radius of the initial beachhead) based on the wave speed that is required for the population to establish and radially expand . We address this critical size threshold qualitatively, as the analytical solution (i.e.,; lewis and kareiva 1993) is for two - dimensional spread, while we work with a simpler one - dimensional model . The minimum critical radius is proportional to the ratio of diffusivity (i.e., diffusion coefficient, d) to the reproductive rate (controlled by r). The inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success . The diffusion process has been extensively analyzed in invasion processes (e.g., fisher 1937; skellam 1951; okubo 1980). In order to incorporate evolutionary factors that may influence invasion success, we develop a quantitative genetic subsystem . This genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population's likelihood of survival . Specifically, we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait (e.g., a trait impacting the component allee effect). From here on, except in the absence of evolution, referring to the allee threshold implies that that value is the initial value, as it changes over time . This quantitative trait influences an organism's ability to survive and reproduce in a small population . The results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . Including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies . Therefore, it is important to investigate the possibility of evolution in the analysis of invasions . The framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast, ecological and slow, evolutionary timescales in order to make these rates comparable (kondrashov and khibnik 1996). In the coupled evolutionary ecology model, the reaction - diffusion eqn . (1) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance . As the population dynamics vary across space, the genetic subsystem describes the rate of change of the trait mean (i.e., the allee parameter) at each location x by: (pease et al . 1989; garca - ramos and kirkpatrick 1997; kirkpatrick and barton 1997; hare et al . The first term on the right hand side reflects the force of local directional selection, where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function (i.e., per - capita growth rate:) with respect to the trait, a (lande 1976; falconer 1989). Thus,, where we assume that individual fitness approaches the population mean fitness, as most individuals are close to the average phenotype (webb 2003). This suggests that the genetic variance () is small (and constant in this model). This small parameter for the genetic variance can be used to couple the fast ecological timescale, t, with the slow evolutionary timescale, = t (kondrashov and khibnik 1996; webb 2003). Combing these two components of genetic variance and selection, quantifies the effect of natural selection on the local mean value of the quantitative trait (the allee parameter; lande 1976; falconer 1989). In order to account for the influence of migration on the trait's local mean, the latter two terms in eqn . The middle term takes into account asymmetrical gene flow caused by the variation of density across space (pease et al . 1989; garca - ramos and kirkpatrick 1997; kirkpatrick and barton 1997; hare et al . This captures the influence of the mean trait value (i.e., genetic contribution) from more abundant populations to less abundant neighboring locations due to the spatial gradient, as more individuals migrate from areas with relatively high population densities . The last term mirrors the diffusion term from the ecological model, and describes the homogenizing effect of random dispersal . We solved the spatially explicit system numerically using matlab 7.0 (2004, the mathworks, natick, ma, usa) using a finite difference method to incorporate diffusion and gene flow (adapted from garvie 2007). By iterating eqns (1) and (2) forward in time, the population density and allee threshold at each location are updated with diffusion following growth and selection, respectively, while incorporating the spatial gradient . The simulated populations, with and without evolution, behaved as we expected from the model eqns (1) and (2), and adequately approximate / represent the critical conditions that govern this dynamical system . The dynamics of the evolutionary ecology model can be interpreted using the idea of fast and slow timescales (kondrashov and khibnik 1996; webb 2003). Earlier, we assumed that the genetic variation () was small (to use mean fitness as a proxy for individual fitness), which can subsequently be taken advantage of for our analysis of the coupled evolutionary ecological dynamics . When = 0, the situation without evolution, the genetic subsystem is frozen and the population moves towards a stable equilibrium of the ecological subsystem (carrying capacity or extinction) depending on its initial density (greater than or less than the allee threshold respectively; fig . When> 0 but small, the allee threshold evolves relatively slowly and influences the ecological system . Whenever the population is below its carrying capacity (u = 1 for each spatial coordinate x when space is explicit), eqn . (2) is negative, and decreases the mean allee threshold (a), as the intensity of selection is density dependent . Thus, fitness increases as allee effects are suppressed, and selection drives the allee threshold towards zero . If the population density is greater than the allee threshold, but still below the carrying capacity, it will progress towards carrying capacity more rapidly than it would without evolution as a decreases; as the rate at which the population density changes (eqn . 1) is proportional to the difference between u and a. the ecological dynamics are reversed when the population density is below the allee threshold as the population declines towards extinction, but more slowly than it does without evolution . When u <a, eqn . (1) is negative, and the population density approaches extinction more rapidly with a constant (as the difference between u and a increases), than it does with evolution as a decreases (revealing a more pronounced time lag to extinction). During this time lag, as the population slowly declines, the opportunity for evolution to overcome inverse density dependent effects occurs . If the rate of evolution is fast enough, the allee threshold can fall below the population density, causing the rate of change of population density to become positive (where u> a) and the population grows and can successfully invade . The chance that evolution can rescue the population from extinction depends on the relative rates of genetic change in the quantitative trait (i.e., allee threshold) and of population decline (gomulkiewicz and holt 1995); hence the amount of genetic variance greatly impacts the ability to adapt and survive . A nonspatial example of this process, referred to as evolutionary rescue (gomulkiewicz and holt 1995), is shown in fig . 2, where a population is introduced below the allee threshold . Without evolution, the population declines to extinction (fig . 2a, solid line) as the allee threshold remains constant (fig . 2b, solid line). When the population can evolve (fig . 2, dotted line), it declines at first until it can overcome the magnitude of inverse density dependence, and is then able to successfully establish . As it is difficult to measure the allee effect empirically (tobin et al . 2007), we use an extreme value that exaggerates density dependent effects in order to investigate the worst case scenario (a = 0.3, where the population exhibits deterministic decline when its density is less than 30% of its carrying capacity). When evolution is included, we used a small value for the genetic variance, = 0.02, in order to remain consistent with fast - slow dynamics, unless otherwise indicated . Comparison of an invading population introduced at a density below the allee threshold, a = 0.3 (u = 0.25, r = 0.6). The solid line represents the nonspatial system (d = 0) described by eqns (1) and (2) without evolution (= 0) which results in extinction (a) and a constant allee threshold (b). The dotted line indicates population growth (a) when evolution (= 0.02) acts to reduce the allee threshold (b). In general, there is a range of parameter space that permits persistence for a population below the allee threshold in the nonspatial model with evolution (instead of simple decline to extinction). As genetic variance increases, we are essentially relaxing the assumption of fast - slow timescales and allow evolution to occur more rapidly . 3 where initial population densities below the allee threshold require a minimum amount of genetic variance in order to avoid extinction . In this case, the rate of reproduction, r, also influences the potential for evolutionary rescue, as it impacts both population growth and rate of evolution (eqns 1 and 2, respectively). As we relax the assumption of fast - slow timescales, the behavior remains qualitatively the same as that described analytically under a strict fast - slow timescale assumption . Parameter combinations of reproductive rate: r; genetic variance:, and initial population density: u, that result in extinction or evolutionary rescue . In this nonspatial scenario, initial population densities greater than the allee threshold (a = 0.3) always succeed, thus the focus is on the parameter space that allows for evolutionary rescue (i.e., where the population growth changes from negative to positive). As the reproductive rate increases from 0.1 to 1, there is less genetic variance needed for a population to evolve to overcome inverse density dependence as increased reproduction will contribute to suppressing allee effects . Nonetheless, the additional complexities result in qualitatively similar behavior to the nonspatial model . In this case, not only will evolution influence population growth, it affects the wave speed and the critical size threshold, rmin . As the population overcomes allee effects with a decreasing allee threshold, the wave speed accelerates and the critical patch size becomes smaller . Thus, in addition to the initial density of the introduced population and the genetic variance, the initial radius or patch size of the initial invasion area, the ratio of diffusion to reproduction, and gene flow will factor into successful establishment and give rise to a wider range of interactions between the ecology and evolution of this system . (1) (without the evolutionary subsystem) in one - dimensional space, with an initial population density below the allee threshold, declines to extinction (fig . This is contrasted by the results when the evolutionary subsystem is included . With the initial population density below the allee threshold, fig . The same type of rescue occurs for a population that starts near carrying capacity, but occupies an initial spatial size below that which is necessary for a population to successfully establish . Figure 5a shows a rapidly declining population that goes extinct . Under the same circumstance, but where evolution of the allee threshold occurs, fig . 5b shows the population density at first beginning to shrink and then growing and expanding . In addition to the time evolution of population density across space in figs 4 and 5, the evolution of the mean trait value across space illustrates how gene flow and the density dependent selection gradient influences its rate of change and distribution (figs 4c and 5c). As the intensity of selection is density dependent (and we assume constant genetic variance), locations with smaller populations can evolve the trait value more rapidly compared to other areas where allee effects may not be as strong and experience weaker selection . The trait distribution over time, figs 4c and 5c, therefore reflect the population density distribution, but are also influenced by the trait values of the migrants . As individuals disperse out to new locations and push the boundaries of the species range, their trait values are averaged to determine the demographic allee threshold for that spatial coordinate . This demographic allee threshold combines with their local population density to influence individual fitness and population growth (where the distance between the density and mean trait value is the initial degree of maladaptation). Diffusive dispersal of an introduced population at an initial density (bold dashed line) below the initial allee threshold, a = 0.3 (u = 0.25, r = 1, d = 0.1) across a linear, one dimensional habitat . The population collapses over time to extinction (a) where there is no evolution (= 0), and succeeds (b) after an initial decline with evolution (= 0.02). (c) the evolution of the mean value of the allee threshold across space (where the initial distribution is given by the bold dashed line). The population density distribution and corresponding trait values (i.e., allee threshold) are plotted at equal time increments (every 20 of 1200 model iterations). Population density of a diffusion dispersed population across one dimensional space . The initial population density (bold dashed line) is near carrying capacity (u = 0.95, a = 0.3, r = 1, d = 0.5), but introduced below the minimum radius of area determined to be critical for invasion success . (a) is collapsing to extinction without evolution (= 0), whereas (b) shows success of an invader with evolution (= 0.02) after initial decline . (c) the evolution of the mean value of the allee threshold across space (where the initial distribution is given by the bold dashed line). The population density distribution and corresponding trait values (i.e., allee threshold) are plotted at equal time increments (every 20 of 1200 model iterations). We explored when evolutionary rescue occurred across a range of parameter values for the spatially explicit model . According to drake et al . (2005), variability among locations and over time makes it unreasonable to determine precise estimates for the diffusion coefficient, d. we therefore explored a range of values, and present those that best illustrate breadth of behavior . The parameter that controls the reproductive rate, r, was also varied substantially, but as the spatial dynamics depend on the ratio of diffusion to rate of reproduction (resulting in a measure of length); we fixed r and varied d, unless otherwise noted . This was justified as the results of the spatial simulations are qualitatively identical for equivalent ratios . The effects of the critical patch size, initial population density, ratio of diffusion to growth and genetic variation on evolutionary rescue and population dynamics are shown in fig . The ratio of the diffusion coefficient (d) to the reproductive rate (r) determines whether the population will expand or collapse according to the initial radius of the introduced population . The areas under the curves denote combinations of genetic variance and initial population density that result in extinction . Areas above the curves are combinations of genetic and/or demographic conditions that produce inevitable persistence . The parameter space between the vertical dashed lines refers to the different ways population survival is influenced . To the left of the initial allee threshold, the initial population density will either go extinct due to density dependent effects (below the d / r curve), or given enough genetic variation, will be evolutionarily rescued (above the d / r curve). The area to the right of the initial allee threshold [and between the dashed lines in (b) and (c)] is the case where the initial population density is greater than the allee threshold but due to the initial spatial size and the ratio of diffusion to reproduction, the population may go extinct without sufficient genetic variance (below the d / r curve), otherwise it will evolve to overcome the critical patch size effect . For initial population densities thus, the area between the dashed lines in (b) and (c) truly delineates evolutionary rescue when d / r = 1 . The rightmost vertical line moves slightly to the left to the point of intersection of the d / r curve and the x - axis for other values of d / r . Graphs (a), (b), and (c) represent different radii of the linear habitat that the introduced population initially occupies . When the size of the initial population is too small (i.e., a radius of 1), a population at carrying capacity (i.e., u = 1) will go extinct without evolution due to the relative effect of diffusion to reproduction (fig . If evolution occurs rapidly enough (i.e.,> 0.02), the population can overcome inverse density dependent effects and compensate for the loss due to diffusion and rebound from low densities . When the initial radius of the population is increased (fig . 6b, c), the chance of survival and establishment (growth and expansion) of populations above or below the allee threshold increases with initial density and genetic variance . Therefore, the initial radius of the population can significantly impact the likelihood of evolutionary rescue for populations with the same amount of genetic variance . This is demonstrated further in fig . 7a, where the rate of recovery (i.e., the inverse of the time lag before growth becomes positive and the population reaches carrying capacity) for a population near carrying capacity depends on its initial size / radius and genetic variance . Where size and variance are small, rescue never occurs . As these parameters increase, the rate of recovery gradually becomes faster until it essentially plateaus (although with greater variance and initial radius, the rate of recovery may slow slightly if the initial spatial extent is large enough for the population to experience early growth before diffusion causes decline prior to recovery). If the population occupies a large enough spatial extent, it will succeed without evolution (where the genetic variance is zero), however the lag time may be more pronounced depending on the ratio of diffusion to reproduction through the tradeoff between growth and spread (e.g., if spread is relatively fast compared to reproduction, d / r = 1). The population density may thus initially decline across space until reproduction can sufficiently overcome the loss due to diffusion, and the population can grow to carrying capacity . Similar to the nonspatial case, a population (greater than the allee threshold) above the spatial threshold will grow to carrying capacity more rapidly with evolution than without . Rate of recovery in terms of the inverse of the time lag before population growth becomes positive, where one timestep equals 24 iterations of the model . In (a), the initial population density is near carrying capacity (u = 0.95, a = 0.3, d / r = 1), and the initial radius and genetic variance,, varies . Where the rate of recovery is zero, the population goes extinct as it initially occupies an area smaller than the critical patch size (in this case, a radius of 1.4) or does not have sufficient genetic variance to evolve quickly enough to be rescued prior to extinction . Increasing the genetic variance and initial radius will decrease this time lag until the population no longer experiences any negative growth (in this case, for initial radii 3.8 and 0.036; for initial radii> 2.7, the rate of recovery slows slightly due to early growth followed by a transient decline that precedes ultimate recovery). When the initial population density varies (indicating the initial degree of maladaptation where a = 0.3), (b) shows the rate of recovery with the initial radius fixed (as in fig . 5a where the radius = 1 and d / r = 1). In this case, extinction will occur without evolution not only for an initial density below the initial allee threshold, but for any density as the initial radius is below the critical patch size . Hence, a nonzero rate implies evolutionary rescue and a zero rate means extinction . As shown in fig . 7b, when evolutionary rescue is possible, the initial level of maladaptation (a0u0) and the genetic variance () also determine the rate at which evolutionary rescue proceeds . Figure 7b uses parameters (i.e., radius and ratio of d to r) for a population that would decline and go extinct without evolution regardless of the initial density . Hence, it is clear that the amount of time required for a population to begin growing depends on its initial level of maladaptation (to both the critical density and spatial thresholds) and/or genetic variance . As the rate at which this rescue occurs depends on the amount of genetic variance (eqn . 2), it may take an extremely long time (as 0, the rate of recovery 0) for the allee threshold to fall below the population density . In this circumstance, as the population density becomes very close to zero, the rate of change of the allee threshold is greater than that of the population density (as u 0, u/t 0 and a/t -2ra). Thus, theoretically, rescue would always occur (gomulkiewicz and holt 1995). However, to maintain biological realism, when solving this system numerically, we always considered the population extinct when the maximum density (across space, when diffusion is included) becomes reasonably close to zero (i.e., u = 0.0001; we chose this protocol instead of the total population across space due to the diffusion dynamics based on the gaussian dispersal kernel and the pushed wave front behavior). Overall, the numerical results qualitatively hold for a wide range of dimensional parameter values and initial conditions with and without diffusion and in one- and two - dimensional space . Results for two - dimensional space are not shown as they are qualitatively similar to the simpler, one dimensional model . From these results, it is apparent that adaptations that enable organisms to overcome the negative effects of low densities can allow the population to rebound from a trajectory toward extinction to grow to reach carrying capacity . Current management strategies (e.g., reducing population density or size) are based on ecological theory (e.g. Drake et al . 2005), but this evolutionary ecology model suggests that adaptive evolution can enable successful establishment and that ecological considerations alone may not be sufficient . Under the assumptions of an allee effect and diffusive dispersal, the idea of ecological size thresholds fits well with the ecological evidence that a large founding population is a primary cause of successful establishment (lockwood et al . However, by incorporating evolution, we see that the situation is not quite this simple because ecological size thresholds and genetic variance can interact to determine successful establishment . As the ratio of diffusion to reproduction decreases, the spatial constraint on population growth becomes weaker, and less genetic variance is needed to rescue populations with densities below the allee threshold . As the initial spatial radius of introduction increases, furthermore, the rate of this rescue depends on the initial genetic load or maladaptation (i.e., how far the population density is from the allee threshold), as well as the amount of genetic variance . Because bottlenecks during founding events do not always result in highly reduced genetic variability, even small founding populations may have sufficient genetic variation to evolve to overcome allee effects and establish, contrary to solely ecologically based predictions . Additionally, we can draw several general insights about how dispersal impacts selection and evolution of allee effects in an invasion context . As species are transported from their native environment into novel habitats or simply disperse on their own, it is clear that the genetic composition of the local population can influence the rate of evolution and adaptation to the new local conditions . Given enough genetic diversity, local populations can adapt to their local environment, but dispersal may hinder survival across ecological clines as dispersers tend to be maladapted to the new local environment . Essentially, local population persistence depends on the race between the rate of evolution and the degree of maladaptation (gomulkiewicz and holt 1995). In this case, gene flow will play a major role in determining the outcome . As kirkpatrick and barton (1997) and garca - ramos and kirkpatrick (1997) demonstrate, individuals moving from one selection regime from the center of their species range to the periphery can introduce enough maladaptation that the new area becomes a sink environment . On the other hand, holt et al . (2003, 2004) show that immigration can have a positive influence on adaptation to sink environments, in some circumstances . Resolving the disparity between these perspectives requires understanding what is contributing to the severity of maladaptation and the population's ability to overcome it . In our model, dispersal impacts survival ecologically through the critical patch size, and genetically, as individuals may move from areas where they are well adapted (i.e., the population density is greater than the allee threshold or mean trait value) to sink regions, where they are maladapted . As individuals disperse across space, they may be contributing positively in an ecological sense to the quality of their new local environment (by increasing the local population density). However, dispersers are more likely to come from higher density areas where allee effects, and hence selection, are locally weak . These dispersers potentially introduce more maladaptation to their new location, because they increase the average phenotype (allee threshold) in the new location where density is likely to be lower . Interestingly, the evolutionary impacts of migration in this model do not dramatically influence the dynamics . Changes to the local mean phenotype through local selection and simple mixing (i.e., diffusion) actually slightly hastens the evolutionary rescue effect over a model that considers only the impact of local selection . As the selection intensity is density dependent and proportional to u 1 for each point in space, the peripheral individuals faced with stronger selection with lower trait values have a small positive influence on the more dense neighboring populations . The gradient term accounts for asymmetric gene flow due to differential migration from areas of relatively high population densities . However, this term does not alter the overall evolutionary dynamics based on local selection any more than adding the diffusion term, as the negative effects of gene flow and the local rate of evolution (which is relatively fast, based on the selection intensity) essentially cancel each other out . In this context, similar to that of holt et al . (2003, 2004), the immigrants simply contribute to the local population density, which helps prevent extinction long enough for evolutionary rescue to occur locally (i.e., positive population growth; note that whereas holt et al . (2003) attribute the main effect of immigration to the contribution of variation, this is not the case in our model, as we assume constant genetic variance). (2004) where immigration has a demographic effect on increasing fitness that can essentially outweigh the the primary determinant of invasion success depends on positive population growth at the center of the introduced range . This result comes from the allee effect [and the solution to the pde (1)] by forcing a pushed travelling wave front (lewis and kareiva 1993), where the wave speed causes population expansion, contraction, or propagation failure (i.e., pinning; keitt et al . 2001). Intuitively, aggregation - like behavior emerges based on the strength of the allee effects . Individuals that disperse too far from the whole are likely to die before they can pull others in their vicinity . In this regard, growth occurs from the inside out, where the population seemingly spills out and overflows to expand its range . Consequently, in this study, and for biological invasions that exhibit similar dynamics, it is more important to focus on the center of the invader's range and whether the initial beachhead can survive (through evolutionary rescue), than the fate of peripheral populations at the wave front when determining the importance of evolution on invasion success . The overall dynamics are qualitatively similar in the parameter space that allows for evolutionary rescue to occur . Even though gene flow and spatial structure do not dramatically influence the establishment of an introduced population, additional invasion criteria need to be considered . When analyzing the model behavior in a spatially explicit context, there is an ecological tradeoff between growth and spread that affects establishment and the rate of recovery . As previously mentioned, reproduction needs to compensate for the loss due to diffusion . Including evolution and suppressing allee effects, actually contributes to the acceleration of the wave front (i.e., enhancing dispersal speed). A population then can more rapidly disperse as it evolves, and may become more of an invasion threat as long as this range expansion does not reduce their density too quickly . Whereas this increasing wave speed can lead to a slightly longer lag phase prior to positive population growth, the population will likely be inevitably rescued because this effect primarily influences the dynamics at the periphery and is offset by the reduction in the critical invasion area (rmin). Although there is no range contraction (as there is always a positive wave velocity with unbounded expansion due to the parameter values and absence of environmental heterogeneity or range limitations; filin et al . 2008), as the critical patch size (rmin) becomes smaller with the decreasing allee threshold, rescue occurs more readily at the range center as the critical patch size threshold criteria weakens and the behavior approaches that of the nonspatial model . This may seem like an oversimplification of the global dynamics; however these conclusions are valid in the context of this investigation which focuses on the establishment phase rather than subsequent range expansion and spread . Recognizing that evolution can significantly affect the establishment success of invasive species is becoming more widely accepted, influencing the ways in which invasion biologists conduct their research (see the other articles in this issue). Specifically, adaptations that diminish allee effects and evolutionary responses to density dependence are beginning to emerge as viable explanations for sustaining vulnerable populations at low density and size (gascoigne et al . As it is difficult to conclusively support this claim empirically (as the origin of the adaptation or the associated cost may be unknown; courchamp et al . 2008; gascoigne et al . 2009), mathematical models that incorporate evolution and compare the effects of various strategies (e.g., mitigating component allee effects) can help decipher the mechanisms that both limit and facilitate population growth . Two such models that incorporate adaptations to component mate - finding allee effects compare the efficiency and survival of populations at various densities that attract mates with or without a sexual pheromone (jonsson et al . Another study suggests that broadcast spawners that evolve their gamete morphology and performance under sperm limitation (at low density) bear a cost of decreased fitness at high density due to hybridization and competition (levitan 2002). In these cases, particular strategies are shown to influence population viability in addition to an associated tradeoff, whereas our investigation provides broad, albeit simplistic, results dealing with generalized demographic allee effects and evolution . In order to understand how the results of this simplistic model extend to more realistic and complex evolutionary scenarios, spatially explicit, individually - based stochastic simulation of the introduced populations should be developed to investigate more closely the mechanisms that allow these population level dynamics to emerge . In particular, tracking the mean value of a component allee effect is sufficient to illustrate how evolution can overcome inverse density dependence and result in invasion . However, this approach may not be sufficient to make the specific quantitative predictions necessary for management of invasive species . This is due to the simplifying assumption of constant genetic variance based on mutation - selection balance (lande 1976). Complex simulations could relax this assumption and permit genetic variation to change via mutation, selection, and drift, in tandem with the demographic processes in a heterogeneous environment, and explicitly investigate the costs associated with avoiding allee effects . Hence, future models should incorporate how propagule pressure (size and frequency of introduction events) impacts genetic variation and how more realistic genetic architectures contribute to the evolutionary trajectory of invasive species . Although there is still much more work to be done to elucidate the factors that determine establishment success of founder populations, this theoretical approach has the promise to provide evidence in support of our working hypothesis that adaptive evolution can mitigate allee effects and be an important driver of biological invasions.
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Genital infection due to chlamydia trachomatis is one of the most prevalent bacterial sexually transmitted infections (stis). According to the who estimates, globally 92 million new cases of c. trachomatis infection occur each year and about two - thirds of these cases occur in the developing world, where diagnostic and treatment services are scarce [2, 3]. Most epidemiological data on chlamydia trachomatis infection (cti) is from industrialized nations and reliable data from the resource poor developing nations is not available where the disease burden is concentrated . However, it is important to document laboratory - confirmed incidence and prevalence of cti from the developing world as well . The available indian data show a wide variation in ct prevalence with infection rates in indian women ranging from 3.3% to 33% depending on the population sampled [413]. Infection with this agent is usually asymptomatic in up to 80% of women which makes diagnosis and detection all the more difficult . Left undetected and untreated the infection may evolve into pelvic inflammatory disease and may result in serious sequelae, such as ectopic pregnancy and infertility [14, 15]. Cti in women has also been linked to adverse pregnancy outcomes like recurrent miscarriage and preterm labor and may cause conjunctivitis, nasopharyngitis, and pneumonia in newborns by vertical transmission . Because the infection is easily treatable with antibiotics, early detection and treatment of infected individuals are the key to prevent adverse sequelae among those infected and reduce c. trachomatis transmission . Thus, it is important to screen adolescents and sexually active women for cti even if they are asymptomatic [17, 18]. But, in the developing countries with the exception of sporadic testing, screening for chlamydia is rarely done . Epidemiological studies have also shown that untreated genital chlamydia infection can lead to an increased risk for heterosexual acquisition of hiv . Hence, screening for cti done in high risk populations can assist in designing hiv risk reduction strategies . On the other hand, immunosuppression due to hiv may lead to more aggressive chlamydia disease conditions like pid in hiv seropositive women . Thus, screening for cti in hiv seropositive women is highly recommended to prevent morbidity associated with the disease and devastating clinical consequences . Different diagnostic modalities for detection of cti like serology, culture method, elisa for antigen and antibody, direct fluorescence assay (dfa) and nucleic acid amplification tests (naats) have been used in the last 20 years but none of them are 100% sensitive . Table 1 shows the prevalence of cti detected by using different diagnostic techniques in new delhi population . Polymerase chain reaction is an accurate, rapid, and reliable method for the detection of chlamydia trachomatis . Real - time pcr has increasingly been used and is easier to perform and faster, and since it is performed in a closed system it is less prone to contamination than the conventional pcr . Keeping the above background in mind this study was undertaken to generate reliable data regarding prevalence of cti in hiv - infected and hiv - uninfected women by real - time pcr, the most sensitive and specific test available currently for diagnosing genital chlamydia infection . The primary objective of this study was to establish the need for screening hiv seropositive women for cti . Study subjects were recruited as follows: thirty adult hiv seropositive women with symptoms suggestive of rtis (study group a1),thirty adult hiv seropositive women without symptoms suggestive of rtis (study group a2),thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis (control group b1),thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis (control group b2). Thirty adult hiv seropositive women with symptoms suggestive of rtis (study group a1), thirty adult hiv seropositive women without symptoms suggestive of rtis (study group a2), thirty age and sex matched adult hiv seronegative women with symptoms suggestive of rtis (control group b1), thirty age and sex matched adult hiv seronegative women without symptoms suggestive of rtis (control group b2). All the study subjects were enrolled from the integrated counseling and testing center (ictc) for hiv / aids, department of microbiology, maulana azad medical college, which is attached to the lok nayak hospital, new delhi . This study was conducted prospectively between july 2010 and january 2011 at the hiv molecular laboratory of the department of microbiology, maulana azad medical college . This was a cross - sectional analysis to determine the prevalence of chlamydia trachomatis infection by using real - time pcr in hiv seropositive and seronegative, symptomatic, and asymptomatic women visiting the ictc of our department . Subjects were enrolled in this study following institutional ethical committee clearance and a written informed consent of all participants . Each participant was interviewed using a questionnaire concerning general sociodemographic information, personal details, and clinical symptoms . Subjects having one or more of the following symptoms were considered symptomatic for rti: vaginal discharge, vesicular and/or nonvesicular genital ulcers, inguinal bubo, lower abdominal pain, genital skin conditions, urinary burning or frequency, dysmenorrhea, menorrhagia, and intermenstrual bleeding . Vesicular and/or nonvesicular genital ulcers, lower abdominal pain, genital skin conditions, urinary burning or frequency, dysmenorrhea, menorrhagia, and intermenstrual bleeding . Each study subject then underwent a general physical, per abdomen, per speculum, and per vaginum examination . Diagnosis of hiv infection was done by following the standard protocol at our ictc that employs pretest and posttest counseling and obtains informed consent before hiv testing . Three different rapid tests were used to detect hiv-1 and hiv-2 antibodies (combaids (span diagnostics ltd . ), retrocheck hiv (qualpro diagnostics), and tri - line (rapid diagnostics)) following the manufacturer's instructions . One endocervical swab was collected from all participants according to the instructions provided in the specimen collection and transport kit (amplicor std swab collection and transport set) for detection of chlamydia trachomatis by real - time pcr . Genital c. trachomatis infection was diagnosed by using cobas taqman ct test, v2.0, an in vitro nucleic acid amplification test for the qualitative detection of chlamydia trachomatis dna in female endocervical swab specimens . Specimens were processed using the amplicor ct / ng specimen preparation kit for manual specimen preparation and the cobas taqman 48 analyzer for automated amplification and detection (roche diagnostics). The age and gender profile of all the participants is shown in table 2 . In both the hiv seropositive study group and the hiv seronegative control group the mean age of hiv - infected cases was found to be 30.92 5.7 years and in hiv - uninfected controls 28.52 6.9 years . Table 3 shows the presenting complaints of the symptomatic women in both the hiv seropositive study group and the hiv seronegative control group . Vaginal discharge and lower abdominal pain were the most common presenting complaints amongst the symptomatic participants . Table 4 shows the sti / rti syndromes diagnosed in study subjects on per speculum examination . Vaginitis was the most common syndrome diagnosed in both the study and the control groups . Table 5 shows the correlation between presence of symptoms and cti in the study and the control groups . Chlamydia trachomatis infection was more commonly diagnosed in the asymptomatic hiv seropositive study subjects as compared to the symptomatic hiv seropositive study subjects . In our study the prevalence of cti was higher in hiv seropositive women as compared to hiv seronegative women (or 4.214; 95% ci 0.45738.865) and among the hiv positive asymptomatic as compared to the hiv negative asymptomatic (or 2.111; 95% ci 1.6062.776), although the differences were not found to be statistically significant (table 6). In india stis / rtis and hiv / aids are major public health problems . Incidence and prevalence data have a key role in control strategies for hiv and stis . Comprehensive baseline information on the epidemiology of stis is essential for the design, implementation, and monitoring of successful control programs to reduce their incidence . Routine surveillance of stis / rtis is not carried out in our country due to the lack of laboratory diagnostic facilities, limited resources, stigma, and discrimination associated with stis and poor attendance of sti patients, especially women, in sexually transmitted disease (std) clinics . Taking the above facts into consideration, it is meaningful to have genuine laboratory - confirmed data on the incidence / prevalence of rtis / stis in india . As the asymptomatic nature of stis is well known, there is a need to adopt a specific strategy for the screening of the sexually active population in india to reduce the overall rate of stis, which would, in turn, reduce the risk of hiv infection . Our study presents an insight into the prevalence data of genital chlamydia infections in hiv - infected and hiv - uninfected women visiting the ictc of new delhi's largest tertiary care hospital . The mean age of the hiv seropositive subjects in our study was 30.92 5.7 and that of the hiv seronegative control group was 28.52 6.9 . This compares well with the findings of another study done in baroda, india, to look for the prevalence of rtis in hiv - infected women wherein the mean age for hiv positive women was 30 and that for hiv negative women was 27 . The predominant age group in the hiv positive participants in our study was 2635 years . This association of chlamydia infection with younger age is consistent with studies from other developing countries [32, 33]. This finding supports the fact that young sexually active adults should constitute a priority target group in the sti control program . However, the findings of our study cannot be generalized to all sexually active adults as the study has its limitations of a small sample size and the study participants belonging to a high risk group being recruited from the ictc of our department . 80% of our hiv positive study participants were married which is quiet similar to what has been reported by another study from sub - saharan africa in which 85.3% of the hiv - infected women were married . This finding highlights the importance of concurrently screening and treating spouses / sexual partners to decrease the sti burden in the country but this is challenging due to the lack of knowledge and cooperation from husbands especially in the indian setup . In the present study, vaginal discharge and lower abdominal pain were the 2 most frequently reported symptoms by the symptomatic women in both the study and the control groups, confirming the data reported by some previous indian studies [22, 29]. Another notable observation of our study was that vaginitis was the most common clinical finding detected on per speculum examination in both the hiv positive (50%) and the hiv negative groups (56.67%) which is in accordance with the observations of garg et al . And balamurugan and bendigeri where a majority of women on clinical examination had vaginitis of 94.6% and 36.9%%, respectively [22, 35]. The present study also validates the fact that chlamydia infections are usually asymptomatic as 10% of the asymptomatic women were diagnosed with cti while only 3.3% of symptomatic women had lab - confirmed cti in the hiv positive study group . Once again this emphasizes the importance of routine screening of at risk young sexually active women . In our study cti was detected in 6.67% (4/60) hiv - infected women and in 1.67% (1/60) hiv - uninfected women by real - time pcr although this difference was not statistically significant but the odds ratio was 4.214 . A study from cuba reported c. trachomatis infection in 10% of hiv - infected cases and 6.6% of hiv - uninfected cases by nested pcr but the difference was not statistically significant while the odds ratio was 3.39 which is in concordance with our study . Studies conducted by natividad - villanueva et al . In usa and seck et al . In senegal reported c. trachomatis infection in 3.33% and 2.1% of hiv - infected females, respectively [38, 39]. Larger studies are required to validate our observation as our study was restricted to a small number of cases . This study clearly shows that cti is more prevalent among hiv - infected females (with or without symptoms of rti) as compared to hiv - uninfected females . Our study also stresses the usefulness of screening asymptomatic hiv - infected and hiv - uninfected females for cti by risk assessment and diagnostic testing periodically to prevent the occurrence of adverse outcomes associated with the disease and also to check further spread of infection in the community . For countries like india that still do not currently have an active chlamydia screening program in place, randomized controlled trials are required to delineate the benefits of screening in the sexually active population as our study participants represent a high risk group . It is also important that any test adopted in a national screening program be used in the primary care setting by practitioners without the need for expensive training and equipment . However, since use of real - time pcr is not feasible in most hospitals in developing countries efforts should be made to develop a simple, cost - effective, sensitive, and specific point of care test to identify and treat women with cti for prevention of sequelae and hiv transmission.
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Multilevel cervical spondylotic myelopathy (mcsm) is a serious disease which can lead to spinal cord dysfunction and a substantial decrease in quality of life . In addition, mcsm requiring multilevel cervical spine surgery can be accompanied by extensive blood loss . As spinal surgery has become increasingly complex, so control of perioperative bleeding has become an important clinical issue for spine surgeons . Excessive blood loss can lead to a range of comorbidities such as anemia, hypotension, hematoma formation, and inadequate oxygenation of organs, thereby affecting patient outcome . Excessive blood loss often requires allogeneic blood transfusion, the risks of which are numerous and among which immunological reactions and transmission of viruses are considered the most serious . In addition, hematoma formation within a few millimeters of the spinal canal can cause considerable neurological damage . Although occurrences of postoperative spinal hematoma formation requiring emergency surgery are rare, it is nevertheless important to control perioperative bleeding to decrease its incidence . Tranexamic acid (txa) is a synthetic derivative of the amino acid lysine which operates through competitive inhibition of the activation of plasminogen to plasmin by binding specific sites on both plasminogen and plasmin, thereby retarding fibrinolysis, the degradation of blood clots . It has been widely used in various medical fields such as cardiac surgery, gynecology, dentistry, urological surgery, and liver transplantation to reduce the perioperative blood loss, as it is relatively inexpensive and has not been cited in the literature as causing any significant untoward side effects . In addition, txa has been reported to reduce blood loss and transfusion requirements during orthopedic surgery, most commonly in knee and hip joint replacement . Although the benefits of txa in spinal surgery have been reported indeed, there are no studies of txa in cervical laminectomy with lateral mass screw fixation and bone grafting (clf) so far . Clf has been widely used for many years for treating mcsm caused by multilevel cervical spinal cord compression . Although some authors suggest that laminoplasty is superior to laminectomy with lateral mass screw fixation and bone grafting regarding preserved range of motion, indications for the use of clf are broader than that of laminoplasty and recent studies showed good outcomes for clf . Thus, the aim of this study is to evaluate the efficacy and safety of txa in controlling blood loss during clf for the treatment of mcsm . A retrospective comparative analysis was performed in patients with mcsm undergoing clf of vertebrae c3 to c6 . Patients with cirrhosis of the liver, serious cardiac disease, chronic renal failure, cancer, allergy to txa, a history of thromboembolic disease (deep vein thrombosis, ischemic heart disease, pulmonary embolism, transient ischemic attack, strokes, or subarachnoid hemorrhage), bleeding disorders, hypercoagulation status, disseminated intravascular coagulation, pregnancy, combined anterior and posterior spinal fusions, patients receiving antiplatelet and/or anticoagulant therapy at the time of the study, and treatments of vertebrae outside of c3 to c6 were excluded from the study . Between november 2014 and april 2016, 119 clfs fitting the inclusion criteria were performed in our center . Each surgeon (hao, liu, he, wu, wang, zheng, and zhao) in this study had more than 20 years of experience in spinal surgery . The control group consisted of 46 patients who underwent clf for the treatment of mcsm in our institution without being given txa . In both groups, we analyzed patient demographic trait (age at surgery, gender, body weight, height, and body mass index) and duration of surgery which was defined as the time from the initial incision to the completion of wound closure . The quantity of intraoperative and postoperative blood loss and the preoperative and day 1 postoperative hematological data for each patient were obtained in the 2 groups . Total blood loss was calculated as the sum of intraoperative and postoperative blood loss (the quantity during the 1st 16 hours). The spinous processes, lamina, and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision . Laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side, prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae (c3c6). The excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material . Lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique . All patients in the txa group were given a dose of 15 mg / kg of txa (transamin; daiichi pharmaceutical, tokyo, japan) before a skin incision was made, followed immediately with a maintenance dose of 100 mg / hour, and continued until wound closure . No patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug . Continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value . Statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables . All analyses were performed using ibm spss statistics for windows, version 19.0 (ibm corp ., united states of america). The spinous processes, lamina, and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision . Laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side, prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae (c3c6). The excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material . Lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique . All patients in the txa group were given a dose of 15 mg / kg of txa (transamin; daiichi pharmaceutical, tokyo, japan) before a skin incision was made, followed immediately with a maintenance dose of 100 mg / hour, and continued until wound closure . No patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug . Continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value . Statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables . All analyses were performed using ibm spss statistics for windows, version 19.0 (ibm corp ., united states of america). There was no statistical difference in demographic values (patients age, gender, body weight, height, and body mass index) between the 2 groups (table 1). No statistically significant difference in duration of surgery was observed between the 2 groups, with a mean time of 155.72 15.59 and 153.52 11.91 minutes in the control and txa groups, respectively (p = 0.387). There was significantly less intraoperative blood loss in the txa group compared to the control (fig . 1), at 179.66 81.45 and 269.13 94.68 ml, respectively (p <0.001). The txa group had significantly less postoperative blood loss during the 1st 16 hours compared to the control group (108.08 44.31 vs 132.83 49.39 ml, p = 0.005). Total blood loss in the control group (401.96 127.88 ml) was significantly higher than that in the txa group (287.74 115.40 ml, p <0.001). Changes in blood hemoglobin (hgb) content and hematocrit (hct) were not statistically significantly different between the 2 groups: preoperative hgb: 138.28 9.12 g / l (control) and 140.42 10.05 g / l (txa); postoperative hgb: 122.35 10.74 g / l (control) and 125.21 11.29 g / l (txa); preoperative hct: 41.78 2.62% (control) and 42.07 2.08% (txa); and postoperative hct: 36.50 3.22% (control) and 37.32 2.41% (txa). Although the hgb and hct values in the txa group were all higher than those of the control group, the differences were not statistically significant (fig . No patient required an allogeneic blood transfusion during or after the surgery in either group and no serious intra- or postoperative complications, for example, dural tear, infection, epidural hematoma formation, deep - vein thrombosis, pulmonary embolism, allergic reaction, renal failure, or cardiopulmonary complications were observed in either group . Furthermore, no minor side effects associated with the use of txa such as nausea, vomiting, headache, or diarrhea occurred in either of the groups . Demographic parameters of patients in each group . The txa (tranexamic acid) group had significantly less intraoperative, postoperative, and total blood loss compared to the control group . There were no significantly different in blood hgb hct between the control and txa groups . Hgb = hemoglobin, hct = content and hematocrit, txa = tranexamic acid . Numerous studies have supported the use of txa in orthopedic surgery . Although it has been evaluated in many studies, the safety and efficacy of txa for blood loss reduction in spinal surgery has not been clearly demonstrated, especially for clf . Clf for the treatment of mcsm has proved to be a successful technique for restoring normal cervical lordotic alignment, recovering neurological function, and decreasing the morbidity of c5 palsy and axial pain . The principal disadvantage of this approach is a significant decrease in motion due to the fixation of the target vertebrae . Laminoplasty allows a better range of neck motion although it becomes more restricted in flexion and extension over time . For patients with mcsm combined with simple instability or correctable kyphosis, clf is a more suitable method than laminoplasty, even if the consequence is neck stiffness . Fibrinolysis increases transiently when patients undergo surgery, and it has been shown that it contributes to perioperative blood loss during spinal surgery . Txa inhibits fibrinolysis by blocking the lysine - binding sites of plasminogen, plasmin, and tissue plasminogen activator . Since fibrinolysis is activated immediately during and after surgery, txa should be administered before surgery begins . The half - life of txa is approximately 80 minutes in patients with normal renal function . Pharmacokinetic evidence suggests a loading dose of 10 to 15 mg / kg followed by a maintenance dose of 1 mg / kg / hour or repeated dosing . Li et al conducted a meta - analysis of 6 randomized controlled trials and believed that higher txa dosage (15 yang et al conducted a meta - analysis of 9 randomized controlled trials which had a similar sample to li's study . The 2 meta - analyses arrived at a similar conclusion although the quantity of blood loss and incidence of blood transfusion were different . Elwatidy et al used a high single dose (30 mg / kg) of txa and found that it reduced total blood loss and incidence of blood transfusion . Raksakietisak et al reported that 2 doses of txa (15 mg / kg) reduced perioperative blood loss and incidence of blood transfusion among low - risk adult patients undergoing elective complex thoracolumbar spine surgery ., we evaluated the effects of txa with a dose of 15 mg / kg followed by a maintenance dose of 100 mg / hour . The txa treatment group lost significantly less blood than that of the control group, including during the intra- and postoperative periods . Total blood loss in the txa group was 28% less than that of the control group . Although the hgb levels and hct in the txa group were higher than that of the control group, the differences were not significant, possibly because the sample size was too small and the perioperative blood loss in clf relatively minor . A theoretical anxiety associated with the use of txa is its potential for inducing thromboembolic complications . However, many studies have shown that the administration of txa does not increase this risk . In our study, there were no clinical symptoms or signs of thromboembolic events, such as deep - vein thrombosis or symptomatic pulmonary embolism . Thus, the use of txa in patients undergoing clf should be regarded as safe . Additionally, we are unsure whether the complexity of the surgery has an effect on the benefit of txa . A prospective randomized controlled study would better determine the safety and efficacy of txa during laminectomy with lateral mass screw fixation and bone grafting . In this study, blood loss (both intra- and postoperative) in the txa group was significantly lower than that in the control group, and no major intraoperative complications occurred . Our results indicate that the use of intravenous txa is both safe and effective in reducing blood loss in clf . Prophylactic txa may provide the benefit of limiting excessive blood loss in posterior approach cervical spinal surgery . A future prospective randomized controlled trial will provide superior evidence of the efficacy and safety of txa.
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Hip fracture is a moderate musculoskeletal trauma that mainly affects the older population with comorbid conditions . The number will increase markedly in coming years due to the ageing of the population . Comorbidity and the double trauma may dispose them to serious postoperative adverse outcomes and a high mortality dominated by cardiovascular events [25]. Myocardial injury may be difficult to diagnose because of impaired communication, limitations of clinical manifestation and non - specific electrocardiographic (ecg) changes [6, 7]. The isoenzyme myocardium - specific creatine kinase (ck - mb) is expressed in the myocardium and ck in the skeletal muscle cells . These two enzymes have traditionally been analysed in plasma to distinguish myocardial injury and skeletal muscle injury [8, 9]. Troponins have been shown to be more specific and sensitive to cardiac injury [10, 11]. Increased plasma levels of troponin have also been reported in pulmonary embolism, septicaemia and following major orthopaedic and cardiac surgery . In those conditions high plasma troponin levels have been associated with severe adverse outcomes and increased mortality [7, 1215]. In this study we wanted to test the hypothesis that fatal outcome following hip fracture the study was approved by the regional ethics committee and national medical authorities and conducted in accordance with the helsinki declaration . A total of 302 consecutive patients over 75 years of age with dislocated hip fractures were enrolled in the study at elverum (ech) and buskerud (bch) central hospitals during the period 20052009 . Comorbidity was routinely assessed according to the classification of the american society of anesthesiologists (asa). A hemiprosthesis was inserted through a lateral incision and fixed with or without bone cement (landos titan or landos corail, depuy, warsaw, in, usa). Thromboprophylaxis (low molecular weight heparins) was administered routinely preoperatively (on hospital admission) with dalteparin 5000 iu s.c . Blood samples were obtained from an antecubital vein, collected preoperatively (1) and postoperatively [within 24 hours (0)] and on days one [2448 h (+ 1)] and four (+ 4). Troponin t (tnt) and ck - mb were measured by electrochemiluminescence immunoassay (elica, roche, basel, switzerland and abbott, abbott park, il, usa). Ck was measured by absorption photometry (roche, basel, switzerland and abbott, abbott park, il, usa). Values for tnt were only measured at the ech study centre (n = 146). Differences between mortality groups were tested using a two - sample t test or chi - square test for continuous or categorical data, respectively . Non - parametric mann - whitney tests were done for tnt due to skewed distribution . A linear mixed model for repeated measurements with a random intercept and bonferroni adjusted pairwise post hoc comparisons were used to analyse the biochemical markers with respect to mortality and repeated venous blood analyses . Univariate and stepwise multivariate logistic regression analyses were used to estimate unadjusted and adjusted odds ratios with respect to survival at the three month follow - up . Results from multivariate logistic regression were based on models including only significant terms from stepwise regression to maximise the number of patients included . There were 229 women and 72 men, with a mean age of 84.7 (sd 5.1) and 83.7 (sd 4.7) years, respectively . By three months, 59 of 302 (19.5%) had died, 62% women and 38% men . Table 1demographic and clinical characteristics of patients at 3-month follow - upcharacteristicsalive (n = 243)dead (n = 59)p valuesage, years (mean sd)84.1 (5.1)86.2 (4.6)0.004sex female193 (79.4%)37 (62.7%)0.005 male50 (20.6%)22 (37.3%)mobility not mobile37 (15.8%)10 (17.2%)0.109 living aid23 (9.8%)5 (8.6%) crutches51 (21.8%)21 (36.2%) no aid123 (52.6%)22 (37.9%)asa score i10 (4.2%)0 (0.0%)<0.001 ii103 (42.9%)9 (15.3%) iii116 (48.3%)38 (64.4%) iv11 (4.6%)12 (20.3%)asa american society of anesthesiologistsminor deviations between total number of patients in categories compared to number alive or dead are due to missing data . Missing data are assumed to be completely at random demographic and clinical characteristics of patients at 3-month follow - up asa american society of anesthesiologists minor deviations between total number of patients in categories compared to number alive or dead are due to missing data . Missing data are assumed to be completely at random asa, male sex and age were significantly associated with mortality within three months (table 1). Within one day after surgery, the plasma levels of ck and ck - mb increased nearly threefold (inverse for the ratio). On days 1 and + 1, ck and ck - mb values were significantly higher among those who died compared to those who survived (p = 0.001 and p = 0.031, respectively) (fig . 1). On the fourth postoperative day, tnt plasma levels rose twofold in the mortality group and remained unchanged in the alive group . The plasma levels were significantly higher (p <0.05) in the mortality group at all sampling times except at day 0 (fig . 1mean and 95% confidence intervals for troponin t, creatine kinase (ck), myocardium - specific creatine kinase (ck - mb) and ratio of ck - mb to ck before surgery, perioperatively and 1 and 4 days after surgery for 3-month mortality . Statistical significance (p <0.05) between mortality groups is indicated by an asterisk . Statistically significant (p <0.05) differences between before surgery and during follow - up are indicated by the connecting lines . Day 1 1 day before surgery, day 0 within 24 h after the operation, day + 1 2448 h postoperatively, day + 4 4 days after surgery mean and 95% confidence intervals for troponin t, creatine kinase (ck), myocardium - specific creatine kinase (ck - mb) and ratio of ck - mb to ck before surgery, perioperatively and 1 and 4 days after surgery for 3-month mortality . Statistical significance (p <0.05) between mortality groups is indicated by an asterisk . Statistically significant (p <0.05) differences between before surgery and during follow - up are indicated by the connecting lines . Day 1 1 day before surgery, day 0 within 24 h after the operation, day + 1 2448 h postoperatively, day + 4 4 days after surgery the tnt plasma concentrations were split into three equal - sized data subsets, i.e. 0.01, 0.010.04 and> 0.04 g / l, and the ratios of ck - mb to ck concentrations were split into two equal - sized data subsets, i.e. Below and above 0.02 . / l before surgery correlated with three month mortality [odds ratio (or) 10.9, 95% confidence interval (ci) 2.254.0, p = 0.003] (table 2). Stepwise multivariate logistic regression with age, sex, asa category, levels of tnt and ratio of ck - mb to ck concentrations were performed . High tnt plasma concentration was associated with increased mortality (or 6.1 95% ci 623.1, p = 0.008) at day four after surgery . No statistically significant association was found for the ratio of ck - mb to ck during the entire sampling period when adjusted for age, sex and asa (table 3). Similar regression analyses were done for ck and ck - mb . On day + 1, the ck - mb was associated with mortality (or 1.1, 95% ci 1.021.2, p = 0.012). We found that our hypothesis, stating no predictive value from cardio - muscular plasma enzymes with regard to early mortality in patients with hip fracture, was false . Table 2results from univariate logistic regression models predicting 3-month mortality (death)variablesunadjusted or (95% ci)p valuesage1.1 (1.01.1)0.004if male2.4 (1.34.4)0.006asa3.7 (2.26.1)<0.001if ck - mb / ck> 0.02 day 11.6 (0.83.4)0.225 day 00.7 (0.31.6)0.413 day + 11.5 (0.82.9)0.239 day + 41.2 (0.62.4)0.620tnt day 10.011.0 (reference)0.010.041.5 (0.46.0)0.532>0.0411.0 (2.254.6)0.0030.011.0 (reference) day 00.010.041.5 (0.45.4)0.556>0.042.9 (0.711.0)0.1270.011.0 (reference) day + 10.010.041.6 (0.47.4)0.512>0.046.0 (1.721.1)0.0050.011.0 (reference) day + 40.010.043.3 (0.715.0)0.126>0.047.0 (1.925.6)0.003or odds ratio, ci confidence interval, asa american society of anesthesiologists, ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase, day 1 1 day before surgery, day 0 within 24 h after surgery, day + 1 2448 h postoperatively, day + 4 4 days after surgerytable 3results from multivariate stepwise logistic regression models predicting 3-month mortality (death)variablesday 1day 0day + 1day + 4adjusted or (95% ci)p valuesadjusted or (95% ci)p valuesadjusted or (95% ci)p valuesadjusted or (95% ci)p valuesage1.1 (1.01.2)0.0061.1 (1.01.2)0.0061.1 (1.01.2)0.0011.1 (1.01.2)0.027if male2.4 (1.24.6)0.0132.4 (1.24.60.0132.7 (1.45.1)0.002asa3.3 (2.05.6)<0.0013.3 (2.05.6)<0.001ck - mb / cktnt 0.011.0 (reference) 0.010.041.9 (0.49.4)0.450> 0.046.1 (1.623.1)0.008or odds ratio, ci confidence interval, asa american society of anesthesiologists, ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase, day 1 1 day before surgery, day 0 within 24 h after surgery, day + 1 2448 h postoperatively, day + 4 4 days after surgery results from univariate logistic regression models predicting 3-month mortality (death) or odds ratio, ci confidence interval, asa american society of anesthesiologists, ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase, day 1 1 day before surgery, day 0 within 24 h after surgery, day + 1 2448 h postoperatively, day + 4 4 days after surgery results from multivariate stepwise logistic regression models predicting 3-month mortality (death) or odds ratio, ci confidence interval, asa american society of anesthesiologists, ck - mb / ck ratio of myocardium - specific creatine kinase to creatine kinase, day 1 1 day before surgery, day 0 within 24 h after surgery, day + 1 2448 h postoperatively, day + 4 4 days after surgery this prospective study on 302 elderly patients with hip fracture disclosed that 19.5% had died within three months . Preoperatively obtained basic patient information was shown to be of particular importance to assess the risk of postoperative mortality . Asa score on comorbidity, male sex and age correlated significantly with three month mortality, also described by other investigators [1719]. Autopsy studies have shown that cardiovascular events are the main cause of death after hip fracture surgery [20, 21]. Autopsies are rarely done today and the direct cause of postoperative death has not been possible to establish in this or in other recently conducted studies . In the elderly perioperative myocardial ischaemia is often clinically silent, without haemodynamic or notable ecg changes [2225]. Over the years, biochemical plasma markers have been used to distinguish myocardial injury from skeletal muscle damage, an approach also adopted in this study . Potential heart muscle damage was investigated in this study with ck - mb and tnt analyses . We found small and inconsistent differences for ck and ck - mb plasma levels between those who died and those who survived . The ratio of ck - mb to ck was decreased from hospital admission (day 1) to day 0 and stabilised, indicating that skeletal muscle damage dominated and that any myocardial injury remained undetected . This analysis suggests that ck and ck - mb are unspecific enzymes that are not distinct for skeletal and cardiac muscle damage following a hip fracture and are not feasible as prognostic markers of mortality, a finding consistent with other investigators . Release of troponins into the circulation is considered to specifically reflect cardiac injury . In our study we analysed tnt and found that the plasma concentration was significantly higher on the fourth postoperative day in patients that subsequently died compared to those who were alive . G / l had a six times higher risk of dying vs those with normal plasma levels . This calculation was robust when correction was done for age, sex and comorbidity (asa score). These results fit with other reports that showed a second wave of troponin elevation several days after surgery which correlated with postoperative mortality [13, 29]. In summary, this study showed that basic clinical information on sex, age and comorbidity (asa score) and a high postoperative plasma concentration of tnt> 0.04 g / l are robust predictors of three month postoperative mortality in the elderly undergoing hip fracture surgery . This information may be of importance for therapeutic and post - hospital health care intervention.
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A young man was referred with a chief complaint of blurred vision and pain of the left eye 1 month after herpetic encephalitis in the left frontotemporal lobe . The patient had multiple foci of retinitis in the retinal periphery associated with vitritis, blot retinal hemorrhage and retinal arteriolitis . The impression of acute retinal necrosis was confirmed by polymerase chain reaction of aqueous humor by detecting herpes simplex virus type 2; therefore, the patient received intravenous acyclovir . Herpetic encephalitis may be a risk factor for acute retinal necrosis . The virus may reach the eye by the trans - axonal route . Acute retinal necrosis (arn) is one of the clinical presentations of herpes simplex virus (hsv). This disease may present several years after a primary infection, or it may occur following systemic herpetic infection such as herpetic dermatitis . The prevalence of the disease is equal in both sexes and it occurs in the 5th-7th decade of life . American uveitis society criteria for the diagnosis of arn syndrome are: one or more foci of retinal necrosis with discrete borders located in the peripheral retina, rapid progression in the absence of antiviral therapy, circumferential spread, occlusive vasculopathy with arteriolar involvement, prominent vitritis, and anterior chamber inflammation . Optic neuropathy, scleritis, and pain are supportive but not required . In most patients, the diagnosis is made clinically . Polymerase chain reaction (pcr) analysis of the aqueous humor can detect the cause of arn specifically . Pcr is very helpful in the detection of the varicella zoster, hsv type 1 and 2 . Patients with arn due to hsv-1 and varicella zoster virus tend to be older, whereas those with arn due to hsv-2 tend to be younger [3, 4]. In this article, we report one case of unilateral arn 1 month after the herpetic encephalitis . The patient is a 25-year - old man who was admitted with a chief complaint of decreased vision and pain in left eye for the previous 4 days . The patient also complained of perception of floating objects in the visual field of the left eye . The patient had no past medical history indicating an immunocompromised state; he had been admitted to the neurology ward due to herpetic encephalitis 1 month previous to the occurrence of visual symptoms . Herpetic encephalitis diagnosis was made by brain mri (fig . 1) and pcr analysis of the cerebrospinal fluid . He did not report any underlying disease or drug consumption except oral acyclovir 800 mg every 8 h. at physical examination, the right eye was normal . In the left eye, visual acuity was 0.1, papillary reflex was normal and marcus gunn sign was negative . Slit lamp examination of the left eye revealed conjunctival hyperemia and ciliary injection, diffuse fine keratic precipitates in the corneal endothelium, 3 + cellular reaction in the anterior chamber, 3 + cellular reaction in the vitreous without snowball or snowbank opacities, or vitreous hemorrhage . In fundoscopic examination, multiple foci of retinitis and retinal necrosis 2) associated with arteriolar involvement (arteriolitis), blot retinal hemorrhage and optic disc hyperemia . Pcr analysis of aqueous humor revealed hsv-2, so the patient was admitted with the diagnosis of arn and received intravenous acyclovir for 10 days (10 mg / kg / day). After 48 h of antiviral therapy, a systemic corticosteroid (prednisolone 1 mg / kg / day) was introduced and subsequently tapered over several weeks to treat active inflammation . The patient also received aspirin to treat an associated hypercoagulable state . With this treatment, no new lesion developed and opacity of the media decreased; so prophylactic barrier laser photocoagulation in the areas of healthy retina at the posterior borders of the necrotic lesion prevented retinal detachment . Intravitreal antiviral agents such as ganciclovir and foscarnet were not used in this patient because the disease was controlled by intravenous acyclovir . After discharge from hospital, oral acyclovir 400 mg every 5 h was started . During follow - up, the inflammation decreased and the patient had no vitreous traction or retinal detachment or other eye involvement . The brain and retinal involvement were on one side; after 1 month of left frontotemporal lobe involvement of the brain, the retina was involved on the left side . Some of the studies report arn several years after neonatal and infantile herpetic encephalitis [5, 6]; therefore, herpetic encephalitis may be a risk factor for arn development . Other articles report that the virus accesses the retina from the brain by the trans - axonal route; consequently, this virus can cause recurrent episodes of arn [8, 9]. These reports may necessitate a prophylaxis with antiviral agents after herpetic encephalitis to prevent arn . In a study by pavsio et al ., the role of long - term prophylaxis with acyclovir in children who had herpetic encephalitis is an important issue . Prophylaxis could be considered after hsv encephalitis to prevent arn or after arn to prevent second eye involvement . Acyclovir used as prophylaxis for recurrent genital hsv infection in adults for 5 or more years has been associated with minimal toxicity and the selection of resistant strains has not been demonstrated, but there is little experience with the duration of prophylaxis that would have been necessary to prevent arn in our patient . With this report we reemphasize the correlation between these two diseases . On the other hand, arn may be an indication of possible central nervous system involvement and neuroimaging may be necessary in all cases of arn to rule out herpetic encephalitis.
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Inspection of the database of clusters of orthologous groups of proteins (cogs) revealed only one family of such proteins that is represented in most of the sequenced bacterial, archaeal and eukaryotic genomes . The prototype of this family is the rhomboid (rho) protein from drosophila melanogaster, a developmental regulator involved in epidermal growth factor (egf)-dependent signaling pathways [2 - 4]. Not only were homologs of rhomboid detected in prokaryotes and eukaryotes, but the pattern of sequence conservation in this family appeared uncharacteristic of nonenzymatic membrane proteins, such as transporters . Specifically, several polar amino - acid residues are conserved in nearly all members of the rhomboid family, suggesting the possibility of an enzymatic activity . As three of these conserved residues were histidines, it has been hypothesized that rhomboid - family proteins could function as metal - dependent membrane proteases . Recently, however, it has been shown that rho cleaves a transmembrane helix (tmh) in the membrane - bound precursor of the tgf-like growth factor spitz, enabling the released spitz to activate the egf receptor, and that a conserved serine and a conserved histidine in rho are essential for this cleavage . Thus, it appears that rhomboid - family proteins are a distinct group of intramembrane serine proteases . Altogether, the genome of drosophila encodes seven rho paralogs (now designated rho1 - 7, with the original rhomboid becoming rho-1), at least three of which are involved in distinct egf - dependent pathways, apparently through proteolytic activation of diverse ligands of the egf receptor . The newly discovered intramembrane proteolytic activity of rho places the rhomboid family within the framework of regulated intramembrane proteolysis (rip), a new paradigm of signal transduction, which appears to be prominent in all forms of life . Under rip, signaling proteins undergo site - specific proteolysis within tmh, resulting in the release of active fragments, which are the actual effectors in signal tranduction cascades . Until recently, the only characterized cases of rip in eukaryotes involved presenilin-1, an aspartyl protease, which cleaves a transmembrane helix in type-1 membrane proteins such as amyloid -precursor protein (app), notch and ire1, and the metalloprotease s2p, which cleaves a tmh in a type-2 transmembrane protein, the sterol - dependent transcription factor srebp . Notably, s2p has highly conserved bacterial homologs, and the protease domain of presenilins also might be homologous to bacterial and archaeal type iv prepilin peptidases, although, in this case, the sequence similarity is low . In the case of the rhomboid family, the existence of homologs of rho in most prokaryotes is particularly remarkable because animal rho proteins are involved in signaling pathways that are not found outside metazoa, which seems to make functional conservation in prokaryotes a remote possibility . The only prokaryotic protein of the rhomboid family that has been characterized experimentally in considerable detail is aara from the bacterium providencia stuartii . This protein is involved in the export of a quorum - sensing peptide, a function that, in physiological terms, resembles that of rho, although the signaling molecules, other than rho and aara, are obviously unrelated . In a striking recent development, two independent research groups have shown that several bacterial rhomboid - family proteins, including aara, can cleave the egf receptor ligands (spitz, keren and gurken) that are normally cleaved by rho paralogs . The cleavage depended on the conserved serine and histidine residues and, moreover, transgenic flies that expressed aara developed a phenotype indistinguishable from that induced by overexpression of rho, whereas rho could substitute for aara in providencia stuartii . These unexpected findings demonstrated the conservation of a rip mechanism producing extracellular signals in eukaryotes and prokaryotes . Eukaryotic rhomboid family proteins seem to show considerable functional variability; in particular, cross - talk might exist between different rip pathways . A distinct representative of the rhomboid family has been shown to physically interact with presinilins 1 and 2, and was accordingly named presenilins - associated rhomboid - like protein (parl). The yeast ortholog of parl has been suggested to participate in the processing of cytochrome c peroxidase precursor during its import into the mitochondrion . The near ubiquity of the rhomboid family among bacteria, archaea and eukaryotes, along with the remarkable functional conservation, suggests that a signaling mechanism mediated by rhomboids might have functioned already in the last common ancestor of all extant life forms, with subsequent loss in several lineages . To address this possibility although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low (around 10 - 15% identity in the conserved region), the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives . The conserved core of the rhomboid family consists of six conserved tmhs (figure 1). The predicted catalytic serine is located in tmh5, whereas the predicted catalytic histidine is in tmh7; tmh3 contains two additional histidines and an asparagine, which are conserved in the great majority of the rhomboid - family proteins (figure 1). The roles of these conserved residues are not known, but, given the remarkable evolutionary conservation, it seems likely that they also contribute to catalysis; indeed, it has been shown that the conserved asparagine is required for the cleavage of spitz by rho . When examining the multiple alignment of the rhomboid superfamily proteins, we noticed that several eukaryotic members appear to be inactivated proteases, as indicated by the loss of the predicted catalytic serine or histidine (figure 1, and data not shown); these inactivated forms could be regulators of active rhomboid proteases . Several other proteins lack one or more of the conserved residues in tmh3; it remains unclear whether or not these are active proteases . Bacterial and archaeal members of the rhomboid superfamily contain six tmh, whereas the eukaryotic members typically have an additional seventh tmh, which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below . The phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life, but is not necessarily essential for cell function . Rhomboids are missing in the microsporidian encephalitozoon cuniculi, a eukaryotic intracellular parasite with a highly degraded genome, the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium, and several bacterial species, primarily parasites with small genomes but also species with moderately sized genomes, such as xylella fastidiosum (see cog0705 at). In two instances, a representative of the rhomboid family is present in only one of a pair of relatively close genomes (present in t. acidophilum but missing in t. volcanium; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi), which suggests relatively recent, repeated losses of this gene . Most of the prokaryotic species have a single gene coding for a rhomboid - family protein, although some have two or three paralogs (see cog0705); in contrast, eukaryotes show expansion of the rhomboid family, with seven members in drosophila, and as many as 13 in arabidopsis . The multiple alignment of the 6-tmh core of the rhomboid family (figure 1) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood (ml) method (see materials and methods). Only the conserved regions including the tmh and short adjacent stretches shown in figure 1 were used as the input for tree building, whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues (except for the bayesian analysis, which used the complete alignment; see materials and methods). The phylogenetic tree of the rhomboid family presents a complex and unexpected picture (figure 2). Neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades . Instead, the eukaryotic rhomboids are split between two major subfamilies, which are positioned in the midst of different prokaryotic branches (figure 2). The first subfamily, which includes six of the seven drosophila rhomboids, clusters with a distinct prokaryotic assemblage, consisting primarily of gram - positive bacteria as well as a subset of archaea; this clade is strongly supported by bootstrap analysis (figure 2). The proteins in this group of eukaryotic rhomboids, which we designated the rho subfamily, typically have an extra tmh added carboxy - terminally to the 6-tmh core; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core (figure 2). The second eukaryotic subfamily, which we designated the parl subfamily, after parl, the human ortholog of drosophila rho7, resides within a large, heterogeneous prokaryotic cluster (figure 2). Within this subfamily, parl and its orthologs from other animals and from fungi have distinct domain architecture, with an extra tmh added to the amino terminus of the core, whereas the rest have only the core (a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein; figure 2). Thus, the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . Within these two major eukaryotic subfamilies, several lineage - specific expansions of paralogs are noticeable, in insects, mammals and plants (figure 2). Archaeal rhomboids are scattered over the phylogenetic tree, with two major clusters and, in addition, three isolated proteins joining different bacterial branches (figure 2). There is no indication of an affinity between any of the archaeal and eukaryotic rhomboids . Although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages, there are also several clusters that have an odd composition, such as the grouping of proteobacterial and gram - positive species; some of these clusters are well supported by bootstrap (see clusters 1 - 4 in figure 2). This concern is particularly serious for highly divergent families of membrane proteins, such as the rhomboids, in which parallel amino - acid substitutions are likely . Therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests . First, we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa (kh) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated . Specifically, the kh test was used to evaluate two alternative topologies, in which the rho and parl subfamilies formed a clade, and two topologies, in which the rho subfamily formed a clade with archaeal rhomboids (figure 2 and table 1). Each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 (see table 1). In addition, a tree of the rhomboid family was constructed using the bayesian inference method, which has recently become a practical alternative to the more traditional methods of phylogenetic analysis . The tree produced using the mrbayes package showed the same major clades as the tree in figure 2 (data not shown); moreover, clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities (figure 2). We also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method . The resulting tree contained the same major clades as the trees constructed using ml and mrbayes; however, the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach (data not shown). The alternative phylogenies reflected two distinct hypotheses: first, clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2; and second, monophyly of the eukaryotic rhomboids (figure 3). The phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests (table 2). The hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly (p <0.05) worse than the best tree according to each of the tests (table 2). The concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and, in particular, for the polyphyly of eukaryotic rhomboids . The phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the' standard model' scenario, with the major split between the archaeo - eukaryotic and bacterial lineages nor the' mitochondrial' scenario, which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont . Neither can this tree be explained by postulating a small number of lineage - specific gene losses . The parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer (hgt) and lineage - specific gene loss events . In particular, in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms, phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor (luca). Instead, the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt, and then lost in some lineages . In particular, at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids, one of them yielding the rho subfamily and the other one the parl subfamily, with a possible additional hgt in plants (figures 2,3). Given the broad phyletic representation of both subfamilies of eukaryotic rhomboids, both the rho subfamily and the parl subfamily must have been acquired through hgt at an early stage of eukaryotic evolution, definitely before the divergence of the major crown - group lineages . This early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts . An alternative to this multiple - hgt scenario is that luca already had multiple, paralogous rhomboids, which evolved by a series of ancient gene duplications, and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs . Although this cannot be ruled out formally, this hypothesis implies the existence of an elaborate signaling system in luca and, accordingly, suggests that luca was a complex organism, which might have had as many genes as modern bacteria . Theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution . A complex luca with around 2,000 genes is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt . However, explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be' compatible with life', that is, it includes genes responsible for most, if not all, essential cellular functions . We currently believe that this is the most realistic, albeit inevitably imprecise, reconstruction of luca's gene set . With respect to the rhomboid family and other families whose phylogenetic trees show similar patterns, this makes the multiple - hgt interpretation the scenario of choice . Further theoretical, comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument . The multiple - hgt interpretation of the evolutionary history of the rhomboid family, while supported by the above argument, seems, at least at first glance, distinctly counter - intuitive, given that this family is nearly ubiquitous among extant life forms . Indeed, when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [31 - 33], there is no chance that such a widespread family is not assigned to luca . It should be realized, however, that these approaches are inherently probabilistic, and extensive hgt can fool them . For the rhomboid family, the multiple - hgt mode of evolution seems to be particularly plausible . It seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein, probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria . The protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs (figure 4). This would enable the transition from simple transport to the rip mode of controlled export of signaling molecules . Emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed, rather than eliminated, after hgt . In terms of the evolution of sequence itself, the requirements for the conservation of the protease activity apparently' locked' the rhomboid family in a regime of relatively slow evolution, which ensures significant sequence similarity between all family members (figure 1). The scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes, including intramembrane proteases involved in rip, such as presenilins and their homologs and the archaeo - eukaryotic signal peptide peptidase . Although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low (around 10 - 15% identity in the conserved region), the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives . The conserved core of the rhomboid family consists of six conserved tmhs (figure 1). The predicted catalytic serine is located in tmh5, whereas the predicted catalytic histidine is in tmh7; tmh3 contains two additional histidines and an asparagine, which are conserved in the great majority of the rhomboid - family proteins (figure 1). The roles of these conserved residues are not known, but, given the remarkable evolutionary conservation, it seems likely that they also contribute to catalysis; indeed, it has been shown that the conserved asparagine is required for the cleavage of spitz by rho . When examining the multiple alignment of the rhomboid superfamily proteins, we noticed that several eukaryotic members appear to be inactivated proteases, as indicated by the loss of the predicted catalytic serine or histidine (figure 1, and data not shown); these inactivated forms could be regulators of active rhomboid proteases . Several other proteins lack one or more of the conserved residues in tmh3; it remains unclear whether or not these are active proteases . Bacterial and archaeal members of the rhomboid superfamily contain six tmh, whereas the eukaryotic members typically have an additional seventh tmh, which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below . The phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life, but is not necessarily essential for cell function . Rhomboids are missing in the microsporidian encephalitozoon cuniculi, a eukaryotic intracellular parasite with a highly degraded genome, the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium, and several bacterial species, primarily parasites with small genomes but also species with moderately sized genomes, such as xylella fastidiosum (see cog0705 at). In two instances, a representative of the rhomboid family is present in only one of a pair of relatively close genomes (present in t. acidophilum but missing in t. volcanium; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi), which suggests relatively recent, repeated losses of this gene . Most of the prokaryotic species have a single gene coding for a rhomboid - family protein, although some have two or three paralogs (see cog0705); in contrast, eukaryotes show expansion of the rhomboid family, with seven members in drosophila, and as many as 13 in arabidopsis . The multiple alignment of the 6-tmh core of the rhomboid family (figure 1) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood (ml) method (see materials and methods). Only the conserved regions including the tmh and short adjacent stretches shown in figure 1 were used as the input for tree building, whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues (except for the bayesian analysis, which used the complete alignment; see materials and methods). The phylogenetic tree of the rhomboid family presents a complex and unexpected picture (figure 2). Neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades . Instead, the eukaryotic rhomboids are split between two major subfamilies, which are positioned in the midst of different prokaryotic branches (figure 2). The first subfamily, which includes six of the seven drosophila rhomboids, clusters with a distinct prokaryotic assemblage, consisting primarily of gram - positive bacteria as well as a subset of archaea; this clade is strongly supported by bootstrap analysis (figure 2). The proteins in this group of eukaryotic rhomboids, which we designated the rho subfamily, typically have an extra tmh added carboxy - terminally to the 6-tmh core; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core (figure 2). The second eukaryotic subfamily, which we designated the parl subfamily, after parl, the human ortholog of drosophila rho7, resides within a large, heterogeneous prokaryotic cluster (figure 2). Within this subfamily, parl and its orthologs from other animals and from fungi have distinct domain architecture, with an extra tmh added to the amino terminus of the core, whereas the rest have only the core (a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein; figure 2). Thus, the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . Within these two major eukaryotic subfamilies, several lineage - specific expansions of paralogs are noticeable, in insects, mammals and plants (figure 2). Archaeal rhomboids are scattered over the phylogenetic tree, with two major clusters and, in addition, three isolated proteins joining different bacterial branches (figure 2). There is no indication of an affinity between any of the archaeal and eukaryotic rhomboids . Although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages, there are also several clusters that have an odd composition, such as the grouping of proteobacterial and gram - positive species; some of these clusters are well supported by bootstrap (see clusters 1 - 4 in figure 2). This concern is particularly serious for highly divergent families of membrane proteins, such as the rhomboids, in which parallel amino - acid substitutions are likely . Therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests . First, we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa (kh) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated . Specifically, the kh test was used to evaluate two alternative topologies, in which the rho and parl subfamilies formed a clade, and two topologies, in which the rho subfamily formed a clade with archaeal rhomboids (figure 2 and table 1). Each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 (see table 1). In addition, a tree of the rhomboid family was constructed using the bayesian inference method, which has recently become a practical alternative to the more traditional methods of phylogenetic analysis . The tree produced using the mrbayes package showed the same major clades as the tree in figure 2 (data not shown); moreover, clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities (figure 2). We also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method . The resulting tree contained the same major clades as the trees constructed using ml and mrbayes; however, the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach (data not shown). The alternative phylogenies reflected two distinct hypotheses: first, clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2; and second, monophyly of the eukaryotic rhomboids (figure 3). The phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests (table 2). The hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly (p <0.05) worse than the best tree according to each of the tests (table 2). The concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and, in particular, for the polyphyly of eukaryotic rhomboids . The phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the' standard model' scenario, with the major split between the archaeo - eukaryotic and bacterial lineages nor the' mitochondrial' scenario, which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont . Neither can this tree be explained by postulating a small number of lineage - specific gene losses . The parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer (hgt) and lineage - specific gene loss events . In particular, in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms, phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor (luca). Instead, the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt, and then lost in some lineages . In particular, at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids, one of them yielding the rho subfamily and the other one the parl subfamily, with a possible additional hgt in plants (figures 2,3). Given the broad phyletic representation of both subfamilies of eukaryotic rhomboids, both the rho subfamily and the parl subfamily must have been acquired through hgt at an early stage of eukaryotic evolution, definitely before the divergence of the major crown - group lineages . This early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts . An alternative to this multiple - hgt scenario is that luca already had multiple, paralogous rhomboids, which evolved by a series of ancient gene duplications, and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs . Although this cannot be ruled out formally, this hypothesis implies the existence of an elaborate signaling system in luca and, accordingly, suggests that luca was a complex organism, which might have had as many genes as modern bacteria . Theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution . A complex luca with around 2,000 genes is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt . However, explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be' compatible with life', that is, it includes genes responsible for most, if not all, essential cellular functions . We currently believe that this is the most realistic, albeit inevitably imprecise, reconstruction of luca's gene set . With respect to the rhomboid family and other families whose phylogenetic trees show similar patterns, this makes the multiple - hgt interpretation the scenario of choice . Further theoretical, comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument . The multiple - hgt interpretation of the evolutionary history of the rhomboid family, while supported by the above argument, seems, at least at first glance, distinctly counter - intuitive, given that this family is nearly ubiquitous among extant life forms . Indeed, when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [31 - 33], there is no chance that such a widespread family is not assigned to luca . It should be realized, however, that these approaches are inherently probabilistic, and extensive hgt can fool them . For the rhomboid family, the multiple - hgt mode of evolution seems to be particularly plausible . It seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein, probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria . The protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs (figure 4). This would enable the transition from simple transport to the rip mode of controlled export of signaling molecules . Emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed, rather than eliminated, after hgt . In terms of the evolution of sequence itself, the requirements for the conservation of the protease activity apparently' locked' the rhomboid family in a regime of relatively slow evolution, which ensures significant sequence similarity between all family members (figure 1). The scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes, including intramembrane proteases involved in rip, such as presenilins and their homologs and the archaeo - eukaryotic signal peptide peptidase . The rhomboid family might be the most widespread and conserved group of integral membrane proteins . In and by itself, this would suggest that this family is part of the gene repertoire of luca . However, phylogenetic analysis suggests a different scenario, one of emergence in a bacterial lineage with subsequent multiple, independent hgt events and gene losses . Although caution is due in the evolutionary interpretation of phylogenetic trees for large families, particularly when membrane proteins with a relatively small number of conserved positions, such as the rhomboids, are involved, the multiple - hgt scenario seemed to be supported by several methods of tree analysis and statistical tests . Eukaryotes probably acquired their two major rhomboid subfamilies, rho and parl, as the result of two independent, early hgt events . These events, which might have introduced rip as a means of intercellular communication, could have been pivotal in the evolution of eukaryotic multicellularity along the lines discussed previously with regard to the apparent bacterial origin of key components of eukaryotic programmed cell death machinery . Subsequent evolution of rhomboids in eukaryotes proceeded by lineage - specific expansion of paralogs followed by diversification through the addition of an extra tmh in different positions relative to the catalytic core, some limited domain accretion (see figure 2) and sequence divergence . Phylogenetic analysis of the rhomboid family described here carries a general message for studies aimed at the reconstruction of ancestral life forms, particularly luca . Although most of the (nearly) ubiquitous protein families probably do derive from luca, explicit phylogenetic analysis is required to ascertain this in each case . The nonredundant (nr) protein sequence database at the national center for biotechnology information (nih, bethesda) was searched iteratively using the psi - blast program with multiple starting queries . Psi - blast was normally run with expectation (e) value of 0.01 as the cut - off for inclusion of sequences into the position - specific scoring matrix . Multiple alignments of protein sequences were constructed using the clustalw program and manually adjusted on the basis of the examination of psi - blast search outputs and the superposition of the predicted tmhs, which were identified using the programs tmpred and tmap . Phylogenetic trees were built using the least - squares method implemented in the fitch program of the phylip package, with subsequent local rearrangement using the protml program of the molphy package to obtain the maximum likelihood tree . The reliability of the tree topology was assessed using the rell (resampling of estimated log - likelihoods) bootstrap method of molphy, with 10,000 replications . Alternative placements of selected clades in maximum - likelihood trees were compared by using the rearrangement optimization method (kishino - hasegawa test) as implemented in the protml program [43 - 45]. Maximum parsimony trees were constructed using the heuristic search option of paup * . In addition, trees were constructed by bayesian inference using the markov chain monte carlo method as implemented in the mrbayes package . The complete alignment information, including columns with gaps, was used for the mrbayes analysis . Constraint trees were imported into paup * and subjected to neighbor - joining search to generate the phylogenies corresponding to alternative hypotheses . These phylogenies were compared using the kh, templeton (wilcoxon signed - ranks) and winning - sites (sign) tests implemented in paup*. L.p . Is supported by a grant from the natural sciences and engineering research council of canada . The alignment includes the majority of the detected rhomboid family proteins; some closely related sequences were omitted . Only the six conserved (predicted) transmembrane helices (tmh) and short surrounding regions are shown . The boundaries of the predicted tmh are indicated by gray shading and overline and they are numbered 1 - 6 . The number of amino - acid residues in the omitted terminal and internal regions are indicated . The consensus shows amino - acid residues present in at least 90% of the aligned sequences; h stands for hydrophobic residues (a, c, i, l, v, m, f, y, w in the single - letter amino - acid code) and s for small residues (g, a, s, d, n, v). The proposed catalytic serine (tmh4) and histidine (tmh6) as well as conserved residues in tmh2 with possible ancillary roles in catalysis are highlighted in color . The proteins are identified with the gene identification (gi) number from the nonredundant database and an abbreviated species name . Bacterial species are color - coded green, eukaryotic species blue and archaeal species yellow . Species name abbreviations: aerpe, aeropyrum pernix; agrtu, agrobacterium tumefaciens; anoga, anopheles gambiae; arath, arabidopsis thaliana; arcfu, archaeoglobus fulgidus; bacsu, bacillus subtilis; brume, brucella melitensis; caeel, caenorhabditis elegans; caucr, caulobacter crescentus; chlte, chlorobium tepidum; cloac, clostridium acetobutilicum; corgl, corynebacterium glutamicum; deira, deinococcus radiodurans; dicdi, dictyostelium discoideum; drome, drosophila melanogaster; escco, escherichia coli; haein, haemophilus influenzae; halsp, halobacterium sp . ; homsa, homo sapiens; lacla, lactococcus lactis; lisin, listeria innocua; metja, methanoccocus jannaschii; metka, methanopyrus kandleri; metma, methanosarcina mazei; meslo, mesorhizobium loti; mycle, mycobacterium leprae; myctu, mycobacterium tuberculosis; neucr, neurospora crassa; nossp, nostoc sp . ; prost, providencia stuartii; pyrab, pyrococcus abyssi; pyrae, pyrobaculum aerophilum; ralso, ralstonia solanaraceum; sacce, saccharomyces cerevisiae; schpo, schizosaccharomyces pombe; sinme, sinorhizobium meliloti; strco, streptomyces coelicolor; strpn, streptococcus pneumoniae; sulso, sulfolobus solfataricus; sulto, sulfolobus tokodaii; synsp, synechocystis sp . ; theac, thermoplasma acidophilum; thema, thermotoga maritima; thete, thermus thermophilus; vibch, vibrio cholerae; xanca, xanthomonas campestris; xylfa, xylella fastidiosa . The sequences and their regions used to construct the tree are exactly those shown in figure 1 . The color coding and abbreviations are as in figure 1 . The two major eukaryotic subfamilies are denoted as rho and parl (see text) and four clusters containing unexpected (from a phylogenetic viewpoint) sets of species are denoted 1 - 4 . The clades that were investigated in the kh test are denoted a through d. although the tree is shown in a pseudorooted form for convenience, this is an unrooted tree . Internal nodes with at least 70% rell bootstrap supported are denoted by black circles and nodes with a 50 - 70% support by blue circles . The posterior probabilities reported by the mrbayes program are indicated for some key internal branches . The rho and parl subfamilies are denoted; the remaining clusters include prokaryotic rhomboids designated as in figure 2 (with' a' added to the gi number). Within each cluster, the trees are unrooted, although shown in a pseudorooted form . A hypothetical scenario for the origin and dissemination of the rhomboid family proteases . The figure schematically shows the proposed three stages of evolution of the rhomboid family . In (a), the progenitor of the rhomboid family functions as a transporter for a regulatory peptide in some bacterial lineage . In (b), the catalytic site of the intramembrane protease evolves, allowing the switch to rip as the mechanism of the regulatory peptide release . In (c), the emergence of rip is followed by a burst of hgt . The transmembrane helices of rhomboid are designated as in figure 1; their topology in the membrane is based on that proposed in . The catalytic histidine and serine are shown and connected by a dotted line to indicate the proposed charge - relay system of the protease; possible ancillary catalytic residues are not shown . Log - likelihood analysis of possible placements of selected branches of maximum likelihood trees for the proteins analyzed * a - d, clades that were subjected to local rearrangements in the tree as indicated in figure 2 and discussed in the text . Bootstrap probability of the given tree calculated using the rell method (resampling of estimated log - likelihoods). Statistical comparisons of the best neighbor - joining tree with the hypothesis 1 and hypothesis 2 trees * probability of getting a more extreme test statistic under the null hypothesis of no difference between the two trees (two - tailed test).
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An infliction in the life expectancy figure of patients with sickle cell disease (scd) occurred around the middle of the 1970s (fig . This minimal increase in life expectancy coincided with my appointment at thomas jefferson university as the associate director of the newly created adult sickle cell center . The number of adults at that time was small and the transition from pediatrics to adult programs was at the age of 18 years . The trickle of patients increased gradually and we were faced with adolescent and young adult african american patients who were in a state of confusion . Stripped from the protective sphere of the pediatric world and the empathy of their pediatric hematologists and the pediatric ancillary staff, they were in a state of fear, anxiety, depression and, worst of all, severe pain . The fact that most patients were barely educated, many without a high school degree, unemployed, mediocre health coverage, and dysfunctional family structure conferred a logarithmic dimension to the problem . The arrow indicates the infliction point where life expectancy of patients with sickle cell disease began to increase . The steady stream of admissions of patients with acute painful vaso - occlusive crises (vocs) to the emergency department (ed) and hospital were not welcome by most providers, hospital administration, the house and nursing staffs . There was subtle resentment of the patients that sometimes extended to the hematologists who showed compassion to the patients . Soon labels such as drug addicts, drug - seeking behavior, and hospital hopping and frequent flyer emerged . Listening to and believing the patients and keeping detailed records of ed and hospital admissions and the analgesics prescribed, revealed that most patients genuinely do not respond to a certain analgesic or a certain dose . Increasing the doses of an analgesic or switching to another drug solved the problem in most patients . Accordingly and with the approval of the institutional review board (irb), i issued an identification wallet - sized, plasticized card that was carried by patients and presented to the provider treating their voc in the ed, hospital or any other medical facility . Information printed on both sides of the card included: 1) demographic data and a recent photograph; 2) hematological data including reticulocyte count; 3) medical data including the type of scd, its complications and co - morbidities if present; 4) all medications being taken by the patient and the recommended treatment of vocs including the name, dose, and the route of administration of the analgesics in question; and 5) my name and contact information for answering questions if needed . It was not expensive to issue these cards . A polaroid camera available at that time and a laminator were the only equipments needed to issue these cards . Later on, information on the card was computerized and a printed copy was given to the patient . The patients were very compliant in carrying it as faithfully as they carry their medical cards . Some providers liked it very much because it facilitated having a concise history about the patients . While this controversy was brewing, interesting developments in basic science were in progress to understand the pharmacodynamics and pharmacokinetics of opioids . In the 1970s, it was hypothesized that opioids have receptors to bind to and activate in order to relieve pain by blocking or minimizing the transmission of painful stimuli and raising the pain threshold . It did not take long after that to identify opioids as ligands that bind to stereospecific and saturable receptors in the central nervous system and other tissues [3, 4]. In addition, recent elegant studies [6 - 10] have revealed a helical structure of the opioid receptors, which forms pockets in which the corresponding opioid (ligand) fits snugly (fig . Recognition is highly specific, such that only l - isomers of certain opioids exert analgesic activity . Physiologically, by binding to receptors, opioids initiate a series of biochemical events including activation of g proteins, inhibition of adenylate cyclase, and extrusion of potassium ions, resulting in hyperpolarization of cell membranes [14 - 16]; this delays or prevents transmission of painful stimuli . Thus, the riddle why some patients respond to one opioid but not another had a pathophysiologic explanation . (a) morphine - like molecule (yellow) in the deep pocket (blue) of the -opioid receptor . (b) -opioid receptors from an intimate pair when crystallized with a ligand (yellow) such as morphine . Knowing how an opioid molecule (yellow) sticks in the pocket of its receptor (blue) parallel to the progress in the pharmacodynamics of opioids mentioned above, a concomitant advance in the pharmacokinetics of opioids was bubbling to the surface . Phase i involves the cyp enzymes and phase ii metabolism conjugates the drug to hydrophilic substances, such as glucuronic acid, sulfate, glycine, or glutathione . Morphine, hydromorphone and oxymorphone are metabolized by glucuronidation, whereas the majority of the other opioids are metabolized by the cytochrome p450 isoenzyme system . The net effect of an opioid depends on the availability of enzyme(s) to convert it into metabolites that could be active or inactive . Briefly, the cyp2d6 genotypes are categorized into phenotypes based on the activity of the variant enzymes . Ultrarapid metabolizers (ums) have greater than normal activity due to duplication or triplication, of active alleles [20 - 23], extensive metabolizers (ems) have normal enzyme activity, intermediate metabolizers (ims) have decreased enzyme activity, and poor metabolizers (pms) have absent or little enzyme activity . Patients who are ums of fentanyl would rapidly convert it into inactive metabolites with minimal or absent analgesic effect requiring increasing the dose of fentanyl . On the other hand, patients who are pms of fentanyl would experience prompt relief with relatively small doses of fentanyl but higher doses could be toxic due to the accumulation of unmetabolized fentanyl . The cyp3a4 enzyme metabolizes more than 50% of all drugs; consequently, opioids metabolized by this enzyme have a high risk of drug - drug interactions . Together, current data on the pharmacodynamics and pharmacokinetics of opioids show great variability of genotypes among patients and extreme variability in individual responses to opioids . Determining the pharmacogenetics profile of each patient facilitates the choice of drugs that would be efficacious for that patient and avoid those drugs associated with harmful drug - drug interaction . This approach in diagnostics and therapeutics ushers in the dawn of a new field for the management of individual patients based on their unique pharmacogenetics, phenotypic and biomarker characteristics . This future approach is referred to as personalized medicine or, more recently, precision medicine . We hope that this methodology would be approved and sponsored by the insurance companies for patients with scd . In the meantime, listening, believing and respecting the patient with sickle cell pain should be maintained for now as the approach to individualized therapy.
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The potential of rnai to silence any gene has made it an attractive therapeutic modality . However, the main obstacle to rnai in the clinic is delivery . To be effective, sirnas must be transported through the body, bind, and be taken up by target cells where they must traverse the plasma membrane and gain access to the cytosolic compartment, where the rnai machinery resides . To be useful, ease of formulation and administration, overall cost, and any associated toxicities are essential considerations . Dcs are heterogeneous, with subsets defined phenotypically, functionally and by location (reviewed in ref . 2). A delivery vehicle that knocks down expression of specific genes in a distinct dc population(s) would be a valuable tool for targeting diverse diseases including cancers, infectious diseases, autoimmunity and as a vaccine component . Therefore, a platform that delivers sirnas to specific dc subsets in situ would be useful for inhibiting or activating immune responses . Lipid nanoparticles (lnps) are one of the most advanced platforms for sirna delivery to hepatocytes, and are under clinical evaluation for conditions that require hepatic gene silencing . These lnps typically contain ionizable cationic lipids (pka ~6.5) that bind nucleic acids via electrostatic interactions at low ph, but are charge neutral at ph 7.4 . As a well - perfused organ furthermore, lnp uptake by hepatocytes is mediated by association with serum apoe leading to efficient uptake via low - density lipoprotein receptors in the liver . Following cellular uptake of the lnp, the ionization of the lipid within acidic endosomes is thought to promote sirna escape to the cytosol . Importantly, these lnps are associated with minimal toxicity, including little induction of proinflammatory cytokines following administration of physiologically relevant doses . In contrast to the liver, sirna delivery to extra - hepatic cells is challenging . In particular, immune cells such as dcs are relatively resistant to sirna uptake in vitro and in vivo . Although designed for hepatic gene silencing, the efficacy of these lnps and their derivatives has been assessed for rnai - mediated gene silencing in macrophages (mos) and dcs in vitro and in vivo . Lnp uptake and modest gene silencing was achieved at high doses of lnps (~ 5mg / kg). Together with a complementary study, this work demonstrates the feasibility of gene silencing in immune cells using lnp technology . Clearly, however, lnp formulations must be modified to achieve optimal gene silencing in immune cells . Enhanced uptake of lnps by primary hepatocytes is mediated by apolipoprotein e binding to the neutral lnps . This results in recognition by receptors including the low - density lipoprotein receptor and scavenger receptors largely expressed on hepatocytes . The mechanism of uptake strongly suggests that retargeting of these particles to other receptors or other tissues is possible . Indeed, akinc et al . Demonstrated that lnps modified with n - acetylgalactosamine (galnac) were retargeted to the asialoglycoprotein receptor (asgpr) expressed by hepatocytes in vivo . For delivery to nonhepatic cells, we have shown in vitro that anti - transferrin receptor aptamers can be used to redirect similar lnps . More recently, liang et al . Described a similar approach using aptamer - coated lnps to target osteoblasts in vivo . For uptake by immune cells, a recent study coated lnps with a full - length anti - cd4 antibody for sirna delivery to primary cd4 t cells . Lnp uptake and gene silencing was observed in cd4 t cells in various organs including the spleen, lymph nodes, and blood . These studies demonstrate the potential for attaching exogenous ligands to lnps for delivery to hepatic, and importantly nonhepatic primary cells in vivo . Drawing from these works, we reasoned that this lnp platform could be a useful foundation for the delivery of sirnas to dcs to modulate immune responses . To achieve this, we modified lnps using a single chain antibody (scfv) specific for the dc receptor dec205, a c - type lectin expressed at high levels on cd8 dcs . Dec205 dcs mediate cross - presentation of antigen resulting in modulation of cd8 t - cell responses . Therefore, inhibition of gene expression by dec205 dcs is a potentially powerful approach for regulating cd8 t - cell activation . Using this approach, we assessed the ability of dec - lnps (lnps coated with scfv specific for murine dec205, containing sirnas specific for costimulatory molecules) to inhibit immune responses . Injection of dec - lnps resulted in preferential uptake of the dec - lnps by splenic dec205 dcs . Furthermore, when coadministered with adjuvant, lnps containing sirna targeting cd40, cd80, and cd86 reduced expression of these costimulatory molecules to levels similar to those observed in immature dcs . Most importantly, dcs isolated from mice injected with a low dose (~0.6 mg / kg) of these dec - lnps, were able to suppress a robust mixed lymphocyte reaction (mlr), demonstrating the functional efficacy of this approach . Interestingly, when we performed experiments using 2' fluoro (2'f) modified sirna, a formulation reported to be nonimmunostimulatory and nonimmunogenic, significant immune activation of the targeted dcs was detected . This effect was not observed in assays performed with human peripheral blood mononuclear cells (pbmcs) or monocyte - derived dcs (modcs) and could be mitigated through selective 2-o - methyl (2ome) modification of the sirna . Overall, our study demonstrates the functionality of lnps modified for receptor targeting for sirna delivery to dcs . Additionally our results suggest that specific cell targeting can mediate immunological responses to sirna formulations . While care needs to be taken in designing and evaluating targeted approaches using lnps, we found that 2ome modification of the sirna was sufficient to negate immune activation . Our approach enhances the efficacy of sirna - mediated gene silencing by ~10-fold when compared to nontargeted delivery . Therefore, this targeted strategy should prove effective for regulating multiple immune - mediated diseases . Lnps containing sirnas specific either for cd80, cd86, cd40, or a control (nontarget) sequence were synthesized by extrusion using a mixture of 1,2-distearoyl - sn - glycero-3-phosphocholine (dspc):dlindma: dspe - peg: cholesterol at a ratio of 15:40:5:40 (based on refs . Murine anti - dec205 scfv with a c - terminal cysteine was attached to the lipid dspe - peg by a maleimide group (figure 1a, b). Following synthesis, we subjected lnps to quality control to ensure consistency between batches . Dynamic light scattering was used to determine diameter and polydispersity of lnps, and cryo - electron microscopy confirmed lnp size and their unilamellar structure (figure 1c, d). The efficiency of sirna incorporation and scfv binding to dspe - peg was also assessed (figure 1c, e). To show specificity of binding to dec205, scfv - lnps containing dy547-labeled sirna were cultured with either parental (dec205) or dec205 cho cells . As seen in figure 2a, at 4 c dec205 cells bind dec - lnps approximately fourfold better compared with nontargeted lnps (nt - lnps: lnps not coated with any scfv), or lnps coated with an isotype scfv (iso - lnps; cho - dec205 panel). When similar assays were performed at 37 c to allow cell uptake, cell staining increased by at least fivefold . Little uptake was observed when lnps (targeted or nontargeted) were incubated with parental cho cells (cho panel). Similar results were obtained when bone marrow - derived dcs (bmdcs), derived from wild - type mice (b6) were incubated with dec - lnps . We note that as dec205 is expressed at lower levels on bmdcs compared with cho - dec205, less dec - lnp binding and uptake was observed in bmdcs (~2-fold enhancement compared with nt- or iso - lnps; b6 bmdc panel). To further confirm target - specific uptake we performed experiments using bmdcs derived from dec205 mice, and no specific cell binding or uptake the dec205 antibody is internalized via receptor - mediated endocytosis, and is targeted to late endosomes and lysosomes . Confocal microscopy confirmed that similar to dec205 antibody, dec - lnps were targeted to lamp-1 late endosomes or lysosomes (figure 2b). Having demonstrated dec205-mediated uptake of dec - lnps in vitro, we investigated their localization in vivo . B6 mice were injected systemically (intravenous, i.v .) With fluorescently labeled scfv - lnps . As the dec205 receptor is endocytosed when it binds its ligand, we could not use this molecule to identify dec205 dcs . Therefore, we used cd8, a protein that is coexpressed on dec205 dcs, as a surrogate marker for tracking these cells . More than 50% of cd11ccd8 cells took up dec - lnps, and uptake was largely restricted to the cd8 dc population, with little uptake observed by cd8 dcs (figure 3a, b). Specificity was demonstrated by comparison of uptake of dec - lnps and iso - lnps . Significant uptake was only observed in dec205 dcs following dec - lnp injection, and other splenic immune cells took up little dec- or iso - lnps (figure 3a, c). When dec - lnps were injected into dec205 mice, no uptake was detected (figure 3d). To show that dec - lnps were competent for specific gene silencing, we generated dec - lnps containing a sirna specific for cd80 (having identified the most effective sirna sequence; see supplementary figure s1) and injected into b6 mice . Cd11cdec205 cells took up similar amounts of lnps following injection with dec - lnp containing either cd80- or control - sirna (lnp uptake panel, mean fluorescence intensity (mfi): dec - lnp - control = 443 versus dec - lnp - sicd80 = 464). In mice injected with dec - lnp encapsulating cd80-specific sirna, cd80 protein expression approached basal levels, and was reduced by ~2-fold when compared with dec - lnp containing control - sirna (figure 4a). A 75% reduction in cd80 mrna was observed in dec205 dcs that had taken up dec - lnps containing cd80-specific sirna (compared with dec - lnps containing control sirnas; figure 4b). We confirmed that gene knockdown was rnai - mediated using 5 race to detect sirna - directed mrna cleavage products (figure 4c). Sequencing of the polymerase chain reaction (pcr) fragment verified that it was derived from cd80 mrna and that the cut site corresponded with nucleotide positions 1011 of the sirna guide strand (not shown). Interestingly, while the data in figure 4 demonstrate the ability of our dec - lnps to effectively knockdown cd80 expression in dcs in vivo, we observed significant dc activation following injection of dec - lnps containing control sirnas (figure 4a, right panel). This observation was surprising considering these experiments were performed using sirnas containing 2f modified rna, previously reported to have reduced immunostimulatory activity . To better assess this effect, we synthesized a series of luciferase - specific control sirnas (luc) containing 2oh rna (unmodified), 2f pyrimidines or selected 2ome modifications (see methods for modification strategy; based on refs . Lnps were generated and their ability to stimulate immune responses using multiple assays was examined . First, b6 mice were injected with dec - lnps that contained either unmodified, 2f or 2ome modified luc - specific sirnas in the absence of any adjuvant . One day later, splenic dcs were analyzed for the expression of costimulatory receptors (supplementary figure s2a). As expected, dcs isolated from mice that had received unmodified sirnas demonstrated significant upregulation of cd40, cd80, and cd86 . Consistent with our previous results (figure 4a), animals treated with dec - lnps containing 2f modified sirnas also upregulated these costimulatory molecules . Importantly, and similar to work by other groups, we found that 2ome modified sirnas induced minimal immune activation . To attempt to circumvent the need for a time and resource intensive in vivo assay, we wanted to determine whether an in vitro assay could be used to detect immunostimulatory sirnas . However, when we cultured b6-derived bmdcs with dec - lnps (containing cd80-specific sirnas) no difference between 2oh modified sirna, 2f or 2ome modified sirna was detected (supplementary figure s2b). We also used nt - lnps to assess the effects of various sirna formulations on human pbmcs and modcs using standard preclinical assays (supplementary figure s2c). Pbmcs or modcs were cultured for 24 hours at which time cells were assessed for induction of apoptosis and culture supernatants were tested for the presence of proinflammatory cytokines (see methods). Apoptosis was not observed under any conditions tested (data not shown). As expected, pbmcs cultured with the highest concentration of lnps containing unmodified sirnas elicited the secretion of several cytokines . 2f and 2ome luc sirnas induced production of one cytokine (il8), also at the highest concentration tested . No cytokine production was observed following culture of modcs with lnps encapsulating unmodified, 2f or 2ome sirnas . Taken together, these experiments suggest that the assays tested show marked differences in sensitivity . Surprisingly, bmdcs and modcs were the least receptive for detecting immunostimulatory sirnas . Conversely, dec - lnp injection was useful for the identification of sirnas with immune - activating potential . From these results, we used sirnas containing selective 2ome substitutions for all subsequent studies . To target autoimmune diseases, reducing the expression of several costimulatory molecules therefore, we next determined the ability of dec - lnps that encapsulated 2ome modified cd86 specific sirnas, to reduce gene expression following i.v . Administration (optimal sirna identified as previously; supplementary figure s1). As the sirnas were 2ome modified, mice were coinjected with lps to stimulate expression of costimulatory molecules . Analysis of splenic dec205 dcs 1 day following dec - lnp injection showed reduction of cd86 protein to near steady - state levels (figure 5a). Total rna was isolated from dec205 dcs that had taken up dec - lnps encapsulating either cd86 or control sirnas . An approximately 70% reduction in cd86 mrna levels was observed from dcs isolated from mice that received dec - lnps containing cd86 sirna in comparison to dcs derived from control sirna treated mice . To verify knockdown occurred via the rnai pathway, we next tested whether combining sirnas in dec - lnps would induce gene silencing equivalent to that observed with single sirnas . A combination of 2ome modified sirnas targeting cd80, cd86 and cd40 were incorporated into dec - lnps and injected into b6 mice along with adjuvant (lps) to activate the dcs . Reduction of each of the targeted costimulatory molecules was observed, with cd80 and cd86 knocked down to basal levels (figure 5c). Gene silencing was similar to that achieved following treatment with dec - lnps containing single sirnas (e.g., ~50% for cd86: compare cd86 dec - lnp, figure 5a with mix dec - lnp, figure 5c right panel). To determine functional relevance of this knockdown, we used the robust mlr assay . B6 mice were injected with dec - lnps encapsulated with either a mix of cd80, cd86, and cd40 or control sirnas . After 24 hours, splenic cd8 dcs that had taken up dec - lnps were isolated, irradiated, and cultured with splenic t cells derived from a balb / c mouse . Maximal proliferation was observed when dcs were isolated from mice injected with lps only, or lps plus dec - lnps containing control sirnas . In contrast, proliferation of t cells cultured with dcs derived from mice injected with lps plus dec - lnps containing cd80, cd86, and cd40 sirnas was significantly reduced (figure 5d). Currently, the most advanced lnp platforms use the ionizable cationic lipid dlindma, or its dlin - kc2-dma and dlin - mc3-dma derivatives . Following systemic administration, these lnps accumulate in first pass organs, i.e., liver and spleen, making them attractive vehicles for hepatic gene knockdown . Furthermore, these lnps have been shown to safely and effectively reduce expression of hepatocyte genes in clinical trials . However, there is only very limited data demonstrating targeted delivery of potentially clinically relevant lnps to nonhepatic cells . We have shown that conjugation of a scfv, specific for the dc receptor dec205, is sufficient to direct delivery of dlindma - formulated dec - lnps to splenic dcs . Similar to conventional nt - lnps, dec - lnps incorporated sirnas with high efficiency (> 80%), and were of uniform diameter (~100 nmol / l). We found that coating lnps with only ~50 scfv was sufficient for dc delivery (figures 1, 2, and 3). We showed that dec - lnp uptake correlated well with dec205 receptor density: dcs> b cells, t cells and macrophages (dec205 expression is 1050-fold less than on dcs). Use of dec205 mice further confirmed that uptake occurred via the dec205 receptor (figures 2a and 3d). We also showed that the intracellular pathway accessed by dec - lnps was consistent with the dec205 pathway (figure 2b). Importantly, we demonstrated that targeted delivery resulted in effective rnai - mediated gene silencing of one, or several genes, to essentially basal expression levels (figures 4 and 5a c). This reduction in gene expression was sufficient to inhibit a robust mlr (figure 5d). Coated lnps formulated with the cationic lipid ddab with full - length dec205 antibody for sirna delivery and demonstrated the ability to reduce expression of the costimulatory molecule cd40 in dec205 dcs . While some gene knockdown was achieved, this lipid is known for its adjuvant qualities, and is being developed for applications that require immune response stimulation . The lnps also proved relatively inefficient at sirna loading (10% compared with 85% for dlindma lnps). The sirnas used were also unmodified, which could serve as another possible source of immune stimulation . Therefore, the potential for this formulation appears to be limited . More recently, ramishetti et al . Utilized a full - length antibody targeting cd4 to enhance t - cell uptake and induce sirna - mediated gene silencing using a clinically relevant lnp formulation similar to the one we employed in our work . Interestingly, both this study and the work from zheng et al . Chose to utilize full - length antibodies for lnp targeting, which may limit their utility when considering the immunogenicity of whole antibodies and their rapid clearance from the circulation by fc - mediated uptake by macrophages . In fact, in preliminary studies, we coated lnps with full - length dec205 antibody and failed to observe uptake by dec205 dcs . Using our dec - lnps mg / kg) comparable to that study which used n - acetylgalactosamine (galnac)-coated lnps to target hepatocytes via the asialoglycoprotein receptor (asgpr) in apoe knockout mice . In contrast, when the ability of nt - lnps to silence antigen presenting cells (apcs), including dcs, was investigated a dose of 5 mg / kg was required to achieve partial silencing: ~10-fold higher when compared with our dec - lnps . Furthermore, the authors used the dlindma derivative, dlin - kc2-dma, which displays improved silencing ability in hepatocytes (relative ed50 dlin - kc2-dma: 0.1 mg / kg versus dlindma: 1 mg / kg). As these lnps were formulated for effective gene silencing in hepatocytes, biodistribution studies show that nt - lnps are found in the spleen, although at ~50-fold less than in the liver . While targeting ligands have been shown to have little effect on the overall biodistribution of nanoparticle formulations, they can enhance cell - specific uptake . Therefore, while high doses of nt - lnps can confer partial gene silencing in apcs, at low lnp concentrations, we find that a targeted approach is required to achieve knockdown in a specific dc subset . Injection of low - dose nt - lnps failed to result in detectable uptake / gene silencing . As the rnai pathway is resident in the cytoplasm, the intracellular pathway used following receptor ligation is an important consideration . We chose the well - characterized dec205 receptor for targeting our lnps . Following binding, the internalized receptor is routed to late endosomes and associated cargo gains access to the cytoplasm . The dlindma lipid fuses with anionic phospholipids present in the endosomal membrane resulting in release of encapsulated cargo into the cytoplasm . However, it is likely that different routes of uptake will affect the efficiency of delivery and subsequent gene silencing . Thus, a logical extension to our approach is to determine whether we observe improved gene silencing using other dc specific targeting agents and/or more effective dlindma derivatives . We are currently investigating the efficacy of dec - lnps formulated with the dlin - mc3-dma (ed50 0.03 mg / kg). Having demonstrated that we could efficiently target sirnas to dcs, the lack of toxicity of these lnps and their cargo had to be established . We observed significant dc activation following injection of targeted lnps containing unmodified and 2f modified sirnas, and incorporation of 2ome modifications at distinct residues was required to maintain costimulatory molecules analyzed at basal levels (figure 4a and supplementary figure s2a). As injecting mice to determine immune stimulation is time and resource intensive, we assessed immune stimulation in murine and human tissue culture systems . Murine bmdcs were refractory to lnp stimulation suggesting they would not serve as a useful surrogate for gauging immunogenicity of lnps . Taken together, these assays demonstrate a requirement for in vivo analysis to uncover the stimulatory capacity of the lnps . Recently, a human whole blood cytokine assay has been described that showed similar activation profiles elicited by lnps when compared with in vivo injection, potentially circumventing a requirement for cumbersome in vivo analyses . For the purposes of inhibiting a multifactorial immune response, such as that observed in graft rejection or autoimmunity, the simultaneous knockdown of several (co)stimulatory genes this approach has been used to demonstrate that combining antibodies targeting cd80 and cd86 can reduce disease severity in experimental autoimmune encephalomyelitis and in antigen - induced arthritis . Transfer of dcs treated ex vivo with cd40, cd80, and cd86 antisense oligonucleotides (as - odn) into nod mice delayed type 1 diabetes (t1d) onset . A follow - up study demonstrated that multiple injections of microspheres containing cd40, cd80, and cd86 as - odns were sufficient to prevent t1d, possibly due to reduced expression of the costimulatory molecules by splenic dcs . We achieve significant reduction in expression of cd40, cd80, and cd86 on splenic dcs using our dec - lnps following systemic delivery, and ongoing studies are addressing the durability of gene knockdown and the potential use of these dec - lnps for treating autoimmune diseases . In summary, we have shown that a clinically relevant lnp can be modified for cell - specific delivery of sirnas . In vivo administration did not produce overt inflammatory responses, and we achieved significant rnai - mediated gene - specific knockdown at low sirna doses . These attributes make this an attractive platform to warrant further investigation as a potential therapeutic agent for various autoimmune diseases . Furthermore, judicious selection of the ligand coating the lnp opens up the potential for targeting multiple cell types . Mice . C57bl/6 (b6) and balb / c mice were purchased from taconic (hudson, ny). Dec205 mice were bred to homozygosity on the b6 background (dec205), and validated by pcr and flow cytometry . Mice were housed in the barrier facility at the institute for animal studies, albert einstein college of medicine (bronx, ny) according to institutional animal care and use committee (iacuc) guidelines . The single - chain fragment variable (scfv) antibodies constructed for dec205 (clone nldc145) and isotype (clone gl117) were cloned from antibody constructs, as previously described . The variable regions of the heavy and light chain sequences were fused with a flexible linker (g4s)6 separating them and cloned into a pet28a (emd millipore, billerica, ma) bacterial expression vector . Further modifications to the constructs included a strep - tag - ii (wshpqfek) at the n - terminus and a 10-residue histidine tag (h10) with a short flexible linker (g4s) near the c - terminus . A c - terminal unpaired cysteine residue was introduced for scfv conjugation to the lipid dspe - peg - mal . The scfv constructs were transformed into bl21 (de3) plyss competent cells (promega, fitchburg, wi). The scfv dec205 or isotype were expressed in escherichia coli strain bl21 (de3) plyss as insoluble inclusion bodies . Following bacterial growth in luria broth (lb) medium and induction of protein expression with isopropyl 1-thio - d - galactopyranoside cells were lysed, and insoluble protein pelleted by centrifugation . Solubilized inclusion bodies were passed over a ni - nta column and the bound protein was eluted with 8 m guanidine hydrochloride . The purified proteins were refolded by rapid dilution in refolding buffer, and purified by size - exclusion chromatography using superdex 200 (ge healthcare life sciences, pittsburgh, pa). Proteins were buffer - exchanged in phosphate - buffered saline (pbs) and concentrated . Scfvs were tested for endotoxin contamination using a kinetic chromogenic limulous amoebocyte lysate (lal) assay (kinetic - qcl lal assay, lonza, walkersville, md). The free cysteine engineered onto the n - terminus of the scfv was reduced using tris-(2-carboxyethyl) phosphine hydrochloride (life technologies, carlsbad, ca). Following buffer exchange into pbs, the reduced scfv was reacted with a 10-fold molar excess of alexafluor488 (af488)-c5-maleimide according to manufacturer instructions (life technologies, carlsbad, ca). Polyacrylamide gel electrophoresis (sds - page) and coomassie staining and storm molecular phosphorimager scanning of the stained gel (ge healthcare, piscataway, nj). Preparation of lnps . Lnps were formed using the following lipids at a 15:40:5:40 mol%: dspc (avanti polar lipids, alabaster, al), cholesterol (chol; sigma - aldrich, mo), 1,2-distearoyl - sn - glycero-3-phosphatidylethanolamine - n-(maleimide-(polyethylene glycol)-2000) (dspe - peg - mal, avanti polar lipids, alabaster, al), and 1,2-dilinoleyloxy-3-dimethylaminopropane (dlindma) synthesized by the chemical biology core facility at albert einstein college of medicine as previously described . A fluorophore conjugated to a lipid, 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(lissamine rhodamine b sulfonyl) (dope - rhodamine b; avanti polar lipids, alabaster, al) was incorporated (0.1%) to monitor lnp delivery . Lnps were formed using a modified version of the spontaneous vesicle formation by ethanol dilution method . The lipid mixture was extruded (mini - extruder and 1 ml gas - tight syringes, avanti polar lipids, alabaster, al) sequentially with polycarbonate 200 and 80 nm pore size membranes . The extruded lipid mixture was mixed via rapid pipetting and vortexing with sirna, followed by dialysis into pbs . For attachment of scfv, lnps were mixed with reduced scfv at room temperature for 1 hour, and free maleimide groups were then quenched with -mercaptoethanol (me). Tangential flow filtration (microkros filter module) with a 500 kda mwco (spectrum laboratories, rancho dominguez, ca) was used for buffer exchange into pbs and concentration of lnps . Lnps were assayed for size and polydispersity by dynamic light scattering (dynapro plate reader, wyatt technology, santa barbara, ca). Cryo - electron microscopy confirmed lnp size, geometry and lamellarity (analytical imaging facility; albert einstein college of medicine, bronx, ny). Sirna incorporation was determined by quant - it ribogreen rna assay (life technologies, carlsbad, ca). To determine scfv conjugation efficiency, lnps were resolved on sds - page gels . A 3 kda difference in migration of the scfv was indicative of binding to dspe - peg - mal (unconjugated scfv ~30 kda, scfv - dspe - peg - mal ~33 kda). A cholesterol quantification kit (abcam, cambridge, ma) was use to determine the total lipid concentration . Based on lipid concentration, the surface area per lipid head group (~0.6 nm), lnp diameter and their unilamellar structure, ~5060 scfvs are conjugated to each lnp under the conditions outlined above . Sirna synthesis . Modified and unmodified individual sirna sequences were synthesized on an expedite 8909 dna synthesizer (biolytic, fremont, ca) and purified by standard protocols, as described . Dylight 547-phosphoramidite was attached to the 5 end of the sense strand for synthesis of fluorescently labeled sirna (dy547-sirna; glen research, sterling, va). Sirnas were modified by 2f substitutions at every pyrimidine or by 2ome substitutions at the bolded nucleotides: cd40 sense 5-gaagauuaucccggucauauu-3; cd40 anti - sense 5-uaugaccg; ggauaaucuucuu-3; cd80 sense 5-gaauuaccuggcaucaauauu-3; cd80 anti - sense 5-uauugaugccagguaauucuu-3; cd86 sense 5-caacuggacucuacg acuuuu-3; cd86 anti - sense 5-aagucguagaguccaguuguu-3; control sense 5-uagcgacuaaacacaucaauu-3; control anti - sense 5-uugauguguuuaguc gcuauu-3. Luciferase sense 5-gauuauguccgguuauguauu-3; luciferase anti - sense 5-uacauaaccggacauaaucuu-3. Lnp binding, uptake, and intracellular localization . Cho cells, cho cells that stably express dec205 (cho - dec), a dec205 b lymphoma line (a20 cells) and bmdcs, derived from b6 and dec205 mice, were used to assess lnp binding and uptake . Cells were grown in complete medium (dulbecco's modified eagle's medium, 5% fbs, 10 mmol / l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 20 mmol / l l - glutamine). Bmdcs were generated from bone marrow derived from the femur and tibia as previously described . Adherent cells (a20, cho) were harvested using ethylenediaminetetraacetic acid to preserve receptor expression and were incubated at 4 c, 30 minutes with 500 nmol / l scfv - lnps (containing either 0.1% dope - rhodamine b or cy3-labeled sirnas). For uptake, cells were washed with fluorescence - activated cell sorting buffer (pbs containing 0.5% bsa, 0.1% na n3, 2 mmol / l edta) and analyzed by flow cytometry (lsrii, becton dickinson, franklin lakes, nj; flowjo, ashland, or). To monitor intracellular localization, a20 cells were incubated with scfv - lnps, containing sirnas labeled with dylight 547 (ge dharmacon, lafayette, co), 1 hour, 37 c . Following one wash with pbs, cells were pipetted onto poly - l - lysine coated coverslips and allowed to adhere . Adhered cells were fixed with 4% paraformaldehyde, washed with pbs and stained with anti - lamp1 (clone 1d4b; ebioscience, san diego, ca) and anti - eea1 (clone c45b10; cell signaling technology, danvers, ma). Stained cells were mounted with vectashield mounting media containing 4,6-diamidino-2-phenylindole (dapi) (vector laboratories, burlingame, ca) and samples were visualized on a leica sp5 aobs confocal microscope (leica, buffalo grove, il). Image processing (linear adjustments to brightness and contrast) was performed on image files using nih imagej software (bethesda, md). Lnps (0.61 mg / kg) were injected (retroorbital (r.o .)) Into b6 mice and 25 ng ultrapure lps (invivogen, san diego, ca) was injected r.o . One day later, spleens were harvested, collagenase treated to maximize dc yield, and single cell suspensions subjected to red cells lysis . Cells were stained with cell surface markers and analyzed by flow cytometry to determine lnp uptake and expression of costimulatory molecules . Antibodies were used for detection of the following cell surface markers: cd11c, cd8, dec205, cd40, cd80, cd86, cd19, b220, cd3, tcr, f4/80, cd11b, h2k, dcir2, cd49b, cd45.1 . Cell viability was monitored using a live / dead exclusion dye, i.e., live / dead blue (life technologies, carlsbad, ca). Cells were acquired using an lsrii cytometer and data analyzed using flowjo software . To measure gene knockdown, cd11ccd8 splenocytes that had taken up lnps (i.e., sirna - dy547) were sorted by flow cytometry (facs aria iii, becton dickinson, franklin lakes, nj) into rnaprotect (qiagen, valencia, ca) to stabilize rna . Total rna was isolated using the rneasy rna isolation kit (qiagen, valencia, ca), reverse transcribed using superscript iii (life technologies, carlsbad, ca) and random hexamers (idt, coralville, ia), according to manufacturer's instructions . Quantitative pcr was performed using platinum taq polymerase (life technologies, carlsbad, ca) and sybr green to detect amplified products on an iq5 icycler (biorad, hercules, ca). Reactions were performed in triplicate with the following primers: u6 forward: 5-ctcgcttcggcagcacatatacta-3; u6 reverse: 5-acgaatttgcgtgtcatccttgcg-3; cd40 forward: 5-cggctgtgcgcgctatg-3; cd40 reverse: 5-ggctgagaattcgcctgagtc-3; cd80 forward: 5-ctactctcttatcatcctgggcct-3; cd80 reverse: 5-cccggaagcaaagcaggtaatcct-3; cd86 forward: 5-gccacccacaggatcaattatcct-3; cd86 reverse: 5-aaagagagaggctgttggagatac-3. Relative amounts of mrna were normalized to u6 mrna and primer specificity verified by melt curve analysis . To detect rnai - mediated cleavage products, total rna was isolated from cd11ccd8 splenocytes that had taken up lnps, i.e., sirna - dy547 (as for gene knockdown by qpcr), ligated at the 5 end to an adapter rna oligonucleotide and first strand cdna was synthesized using a gene - specific primer (gsp). Pcr amplification using an adapter - specific primer and a nested gsp primer will amplify the cleavage product and identify the cut site which corresponds to position 10/11 of the sirna guide strand if mediated via rnai . The primers and oligonucleotide sequences used: adapter rna: 5-cgacuggagcacgaggacacugacauggacugaaggaguagaaa-3; gsp forward: 5-cgactggagcacgaggacactga-3; cd80 gsp reverse: 5-tgcgccgaatcctgccccaa-3; cd80 reverse nested: 5-atcaggagggtcttctgggg-3; cd86 gsp reverse: 5-agtaactgaagctgtaatctccttccaata-3; cd86 reverse nested: 5-ctgtgacattatcttgtgatatctgcatgt-3. Analysis of immune stimulation . Cd11c murine bmdcs (day 6; macs purified, miltenyi biotec, san diego, ca) were cultured with dec - lnps and monitored for activation (cd40, cd80, cd86) 1 day later by flow cytometry . In vivo stimulation was assessed by injection of dec - lnps r.o . Into b6 mice, followed by analysis of activation markers (cd40, cd80, cd86) on splenic dcs after 24 hours by flow cytometry . The pbmc fraction was used directly or following purification of cd14 cells (stemcell technologies, vancouver, british columbia, canada), monocytes were cultured for 5 days in the presence of granulocyte - macrophage colony - stimulating factor and il4 to generate modcs . Nt - lnps (5, 1, 0.2, 0.04, 0.008 mol / l) were cultured with pbmcs or modcs overnight and cell culture supernatants collected for cytokine analysis (ifn, il1, il2, il4, il5, il6, il7, il8, il10, il12, il13, tnf, granulocyte - macrophage colony - stimulating factor) using a luminex multiplex format (life technologies carlsbad, ca). Cd11ccd8 splenic dcs were isolated by flow cytometry from b6 mice injected the previous day with dec - lnps containing either a 1:1:1 mix of cd40, cd80, and cd86 sirnas or control sirnas . Splenic t cells from balb / c mice were isolated by macs using a pan t cell isolation kit ii (miltenyi, san diego, ca). Macs sorted balb / c t cells were carboxyfluorescein succinimidyl ester labeled according to published protocols, and carboxyfluorescein succinimidyl ester - labeled t cells were cocultured with the irradiated b6 dcs . Three days later, dilution of carboxyfluorescein succinimidyl ester was monitored by flow cytometry to determine t - cell activation.
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Sevoflurane does not sensitise the myocardium to arrhythmogenic effects of catecholamines and has the least effect on cardiac conduction system . Despite all the research demonstrating its safety, adverse cardiac arrhythmias such as nodal rhythm with bradycardia, prolongation of qtc interval, precipitation of torsade de pointes and isorhythmic atrioventricular dissociation (isravd) have been reported with sevoflurane anaesthesia . A 60-year - old woman weighing 60 kg with carcinoma in the right breast was scheduled for elective modified radical mastectomy . She was a known hypertensive since 4 years, on treatment with oral metoprolol 50 mg twice daily and amlodipine 10 mg once daily, with no other comorbidities . Preoperative pulse rate (pr) was 60/min and blood pressure (bp) was 140/70 mmhg . Preoperative 12 lead electrocardiograms (ecg) showed sinus rhythm with a rate of 60/min . Diazepam 10 mg was administered orally the night before and 60 minutes before surgery, along with morning dose of metoprolol and amlodipine . Anaesthesia was induced with 8% sevoflurane in 100% oxygen after administering 1.5 mg morphine intravenously . During induction, sevoflurane concentration was reduced to 5% and satisfactory proseal laryngeal mask airway insertion (size 3) was accomplished with suxamethonium 100 mg . Capnograph showed a square wave form . Immediately following induction, ecg trace in lead ii showed dissociated upright p waves which remained within qrs for several beats, then reappeared and recede from qrs complex . Ecg also showed intermittent electrical alternans [figure 1] and bp dropped to 86/50 mmhg . Meanwhile, anaesthesia was maintained with sevoflurane 2% and 50% nitrous oxide in 50% oxygen . End - tidal carbon dioxide (etco2) was maintained between 35 and 37 mmhg . Pr (40 - 50 beats / min) and rhythm (isravd) remained unaltered throughout the procedure that lasted for 3 hours . Atropine 0.6 mg and mephenteramine 3 mg were administered intravenously when the pr dropped below 40/min and systolic bp below 90 mmhg . A discrepancy was observed between the hr recorded by the ecg and pr recorded by the pulse oximeter . Hr was 50 to 60 beats / min but pr was 38 to 40 beats / min . At the end of surgery, sevoflurane was discontinued and neuromuscular blockade was reversed with a mixture of atropine (1.2 mg) and neostigmine (2.5 mg). Few minutes after removal of the lma, rhythm reverted to sinus with pr of 50/min and bp was 118/79 mmhg . Metoprolol and amlodipine were discontinued and enalapril 5 mg once daily was started by the cardiologist postoperatively . Patient was observed in intensive care unit for the next 48 hours . Throughout the postoperative course, hr and rhythm remained normal . Real - time picture from the monitor shows electrical alternans with absent p waves in ecg trace . The difference in heart rate (ecg trace) and pulse rate (pulse oximetry trace) is seen due to electrical alternans . Real - time picture from the monitor shows the inspired and expired concentration of sevoflurane a 5-year - old boy weighing 15 kg was scheduled for left inguinal hernia repair . Premedication administered was 7 mg of oral midazolam, 30 minutes before surgery . In the operating room, baseline pr was 88/min with sinus rhythm; spo2, 99% on room air and nibp, 96/62 mmhg . Inhalation induction of anaesthesia was performed with sevoflurane in 100% oxygen (fresh gas flow 5 l / min) through t piece circuit with child breathing spontaneously at a rate of 20 to 24/min . Inspired concentration of sevoflurane was increased in 2% increments every minute up to 8% concentration . The etco2 was between 31 and 33 mmhg, as measured through the angle piece interfaced between the face mask and the breathing circuit . After breathing 8% sevoflurane for 2 minutes sudden drop in hr to 56/min was noticed and the ecg revealed the presence of junctional rhythm with absent p waves and regular narrow qrs complexes . At that moment, spo2 was 99% and bp was 66/36 mmhg . Intravenous fentanyl (30 g) was administered and anaesthesia was maintained with 1.5% isoflurane in a mixture of 50% nitrous oxide in oxygen (fresh gas flow rate 5 l / min). A 60-year - old woman weighing 60 kg with carcinoma in the right breast was scheduled for elective modified radical mastectomy . She was a known hypertensive since 4 years, on treatment with oral metoprolol 50 mg twice daily and amlodipine 10 mg once daily, with no other comorbidities . Preoperative pulse rate (pr) was 60/min and blood pressure (bp) was 140/70 mmhg . Preoperative 12 lead electrocardiograms (ecg) showed sinus rhythm with a rate of 60/min . Diazepam 10 mg was administered orally the night before and 60 minutes before surgery, along with morning dose of metoprolol and amlodipine . Anaesthesia was induced with 8% sevoflurane in 100% oxygen after administering 1.5 mg morphine intravenously . During induction, sevoflurane concentration was reduced to 5% and satisfactory proseal laryngeal mask airway insertion (size 3) was accomplished with suxamethonium 100 mg . Capnograph showed a square wave form . Immediately following induction, ecg trace in lead ii showed dissociated upright p waves which remained within qrs for several beats, then reappeared and recede from qrs complex . Ecg also showed intermittent electrical alternans [figure 1] and bp dropped to 86/50 mmhg . Meanwhile, anaesthesia was maintained with sevoflurane 2% and 50% nitrous oxide in 50% oxygen . End - tidal carbon dioxide (etco2) was maintained between 35 and 37 mmhg . Pr (40 - 50 beats / min) and rhythm (isravd) remained unaltered throughout the procedure that lasted for 3 hours . Atropine 0.6 mg and mephenteramine 3 mg were administered intravenously when the pr dropped below 40/min and systolic bp below 90 mmhg . A discrepancy was observed between the hr recorded by the ecg and pr recorded by the pulse oximeter . Hr was 50 to 60 beats / min but pr was 38 to 40 beats / min . At the end of surgery, sevoflurane was discontinued and neuromuscular blockade was reversed with a mixture of atropine (1.2 mg) and neostigmine (2.5 mg). Few minutes after removal of the lma, rhythm reverted to sinus with pr of 50/min and bp was 118/79 mmhg . Metoprolol and amlodipine were discontinued and enalapril 5 mg once daily was started by the cardiologist postoperatively . Patient was observed in intensive care unit for the next 48 hours . Throughout the postoperative course, hr and rhythm remained normal . Real - time picture from the monitor shows electrical alternans with absent p waves in ecg trace . The difference in heart rate (ecg trace) and pulse rate (pulse oximetry trace) is seen due to electrical alternans . A 5-year - old boy weighing 15 kg was scheduled for left inguinal hernia repair . Premedication administered was 7 mg of oral midazolam, 30 minutes before surgery . In the operating room, baseline pr was 88/min with sinus rhythm; spo2, 99% on room air and nibp, 96/62 mmhg . Inhalation induction of anaesthesia was performed with sevoflurane in 100% oxygen (fresh gas flow 5 l / min) through t piece circuit with child breathing spontaneously at a rate of 20 to 24/min . Inspired concentration of sevoflurane was increased in 2% increments every minute up to 8% concentration . The etco2 was between 31 and 33 mmhg, as measured through the angle piece interfaced between the face mask and the breathing circuit . After breathing 8% sevoflurane for 2 minutes, the child did not show any motor response to jaw thrust . Sudden drop in hr to 56/min was noticed and the ecg revealed the presence of junctional rhythm with absent p waves and regular narrow qrs complexes . At that moment, spo2 was 99% and bp was 66/36 mmhg . Intravenous fentanyl (30 g) was administered and anaesthesia was maintained with 1.5% isoflurane in a mixture of 50% nitrous oxide in oxygen (fresh gas flow rate 5 l / min). American college of cardiology / american heart association recommends using volatile agents during non - cardiac surgery for the maintenance of general anaesthesia in haemodynamically stable patients at risk for myocardial ischemia . Anticipating a difficult airway and considering the patient's cardiac status, and associated hypertension, isravd was recorded during induction of anaesthesia with sevoflurane which reverted back to sinus rhythm after discontinuation of sevoflurane during emergence . 2 pacemakers (sinus and av nodes) fortuitously discharge at the same or nearly similar rate, without antegrade or retrograde conduction across the av node . Either slowing of the sinus node discharge rate or the emergence of a slightly faster subsidiary pacemaker controlling the ventricles is the common initiating event . P waves approaches the narrow qrs complex, disappears within it for several beats, then reappears and recedes from the qrs and this sequence may be repeated . The same was observed in the ecg recording in the present case [figure 1]. From the limited number of experimental studies, the development of isravd and the particular electrocardiographic patterns resulting from there are dependent upon relative discharge rate of the dominant atrial and subsidiary pacemakers, presence or absence of retrograde conduction, chronotropic response to atrial stretch, baroreceptor - mediated autonomic nervous system output and responsiveness of the sinus node . High incidence of nodal rhythm (20%) and bradycardia with use of sevoflurane has been reported in unpremedicated infants . The onset of nodal rhythm was significantly earlier in the high - concentration technique than with incremental induction technique . On the other hand, nakaigawa et al . Demonstrated that up to 2 mac (minimal alveolar concentration) sevoflurane does not affect cardiac conduction system significantly . In both our patients, 8% sevoflurane was used for induction that possibly resulted in isravd and junctional rhythm . Isravd was possibly precipitated by the volatile agent affecting ca release from the sarcoplasmic reticulum, resulting in depression of ca slow inward current in the myocardium . In patients on ca channel and -adrenergic blocker therapy, the arrhythmogenic potential of sevoflurane is likely to be enhanced as ca channel blockers inhibit the entry of ca into the cell or its mobilisation from intracellular stores, while b - adrenergic blockers depresses sinus rate and av node conduction . Reported a case of intraoperative torsades de pointes ventricular tachycardia and ventricular fibrillation during sevoflurane anaesthesia, which persisted with dc shock but reverted to sinus rhythm after 10 minutes of discontinuation of sevoflurane and after the patient was awakened . A discrepancy was observed between the hr and the pr due to intermittent electrical alternans in the ecg . It is therefore wise to rely on the pr rather than the hr in such cases to treat bradycardia . Rarely, sevoflurane - induced suppression of baroreceptor reflex activity might result in desynchronisation between the atria and ventricle causing haemodynamic instability which may require electrical pacing . To conclude, sevoflurane in high concentration (8%) should be used with caution in children and in conditions where primary pacemaker activity is suppressed, as in elderly . Caution should also be exercised with its use in patients on ca channel and b blocker therapy.
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According to the mca, only a compromised ability to make a decision in accordance with certain procedural norms, loosely defined with reference to the ability to understand, retain and use or weigh relevant information, or an inability to communicate a decision, properly grounds judgments of incapacity (s. 3(1)). In addition, the identified problems of decision - making must be attributed to an impairment of, or disturbance in the functioning of, mind or brain (s. 2(1)). This law aims to protect the individual's right to pursue their own ends by adopting a capacity test that focuses on the decision - making process rather than the substance of the patient's decision . Influential liberal thinking holds that it is not the place of the law to pursue certain ends rather, the law should aim to provide a space for individuals to pursue their own ends (plant, 2011) and this position is reflected in english common law relating to mental capacity:[in assessments of capacity] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient, however grave the consequences . The view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind.whether the patient's decision is consistent with proposed substantive norms in relation to what they should pursue for example that people should pursue good health, or that they should want to live is said to be beyond the proper scope of the incapacity test . In adopting this position, english law allows that even the most controversial treatment decisions might be explained by the fact that the patient's evaluation of the situation diverges from a clinical perspective, and perhaps many people's evaluation of the situation . [in assessments of capacity] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient, however grave the consequences . The view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind. In practice then, questions of mental capacity might be thought of as boiling down to a question about whether a decision can be explained in terms of the patient's particular motivating commitments (their desires, values, projects, ideas about a good life); or whether the decision is properly explained in terms of a problem in decision - making, which is due to a dysfunction of mind or brain . Is the patient's controversial decision understandable in the light of their commitments? Or is the patient's ability to understand, retain, and use or weigh what the likely costs and benefits of treatment will be for them, currently compromised? Scrutinizing a patient's commitments in the context of a capacity assessment raises moral concerns because the possibility that a choice can be explained by idiosyncratic commitments plays a significant role in protecting the patient's right to make controversial decisions . Perhaps the most straightforward approach to conceiving of commitments, and the one most likely to protect this right, is to say that the person's commitments are their current commitments those they recognize at the time of the decision . The problem with conceiving of commitments in this way is that it fails to recognise certain problems of practical decision - making to do with motivational coherence over time . Someone with bipolar disorder, for example, might clearly be choosing or acting in accordance with their current desires during a manic phase, but this perspective fails to capture what seemingly goes wrong with practical decision - making in such cases . As a result, if this understanding of commitments is adopted in assessments of mental capacity the reach of this area of law arguably does not extend as far as it should a person may be judged to have mental capacity where this seems doubtful . Psychopathologies characterised by this kind of problem seem better accounted for in a legal structure that conceives of mental capacity, and in particular, commitments, in diachronic terms . A diachronic perspective might also help illuminate some other hard cases that are not normally characterised in terms of problems of motivational coherence over time . For example, someone with anorexia nervosa may be choosing rationally in accordance with their current commitments especially a strong desire not to gain weight, or to exert extreme control over what they eat and so may be judged to have capacity on these grounds . However, an assessment of their evaluative capacities in diachronic terms might yield the conclusion that their decision - making ability is seriously compromised (craigie, 2011). Their future self may come to the view that they should have been motivated in ways that they could not recognise or could not act on at the time, and this might be a fairly robust feature of people with a diagnosis of anorexia nervosa . This perspective in relation to questions of mental capacity chimes with the suggestion that particularly in psychiatry, colloquialisms [such as] you will thank me later sometimes become the unwritten rules within which the merits of a patient's consent are assessed (gostin, 1981, p. 742). Such a perspective may also offer a solution in cases where the patient does not have a psychiatric diagnosis . Mackenzie and watts discuss a case of a woman who was judged to have the capacity to refuse life - saving treatment, and subsequently died, in a situation where she had recently been left by her husband, following the amputation of both her legs . They argue that the trauma of these events and her subsequent inability to imagine a life without her partner clouded the woman's judgment in relation to treatment:the tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life, and a decision to accept the lifesaving treatment might have resulted. (2011, p. 33; for a full description of the case, see halpern, 2012)differences between these cases suggest that taking a diachronic perspective in assessments of mental capacity allows for decision - making to be compromised in at least two ways . In the bipolar disorder and anorexia nervosa cases a diachronic perspective is used to argue that a person's evaluative capacities may be compromised the capacities that determine the desires or values that motivate a decision . In the emotional trauma case, which might equally be applied to cases of depression, a diachronic perspective is used to identify a problem in the person's ability to assess whether what they want or value is achievable . According to jodi halpern's original analysis of the emotional trauma case, the woman should have been judged to lack capacity on grounds that her beliefs about the future were unresponsive to evidence, rendering her unable to think through alternatives (halpern, 2012, pp . The tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life, and a decision to accept the lifesaving treatment might have resulted. (2011, p. 33; for a full description of the case, see halpern, 2012) one problem with adopting a diachronic perspective in assessments of mental capacity is that whether questions of procedural rationality are properly understood in diachronic terms remains a contested issue in the fields of economics, psychology and philosophy . The functional element of the mca's incapacity test concerns problems in meeting certain procedural requirements problems in the ability to understand, retain, use or weigh relevant information . Sceptics deny that at least some norms derived from a diachronic perspective fall into this category . They hold that such requirements are substantive rather than procedural in nature, so i turn now to this theoretical concern . Much of the normative and descriptive work on a diachronic perspective in personal decision - making has taken place in the fields of economics and psychology, but these fields have tended to adopt contrasting positions on the normative question . Economists often treat a person's weighting of the future as a preference like any other, in a framework that assumes that preferences are not available to rational criticism . Prudence to refer to taking a diachronic perspective in decision - making:prudence cannot on this view be explained merely by the perception that something is in one's future interest; there must be a desire to further one's future interests if the perception is to have an effect there seems little doubt that most people have the desire that makes prudence possible [however, according to this view we are not] in any sense required to possess the desires in question: consequently we are not required to act on the specified considerations . If one lacks the relevant desire, there is nothing more to be said . (nagel, 1970, p. 28)psychologists, on the other hand, often assume that people are rationally required to give significant weight to the future personal consequences of choices, the strongest version being that people ought to be temporally neutral in their decision - making . It is widely accepted within this camp that there are at least some good reasons to give less weight to future consequences, particularly in order to take into account an expected depreciation in their future value . . However, foreseeable changes in one's circumstances can also be relevant: 50 might be much more valuable to a final year law student than 100 will be for them in the following year, when they reasonably expect to be working for a corporate lawyer . On these grounds it might be rational for the student to choose 50 now, foregoing 100 in a year's time . Likewise, it is argued that uncertainly with respect to future consequences can provide a good reason to choose a certain outcome now, over a more valuable but uncertain outcome in the future (baron, 1994, pp . I am using the term temporally neutral to refer to decisions that give equal weight to future and present utility, excepting the above kinds of considerations . The pressing question is why people ought to be impartial with respect to all parts of their lives once these kinds of consideration are taken into account . Prudence cannot on this view be explained merely by the perception that something is in one's future interest; there must be a desire to further one's future interests if the perception is to have an effect there seems little doubt that most people have the desire that makes prudence possible [however, according to this view we are not] in any sense required to possess the desires in question: consequently we are not required to act on the specified considerations . If one lacks the relevant desire, there is nothing more to be said . (nagel, 1970, p. 28) the arguments used to defend this position in the psychological literature tend to take the form that a temporally neutral orientation advances the goals of practical decision - making . Choosing an outcome merely because it will happen sooner is said to be irrational because if one option achieves your goals better, then you should choose that one, regardless of when you decide (baron, 1994, p. 514). Using the classic marshmallow experiments as an example, if a child would rather eat two marshmallows than one, then they should choose to wait and be given two marshmallows sometime later rather than one marshmallow now because overall this achieves the child's goals better . This kind of argument considers the question of goal satisfaction from a position that is not situated at a particular point in time, and prescribes that people ought to take this perspective in their decision - making: they should be just as concerned about themselves a year from now as they are about themselves this minute (p. 516). Individuals should give weight to the implications for their future self, so the argument goes, for their own overall good . Nagel argues that prudence is a requirement of practical reason, which is secured because the personal consequences of a man's action concern his future (1970, p. 42; his italics). According to nagel, this constraint on practical decision - making follows from the person's awareness that they persist over time . It reflects the individual's conception of himself as a temporally persistent being: his ability to identify with past and future stages of himself and to regard them as forming a single life . Failure to be susceptible to prudence entails radical dissociation from one's future, one's past, and from oneself as a whole, conceived as a temporally extended individual (p. 58). It is a mistake in practical reasoning because it comes at the cost of dissociation from one's temporally extended self (p. 69). The conception of self as extended throughout a human lifetime is powerful in the context of questions of procedural rationality because it offers grounds for claiming that a person ought to give weight to considerations that they may not currently care about . It offers an answer to what i have characterised as the economists' view, that if a person has no interest in the future personal consequences of their choice there is nothing more to be said, rationally speaking . Applied to the context of treatment decision - making, this kind of perspective is powerful because it offers grounds for calling a patient's mental capacity into question, with reference to their ability to consider their future and value it appropriately . Rejections of this kind of rational requirement are therefore often based on a rejection of the idea of the self as something that necessarily extends throughout a lifetime, and derek parfit's account of personal identity is often used to make this argument (baron, 1994, p. 516; frederick, 2006; maclean, 2006). Parfit holds that the persistence of a person over time must be understood in terms of their psychological continuity the connectedness of psychological features such as memories, character, interests and preferences across a lifetime (parfit, 1983; 1984). On this view, whether the future inhabitant of one's body will be the same person can be a matter of degree . Psychological features sometimes change so radically over a human lifetime that a person now will not necessarily be the same person who occupies that body in the future . And not giving weight to the interests of a future self is irrational only to the degree that one shares the relevant psychological characteristics with that self . More recently, galen strawson has defended a similar position in his rejection of narrative accounts of the self . Strawson argues that although experiencing oneself as one person throughout a lifetime what he calls a diachronic self - experience may be more common, some people experience themselves in what he describes as a more episodic way . Unlike the more diachronic person, the more episodic person has little or no sense that the self that one is was there in the (further) past and will be there in the future, although one is perfectly aware that one has long - term continuity considered as a whole human being (strawson, 2005, p. 65). [predominantly] episodic individuals may sometimes connect to charged events in their pasts in such a way that they feel those events happened to them embarrassing memories are a good example and anticipate events in their futures in such a way that they think those events are going to happen to them thoughts of death can be a good example however, the episodic person has no great or special interest in [their] past, and nor do they have a great deal of concern for [their] future (p. 67). Strawson holds that even a strongly episodic self is within the normal range of human experience, and that it is normatively on a par with a more diachronic self - experience . On his view, theorists such as nagel assert that selves extend over a lifetime, and diachronic norms of procedural rationality are derived from this claim . Parfit and strawson dispute this assumption, and conclude that disregard for the future personal consequences of a choice may simply reflect an alternative way of living a human life, at least when we consider the question of reasons for action in non - moral terms . However, all of the theorists canvassed thus far reason from premises about the extension of self over a lifetime, to conclusions about what reasons individuals have, and it will be argued here that this conception of the relationship between the requirements of practical rationality and the metaphysics of self is the wrong way around . As described above, the kind of requirement in question is often defended by nagel and others on the grounds that not giving weight to the future undermines one's own interests . However, at times a different kind of argument is suggested in nagel's work . The suggestion seems to be that a unified self over a lifetime is not something that is automatically present, but rather something that is generated by giving weight to future personal consequences in decision - making . The proposed requirement is justified, not because it deploys the correct conception of self, or because one's future self will otherwise regret the decision, but because adherence plays a role in bringing the self into being, by enabling the decision - maker to reach towards something outside himself (nagel, 1970, p. 43). Michael bratman is particularly clear about the role that he proposes giving weight to the future plays in the generation of the self, and how this in turn grounds a diachronic perspective on questions of procedural rationality, so it is his view that i will focus on here . Self - governing policies which are integral to temporally extended agency lie at the core of human agency because it is through these that a person's identity emerges . These processes are essential for understanding what it is for an agent to take a stand what it is for an agent to recognise certain desires as one's own and it is this feature in particular that distinguishes human agency from the agency of other purposive creatures (bratman, 2000, pp . Adopting broadly the same approach to personal identity as parfit begins with, bratman suggests that the forward - looking psychological ties that play a role in constituting a unified self over time can be generated through reflective and planning processes: i can help ensure appropriate psychological continuities and connections by sticking with and executing my prior plans and policies, and by monitoring and regulating my motivational structures in favor, say, of my continued commitment to philosophy such processes play an important role in the constitution and support of continuities and connections characteristic of the identity of the agent over time. Indeed, bratman suggests, this is what plans and policies are for (2000, p. 47 by emphasising the role that planning and policy - making play in generating the psychological ties that are proposed to underpin personal identity over time, bratman describes a picture of human agency in which the agent plays an active role . The temporally extended self develops through the agent's exercise of certain psychological capacities . On this point bratman makes a significant departure from parfit and strawson, who seem to hold that the degree to which the relevant psychological ties extend over time is a fixed fact about the individual, from which rational requirements on their action may be derived . The claim that engaging in planning and policy - making processes is rationally required is defended primarily on the non - instrumental grounds that these processes play a central role in developing and sustaining the self . Bratman's argument takes the form of an answer to the problem that agents who reflect on their first - order desires and form second - order desires do not yet exhibit what is distinctive about human agency, because there is nothing to distinguish these new desires as those that the agent identifies with; second - order desires seem to be just one more wiggle in the psychic stew (bratman, 2000, p. 38). The solution that bratman offers is that where an agent stands with respect to a particular first - order desire is established through the exercise of planning and policy - making practices: self - governing policies might, so to speak, crystallize pressures from various elements of one's psychic stew into a more decisive attitude that can, in the relevant context, establish where one stands (2000, p. 51). These mental processes that underpin temporally extended agency give authority to particular desires, so it is through these that the self the distinctive feature of human agency emerges . On this view, a compromised ability to engage in planning and policy - making processes threatens not the interests of the future self, but the very constitution of the self . This is what grounds the procedural requirement that one must give weight to the future personal consequences of actions . And this, we might suggest, is what makes the ability to consider and value the future an appropriate consideration to include in a mental capacity test . Bratman's account offers a story about the self that fits well with a developmental understanding of personhood that is often appealed to in contemporary medical law and ethics . His account of the relationship between temporally extended agency and the self also fits well with the difficulties associated with certain psychopathologies . It is no accident, i suggest, that disorders such as dementia, bipolar disorder and depression are associated with seriously compromised capacities for diachronic agency . Among many other factors, in a complex picture, problems of diachronic agency surely make a contribution to our sense that these constellations of behavioural and experiential features are appropriate targets for the label of disorder . A sense of the self as compromised in the context of problems of diachronic agency is also found in first - person reports of dementia (addis and tippett, 2004; however, see caddell and clare, 2012) and depression (fuchs, 2008; ratcliffe, 2012). It would seem that the functioning of capacities for diachronic agency is connected to our sense of others as having well - functioning human agency, as well as our subjective sense of selfhood, and for these reasons i find it implausible that a strongly episodic self - experience is just a less common way of living a human life . Strawson defends a view along these lines, claiming that he himself is strongly episodic (in his sense). Yet he has a successful academic career which could not have been achieved without meeting a wide range of commitments that each extended over considerable periods of time, for example in completing academic qualifications and writing books . It seems to me that these achievements evidence considerable skills in diachronic agency . While for strawson a diachronic self - experience is connected to a narrative conception of the self, diachronicity in the sense concerned here a life lived in the moment could still be a life lived with diachronic agency, as i will argue at the end of the following section . I now turn back to questions of mental capacity and what implications a diachronic perspective such as bratman's would have . The motivation for investigating these theoretical questions about procedural rationality was the idea that a diachronic perspective might enable a mental capacity test such as that adopted in the mca to deal with certain hard cases, particularly in the context of mental disorder, without undermining its value - neutral approach . The arguments above suggest that a diachronic perspective on questions of mental capacity is consistent with a procedural understanding of the functional element of the mca's incapacity test . Given this, could a diachronic understanding of what it is to have mental capacity help by justifying the inclusion of a requirement such as the ability to consider the future as a part of what it means to be able to understand, retain, use or weigh information in assessments of mental capacity? Something like this requirement already seems to be assumed in the condition that the patient must understand the consequences of the treatment options . The relevant information necessarily concerns the future as well as the present, due to the fact that understanding the likely effects of deciding one way or the other is a part of this requirement (mental capacity act 2005 code of practice, s. 4.16). For example, a person with a severe learning disability might be judged to lack the capacity to consent to a blood test on grounds that they do not understand that the test is necessary for their future physical well - being . The same might be said in a case of a delirious patient who is resisting treatment for a serious injury . In such cases the person's connection to the world, in the sense of their ability to understand how things are likely to be in the future, what to do is limited in a way that can be compared to john stuart mill's paradigm case of justified paternalistic intervention, where a person is about to cross a bridge that unbeknownst to them is unsafe . The person in the example does not understand that they risk falling into water if they attempt to cross . The widely accepted conclusion is that the person is not in a position to assess whether to cross the bridge, and this justifies the use of proportional physical force to stop them from crossing, when this is necessary to inform them about the state of the bridge . The same reasoning might be applied in the case of the woman who has recently suffered a serious emotional trauma, on grounds that her ability to imagine a range of future possibilities is severely limited (halpern, 2012). The patient cannot currently conceive of alternative futures to the desperately unhappy one she imagines, and perhaps this justifies taking the decision out of her hands until she is seeing things more clearly . The problem that returns here is that understanding concerns not simply how things are likely to be, as in the fact that an attempt to cross the bridge is likely to result in a fall into water, or that a certain proportion of people who have both legs amputated learn to walk again using prosthetic limbs . It concerns understanding how the relevant state of affairs will be for this particular patient . This question about the future necessarily involves an evaluative judgment that, according to a value - neutral approach, only the patient is in a position to make . So while it might sometimes be clear that a patient's ability to consider the future is compromised, for example when someone denies that their condition is life - threatening when it clearly is, in many cases assessing understanding in relation to the future will have no equivalent point of reference . In such a context there seems to be no standard against which the functional element of the mental capacity test the part concerning the patient's ability to understand, retain, use or weigh information can be directly assessed . Therefore, any conclusions drawn about this capacity would seem to be based on the fact that the patient has suffered an emotional trauma . Removing a patient's right to make their own treatment decision under these circumstances may seem justified given what we know about how people who lose limbs adapt, as reflected in self - reported measures of well - being, and given the known psychological effects of emotional trauma . However, an appeal to this kind of knowledge in an assessment of mental capacity would involve using generalised observations to draw a conclusion about a particular person's capacity to assess how things will be for them in the future . The legislation is clear that an assessment of decision - making ability should not be based solely on features such as a patient's diagnosis . It must be shown that this particular person's mental capacities are relevantly impaired, not merely that they belong to a diagnostic category . However, this requirement cannot be met in the case of this capacity, because there is no way to directly assess it while remaining value neutral in accordance with the aspirations of this area of law . What about the possibility raised at the beginning of this article, that a person's evaluative capacities might be called into question when viewed from a diachronic perspective? Drawing on bratman's account, planfulenss and self - governing policies are rationally required because of the role they play in developing and sustaining the self . Caring about the interests of the future inhabitant of one's body, and having at least some grip on what those interests will be, seem like prerequisites for engaging successfully in these processes . And perhaps this offers part of an explanation about how decision - making can be compromised in the context of disorders such as depression and bipolar disorder . Very crudely, the idea would be that as part of a much bigger picture the capacities necessary for diachronic agency are not functioning properly and this undermines the constitution of the self . I have argued that, understood in this way, these considerations are procedural rather than substantive in nature . But would the implementation of these considerations in practice nonetheless come into conflict with the goal of preserving patient autonomy? Assessing a person's grip on what interests the future inhabitant of their body will have runs into the same difficulties that occurred in the context of assessing understanding in the emotional trauma case . Without making substantive assumptions judging that a person is likely to lack these capacities in the context of a manic episode may seem justified, because of what we know about the trajectory of bipolar disorder that fairly reliably people's commitments during a manic episode radically undermine their overall interests . But whether this kind of consideration provides grounds for a judgment of incapacity in english law depends on what evidence is considered appropriate for assessing the functional element of the incapacity test . However, as genevra richardson points out, existing case law indicates that courts will take a flexible approach to the legal definition [of capacity] to enable them to reach their preferred outcome (richardson, this volume, p. 00). This may mean that in practice significant weight is given to a person's diagnosis . However, this solution undermines the protections of patient autonomy conferred by making an individual assessment of functional capacities, rather than diagnosis, the proper grounds for infringements on self - determination . A related possibility considered at the beginning of this article was that a diachronic perspective might be used to justify giving weight to a reasonable prediction of what the patient's future self would want in retrospect, in assessments of mental capacity . However, it does not follow from the view offered here, even given the sure knowledge that a patient's future self would retrospectively endorse a medical intervention, that their present self is irrational in refusing it . In the imagined case there is an unambiguous conflict between what the patient currently wants (no treatment) and what the future self will want in retrospect (treatment). Using this conflict to doubt the patient's current capacity to make the treatment decision assumes that the future self's perspective should be given greater weight in the current self's decision - making . However, there is nothing in the present view that would justify resolving the conflict in this way . The proposed account requires that the agent exercise planfulness and self - governing policies in decision - making, which would seem to involve giving equal weight at most, not greater weight, to one's expected future desires . Moreover, humans have a well - established time preference for negative experiences to be in the past rather than the present or future, and this makes it easier for the patient to endorse involuntary treatment when it is in the past (doherty, 2011). This would seem to weigh in favour of privileging the perspective of the self who is facing the infringement on liberty . In the absence of any reasons to believe that the future self's perspective should be privileged, this form of justification for doubting mental capacity looks to be question - begging . Its favouring of the future self's perspective already assumes that the current self's decision - making capacity is relevantly impaired . Favouring the future self's perspective might seem justified on grounds that, for example, evaluative capacities are often compromised in the context of certain diagnoses or during experiences such as emotional trauma . However, at least in theory, this evidence alone does not justify a judgment of incapacity under current english law, for reasons of preserving patient autonomy as argued above . Finally, i turn to the question of caring about future personal interests, especially in the context of life - threatening choices . A central difficulty associated with including a consideration of this kind in questions of mental capacity is the problem of how to assess caring simpliciter, independently of caring about a particular outcome . According to the account offered here, procedural rationality requires that we care about the future personal consequences of choices, but what in the future we should give weight to is left open . Specifying this in assessments of mental capacity would mean that the test was not value neutral . The case of the committed smoker (exemplified by david hockney) can be used to illustrate the point . One criticism often levelled at smokers is that they are irrational because they do not care sufficiently about the future personal consequences of their smoking . However, this criticism assumes that future health, or a long life, is what the person should care about . Considering a person's decision to keep smoking from a value - neutral perspective, which makes no assumptions of this kind, are they being short - sighted? Or do they care about things other than health and long life in a way that makes their decision rational? Bratman's account shows how asking this second question can be consistent with a diachronic perspective on rationality . It is clear that the committed smoker is very likely undermining the health of the future inhabitant of his body . However, it does not follow that he is not engaging in the psychological processes that underpin temporally extended agency . It may be a central feature of the person's life plan that he should not be overly concerned about living a long life . His deep and enduring commitment might be to a hedonistic lifestyle that is incompatible with being concerned about health in old age . For such a person, not living fully enough is a more likely source of future regrets than health - endangering behaviours . Caring about one's future might even require, in such a case, life - threatening or health - threatening choices . Parallel arguments could be made in the case of a decision by a jehovah's witness to refuse a blood transfusion; for a political activist's hunger strike; for a person who risks their life in an attempt to save a loved one; and a freediver's commitment to their dangerous extreme - sports lifestyle . These kinds of decision do not seem characterized by a lack of planfulness about the future . Rather than undermining the sense of oneself as a temporally persisting agent, decision - making in line with these kinds of commitment looks likely to contribute to the overlapping webs of cross - temporal connections and continuities that are proposed to play a role in constituting human agency (bratman 2000, pp . The future self taken into consideration in these cases may be a nearer self than in the case of a person who is invested in living a long life because the artist who is a committed smoker, for example, might not expect to or want to live a long life nothing in the above account enjoins us to aim for a life of a particular length . What it requires is that, at least to some degree, choices be made with a reflectivity about the future, such that insofar as we are agents that agency is temporally extended . This perspective allows us to reconcile the ideas that mental capacity requires that we care about the future personal consequences of our choices, and that life - ending decisions can be made with mental capacity . However, against this theoretical background, resolving questions about whether a patient's capacity to care about the future is compromised would seem to require looking beyond their procedural capacities . Answers to this question will rest on views about whether or not the life - threatening decision is consistent with the person's commitments, understood from a diachronic perspective . These factors are taken into account in an assessment of best interests once it is decided that a patient lacks mental capacity (mca s. 4(6)). However, if the ability to care about the future is included as part of what it means to have mental capacity, then consideration of a patient's commitments from a diachronic perspective will also be central to this prior decision . It has been argued here that a diachronic perspective on mental capacity is relevant to questions about when an infringement on treatment decision - making liberty is justified . However, including considerations derived from a diachronic perspective in assessments of capacity comes into conflict with central patient autonomy - preserving features of the mca . It's worth reflecting, then, on why a diachronic perspective might be morally relevant in this context . I will suggest that the answer lies in a common view about what makes patient autonomy valuable what makes it the case that for the most part, people's treatment decisions, especially their refusals, ought to be respected . The importance of allowing patients to be self - determining in relation to their own medical care is often justified along lines that it protects people's general capacity to lead their lives out of a distinctive sense of their own character, a sense of what is important to and for them (dworkin, 1994, p. 224). Different theorists argue for the value of patient autonomy on importantly different grounds, but a common theme is that treatment decisions should be respected because of their assumed connection to the self . However, in a case where human agency is seriously compromised in the way described by bratman, selfhood is undermined . Capacities for diachronic agency that play a central role in establishing what's important for the person where the person stands are compromised . And in the context of such problems, the importance placed on patient autonomy in liberal societies would seem to give medical professionals a reason to do what they can to restore selfhood, perhaps even if this requires an infringement on personal liberty . This might involve overriding a refusal of treatment, for example, though the justification for doing so would only warrant interventions of a particular kind those that are likely to restore the general capacity that the principle of patient autonomy aims to protect . This suggests that in some cases, especially in the context of mental illness, the importance placed on patient autonomy in contemporary medical ethics presents the law with a dilemma . On the one hand, it seems right that laws which license infringements on decision - making liberty should equally apply to everyone, not just to particular groups; and that if a patient has mental capacity in relation to a particular treatment decision then they should be free to direct the course of their medical care . Consequently, it seems we ought to do away with dedicated mental health laws that legalise detention and involuntary treatment without reference to mental capacity only in the context of a mental disorder . On the other hand, the way in which diachronic agency is compromised in some instances especially in the context of mental disorder seems to justify or perhaps even require an infringement on liberty to restore the self . These grounds are difficult to take into consideration in assessments of mental capacity without compromising features of the mca that are designed to preserve patient autonomy . So while it might be possible to implement the mca in a way that sufficiently accounts for the problems of decision - making and selfhood that are sometimes present in mental disorders, doing so comes at the price of what is seen to be a central virtue of the legislation . In this article my focus was requirements of rationality derived from a diachronic perspective, and the question of what kinds of consideration this perspective would enable mental capacity assessments to include . It was an attempt to explore the limits of the mca's incapacity test, given its value - neutral approach . It was argued that considerations derived from a diachronic perspective are relevant to questions of treatment decision - making liberty, and are consistent with a procedural understanding of the functional element of the test . However, in practice, appeal to these considerations in assessments of mental capacity undermines the role played by the functional element of the test in preserving patient autonomy . While involuntary treatment without reference to mental capacity is often assumed to undermine the principle of respecting patient autonomy, it was argued here that this is not necessarily so . When human agency is seriously compromised, preserving patient autonomy may sometimes require overriding a treatment decision to restore selfhood, on grounds that are not easily accounted for under the mca without compromising the value - neutral aspirations of this area of law . In considering a move to abandon dedicated mental health law, legislators must therefore resolve a bind . The central motivation for such a move is concern about unjustified infringements on liberty in the context of psychiatric diagnoses . The worry is that the protections of patient autonomy in the mca do not currently extend to the treatment of mental disorders, because mental health legislation allows people to be treated for a mental disorder even if they refuse with mental capacity . However, considerations derived from a diachronic perspective that seem relevant to when an infringement on decision - making liberty is justified are especially present in mental disorder . The inclusion of these considerations seems necessary for adequately caring for those with a mental disorder under universal capacity legislation . Yet including these factors in assessments of mental capacity undermines the protections of patient autonomy that supporters of a shift to universal capacity legislation hope to gain.
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Type iii dysbetalipoproteinaemia is a kind of lipid metabolism disorders, caused by apo - e deficiency, which leads to accumulation of chylomicrons and very low density lipoprotein remnants in the plasma.123 lipid metabolism disorders are mostly manifested by xanthomas, which are known as yellowish cholesterol - rich material in large foam cells accumulating in the skins and tendons.14 these yellowish lesions may appear all over the body, on the palm of the hands, sole of the foot, tendons, and even on the eyelids.5 these lesions firstly described with or without hyperlipidaemia in association with monoclonal immunoglobulin . Based on this fact, there are three forms of xanthoma: hyperlipaemic xanthoma, normolipaemic xanthoma and necrobiotic xanthogranuloma.6 xanthomatosis is usually associated with hyperlipidaemia, and morbidity and mortality of this condition are related to atherosclerosis and pancreatitis.7 hyperlipaemic xanthoma lesions are more polymorphic and can include tuberous, tendinous, palmar or eruptive xanthoma . Verruciform xanthoma usually presents as a hyperkeratotic, cauliflower like, verrucous or papillomatous lesion.6 it initially was described as a unique clinicopathologic lesion of the oral mucosa and was subsequently reported in the skin . The epidermal - mucosal changes of hyperparakeratosis, neutrophilic infiltrate and dermal - submucosal foam cell reaction are distinctive features of these xanthomas.8 hereby, we present a rare type of xanthomas calling cauliflower xanthoma in a 43-year - old man with dysbetalipoproteinaemia . A 43-year - old man was presented to outpatient endocrine clinic of tabriz university of medical sciences, tabriz, iran, by his sister for evaluation of skin lesions . He had multiple lesions on shoulders and back, lateral and medial part of dorsal surface of the foot, medial and lateral malleus of both feet, and dorsal and palmar surface of both hands . Siblings of the patient have been followed up in that clinic for clinically diagnosed type - iii dysbetalipoproteinaemia . This diagnosis was based on characteristic palmar, eruptive, tuberous and trauma site xanthomas with typical high levels of both total cholesterol and triglycerides in the plasma . History of coronary artery or other atherosclerotic disorders were negative in index case and siblings . There were not any other complaint, and as a greengrocer, he had an active lifestyle . He had a pathology report of skin lesion biopsy that was performed by a dermatologist few months ago, with presence of lipid laden macrophages consistent with diagnosis of xanthoma . Fasting serum lipid levels reported as: total cholesterol = 507 mg / dl, triglycerides = 470 mg / dl and high density lipoprotein (hdl) cholesterol = 41 mg / dl . He had normal fasting blood sugar, and normal thyroid, renal and liver function tests . Physical examinations revealed numerous xanthomas resembling cauliflower on both knees [figure 1], unusual eruptive, tuberous xanthomas same as previous lesions, on the lateral malleus of both feet [figure 2]. Cauliflower xanthomas on the knees and lateral malleus of both lower extremities cauliflower xanthomas on the lateral malleus of lower extremities there were lots of smaller xanthomas on the right shoulder [figure 3], and cauliflower like xanthomas on the extensor side of the left upper extremity, especially on the elbow [figure 4]. Based on available information, the patient diagnosed as familial dysbetalipoproteinaemia (fdl) and the unique lesions on the lower limb called cauliflower xanthoma . Although siblings of this case had the same disorder, they did nt develop such lesions . Xanthomas on the shoulder and back of the neck xanthomas on the extensor side of the hand by searching in the medical data bases, we cannot find a previous such a lesion . Therefore, we decided to present this case without revealing patient's name, after taking his consent . Considering the fact that the treatment with high dose statin was effective in correction of lipid abnormality and regression of skin lesions in his three siblings, we wanted to try the treatment procedure in this patient but he refused any intervention . Fdl, also known as hyperlipoproteinemia type - iii or broad beta disease, is a rare inherited disorder characterised by improper metabolism of certain lipids, specially plasma cholesterol, triglyceride rich chylomicron and very low density lipoproteins (vldl) remnants.1 presence or absence of the symptoms of this disease depends on two major risk factors: genetic and diet.910 mutations in the gene for apolipoprotein e (apo e) are the main cause of this disease . Replacement of an arginine by a cysteine in position 158 of the 299-amino acid chain of apo e5 is responsible for the defective binding of chylomicron and vldl remnants to cell receptors . Thereafter, slower plasma clearance of these particles occurs, and results in the abnormal accumulation of lipids in the body.9 on the other side, diet has an essential role in the development of the disease . This means that with standard cholesterol diet, symptoms of fdl will not appear, even in the genetically susceptible person.10 xanthomas can be a symptom of fdl . Xanthomas may also be the symptoms of a generalised histiocytosis, or a local fat phagocytosing storage process.11 they are yellowish lesions on the skins and tendons, macroscopically . These macrophages are filled with lipid droplets, which are dissolved and removed from tissue during histologic processing.8 fdl is mostly diagnosed by combination of clinical and laboratory findings.12 most cases are inherited as autosomal recessive trait . Men are more susceptible for fdl probably because of protective impact of estrogen in women . Most of the diagnosed patients were typically young males, with strong family history, characteristic skin lesions, high serum levels of cholesterol and triglycerides and confirming skin histology.113 most of these cases have premature atherosclerosis and other signs of ischaemic disorders.9 the patient in this article was a middle aged man with family history of dysbetalipoproteinaemia and high levels of serum lipids as follows: total cholesterol = 507 mg / dl and triglycerides = 470 mg / dl . He had a rare form of xanthoma all over his body, causing social problems for him . Nicotinic acid, clofibrate, statins or gemfibrozil properly reduce cholesterol and triglycerides in people affected with dysbetalipoproteinaemia.1 the patient mentioned in this article refused receiving any medical intervention . He has just been advised to have low lipid diet, fish oil and regular moderate exercise in order to reducing serum lipid levels . There should be distinctive monitoring of fdl patients including regular checking of their serum lipids . Controlling underlying disorders, and reducing excess calories, saturated fat and cholesterol, is the main aim of treating these patients . According to this article, cauliflower xanthomas could be a symptom of fdl, and it should be considered as a differential diagnosis while approaching to these lesions.
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Exacerbation is an important life - threatening event for patients with copd, and can lead to hospitalization and death.14 patients who suffer frequent and repeated exacerbations within 1 year have a poor prognosis,5 characterized by worsening of health - related quality of life (hrqol),6,7 a rapid decline in lung function,810 and high mortality.11 frequent exacerbators also carry a high risk of further exacerbation and hospitalization.11,12 however, it has been suggested that japanese patients with copd may have fewer exacerbations, and they also may have a higher proportion of elderly patients, those with emphysema, and those with a lower body mass index in comparison to westerners.1215 the prognosis of japanese patients with copd who suffer frequent and repeated exacerbations is unclear . We conducted a 1-year prospective observational trial in a daily - life setting involving 90 japanese patients with copd to investigate whether previous moderate - to - severe exacerbations are associated with future exacerbations in this patient population . We conducted a 1-year prospective observational trial in accordance with good clinical practice (gcp) guidelines and approved by the ethics committee of kurume university and chikugo city hospital (gcp 11 - 127, september 2012august 2014). Consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records . Copd patients were divided into three groups, based on the total number of moderate and severe exacerbations within the last year before enrollment in the study, ie, non- (previous moderate and severe exacerbations, 0/year), infrequent (one exacerbation / year), and frequent (two or more exacerbations / year) exacerbator groups, in accordance with a previous report.16 in addition, patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation (severe exacerbators). The data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations; clinical parameters included age, sex, body mass index, smoking habits, smoking index, comorbidities, duration of copd, 5-grade modified medical research council (mmrc) dyspnea scale score,17 total copd assessment test (cat) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease (gerd) (fssg),20 center for epidemiologic studies depression (cesd) scale score,21 medications, blood pressure and heart rate, lung function and blood parameters, and chest computed tomography . Duration of copd was defined as the period (years) since the patient had been diagnosed by a physician as having copd, emphysema, and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed, each patient was required to regularly visit the hospital every 2 months, and to request emergency admission when the symptoms worsened . Regular respiratory medications were not changed during the period of the study, which was conducted in a daily - life setting . The diagnosis of copd was based on age 40 years, smoking index> 10 pack - years, forced expiratory volume in 1 second (fev1)/forced vital capacity (fvc) ratio of <0.7 after bronchodilator administration, and the spirometric gold (global initiative for chronic obstructive lung disease)-stage classification, ie, stage i (fev1 80% predicted), ii (50% fev1 <80% predicted), iii (30% fev1 <50% predicted), and iv (fev1 <30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas . To exclude any patients with asthma, patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1> 200 ml or> 12% after bronchodilation were excluded.24 diagnosis of asthma copd overlap syndrome (acos) was made on the basis of a history of dyspnea and wheezing attacks at rest, large variations in daily symptoms, a fixed fev1/fvc ratio of <0.7, marked reversibility of fev1 after administration of bronchodilators (> 15% and> 400 ml), and a peripheral eosinophil count of> 600/mm, in accordance with a previous report.25 baseline data for the mmrc scale, total cat, fssg scale, and cesd scale scores were obtained only once, based on a self - report completed by each patient after written informed consent had been obtained.1721 information about comorbidities was obtained from the patients by interview, and the diagnoses were confirmed by physicians . However, patients who had moderate - to - severe comorbidities associated with poor prognosis, such as active malignancies, depression (cesd scale> 16 points),21 liver cirrhosis, digestive ulcers, persistent arrhythmia, congestive heart disease, coronary artery disease, lung fibrosis and bronchiectasis, chronic renal failure requiring dialysis, and central nervous system disorders, including palsy and dementia, were excluded, in accordance with previous studies.26,27 hypertension (systolic> 140 mmhg or diastolic> 90 mmhg blood pressure or use of medications), dyslipidemia (serum low-[14] or high- [<4] 15 mg / l, or use of medications), diabetes (blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy (neuropathy, retinopathy, and nephropathy), and gerd (fssg scale> 7 points)20 were accepted as comorbidities . Previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events . Moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis, such as increased cough and sputum production, a change in sputum color, and worsening of dyspnea from a stable state and beyond normal day - to - day variations, ie, showing acute onset and necessitating a change in regular medication, in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year . Hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy, performance status was 3, and unconsciousness occurred with copd exacerbation.1,23,31 however, pneumonia was also recognized as exacerbation . Mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . However, mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators . Unreported exacerbations were considered to be those to which patients had been insensitive, or those that had been self - controlled in spite of worsening of respiratory symptoms . Data are expressed as mean standard deviation (sd) and number (percentage) of non-, infrequent, and frequent exacerbators at baseline . Characteristics were compared using analysis of variance, fisher s exact test, -test for trend, and tukey subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators, and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . For patients who were observed throughout the study period, the odds ratio (95% confidence interval [ci]) of baseline parameters was determined to predict factors for future risk (subsequent exacerbations once or more and twice or more) using univariate and logistic multivariate regression analyses . Medians of age, body mass index, smoking index, and duration of copd were 68 years, 21.4 kg / m, 53.5 pack - years, and 4 years, respectively, and each median was used as the cutoff value for the analysis . Statistical analysis was performed by the jmp version 9.0 statistical software package (sas institute inc, cary, nc, usa). We conducted a 1-year prospective observational trial in accordance with good clinical practice (gcp) guidelines and approved by the ethics committee of kurume university and chikugo city hospital (gcp 11 - 127, september 2012august 2014). Consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records . Copd patients were divided into three groups, based on the total number of moderate and severe exacerbations within the last year before enrollment in the study, ie, non- (previous moderate and severe exacerbations, 0/year), infrequent (one exacerbation / year), and frequent (two or more exacerbations / year) exacerbator groups, in accordance with a previous report.16 in addition, patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation (severe exacerbators). The data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations; clinical parameters included age, sex, body mass index, smoking habits, smoking index, comorbidities, duration of copd, 5-grade modified medical research council (mmrc) dyspnea scale score,17 total copd assessment test (cat) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease (gerd) (fssg),20 center for epidemiologic studies depression (cesd) scale score,21 medications, blood pressure and heart rate, lung function and blood parameters, and chest computed tomography . Duration of copd was defined as the period (years) since the patient had been diagnosed by a physician as having copd, emphysema, and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed, each patient was required to regularly visit the hospital every 2 months, and to request emergency admission when the symptoms worsened . Regular respiratory medications were not changed during the period of the study, which was conducted in a daily - life setting . The diagnosis of copd was based on age 40 years, smoking index> 10 pack - years, forced expiratory volume in 1 second (fev1)/forced vital capacity (fvc) ratio of <0.7 after bronchodilator administration, and the spirometric gold (global initiative for chronic obstructive lung disease)-stage classification, ie, stage i (fev1 80% predicted), ii (50% fev1 <80% predicted), iii (30% fev1 <50% predicted), and iv (fev1 <30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas . To exclude any patients with asthma, patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1> 200 ml or> 12% after bronchodilation were excluded.24 diagnosis of asthma copd overlap syndrome (acos) was made on the basis of a history of dyspnea and wheezing attacks at rest, large variations in daily symptoms, a fixed fev1/fvc ratio of <0.7, marked reversibility of fev1 after administration of bronchodilators (> 15% and> 400 ml), and a peripheral eosinophil count of> 600/mm, in accordance with a previous report.25 baseline data for the mmrc scale, total cat, fssg scale, and cesd scale scores were obtained only once, based on a self - report completed by each patient after written informed consent had been obtained.1721 information about comorbidities was obtained from the patients by interview, and the diagnoses were confirmed by physicians . However, patients who had moderate - to - severe comorbidities associated with poor prognosis, such as active malignancies, depression (cesd scale> 16 points),21 liver cirrhosis, digestive ulcers, persistent arrhythmia, congestive heart disease, coronary artery disease, lung fibrosis and bronchiectasis, chronic renal failure requiring dialysis, and central nervous system disorders, including palsy and dementia, were excluded, in accordance with previous studies.26,27 hypertension (systolic> 140 mmhg or diastolic> 90 mmhg blood pressure or use of medications), dyslipidemia (serum low-[14] or high- [<4] 15 mg / l, or use of medications), diabetes (blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy (neuropathy, retinopathy, and nephropathy), and gerd (fssg scale> 7 points)20 were accepted as comorbidities . Previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events . Moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis, such as increased cough and sputum production, a change in sputum color, and worsening of dyspnea from a stable state and beyond normal day - to - day variations, ie, showing acute onset and necessitating a change in regular medication, in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year . Hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy, performance status was 3, and unconsciousness occurred with copd exacerbation.1,23,31 however, pneumonia was also recognized as exacerbation . Mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . However, mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators . Unreported exacerbations were considered to be those to which patients had been insensitive, or those that had been self - controlled in spite of worsening of respiratory symptoms . Data are expressed as mean standard deviation (sd) and number (percentage) of non-, infrequent, and frequent exacerbators at baseline . Characteristics were compared using analysis of variance, fisher s exact test, -test for trend, and tukey subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators, and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . For patients who were observed throughout the study period, the odds ratio (95% confidence interval [ci]) of baseline parameters was determined to predict factors for future risk (subsequent exacerbations once or more and twice or more) using univariate and logistic multivariate regression analyses . Medians of age, body mass index, smoking index, and duration of copd were 68 years, 21.4 kg / m, 53.5 pack - years, and 4 years, respectively, and each median was used as the cutoff value for the analysis . Statistical analysis was performed by the jmp version 9.0 statistical software package (sas institute inc, cary, nc, usa). Ninety of 110 patients who provided informed consent were finally analyzed; 32 (35.6%) patients had suffered previous moderate and/or severe exacerbations during the last year . The numbers of patients with non-, infrequent, and frequent exacerbations were 58 (64.4%), 12 (13.3%), and 20 (22.2%), respectively (figure 1). At baseline, frequent exacerbators had a significantly lower body mass index, were less likely to be male or current smokers, had higher mmrc - scale grades and higher total cat score, had a higher proportion of spirometric stages iii and iv, gerd, and use of inhaled corticosteroid (ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy, and lower lung - function parameters, including fev1, fvc, and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators, but not in comparison with infrequent exacerbators (table 1). Frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators (table 1). There was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations (table 1). During the 1-year prospective observation period, one frequent exacerbator, one infrequent exacerbator, and two nonexacerbators died, with causes of death respiratory failure with copd exacerbation, cerebrovascular attack, and malignancies (small - cell lung cancer and colon cancer), respectively, whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . As a result, 78 patients (17 frequent, eleven infrequent, and 50 nonexacerbators) completed the study (figure 1). Figure 2 also shows that among the frequent exacerbators, the number of patients who subsequently suffered severe exacerbations (requiring one or more hospitalizations) was two (11.8%), and frequent (two or more exacerbations / year), infrequent (one exacerbation / year), and no moderate or severe exacerbations were seven (42.1%), six (35.3%), and four (23.5%), respectively, whereas seven (63.6%) of eleven infrequent and 37 (74.0%) of 50 nonexacerbators experienced no further exacerbation . The proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators (odds ratio [95% ci] 2.94 [1.217.17], p=0.0340; 2.94 [1.725.03], p=0.0004, respectively), but not in comparison with infrequent exacerbators (1.51 [0.494.63], p>0.05; 2.01 [0.924.80], p=0.053, respectively). Among five (5.6%) of the severe exacerbators, two (40.0%) had subsequently suffered severe exacerbations . The number of patients who had subsequent frequent, infrequent, and no moderate or severe exacerbations was two (40%), 0, and three (60%), respectively (figure 2). In subanalysis, there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups (p>0.05). In addition, one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period . The numbers of patients who reported pneumonia (n=78) and those who regularly used ics (n=15) were seven (9%) and three (20%), respectively . Patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics (p>0.05). Figure 3a shows that the mean annual frequencies (sd, exacerbations / year) of future total (1.41.2, p=0.0020) and moderate (1.31.1, p=0.0057) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators (0.40.8 and 0.40.7, respectively), but not in infrequent exacerbators (0.91.4 and 0.81.4, respectively). Figure 3b shows the kaplan meier analysis of the period until the first moderate and severe exacerbations . The median (mean sd) periods (days) until the first moderate and severe exacerbations for frequent, infrequent, and nonexacerbators were 266 (23528), 365 (29330), and 365 (32313) days, respectively (log - rank test, p=0.0012). There was no significant difference in the annual frequencies of severe exacerbation (hospitalization) among non- (0.00.3), infrequent (0.10.39), and frequent (0.10.3) exacerbators (p>0.05). The median (mean sd) periods until the first severe exacerbation were 365 (3632), 365 (3650), and 365 (3605) days, respectively (p>0.05; data not shown). Univariate analysis revealed that a low fev1 (<50%) had the highest odds ratio for risk of future exacerbation (one or more exacerbations / year), followed in order by regular use of ics, two or more previous exacerbations in the last year, presence of gerd, pneumococcal vaccination, one or more previous exacerbations in the last year, low body mass index (21.4 kg / m), a high total cat score (10 points), a high mmrc scale grade (2) and older age (68 years), whereas in patients who suffered two or more exacerbations, the highest odds ratio was observed due to the presence of gerd, followed in order by regular use of ics, low fev1 predicted, pneumococcal vaccination, older age, a high mmrc - scale grade, long duration of copd (4 years), and two or more and one or more previous exacerbations in the last year (table 2). However, 13 (86.7%) of 15 patients who had received ics had also received pneumococcal vaccination . The independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted, presence of gerd, and regular use of ics, whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations (table 3). Ninety of 110 patients who provided informed consent were finally analyzed; 32 (35.6%) patients had suffered previous moderate and/or severe exacerbations during the last year . The numbers of patients with non-, infrequent, and frequent exacerbations were 58 (64.4%), 12 (13.3%), and 20 (22.2%), respectively (figure 1). At baseline, frequent exacerbators had a significantly lower body mass index, were less likely to be male or current smokers, had higher mmrc - scale grades and higher total cat score, had a higher proportion of spirometric stages iii and iv, gerd, and use of inhaled corticosteroid (ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy, and lower lung - function parameters, including fev1, fvc, and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators, but not in comparison with infrequent exacerbators (table 1). Frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators (table 1). There was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations (table 1). During the 1-year prospective observation period, one frequent exacerbator, one infrequent exacerbator, and two nonexacerbators died, with causes of death respiratory failure with copd exacerbation, cerebrovascular attack, and malignancies (small - cell lung cancer and colon cancer), respectively, whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . As a result, 78 patients (17 frequent, eleven infrequent, and 50 nonexacerbators) completed the study (figure 1). Figure 2 also shows that among the frequent exacerbators, the number of patients who subsequently suffered severe exacerbations (requiring one or more hospitalizations) was two (11.8%), and frequent (two or more exacerbations / year), infrequent (one exacerbation / year), and no moderate or severe exacerbations were seven (42.1%), six (35.3%), and four (23.5%), respectively, whereas seven (63.6%) of eleven infrequent and 37 (74.0%) of 50 nonexacerbators experienced no further exacerbation . The proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators (odds ratio [95% ci] 2.94 [1.217.17], p=0.0340; 2.94 [1.725.03], p=0.0004, respectively), but not in comparison with infrequent exacerbators (1.51 [0.494.63], p>0.05; 2.01 [0.924.80], p=0.053, respectively). Among five (5.6%) of the severe exacerbators, two (40.0%) had subsequently suffered severe exacerbations . The number of patients who had subsequent frequent, infrequent, and no moderate or severe exacerbations was two (40%), 0, and three (60%), respectively (figure 2). In subanalysis, there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups (p>0.05). In addition, one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period . The numbers of patients who reported pneumonia (n=78) and those who regularly used ics (n=15) were seven (9%) and three (20%), respectively . Patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics (p>0.05). Figure 3a shows that the mean annual frequencies (sd, exacerbations / year) of future total (1.41.2, p=0.0020) and moderate (1.31.1, p=0.0057) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators (0.40.8 and 0.40.7, respectively), but not in infrequent exacerbators (0.91.4 and 0.81.4, respectively). Figure 3b shows the kaplan meier analysis of the period until the first moderate and severe exacerbations . The median (mean sd) periods (days) until the first moderate and severe exacerbations for frequent, infrequent, and nonexacerbators were 266 (23528), 365 (29330), and 365 (32313) days, respectively (log - rank test, p=0.0012). There was no significant difference in the annual frequencies of severe exacerbation (hospitalization) among non- (0.00.3), infrequent (0.10.39), and frequent (0.10.3) exacerbators (p>0.05). The median (mean sd) periods until the first severe exacerbation were 365 (3632), 365 (3650), and 365 (3605) days, respectively (p>0.05; data not shown). Univariate analysis revealed that a low fev1 (<50%) had the highest odds ratio for risk of future exacerbation (one or more exacerbations / year), followed in order by regular use of ics, two or more previous exacerbations in the last year, presence of gerd, pneumococcal vaccination, one or more previous exacerbations in the last year, low body mass index (21.4 kg / m), a high total cat score (10 points), a high mmrc scale grade (2) and older age (68 years), whereas in patients who suffered two or more exacerbations, the highest odds ratio was observed due to the presence of gerd, followed in order by regular use of ics, low fev1 predicted, pneumococcal vaccination, older age, a high mmrc - scale grade, long duration of copd (4 years), and two or more and one or more previous exacerbations in the last year (table 2). However, 13 (86.7%) of 15 patients who had received ics had also received pneumococcal vaccination . The independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted, presence of gerd, and regular use of ics, whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations (table 3). The frequent - exacerbator phenotype is important to consider in the management of copd patients, who require future exacerbation - related hospitalization associated with high mortality.16 a previous western study suggested that frequent exacerbators tend to have had exacerbations during the previous year, a lower fev1, more severe hrqol, a history of gerd, and a higher peripheral white blood - cell count in comparison with infrequent exacerbators and nonexacerbators.12 another western study demonstrated that the characteristics of frequent exacerbators include a more severe mmrc - scale grade, lower fev1 predicted, comorbid cardiovascular disease, depression or osteoporosis, and female sex as independent risk factors.32,33 we conducted the present study to observe moderate and severe exacerbations 1 year before and after baseline to investigate the characteristics of japanese copd patients who were frequent exacerbators . Based on exacerbations during the previous year, we found that frequent exacerbators were more likely to be female, have a lower body mass index, have a significantly lower fev1 predicted, have a higher mmrc - scale grade (lower exercise tolerance), and have a lower total cat score (lower hrqol). We also found that the characteristics of frequent exacerbators were similar between japanese and westerners, except for body mass index, as reported previously.12,16,32 interestingly, univariate analysis did not show that previous frequent exacerbators would become future frequent exacerbators (table 2). Indeed, 60% of previous frequent exacerbators did not suffer subsequent exacerbations, whereas conversely 14% of patients who had not previously suffered exacerbations subsequently did so (figure 2). Investigation of factors predicting the change in frequency of exacerbations is critically important, as they are still unclear.34 a total of 78 (86.7%) of our 90 patients completed the 1-year prospective study period . Among five severe exacerbators, one (20%) died, two (40%) again developed severe exacerbation, and two (40%) therefore, severe exacerbators appear to have a poor prognosis . However, our study - sample size was small and the observational period short . Frequent exacerbators had a significantly higher frequency of future exacerbations and a shorter period until the next exacerbation than nonexacerbators, and also had a significantly poorer prognosis than the latter, thus confirming the findings of a previous study.12 previous studies have demonstrated that over half of copd patients have unreported exacerbations . Such unreported exacerbations are thought to be an important component of hrqol decline.7,35,36 in our present study, to make the conditions for previous and future exacerbations uniform, daily journals for symptoms were not accepted . Previous studies have demonstrated that the frequency of annual moderate or severe exacerbations per patient in japanese individuals may be lower than that in the usa and europe.13,14,3537 in our study, the mean (sd [range]) of previous annual total and severe exacerbations were 0.841.48 (06) and 0.060.23 (01) exacerbations / year, respectively for all patients (data not shown), whereas those of future total and severe exacerbations were 0.711.08 (04) and 0.060.28 (02) exacerbations / year, respectively . Our data for the frequency of exacerbations seem to indicate a slightly lower incidence in japanese than in westerners.1215,31,32,38 previous japanese reports on the frequency of exacerbations have been scarce.13,14 japanese copd patients may be slightly older and thinner on average than westerners.1215,31,32,38 although the discrepancy between japanese and westerners is still unclear, the difference in the frequency of exacerbations may be associated with different populations of phenotypes with emphysema, locality, and understanding of both the physician and the patient about diseases and exacerbations, such as convenient use of ics, and in definition of exacerbation . Patients with copd have a wide variety of numerous comorbidities, which are strongly associated with mortality and exacerbation.1,12,26 we assessed baseline comorbidities based on interviews with patients and the physicians diagnosis, and partly through examinations or questionnaires . Several comorbidities, such as depression21,39 assessed by the cesd questionnaire, acos27,40 based on previous criteria, and congestive status with heart failure and cor pulmonale based on history or medical signs,41 were carefully excluded . Gerd, although not moderate to severe, was a common major comorbidity, and the proportion of frequent exacerbators with gerd was significantly higher than that of nonexacerbators . Gerd was an independent, and the highest, risk factor for future frequent exacerbations, although all patients with gerd were receiving proton - pump inhibitors . Our results confirmed previous reports.12,42 a low fev1 predicted was an independent risk factor for future exacerbations . The guidelines1,43 recommend long - term treatment with ics and pneumococcal vaccination for patients with severe and very severe copd, and frequent exacerbations may not be adequately controlled by long - acting bronchodilators . In our study regular use of ics, but not pneumococcal vaccination, was an independent risk factor for future frequent exacerbations . Sixteen (17.8%) of our patients used ics, although none had received ics without long - acting bronchodilators at baseline . Users of ics included four nonexacerbators with gold stage ii, seven patients with frequent exacerbations, and the remaining five had severe or very severe disease . The risk of ics for respiratory infection including pneumonia is a concern.4446 among four nonexacerbators with gold stage ii and users of ics during the prospective observation period, two patients had frequent exacerbations and one had infrequent exacerbations . This risk must be weighed against the benefits when prescribing ics to patients with copd . First, the study population was small and the observation period rather short for analyzing severe exacerbations and mortality . Balcells et al demonstrated that female sex was the highest risk factor for exacerbation,33 although in the present study there was no difference in the frequency of exacerbations between males and females . Both physicians and patients were aware of the severity and frequency of previous exacerbations at baseline . We carefully removed severe comorbidities, such as cardiovascular disease and depression, in accordance with previous reports.26,27 however, patients with asthma and acos may have been included, because we did not investigate airway responsiveness and inflammation or serum total immunoglobulin (ig) e levels, although we carefully excluded patients with asthma based on symptoms and spirometry . However, no patients had received medications for osteoporosis and low peripheral lymphocyte counts, although latent osteoporosis and hiv infections were not tested for . Fourth, the contents of medicines could not be ethically unified in a clinical setting in japan, and in our study adherence to pharmacological medicines and compliance with inhalation techniques were not assessed or included . The effects of respiratory medications, including inhaled medicines, on previous and future exacerbations were investigated . The presence of gerd, regular use of ics, and low fev1 may be associated with frequent exacerbations.
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Lipoma is a rare tumor but can be found at various sites in the abdominal cavity (fig . 2). If it is of a large size, it can compress adjacent tissues or organs and become heterogeneous . In such cases, imaging is incapable of distinguishing it from liposarcoma.lipoblastoma is a type of a benign tumor with embryonic fat . It is usually found in the extremities and torso whereas abdominal cavity belongs to its rather rare sites . In us, such tumors are echogenic . Lipoblastomatosis is a form of this tumor characterized by aggressive localized growth.hibernoma is a benign tumor, mainly composed of brown fat, in whose cells the numbers of mitochondria responsible for enhanced metabolic activity are impressive . These tumors are rarely encountered in the retroperitoneal space but are also echogenic with well - or ill - defined margins . Their characteristic feature is evident flow in color doppler.other benign tumors with a fat component, such as myolipoma, angiomyolipoma and myelolipoma, apart from teratoma (which is usually benign), tend to be homogeneous with echogenicity increased to various degrees (fig . 3, 4). Only gonadal germ cell tumors, such as teratoma and dermoid cyst that arise from several germ layers, frequently have irregular echotexture due to the presence of fluid sebum, hair, soft tissues and bony elements . That is why they are encountered in solid, cystic or solid - cystic forms (fig . Mature fat tissue can also be found in choristoma which, however, is not a neoplasm . Lipoma is a rare tumor but can be found at various sites in the abdominal cavity (fig . 2). If it is of a large size, it can compress adjacent tissues or organs and become heterogeneous . It is usually found in the extremities and torso whereas abdominal cavity belongs to its rather rare sites . In us, such tumors are echogenic . Hibernoma is a benign tumor, mainly composed of brown fat, in whose cells the numbers of mitochondria responsible for enhanced metabolic activity are impressive . These tumors are rarely encountered in the retroperitoneal space but are also echogenic with well - or ill - defined margins . Other benign tumors with a fat component, such as myolipoma, angiomyolipoma and myelolipoma, apart from teratoma (which is usually benign), tend to be homogeneous with echogenicity increased to various degrees (fig . 3, 4). Only gonadal germ cell tumors, such as teratoma and dermoid cyst that arise from several germ layers, frequently have irregular echotexture due to the presence of fluid sebum, hair, soft tissues and bony elements . That is why they are encountered in solid, cystic or solid - cystic forms (fig . Mature fat tissue can also be found in choristoma which, however, is not a neoplasm . Two views show lipoma (arrows) as a slightly echogenic lesion in the supraperitoneal fat three lipomas (l) in the small bowel mesentery, which in computed tomography showed density ranging from 57 to 74 hounsfield units two views show angiomyolipoma (l) located entirely in the adipose capsule of the right kidney . An arrow points to the site of regrowth in the form of the beak sign myelolipoma in the right suprarenal field (arrow) as a visible hyperechoic mass a heterogeneous mass arising from the retroperitoneal space is a mature teratoma with slight calcifications (arrows) liposarcoma is the only malignancy deriving from adipose tissue that is relatively frequently encountered in the retroperitoneal space (it constitutes nearly 1/3 of sarcomas at this site and 1015% of all neoplasms of this type). According to the who, these tumors can be divided into five histological subtypes depending on the grade of their differentiation (well - differentiated, dedifferentiated, myxoid, round cell, pleomorphic), which is relevant in prognosis . Its presence can be indicated only by uneven margins and heterogeneous enhancement upon contrast agent administration . Poorly differentiated subtypes account for even 40% of local relapses, and metastases are observed in 17% of cases . As has already been mentioned, well - differentiated liposarcoma is difficult to distinguish from lipoma also in sonography . One of the differential criteria can be tumor's reaction to compression with a transducer (fig . More aggressive forms of this tumor, however, tend to be unevenly delineated, present heterogeneous echotexture and infiltrate adjacent tissues (fig . Myxoid liposarcoma is characterized by a cystic structure and should be distinguished e.g. From lymphangioma, teratoma or cystic mesothelioma . No lesion compressibility when pressure is applied with the transducer (arrow) heterogeneous retroperitoneal liposarcoma infiltrates into adjacent tissues (arrows) mesenteric liposarcoma with pathological vascularity finally, immature teratoma must be mentioned . It is a primary germinal tumor that can contain adipose tissue and occurs at similar sites . They are characterized by the predominance of a solid component, and the only sign suggesting a malignancy is poorly circumscribed margins . A chance for malignant transformation cystic lesions can correspond to mesenteric cysts, lymphatic or vascular angiomatosis and even a cystic form of peritoneal mesothelioma, which is considered a benign lesion (fig . The differential diagnosis of these pathologies should include ovarian cystic tumors, endometrial cysts and cystic tumors growing beyond organs, e.g. Of the pancreas, kidneys or liver . Solid tumors found at these sites include: leiomyoma and rhabdomyoma, angiomas as well as stromal, neurogenic and desmoid tumors (fig . Desmoid tumors, also called fibromatosis, are an interesting group of benign tumors that exhibit local aggressiveness and have considerable tendency to recur (in approximately 50% of cases). All lesions were hypoechoic with slightly irregular echo patterns, 59% of cases were well - defined and 41% had irregular margins . The differential diagnosis should include sarcomas, mainly the two types that are most frequently found in soft tissues: histiocytic fibrosarcoma and liposarcoma, i.e. Lesions characterized by higher echogenicity . It is of note that desmoid tumors frequently coexist with familial adenomatous polyposis, which is estimated at 918% of cases . Of all imaging modalities, some authors recommend punch or even surgical biopsy in the event of doubts . In this case, mesenteric lymphangioma as a multilocular lesion without flow (arrow) retroperitoneal neuroblastoma in a 13-year - old (arrows). Aorta, v inferior vena cava desmoid, poorly vascularized tumor (d) in the small bowel mesentery these changes are rarely encountered but they represent a rich spectrum of mainly mesenchymal neoplasms: fibrosarcoma, malignant fibrous histiocytoma, hemangiopericytoma, leiomyosarcoma, rhabdomyosarcoma and gastrointestinal stromal tumors (fig . An epithelioid malignant neoplasm encountered at this site is malignant peritoneal mesothelioma, a lesion that almost exclusively develops in males in the fifth and sixth decade of life . It can assume two morphological forms: irregular peritoneal infiltration without ascites (dry appearance) (fig . 13) and nodular thickening of the peritoneum and greater omentum with ascites (wet appearance). Lesions of this type must be distinguished from papillary serous carcinoma of the peritoneum (a rare neoplasm found in elderly women), abdominal carcinomatosis, in which ascites prevails over implants, and peritoneal tuberculosis, in which there are no tumorous lesions (but ascites is present, the greater omentum is thickened with preserved smooth surface and the mesenteric lymph nodes are enlarged). Lymphomas, however, are characterized by considerable abdominal lymphadenopathy, rare and slight ascites and no omental involvement . Rhabdomyosarcoma with rich vascularity in the retroperitoneal space (arrows) two views present poorly vascularized malignant mesothelioma of the greater omentum (m) this is the most numerous group with prevailing carcinoma foci, mainly deriving from abdominal organs, such as pancreas, stomach, large bowel or ovaries . These lesions spread by direct extension, via the lymphatic system or blood stream and through the peritoneal cavity . Their echotexture can vary . 14) and thickened parietal peritoneum (its normal thickness is up to 1.5 mm) (fig . Echogenic lesions that only slightly differ from the surrounding tissues are the hardest to find (fig . Peritoneal sites at which carcinoma implants tend to develop are: the recto - uterine pouch, right iliac fossa, right paracolic gutter and the region of the upper sigmoid mesentery . Moreover, ovarian carcinoma cells frequently migrate to the supra- and subhepatic area (fig . Well - designed prospective studies have proven ultrasound imaging to be highly useful in detecting abdominal carcinomatosis . It occurred that lesions in the greater omentum were the easiest to detect (91%) whereas those in the small bowel mesentery were the hardest to find (67%). Moreover, ultrasound guidance enables one to obtain diagnostically valuable material for cytological and histological analysis in the vast majority of such cases . Metastasis of malignant melanoma (m) from the interscapular space to the small bowel mesentery peritoneal carcinomatosis of ovarian carcinoma . Thickened hypoechoic parietal peritoneum with signs of vascular flow (arrows) two views show a hyperechoic implant of ovarian carcinoma in the gastrocolonic ligament (arrows) ovarian carcinoma implants in the pouch of morison (arrows) another rare neoplasm encountered in fatty bodies of the abdominal cavity is non - hodgkin lymphoma . The involvement of the small bowel mesentery, with prominent, large mesenteric lymph nodes, is observed to occur in a half of patients with this neoplasm . These lymph nodes, together with the superior mesenteric vessels, create a so - called sandwich image (fig . It is usually a small neuroendocrine neoplasm located in the small bowel . Sometimes, the first morphological sign of its existence is the presence of mesenteric abnormalities in the form of enlarged lymph nodes or a hypoechoic mass . Mesenteric thickening or contraction, which can lead to small bowel obstruction, is more rarely a predominant element of the image . Any doubts in the assessment of these lesions require verification in computed tomography and, sometimes, magnetic resonance imaging . Ultrasound imaging, however, enables precise, percutaneous sampling for cytological and histological analysis, which is worth remembering and applying . Sandwich sign in the small bowel mesentery (arrows) created by enlarged lymph nodes surrounding the superior mesenteric vessels a manifestation of non - hodgkin lymphoma extranodal location of follicular lymphoma in the small bowel mesentery (arrow) lesions in the lymph nodes, which are relatively frequently affected by various neoplastic and non - neoplastic processes, have been presented only briefly since these issues are too broad to be included in this review . Authors do not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication and/or claim authorship rights to this publication.
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The national cancer institute (nci) of the united states of america has proposed a reporting system for thyroid fine needle aspiration (fna) cytology, so - called bethesda system, which became an international standard of thyroid cytology [1, 2]. Following this recommendation, the uk royal college of pathologists (the uk system) [3, 4] and italian societies of endocrinology and the italian society for anatomic pathology and cytology joint with the italian division of the international academy of pathology (the italian system) [5, 6] updated their diagnostic schema comparable with the bethesda system . In 2013, the japan thyroid association (jta) published guidelines for clinical practice for the management of thyroid nodules, including its diagnostic system for reporting thyroid fna cytology as shown in table 1 [79], using criteria similar to those used in the papanicolaou society recommendation, the bethesda system [1, 2], the uk system [3, 4], and the italian system [5, 6]. The jta reporting system of thyroid cytology further recommends risk stratification of follicular neoplasms (fn) into favor benign (low risk: lr), borderline (moderate risk: mr), and favor malignant (high risk: hr), which was adopted from the practice of high - volume thyroid centers in japan . This study examined observer variation in the subclassification of fn among 4 thyroid experts to validate the usefulness and limitations of this characteristic risk stratification of fn recommended in the jta guidelines . It is not our purpose to address in detail all the morphological issues of thyroid cytology . Conventional smear samples of indeterminate diagnosis (n = 389) were selected from files (n = 3843) from the year 2005 at ito hospital, tokyo, japan, by one (k. kameyama) of the authors . Inadequate samples or those with poor preparation and cases under clinical follow - up (no final histologic diagnosis) were excluded . There were 91 (23.4%) surgically treated patients with thyroid nodule under indeterminate cytological diagnoses . These patients visited ito hospital in 2005 and underwent diagnostic surgery or curative surgery in the subsequent years between 2005 and 2008 . There were 48 cases of benign diagnoses, including 29 follicular adenomas (fas), 19 adenomatous nodules (an), and 43 malignant diagnoses, including 13 follicular carcinomas (ftcs), 24 papillary carcinomas (ptcs), 1 poorly differentiated carcinoma, 2 c - cell carcinomas, and 3 malignant lymphomas . Twenty cases of cytological smear samples with follicular patterned lesions were randomly selected by one (k. kameyama) of the authors and they were further analyzed for subclassification of fn and diagnoses by 4 reviewers, as shown in table 2 . The present study is a reproducibility study undertaken by 4 reviewers (kennichi kakudo, kaori kameyama, mitsuyoshi hirokawa, and ryohei katoh) who have special interest in thyroid pathology, all of whom are members of the clinical guideline committee of the jta . They were requested to subclassify fn following the new jta reporting system of thyroid cytology as shown in table 1 . Those 20 cases of smear samples (one representative smear sample each stained by the papanicolaou method) were circulated among the 4 reviewers without clinical information . The 4 reviewers examined cytological samples independently without exchanging opinions and were unaware of the original cytological diagnoses and final histologic diagnoses . Because this study was conceived as an audit of cytology performance and the results are anonymized, institutional ethical committee permission was not required for its conduct . Informed consent of all 4 reviewers was obtained for this study protocol . For statistical analysis of observer concordance, was first introduced as a measure of the level of agreement between pairs of raters and extended to multiple raters, known as composite [11, 12]. Composite statistical analysis was performed by using data analysis and statistical software, version 13 (stata press, college station, texas, usa). Values of can be interpreted as follows: 0.000.20, slight or very weak agreement; 0.210.40, weak to fair agreement; 0.410.60, moderate agreement; 0.610.80, substantial or good agreement; and values over 0.81, optimal or almost perfect agreement . There were 8 malignant cases (2 ftcs, widely invasive; 4 ftcs, minimally invasive; and 2 ptcs) and 12 benign cases (1 an and 11 fas). All diagnoses made by the 4 reviewers on the 20 follicular pattern lesions are shown in table 2 . There were only 6 (30%) cases with a consensus diagnosis among all 4 of the reviewers: 3 in fa and 3 in ftc . Diagnoses of fn and hr were made in only 7 cases (35%), including 4 malignant and 3 benign cases . Composite statistical analysis clearly demonstrated good concordance for the hr diagnosis in malignant cases (= 0.7714) (table 3), while this was not found for the other subcategories in both benign and malignant lesions (<0.4). It is of note that there was no interobserver variation in ftcs, widely invasive (n = 2), and all 4 reviewers classified the 2 cases into the hr subcategory . It is clear that ftcs, widely invasive, have different degrees of cellular abnormality in our study . In contrast, there were significant disagreements among other types of malignancy and benign lesions, which clearly confirmed that the subclassification of fn is not a definite diagnosis but risk stratification useful for triage patients . Concerning ftc, minimally invasive (n = 4), there was only one concordant case among the 4 reviewers and all reviewers made a mr diagnosis in 1 case . There were disagreements between mr and hr in 1 case and between lr and mr in 2 cases . As for ptc (n = 2), the 4 reviewers gave 2 diagnoses each, which included 3 lrs, 3 mrs, 1 hr, and 1 suspicious for ptc . However, it is of note that there was no single case in our 20 cases whose subclassification varied among all 3 subcategories . In benign final histologic diagnosis (n = 12), there were 9 split diagnoses, including 6 cases between lr and mr and 3 cases between mr and hr, but no case between lr and hr . The incidence of lr diagnoses by the 4 reviewers in the 12 benign lesions was 31.3% (15/48) and that of lr diagnoses in the 6 cases of ftc was 16.7% (4/24) (p = 0.186). The incidence of hr diagnosis in the 12 benign lesions was 10.4% (5/48) and that of hr in the 6 cases of ftc was 47.1% (10/24) (p = 0.002). The incidence of malignancy of the hr was high at 60% for reviewer a, 75% for b, 75% for c, and 75% for d (6075%). The incidence of malignancy in the mr was lower than that of hr and was 33.3% for reviewer a, 35.7% for reviewer b, 25% for c, and 30.8% for d (2535.7%). The incidence of malignancy in the lr was lower than that in the hr and was 33.3% for reviewer a, 0% for b, 37.5% for c, and 33.3% for d (037.5%). Although the risks of malignancy in the subcategories of fn differed among the 4 reviewers, it is of note that hr cytological diagnosis was significantly correlated with malignant histological diagnosis, and the risk of malignancy (6075%) was significantly higher than that in the other 2 subcategories (035.7%) in our study (p = 0.002). Although the standardization of terminology and diagnostic criteria is important for accurate communication among patients, clinical doctors, and cytopathologists [110], there are still differences among reporting systems in thyroid cytology as to how to interpret cytological diagnoses and how to decide on the clinical management of patients . In japan, cytopathologists attempted to use an internationally accepted reporting system, but it had to be modified to fit our practice . In japan, all patients with indeterminate cytology undergo further diagnostic procedures, without immediate surgery, to search for higher risk patients who should undergo surgery [79, 1315]. It is because the majority of thyroid carcinomas in indeterminate cytology are indolent and more conservative approaches, other than immediate diagnostic surgery, usually do not create any harm to the patient with malignancy [1620] and diagnostic surgery to all patients with indeterminate cytology results in risks of unnecessary surgery to the patients with benign nodules, more than 80% of the patients [79, 1922]. The proportion of malignancy found at thyroidectomy from patients with indeterminate cytology in this clinical setting will increase in number, and the malignancy rate of patient with indeterminate cytology in our study was calculated as 47.3% as shown in materials and methods . Takezawa et al . From japan retrospectively analyzed their 1606 cytological samples using bethesda system, although it was written in japanese with english abstract, and they identified 115 (7.9%) cases of aus / flus (atypia of undetermined significance / follicular lesions of undetermined significance) and 61 (4.2%) cases of fn / sfn (follicular neoplasms / suspicious for follicular neoplasms). The resection rate of their aus / flus nodules was 30.4% (35/115 cases) and its malignancy rate was 88.6% (31/35 cases), and the resection rate of fn / sfn nodules was 36.0% (22/61 cases) and its malignancy rate was 72.4% (16/22 cases), which were very different from the ranges reported in most of the literatures from western countries using the bethesda system or the uk system [1, 2, 2023]. As it is clear, further triage of patients with indeterminate nodules reduces resection rates and increases malignancy rates in any diagnostic systems including the bethesda system . This clinical management was also true in some other countries [2427], as crippa and dina commented in a letter to an editor that thyroid cytology is the most important but not the only important deciding factor and therefore it must be integrated with other diagnostic procedures . We propose that future thyroid cytology classification schemes should reconsider clinical managements of indeterminate categories and how to reduce unnecessary surgeries for patients with benign thyroid nodules [79]. The risk stratification of follicular pattern lesions into 3 subcategories (cellular follicular lesion, fn favor benign, and fn favor malignant) was suggested by the papanicolaou society in 1996, but it did not become popular in thyroid cytology worldwide, apart from japan [8, 9]. There have been some reports in the literature on risk stratification of fn [10, 2834]. Kelman et al . Reported that 31/52 (60%) nodules with nuclear atypia consistent with fn were malignant and it was 4/9 (44.4%) in fn with atypia by goldstein et al . . Reported that atypical proliferation was more often malignant than follicular group (53% versus 19%) in their indeterminate category (tir3/thy3). Some researchers pointed out that the malignancy rate of fn without atypia is low and assessment later on could be an alternative approach [31, 32] and patients with high - risk cytological features such as nuclear overlapping (crowding) should be advised to have a surgical intervention [3133]. Gerhard and da cunha santos using the papanicolaou society guidelines studied reproducibility between 2 observers in 97 diagnoses . They reported a substantial level of diagnostic interobserver (= 0.71) and intraobserver (= 0.66) reproducibility, although interobserver disagreement in the cytological diagnosis occurred in 23 cases (24.7%) and 18 (41.7%) of them were for fn . In an interobserver reproducibility study using the uk system, kocjan et al . Reported that the statistic was very poor (0.11) for the thy3a category and that for thy4 was 0.17, in contrast to moderate to good agreement for thy1 (0.69), thy2 (0.55), thy3f (0.51), and thy5 (0.61). The observer variation of fn in our study occurred more often between lr and mr (9 cases, 45%), followed by between mr and hr (5 cases, 25%), but it is remarkable to note that none occurred between lr and hr . In other words, discordance is limited to between lr and mr or mr and hr, so we may conclude that the mr subcategory has an essential role in minimizing discordance between lr and hr . Another choice of subclassification of follicular neoplasms would be two categories (low cancer risk and high cancer risk) instead of three categories (lr, mr, and hr), and this modification (two categories) is also described as acceptable in the reporting system recommended by the japan thyroid association [7, 8]. The second conclusion we may draw is that hr in the jta system is a powerful cytological subcategory to be used for the triage of patients for diagnostic surgery because the risk of malignancy of hr is high (6075%) equivalent to suspicious for malignancy category in the bethesda system, with good concordance among the 4 reviewers (= 0.7714). Abele and levine reported their rate of indeterminate category to be 5% of 51,000 adequate fnas and they suggested that the national rate of 15% was in large part due to overdiagnosis . This significant difference in ratio of indeterminate categories may be due to experience of thyroid cytology and not patients' background . Clary et al . Commented in their interobserver variability study that some pathologists make greater use of indeterminate categories such as follicular lesion, favor nonneoplastic or follicular lesion, and favor neoplastic lesion, whereas others show more definitive categorization into benign and neoplastic groups . Cibas et al . Also stated in their report on interobserver variability that cytopathologists with experience of thyroid cytopathology are more likely to make a definitive interpretation (i.e., benign or malignant). This tendency was seen in our present study in which reviewers b and d (whose indeterminate diagnosis rates are about 15% in their practice) made mr diagnosis more often (70% and 65%, resp .) And lr diagnosis less frequently (10% and 15%, resp .) Than those of reviewers a and c (whose indeterminate diagnosis rates are about 5% in their practice). The difference in the prevalence of benign and fn / sfn may explain the different rates of lr (low risk) and mr (moderate risk) among reviews in our study, because incidence of one category may expand or contract depending on the rates of other categories . Some fn / sfn lesions with benign pattern would be classified as benign by different authors and the incidence of fn / sfn of bethesda system in recent 7 series varied from 1.5 to 9.7% and that of benign category was between 54 and 77.4% summarized by ohori and schoedel . Rapid development of molecular analyses on thyroid cytology may lead us possibly in the near future to more accurately identify patients who should be referred to surgery . Until that time comes, thyroid fna cytology remains a main stay in the management of patients with thyroid nodules integrated with other clinical tests, such as ultrasound image diagnosis . As a conclusion the hr subcategory has a high predictive value of malignancy and good agreement among the 4 reviewers which is clinically helpful to triage patients for surgery . We believe that patients with cytological diagnosis of hr subcategory of fn in the jta system should be surgically treated especially if other risk factors coexisted . On the other hand, patients with lr or mr category therefore, surgical resection rate of indeterminate category is low in japan usually less than 50%, particularly in cases with fn.
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It is not uncommon to see bleeding symptoms in patients in outpatient or hospital - based practice . Postpartum hemorrhage complicates pregnancy and accounts for significant morbidity and mortality, particularly in underdeveloped countries.1,2 menorrhagia is a common clinical challenge and is often associated with secondary anemia, excessive fatigue, and a negative effect on health - related quality of life.3 postoperative bleeding is one of the more common complications of surgery . Trauma is a leading cause of morbidity and mortality in the younger population.4 while bleeding symptoms may be commonly seen by physicians of multiple specialties, it is unclear how frequently these symptoms belie an underlying undiagnosed congenital or acquired bleeding disorder . In the us, the most common congenital bleeding disorders include von willebrand disease, which affects approximately 1% of the population (males and females equally),5 and hemophilia a and b combined, which affect approximately 20,000 persons (essentially all males, with rare exception).6 medications can also affect coagulation or platelet function,7 as can certain herbal supplements.8 trauma and surgery can lead to blood loss, and critical reduction in coagulation factors can lead to additional non - surgical bleeding complications (coagulopathic bleeding).9 the most ubiquitous method for evaluating coagulation is prothrombin time (pt)/international normalized ratio (inr) and activated partial thromboplastin time (aptt). Typically, they are ordered to monitor anticoagulant therapy (pt / inr for warfarin, aptt for heparin), to evaluate coagulation preoperatively, or in response to hemorrhagic symptoms . The pt / inr provides an assessment of the extrinsic (tissue factor - dependent) and final common pathways of the coagulation cascade, while the aptt provides an assessment of the intrinsic (tissue factor - independent) and final common pathways.10 an example of a potentially life - threatening cause of unexplained recent onset or acute bleeding associated with a prolonged aptt is acquired hemophilia, with an incidence of approximately 1 to 4 per million / year.11 acquired hemophilia primarily affects older adults,12 an ever - growing segment of the population that presents for medical evaluation and care, but may also occur during pregnancy as postpartum hemorrhage or in association with other underlying diseases, including cancer and autoimmune disorders.13 prompt diagnosis is a primary determinant of prognosis in acquired hemophilia14 because initiation of definitive therapy (ie, hemostatic and immunosuppressive) is delayed until the diagnosis is made . Acquired hemophilia - related bleeding does not respond to the typical management algorithms used for hemorrhaging in a patient and, therefore, is associated with high morbidity and mortality . Severe bleeds occur in up to 90% of patients with acquired hemophilia,11 and the reported overall mortality rate in these patients ranges from 8% to 22%.1517 given the rarity of acquired hemophilia, combined with the general lack of familiarity of nonhematologists with this condition, the diagnosis of acquired hemophilia poses a clinical challenge, even in patients presenting with straightforward bleeding and an isolated, prolonged aptt . A survey was conducted of physicians across a number of specialties to identify potential barriers to the effective recognition and management of this rare but important cause of serious bleeding . The survey, based on an actual case found to be the result of acquired hemophilia, focused on participants stepwise evaluation and management of a case patient who presented to the hospital with recent - onset bleeding . The survey also assessed participants history with regard to consulting hematologists, discovering or diagnosing underlying bleeding disorders, and encountering acquired hemophilia in clinical practice . Findings pertaining to the interpretation and follow - up of abnormal coagulation studies, enlistment of hematology consultation, and the diagnostic decision process in an actively bleeding case patient were the primary areas assessed in this analysis . Results were evaluated across specialties to determine any specialty - specific practice trends that might hinder effective recognition and management of an actively bleeding patient with coagulopathy, including acquired hemophilia . Physicians within the specialties of hematology, hematology / oncology, emergency medicine, geriatrics, internal medicine, rheumatology, obstetrics and gynecology, critical care medicine, and general surgery were randomly sampled from the american medical association physician masterfile . Physicians who were part of the harris interactive online physician panel were invited via email to participate in the survey, while physicians who were not part of the panel were invited via first class mail . The invitation provided a uniform resource locator address and password for one - time use to log on to the survey site, where invitees first encountered questions to determine eligibility to participate in the survey . Eligible physicians were required to be actively practicing, with additional specialty - specific requirements . In particular, hematologists, hematologist / oncologists, general surgeons, and emergency medicine and critical care physicians were required to be practicing at an acute care hospital . For internists and geriatricians, at least 60% of their practice had to consist of patients older than 60 years of age . Obstetricians / gynecologists were required to have an active obstetrics practice . Upon confirmation of eligibility, participants completed the online survey, the average duration of which was approximately 10 minutes . Each specialty was recruited to a final sample of 50 complete responses to ensure a sufficient number of responses to generate reasonable hypotheses about specialists behavior to test in face - to - face interviews . Findings from the surveys completed by the physicians specializing in hematology, hematology / oncology, emergency medicine, geriatrics, internal medicine, rheumatology, and critical care medicine are presented herein . General surgeons and obstetrics and gynecology physicians each completed different surveys from the aforementioned specialties; the findings of those surveys are not included here . Survey questions focused on the diagnostic and general therapeutic approach to the management of a case patient (figure 1) whose presentation and clinical course were based on those of a real patient who experienced significant delays in diagnosis despite the involvement of multiple specialists . At each juncture in the case, participants were given lists of actions that included diagnostic tests (laboratory, radiology, other), consultations, and potential treatments addressing bleeding or anemia (local hemostasis, transfusion). Since familiarity with the upper limits of normal of the pt / aptt tests was being tested in this study, upper limits of normal were not specified, and aptts were 25% to 30% above the upper limits of normal and set at a value that respective specialties would recognize as abnormal based on their experience with ordering and interpreting this laboratory test . The survey did not specify whether laboratory testing, imaging studies, or consultations in this hypothetical case had to be available immediately or locally, and the assumption was that respondents would answer based on their best medical judgment in an ideal practice situation . Rheumatologists were only asked about an initial emergency department / urgent care presentation because it was expected that if a bleeding disorder developed in one of their patients it would present in an outpatient or emergency department / urgent care setting . Conversely, critical care specialists started at presentation 2, since their involvement in a recurrent epistaxis workup would be unlikely . Vital signs were adjusted to make it more likely that a potential intensive care unit admission would require their evaluation . Since emergency medicine physicians would most likely have ordered an imaging study initially to define the patient s retroperitoneal hematoma, we allowed for the option of a general surgery consultation even though the computed tomography findings were never presented . Hematology / oncology respondents were given the option of ordering additional specialty coagulation laboratory tests (eg, mixing studies). Participants were additionally questioned about their experiences diagnosing underlying bleeding disorders and, in the case of nonhematologist specialists, consulting hematologist colleagues . Conversely, hematology and hematology / oncology specialists were asked about being on the receiving end of consultations, specifically from emergency medicine personnel, and the reasons for those consultations . A total of 302 physicians (5051 per specialty) participated in the case - based survey reported herein . Demographics of the specialty groups were consistent: mean age ranged from 45.6 to 50.6 years, and mean years of practice experience ranged from 14.5 to 19.3 . Faced with an older adult female patient with recurrent epistaxis, nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies (pt / inr and aptt) as part of the initial evaluation (figure 2a). Despite abnormal results of the aptt at 42 seconds (with an upper limit of normal typically ranging from 35 to 39 seconds, although not specified in the case), less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps (figure 2b). In contrast, 67% of hematologists would have repeated these studies . Less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results (figure 2b). Rheumatologists were most likely to obtain a consult (43%), although they were presented with an initial aptt that was more abnormal (50 seconds) than the aptt initially presented to the other specialists . After the patient s second presentation several weeks later with bruising and abdominal / back pain, again nearly 90% or more of respondents (including critical care specialists, who began their case review at this juncture in the patient s clinical course) would have ordered both a complete blood and coagulation studies as part of their initial evaluation (figure 2c). When these results revealed a clearly abnormal and markedly changed aptt of 63 seconds, the majority of respondents indicated they still would not have repeated coagulation studies (figure 2d). Approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point, compared with 47% and 50% of emergency medicine and critical care specialists, respectively (figure 2d). Sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point . In addition to the aforementioned hematologic laboratory tests, other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 . Participants across specialties clearly preferred computed tomography of the abdomen (80%84%) over abdominal ultrasound (9%28%), upper gastrointestinal endoscopy (2%16%), or colonoscopy (0%16%). Emergency medicine physicians demonstrated the greatest breadth of testing, with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test . Requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group, which also had the highest proportion of physicians recommending endoscopy (16% each for upper gastrointestinal endoscopy and colonoscopy). For the purposes of assessing multiple specialties, the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . By the end of this observation period, the aptt had further increased to at least twice the upper limit of normal, while the hemoglobin level had decreased . Internists and geriatricians were most inclined to repeat the laboratory studies at this juncture (figure 4). In contrast with previous time points, by this point in the patient s clinical presentation, 73% to 94% of respondents would have consulted a hematologist . The majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors (97%) and fibrinogen / fibrin split products testing (75%) in response to the laboratory results obtained after 12 hours of observation, as part of a diagnostic evaluation of the prolonged aptt . Emergency medicine physicians were given the additional option of consulting general surgery practitioners; 2% would have done so after the first set of laboratory results at the second presentation, and 20% would have done so in response to the second set of laboratory results . The percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a . Across the majority of specialties, less than 20% of physicians would have recommended overnight admission to a general hospital floor, while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home . In contrast with respondents from other specialties, a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility . The majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital . The differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation (figure 5b). While approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results, the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated (worsening anemia and coagulopathy). Based on the laboratory results obtained after 12 hours of observation, nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor, compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation . The difference was most noticeable for physicians in emergency medicine (35%73%), the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service . More than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder, while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one (figure 6a). The percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . Among those physicians who had previously diagnosed or discovered a bleeding disorder, 14% to 77% had specifically encountered acquired hemophilia (figure 6b), although this quantitative survey did not assess whether they understood this disorder . Hematologists and hematologists / oncologists accounted for the highest percentage in this group, while internal medicine specialists accounted for the lowest percentage . Table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation . The distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . In the majority of specialties (emergency medicine, geriatrics, and internal medicine), the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt . In contrast, more than 80% of rheumatologists had consulted a hematologist 3, 4, or 5 or more times for the same reason . The highest percentage (39%) of critical care physicians the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding (table 2), which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed (data not shown). The most common response among critical care specialists to the open - ended question, what are the circumstances around which you would call for a hematology consult? Was an unexplained abnormal lab or bleeding (33 like or similar mentions), followed by any unexplained bleeding disorder conversely, the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 . The majority (57%) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine . The majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding (table 2). Faced with an older adult female patient with recurrent epistaxis, nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies (pt / inr and aptt) as part of the initial evaluation (figure 2a). Despite abnormal results of the aptt at 42 seconds (with an upper limit of normal typically ranging from 35 to 39 seconds, although not specified in the case), less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps (figure 2b). In contrast, 67% of hematologists would have repeated these studies . Less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results (figure 2b). Rheumatologists were most likely to obtain a consult (43%), although they were presented with an initial aptt that was more abnormal (50 seconds) than the aptt initially presented to the other specialists . After the patient s second presentation several weeks later with bruising and abdominal / back pain, again nearly 90% or more of respondents (including critical care specialists, who began their case review at this juncture in the patient s clinical course) would have ordered both a complete blood and coagulation studies as part of their initial evaluation (figure 2c). When these results revealed a clearly abnormal and markedly changed aptt of 63 seconds, the majority of respondents indicated they still would not have repeated coagulation studies (figure 2d). Approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point, compared with 47% and 50% of emergency medicine and critical care specialists, respectively (figure 2d). Sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point . In addition to the aforementioned hematologic laboratory tests, other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 . Participants across specialties clearly preferred computed tomography of the abdomen (80%84%) over abdominal ultrasound (9%28%), upper gastrointestinal endoscopy (2%16%), or colonoscopy (0%16%). Emergency medicine physicians demonstrated the greatest breadth of testing, with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test . Requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group, which also had the highest proportion of physicians recommending endoscopy (16% each for upper gastrointestinal endoscopy and colonoscopy). For the purposes of assessing multiple specialties, the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . By the end of this observation period, the aptt had further increased to at least twice the upper limit of normal, while the hemoglobin level had decreased . Internists and geriatricians were most inclined to repeat the laboratory studies at this juncture (figure 4). In contrast with previous time points, by this point in the patient s clinical presentation, 73% to 94% of respondents would have consulted a hematologist . The majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors (97%) and fibrinogen / fibrin split products testing (75%) in response to the laboratory results obtained after 12 hours of observation, as part of a diagnostic evaluation of the prolonged aptt . Emergency medicine physicians were given the additional option of consulting general surgery practitioners; 2% would have done so after the first set of laboratory results at the second presentation, and 20% would have done so in response to the second set of laboratory results . The percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a . Across the majority of specialties, less than 20% of physicians would have recommended overnight admission to a general hospital floor, while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home . In contrast with respondents from other specialties, a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility . The majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital . The differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation (figure 5b). While approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results, the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated (worsening anemia and coagulopathy). Based on the laboratory results obtained after 12 hours of observation, nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor, compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation . The difference was most noticeable for physicians in emergency medicine (35%73%), the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service . More than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder, while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one (figure 6a). The percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . Among those physicians who had previously diagnosed or discovered a bleeding disorder, 14% to 77% had specifically encountered acquired hemophilia (figure 6b), although this quantitative survey did not assess whether they understood this disorder . Hematologists and hematologists / oncologists accounted for the highest percentage in this group, while internal medicine specialists accounted for the lowest percentage . Table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation . The distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . In the majority of specialties (emergency medicine, geriatrics, and internal medicine), the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt . In contrast, more than 80% of rheumatologists had consulted a hematologist 3, 4, or 5 or more times for the same reason . The highest percentage (39%) of critical care physicians had consulted a hematologist at least 5 times for such a patient . The most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding (table 2), which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed (data not shown). The most common response among critical care specialists to the open - ended question, what are the circumstances around which you would call for a hematology consult? Was an unexplained abnormal lab or bleeding (33 like or similar mentions), followed by any unexplained bleeding disorder conversely, the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 . The majority (57%) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine . The majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding (table 2). Bleeding is commonly encountered in outpatient and hospital - based medical practice and can have a wide variety of underlying causes . While bleeding may be relatively common, most acquired and congenital bleeding disorders are uncommon, and some, such as acquired hemophilia, are rare . Nevertheless, the consequences of failure to recognize promptly and treat properly a bleeding disorder may be significant.18 in the case of acquired hemophilia, morbidity and mortality rates are particularly high: severe bleeding is experienced by up to 90% of affected patients, and mortality rates are as high as 22%.11 this survey provided a step - wise methodology to tease out specialty - specific patterns of interpretation of clinical data to identify barriers to the diagnosis and treatment of underlying bleeding disorders . The sample size obtained across specialties was sufficient to generalize these findings, at least to the point of identifying specific issues for education and development of clinical decision - making pathways . When presented with a clinical picture that includes a recent history or symptoms of active bleeding, clinicians typically obtain coagulation times, such as the pt / inr and aptt, as part of the initial diagnostic evaluation . Proper interpretation of laboratory test results includes recognition of abnormal values and, more important, the potential clinical significance of such results . A common pitfall in the interpretation of coagulation times is failure to appreciate that even mildly abnormal values may represent a serious underlying coagulation deficit . Another important observation is to identify how an abnormal laboratory value may have changed over time, which can be facilitated by the ability of electronic medical record systems to display data trends . In the absence of iatrogenic causes, even a mildly elevated pt / inr or aptt may be indicative of true coagulopathy and should not be ignored or dismissed, particularly when there is evidence of bleeding, as was the case with this patient, even at initial presentation . After excluding laboratory error, the differential diagnosis of an isolated prolongation of aptt includes heparin effect, lupus anticoagulant, and deficiency of or antibody against an intrinsic pathway factor (viii, ix, xi, or xii).19,20 a detailed history, focusing on factors such as heparin exposure,19,20 history of thromboembolism (lupus anticoagulant),21 and prior personal or family history of bleeding,22 may provide diagnostic clues . Laboratory testing should include a 1:1 mixing test of patient plasma with control plasma to determine whether a prolonged aptt is the result of an intrinsic pathway factor deficiency or an inhibitor that continues to block the activity of the intrinsic system even in the presence of control plasma . The majority of inhibitor antibodies identified in this manner will turn out to be lupus anticoagulants . Although far less common, this is the same diagnostic pathway that leads to the identification of the antifactor viii antibodies associated with acquired hemophilia.23 unlike acquired hemophilia, lupus anticoagulants typically do not present with bleeding, and the abnormal aptt is due to interference with phospholipid - dependent coagulation reactions . Once an acquired antifactor viii antibody is suspected, confirmatory testing includes measuring factor viii activity, which should be significantly reduced, and the bethesda assay,24 which is used to quantitate antifactor viii antibodies inhibitor activity . We found a general lack of appropriate consideration and response to the presenting symptom of bleeding and the prolonged aptt throughout this case study . This is consistent with data from the european acquired haemophilia registry (each-2), which reported a median delay of 3 days between onset of bleeding symptoms and the diagnosis of acquired hemophilia and a median delay of one day between the first abnormal aptt test in those same patients and the established diagnosis.23 in addition, we found that emergency medicine and critical care physicians were reluctant to consider a bleeding disorder as the primary explanation for this patient s clinical presentation . The disposition of a patient with active hemorrhage and evidence of coagulopathy should be based on several factors, including the patient s current condition and anticipated clinical course, taking into account the presenting vital signs and evolving laboratory findings . At the time of the patient s second presentation, vital signs were notable for mild tachycardia and a pulse pressure at the upper limit of normal, and subsequent laboratory findings indicated a decreasing hemoglobin level and an increasing aptt . These findings alone prompted hospital admission, although the exact location (general floor versus intensive care unit) of admission may vary, based on the level of monitoring and nurse - to - patient ratios in a particular hospital . Another important variable is the anticipated potential for clinical deterioration, which is based in large part on clinician appreciation of the seriousness of the diagnosis . We found a consistent tendency to consider admission to a general floor bed with the second presentation, even though this was ultimately an unstable, critically ill patient with an undiagnosed bleeding disorder . We also found that the physicians who participated in this survey were reluctant to consult a hematologist as they worked through this case scenario, particularly given that options for additional testing (liver function, disseminated intravascular coagulation) were not available to evaluate for common causes of coagulopathy . Relative to their reported historical experience with hematology consultations during an average practice experience of approximately 20 years, this survey finding was somewhat surprising . Rheumatologists and critical care specialists reported a greater frequency of hematology consultation relative to the other specialties (table 2). We would expect the emergency medicine physicians to be most likely to consult a hematologist, yet 16% of them reported never having consulted a hematologist . The highest percentage (46%) had only consulted a hematologist 1 or 2 times, and almost one quarter of geriatricians and internists had never consulted a hematologist, even though these specialists would be expected to first encounter patients with undiagnosed bleeding disorders, including acquired hemophilia . One potential reason for not seeking hematology consultation might be the lack of availability of hematology / oncology specialists with expertise in coagulation disorders, including in rural and community hospitals . A limitation of this survey was that one cannot interpret the thinking behind the responses of the individual participants . Therefore, 31 qualitative 45-minute interviews were conducted subsequent to the quantitative study and focused particularly on critical care (n = 7), emergency medicine (n = 6), hematology / oncology (n = 4), or hematology (n = 2) physicians to understand the reasoning behind their decisions and depth of knowledge (unpublished data). We found that the physicians focus was generally on finding the source (location) of bleeding and not on finding the underlying reason for bleeding . This could potentially lead to surgical intervention in the face of an underlying bleeding disorder, with subsequent adverse outcomes . In a series of 67 patients with acquired hemophilia at a single center in bonn, germany, 4 of 5 deaths were the result of surgical intervention for bleeding at outside hospitals in the setting of a delayed diagnosis of acquired hemophilia.13 when queried about their experience encountering and/or diagnosing underlying bleeding disorders, particularly acquired hemophilia, more than 85% of physicians in hematology, hematology / oncology, emergency medicine, and critical care medicine reported having ever discovered or diagnosed an underlying bleeding disorder, compared with 65%, 54%, and 47% of rheumatologists, internists, and geriatricians, respectively . While reports of ever specifically having encountered acquired hemophilia were high, it is unclear from this study whether the participants truly understood the diagnosis . This seems unlikely, given the rarity of acquired hemophilia, relative to the reported frequency of having encountered it . Except for hematology and/or oncology (77%) and critical care (36%) specialists, approximately one quarter of surveyed physicians had ever encountered acquired hemophilia . Although they accounted for the highest percentage of physicians who had ever encountered this condition, nearly one quarter of hematologists had never encountered acquired hemophilia . Subsequent unpublished data from the aforementioned qualitative research further suggest that, compared with hematology practitioners, specialists in hematology / oncology, who likely practice mostly oncology, might be able to identify mixing studies and inhibitors but might not fully understand the underlying pathophysiology that constitutes acquired hemophilia, making it hard for them to recognize the condition . Given the survey findings reflecting the infrequency with which most physicians have encountered these conditions, consultation with a hematologist may facilitate the diagnostic evaluation and proper management of a hemorrhaging patient suspected of having an underlying bleeding diathesis, particularly acquired hemophilia . The consulting hematologist can provide specific guidance, leading to the prompt diagnosis and optimal management of an actively bleeding patient with acquired hemophilia, including initiation of immunosuppression, which is usually necessary to eradicate the inhibitor and to prevent additional bleeding episodes . However, this requires a level of familiarity and expertise in treating acquired hemophilia and other rare bleeding disorders that is not often seen outside of an academic hematology practice . This represents yet another barrier to the effective diagnosis and management of this rare yet serious bleeding diathesis . For the hospitalist or intensivist charged with the care of a bleeding patient, immediate stabilization is the initial priority and should take precedence over determination of a specific etiology of bleeding . However, in the presence of underlying coagulopathy, particularly acquired hemophilia or other rare disorders, traditional measures of stabilization may not be as effective as expected . A prolonged pt / inr or aptt in the absence of iatrogenic causes should never be ignored, even with only minimally prolonged values, and determination of the cause of an abnormal coagulation study should carry at least equal weight to looking for the anatomic site of bleeding . Any delay in establishing the diagnosis of a bleeding diathesis such as acquired hemophilia can result in significant morbidity or even death . While hospitalists and intensivists should be able to conduct a thorough differential of bleeding and eliminate most common etiologies, consultation with a hematologist (particularly one with specific expertise in coagulation disorders) may facilitate the evaluation of coagulopathic patients and subsequent interpretation of diagnostic findings, as well as initiation of appropriate treatment . Given the rarity of acquired hemophilia, as exemplified by the findings of this survey, physicians must harbor a high index of suspicion to diagnose this condition promptly in patients who present with recent - onset or acute bleeding . Given the high morbidity and mortality the insights from this survey highlights knowledge and practice gaps that could be the focus of targeted educational initiatives, including diagnostic algorithms, to ensure proper and efficient workup of the abnormal laboratory studies that characterize acquired hemophilia.
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Plant cells need to maintain the functional integrity of their walls during cell morphogenesis and exposure to biotic / abiotic stress . The available evidence suggests that a dedicated plant cell wall integrity (cwi) maintenance mechanism exists (wolf et al ., 2011). While our understanding of the mechanisms regulating stress responses and morphogenesis has increased significantly, our knowledge regarding the processes maintaining cwi is still limited . In the last years several reviews have been published on the plant cwi maintenance mechanism illustrating the increased interest in this area (humphrey et al ., 2007; hematy et al ., 2009; ringli, 2010; seifert and blaukopf, 2010). A recently published review focuses on cwi maintenance during plant cell wall morphogenesis (wolf et al ., 2011). Similarities between the yeast and plant cwi maintenance mechanisms have also been reviewed (hamann and denness, 2011). Therefore, the available knowledge regarding cwi maintenance during plant development and in yeast will be covered here only briefly to provide a conceptual framework regarding cellular processes involved and to illustrate the degree of functional conservation between species . This review will focus on recent developments regarding the role of cwi maintenance during biotic stress responses . It will discuss how cwi maintenance could have a previously unrecognized role in the perception of and response to biotic stress . While both plant and yeast cells are enveloped by cell walls, certain important differences exist that affect the biological role and function of the plant cwi maintenance mechanism . In addition, plant cell walls are structurally and chemically more complex than the yeast cell wall . This means that in plant cells the sheer number of cell wall - related signaling events during development and plant environment interaction could disguise the activity of a dedicated plant cwi maintenance mechanism . The yeast cwi monitoring and maintenance network is quite complex, providing an indication of the possible complexity of the plant cwi maintenance network . By combining inputs from a turgor pressure sensor (sln1), mechano - perception (mid1/cch1), and dedicated cell wall damage (cwd) sensors (wsc1, 2, 3, mid2, mtl1) the yeast cwi maintenance network generates signals that permit highly specific responses to any challenge that impairs the functional integrity of the yeast cell wall . The available phenotypic and genetic data also implicate turgor pressure, mechano - perception, and cwd detection in plant cwi maintenance (hamann et al . Interestingly, arabidopsis histidine kinase1 (ahk1) and cytokinin receptor1/arabidopsis histidine kinase4 (cre1/ahk4) can at least partially rescue a yeast strain with a loss of function allele in sln1 (urao et al . In addition, expression of the arabidopsis thaliana mid1 complementing activity 1 and 2 (mca1,2) genes rescues a mid1-deficient yeast strain suggesting that they could function as stretch - activated calcium channels (nakagawa et al . No functional homologues for the yeast cwd sensors have been identified in plants . In yeast, the signals generated by the sensors are relayed to the response genes via different signaling cascades involving calcineurin (mid1/cch1); rho1/mpk1 (wsc1, mid2), and ypd1 (sln1) (levin, 2005). Transcription factors mediating the response are skn7, rlm1, and swi4/6 (levin, 2005). The response can involve activation of genes required for cell wall biosynthetic processes, remodeling of the cytoskeleton, and cell cycle progression . The available data suggest both organizational and functional similarities between the yeast and plant cwi maintenance mechanism while also highlighting how signals from mechanical and chemical sensors regulate jointly the cwd response . The plant cell wall is comparable to an exoskeleton surrounding the plant cell and providing both structural support and protection from biotic as well as abiotic stresses . It consists of cellulose microfibrils, pectin, hemicelluloses, proteins, and in certain cases lignin (somerville et al ., 2004). Plant cell walls are divided into primary (laid down during cell elongation / differentiation) and secondary (formed after cell morphogenesis is concluded) walls . In parallel, dependent on the presence of certain polysaccharides type i and type ii cell walls are distinguished (popper et al ., 2011). Cellulose microfibrils are the main load bearing elements, which are cross - linked to hemicelluloses and (in vitro) to pectin (dick - perez et al ., 2011). Hemicelluloses and pectin also form direct links creating a matrix in which the microfibrils are embedded like the steel mesh in a concrete wall . Pectic polysaccharides like homogalacturonan (hg) are connected by calcium bridges between dimethyl - esterified parts of the molecules or through borate ester linkages in the case of rhamnogalacturonan ii (rg ii). They are targeted by pathogen - derived cell wall degrading enzymes (polygalacturonases), which generate oligogalacturonides (ogas) from hg (hahn et al ., 1981; kars et al ., 2005). Biologically active ogas consist of chains of 915 galacturonic acid (gala) monomers and can function as signaling molecules (see below). During secondary cell wall formation monolignols (precursors for lignin) are secreted into the cell wall space and randomly cross - linked (vanholme et al ., 2010). The cross - linking is dependent on the availability of reactive oxygen species (ros) generated by laccases and peroxidases . This process reinforces the wall against pathogen infection, waterproofs it, and increases structural integrity (tronchet et al ., 2010; reduction of cellulose biosynthesis during primary cell wall formation through genetic or chemical means leads to lignin production (ellis et al ., 2002; hamann et al ., this highlights the ability of the cell to adapt to changes in cell wall composition and provides evidence for the existence of a cwi maintenance mechanism . Plant cell walls are capable of adjusting their composition and structure in response to pathogen infection (dong et al ., 2008). A mutation in (cellulose synthasea3, cesa3) a subunit of the cellulose synthase complex leads to ectopic production of lignin, i.e., replacement of a missing load bearing cell wall component by another one (cano - delgado et al follow up studies found that inhibition of cellulose biosynthesis during primary cell wall formation either through mutations like constitutive expression of vsp1 (cev1) and ectopic lignification1 (eli1) or inhibitors such as isoxaben results in transcriptional activation of stress response mechanisms; ectopic production of ethylene (et), salicylic acid (sa), jasmonic acid (ja), callose, and ros as well as changes in cell wall composition and structure (ellis et al ., 2002; cano - delgado et al ., 2003; manfield et al ., 2004; hamann et al ., the experiments also showed that the response to cwd consists of early and late stages reminiscent of the response to pathogen infection (denness et al ., 2011). During the early stage, ros- and calcium - based signaling cascades are required for initiating the response to cwd (denness et al ., 2011). Interestingly, 1-aminocyclopropane-1-carboxylic acid (acc, an et precursor) and not et itself seems to be acting as signaling substance during the early response to isoxaben with inhibition of cell expansion being an active process and not simply an automatic consequence of cellulose biosynthesis inhibition (tsang et al ., 2011). During the late stage, responses to cwd - like lignin deposition are initiated and the extent of lignin formation is apparently modulated by a negative feedback loop formed by ros and ja (denness et al ., 2011). A combination of genetic and phenotypic analysis has implicated the nadph oxidases respiratory burst oxidase homologd and f (rbohd, f), the serine / threonine kinase oxidative signal inducible1 (oxi1), mca1, the receptor - like kinase (rlk) theseus1 (the1) as well as the ja biosynthesis genes allene oxide synthase (aos) and jasmonic acid resistant1 (jar1) in the signaling mechanism mediating the response to cwd in arabidopsis seedlings . Interestingly, the cev1 mutation that affects cellulose biosynthesis during primary cell wall formation also causes enhanced resistance to infection by different powdery mildews (erysiphe orontii, e. cichoracearum, and oidium lycopersicum; ellis and turner, 2001). A screen for mutants causing resistance to powdery mildew infection provides further evidence of the close relationship between plant cell walls and pathogen resistance (vogel and somerville, 2000). Three of the powdery mildew resistance (pmr) mutants that have been identified on the molecular level affect genes involved in cell wall biosynthetic processes . Pmr4 encodes a callose synthase, pmr5 a gene of unknown function required for pectin production and pmr6 a pectate lyase (vogel et al . Pmr4 resistance seems to be mediated via hyper - activation of sa signaling, whereas pmr5 and 6 resistance phenotypes are independent of ja, sa, and et signaling . Mutations in irregularxylem1 (irx1/cesa8), 3 (irx3/cesa7), and 5 (irx5/cesa4) impair cellulose biosynthesis during secondary cell wall formation and cause enhanced resistance to the soil borne bacterium ralstonia solanacearum and the necrotrophic fungus plectosphaerella cucumerina (hernandez - blanco et al ., 2007). Mutations affecting cellulose biosynthesis during primary cell wall formation (cesa1, 3, 6) or other components of the secondary cell wall (pmr5, pmr6) did not cause enhanced resistance to the same pathogens . Genetic analysis showed that the enhanced resistance in cesa4, 7, 8 is independent of ja, sa, and et - based signaling mechanisms . Results from expression profiling experiments and genetic analysis using different abscisic acid (aba)mutants(aba insensitive 1 - 1;2 - 1;aba1 - 6) suggest that aba is mediating developmental and pathogen resistance phenotypes caused by the irx mutants . However, it remains to be determined if the aba involvement is direct or a secondary effect due to water stress caused by problems with xylem cell wall formation in the mutants . To summarize, these results suggest distinct resistance signaling cascades are induced by defects in primary and secondary cell wall formation as well as for different secondary cell wall components . They also highlight the direct impact of changes in cell wall composition / structure on the response to pathogen infection . The mode of action of the plant cell wall maintenance mechanism is not well understood . Based on the knowledge from yeast, chemical and physical signals could act as indicators for the functional integrity of the plant cell wall either individually or jointly . By combining these different types of signal the plant cell would receive precise information regarding the state of its cell wall and the exact type of cwi impairment occurring . Physical signals could be generated by stretching of the plasma membrane due to a weakened cell wall that cannot resist the high turgor pressure levels within a plant cell or a plasma membrane that is displaced relatively to the cell wall . These events could be detected by stretch - activated or mechanosensitive channel proteins that lead to calcium influx into the cytoplasm, indicating cwd . Sensor candidates could be encoded by members of the mechanosensitive channels of small conductance (mscs)-like (msl) gene family like msl 9 and 10 affect mechano - perception in protoplasts derived from arabidopsis root cells (haswell et al ., 2008). Another candidate of interest is the putative stretch - activated calcium channel mca1 is required for cwd - induced lignin deposition . Interestingly, all isoxaben - induced cwd phenotypes can be suppressed by provision of osmotic support suggesting that changes in turgor pressure due to a weakened cell wall could result in signal generation via turgor pressure sensors (hamann et al ., 2009; denness et al ., 2011). While ahk1 and 4/cre1 can function as osmosensors in yeast and have been implicated in abiotic stress responses, no clear evidence exists implicating them in cwd perception in plants (urao et al ., 1999; inoue et al ., 2001; tran et al ., 2007). In addition, ahk4/cre1 has been shown to function as a cytokinin receptor(inoue et al ., 2001). Therefore, the question that needs to be resolved at this point is if turgor pressure is a passive element in the process (generating cell wall fragments due to a weakened cell wall) or an active component that is being monitored and provides input into the process . The plant cell wall contains a large number of components that could generate chemical signals (ligands) indicative of cwd or general danger signals . The term damage associated molecular patterns (damps) has been coined to describe such ligands and the number of possible damps originating in plant cell walls is rather large (zipfel, 2009). Here i will focus on the best - characterized group of signals, which are probably ogas . They can be generated through degradation of hg by pathogen - derived enzymes (kars et al . Ogas have been shown to induce gene expression changes, stomatal closure, production of et, and ros as well as cell wall reinforcement (denoux et al ., 2008; ferrari et al ., 2008). A hybrid kinase consisting of the extra cellular domain of wall - associated kinase1 (wak1) and the intracellular domain of elongation factor tu receptor (efr) kinase can bind ogas and activate defense responses (brutus et al ., 2010). Wak1 belongs to a family of five wak genes encoding plasma membrane - localized ser / thr kinases that have been implicated in response to pathogen infection and regulation of cell elongation (kohorn et al ., 2011). The effects of the chimeric wak1 kinase on pathogen resistance suggest that ogas and waks represent an in vivo ligand receptor pair . Results from the analysis of a dominant active wak2 allele suggest the cwd signals perceived by waks could be relayed to downstream response genes through mapkinase6 (mpk6) (kohorn et al . Interestingly, wak2 has also been implicated in regulation of invertase activity and turgor pressure during cell elongation (kohorn et al ., 2006). However, there is currently no confirmation that waks are actively involved in cwi maintenance . In arabidopsis, more than 600 rlks have been identified and a large number of them have been implicated in developmental and stress response processes (shiu and bleecker, 2001). I will focus here on several kinases that have been implicated in cwd perception and/or pathogen response . Most of the rlks implicated in cwi maintenance [the1, hercules1 (herk1), feronia (fer)] belong to the catharanthus roseus rlk1 (crrlk1)-like protein family, which has 17 members in arabidopsis . The1 was isolated as a suppressor of the cellulose - deficient cesa6 procuste (pre) mutant, which exhibits a hypocotyl elongation defect (hematy et al ., 2007). Subsequently it has been shown that the1 is required for cellulose biosynthesis inhibition - induced ros production and lignification in the root elongation zone (denness et al ., 2011). The1, herk1, and fer have been implicated in brassinosteroid - induced cell elongation (guo et al ., 2009; deslauriers and larsen, 2010). Both fer and nortia / mildew resistance locus o 7 (ml07; a seven - transmembrane domain protein involved in powdery mildew resistance) are required for successful fertilization and resistance to infection by golovinomyces (syn . Interestingly, ropgef (guanine - exchange factors) proteins have been identified as targets of fer activity (duan et al ., 2010). Ropgefs are required for the activation of rho gtpases, which in turn activate nadph oxidases like rbohd / f . These results suggest the same molecular components could mediate cell - cell interaction during development and plant heterotrimeric g - proteins (g, g, g) form a highly conserved signaling complex that has been implicated in signal transduction during development and stress responses in mammals, yeast, and plants (digby et al ., 2006; temple and jones, 2007). In arabidopsis, five genes gpa1 (g), agb1 (g), agg1, 2, 3 (g1, 2, 3) encode the subunits of the complex (thung et al ., 2011). Recently, it has been reported that mutations in agg1, 2, and agb1 apparently cause enhanced susceptibility to infection with p. cucumerina (delgado - cerezo et al ., 2011). A combination of metabolomic and microarray - based expression profiling studies of the mutants established that the pathogen phenotype is independent of sa, ja, aba, and et signaling cascades . Interestingly, a large number of cell wall biosynthetic / modifying genes are mis - regulated in agbl and aggl2 plants . Analysis of cell wall composition / structure in these plants found reduced xylose contents in the mutants compared to wildtype . To summarize, the available evidence supports the notion that the plant cell wall is an integral component contributing to pathogen response mechanisms and illustrates the influence of cell wall defects on infection . Specific signaling cascades seem to mediate the response to particular cell wall defects, which in turn affect the response to necrotrophic or biotrophic pathogens . More importantly the data presented above allow correlation between certain types of cell wall defects, and not only signaling cascades but also resistance phenotypes . Mutations in cesa4, 7, 8, agg1, 2, and agb1 affect resistance to necrotrophs and are independent of phytohormone - based signaling cascades . Pmr5, 6 plants exhibit resistance to biotrophs and also do not rely on phytohormone - based signaling cascades . Pmr4 affects resistance to biotrophs and resistance depends on the integrity of the sa signaling cascade . The cell wall composition / structure changes could prevent pathogen colonization simply because the infection machinery of the pathogen is too specialized to breach the chemically modified cell wall . Another option is that the cell wall mutants cause defects similar to those occurring during infection by particular pathogens, i.e., simulate infection by necrotrophs or biotrophs . This would cause early / constant activation of the cwi maintenance / defense mechanism, which primes plant immunity thus making successful infection more difficult . The latter would explain both the specificity of the responses observed and dependence on particular signaling mechanisms . Therefore, studies focusing on the effects of particular cell wall defects on pathogen resistance and the mode of action of the cwi maintenance mechanism could facilitate research into biotic stress response . The reason being, that by removing the potentially multiple effects of the pathogen during infection, they reduce the complexity of the interaction and should therefore allow novel insights into the mechanisms responsible for detection of infection and/or physical damage . The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Genomic dna hypomethylation has been observed in the peripheral blood mononuclear cells of leukemia patients and in tumor cells of patients with b - cell lymphoproliferative diseases (1). Dna methylation may affect karyotypic stability, may influence euchromatin - heterochromatin interactions, and has been correlated with disease progression (2). On the other hand, for example, some patients with lymphomas do not express tumor suppressor genes because the promoters of these genes are methylated ., bone marrow produces a large number of abnormal white blood cells, which overwhelm the other types of blood cells, including red blood cells and platelets, thus impairing the production of normal white blood cells . In clinical classification, leukemia can be classified as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml). Cml is a clonal disease of stem cell origin that is characterized by the presence of the philadelphia chromosome (ph+), which has been named t(9,22)(q34:q11). Its fusion gene product, bcr - abl, is a constitutively active tyrosine kinase . Nevertheless, the differential factors through which they produce a different type of leukemia are not yet completely understood . In hematopoietic malignancies, hypermethylation of several genes including e - cadherin, dap kinase, estrogen receptor (er) alpha, and p15 are associated with gene inactivation (7 - 10). Genes, such as dab2ip, dlc-1, h - cadherin, id4, integrin 4, runx3, sfrp1, and shp1, has been identified as being implicated in aberrant dna methylation during development of human malignancy (11 - 18). In order to gain insight into the differential epigenetic alterations in leukemia, we investigated the methylation statuses at selected locus of these genes in aml and cml patients using a methylation - specific polymerase chain reaction (msp). Diagnostic bone marrow samples were obtained from 23 patients with aml and 21 patients with cml . The samples were gathered by the division of hematology / oncology (department of internal medicine, korea university medical center, seoul, republic of korea) and analyzed by pathologist . Institutional review board approval and informed consent were obtained (kumc - irb-2006011-p-1, kumcirb-2006012-p-2). Of the aml patients, 17 males and 6 females were included, with ages ranging from 26 to 78 yr, at a median age of 45.61 (sd, 15.56) yr . Of the cml patients, 11 males and 10 females were included, with ages ranging from 18 to 75 yr (meansd, 49.9116.53). As controls, 22 normal peripheral bloods were obtained from healthy volunteers (11 males and 10 females) ranging from 20 to 78 yr of age (meansd, 45.3620.64). Chemical modification was performed as described previously, with minor modifications (19). In brief, 1 g of genomic dna was denatured by incubation with 0.2 m naoh for 10 min at 37, followed by the addition of 550 l of 3 m sodium bisulfite (ph 5.0) (sigma, st . Louis, mo, u.s.a .) And 10 mm hydroquinone (sigma), which was brought to a final volume of 600 l . The mixtures were incubated at 55 for 16 hr, and the modified dna was then desalted with the wizard clean - up system (promega corp ., we performed polymerase chain reaction (pcr) using specific pcr primers capable of distinguishing between methylated and unmethylated dna sequences . The primers for the unmethylated and methylated dna sequences, pcr product size, and annealing temperature are shown in table 1 . The msp primer sets were selected at the 5'-cpg island regions of genes using the methprimer software (www.urogene.org). The pcr conditions were as follows: initial denaturation and hot start at 95 for 5 min, and cycles consisting of 30 sec at 95, 30 sec at the annealing temperature, and 30 sec at 72. in addition, sck, a human cholangiocarcinoma cell line, was used as positive control for integrin 4 gene (20). Msp results were analyzed as a dichotomous variable based on the presence or absence of gene methylation . The msp results of tumor and normal samples were compared and analyzed with the pearson chi - square test (version 12; spss inc ., diagnostic bone marrow samples were obtained from 23 patients with aml and 21 patients with cml . The samples were gathered by the division of hematology / oncology (department of internal medicine, korea university medical center, seoul, republic of korea) and analyzed by pathologist . Institutional review board approval and informed consent were obtained (kumc - irb-2006011-p-1, kumcirb-2006012-p-2). Of the aml patients, 17 males and 6 females were included, with ages ranging from 26 to 78 yr, at a median age of 45.61 (sd, 15.56) yr . Of the cml patients, 11 males and 10 females were included, with ages ranging from 18 to 75 yr (meansd, 49.9116.53). As controls, 22 normal peripheral bloods were obtained from healthy volunteers (11 males and 10 females) ranging from 20 to 78 yr of age (meansd, 45.3620.64). Chemical modification was performed as described previously, with minor modifications (19). In brief, 1 g of genomic dna was denatured by incubation with 0.2 m naoh for 10 min at 37, followed by the addition of 550 l of 3 m sodium bisulfite (ph 5.0) (sigma, st . Louis, mo, u.s.a .) And 10 mm hydroquinone (sigma), which was brought to a final volume of 600 l . The mixtures were incubated at 55 for 16 hr, and the modified dna was then desalted with the wizard clean - up system (promega corp ., we performed polymerase chain reaction (pcr) using specific pcr primers capable of distinguishing between methylated and unmethylated dna sequences . The primers for the unmethylated and methylated dna sequences, pcr product size, and annealing temperature are shown in table 1 . The msp primer sets were selected at the 5'-cpg island regions of genes using the methprimer software (www.urogene.org). The pcr conditions were as follows: initial denaturation and hot start at 95 for 5 min, and cycles consisting of 30 sec at 95, 30 sec at the annealing temperature, and 30 sec at 72. in addition, sck, a human cholangiocarcinoma cell line, was used as positive control for integrin 4 gene (20). Msp results were analyzed as a dichotomous variable based on the presence or absence of gene methylation . The msp results of tumor and normal samples were compared and analyzed with the pearson chi - square test (version 12; spss inc ., the multiple genes were found to be methylated in bone marrow from patients with aml or cml . Specifically, the frequencies of promoter hypermethylation at selected locus in the 23 aml samples were: 78.3% (18/23) for shp1, 65.2% (15/23) for id4 and sfrp1, 26.1% (6/23) for h - cadherin, 8.7% (2/23) for dlc-1, and 4.3% (1/23) for dab2ip and runx3 . The frequencies of dna hypermethylation at selected locus in the 21 cml samples were: 28.6% (6/21) for shp1, 19.0% (4/21) for h - cadherin, 14.3% (3/21) for id4, 9.5% for (2/21) for sfrp1, and 0% (0/21) for dab2ip, dlc-1, integrin 4, and runx3 (fig . However, promoter hypermethylation of the 22 normal peripheral bloods was observed less frequently (table 2). There was a statistically significant difference between normal peripheral bloods and aml with respect to the frequencies of methylation of id4, sfrp1, and shp1 (pearson chi - square test; p<0.0001, p<0.0001, and p<0.0001, respectively) and between normal peripheral bloods and cml with respect to the frequencies of shp1 methylation (pearson chi - square test; p=0.007). Furthermore, there was a statistically significant difference between the dna methylation frequencies of aml patients and cml patients . The frequencies of dna methylation of id4, sfrp1, and shp1 were higher in aml compared to those in cml (p=0.001, p<0.0001, and p=0.001, respectively) (table 2). In contrast, no statistical differences between aml and cml were detected in other genes such as dlc-1, dab2ip, h - cadherin, integrin 4, and runx3 . Promoter methylation results at selected locus of eight genes with msp method were shown in fig . The results of the present study demonstrate the substantially increased frequency of promoter hypermethylation in id4, sfrp1, and shp1 genes in aml compared to cml . Leukemia, a heterogeneous group of hematopoietic malignancies that occur worldwide, includes acute and chronic myeloid leukemia . Despite many important advances in understanding the different biological and cytogenetic aspects of acute and chronic leukemia, it is understood that some kinds of leukemia present specific cytogenetic alterations (21). Different types of leukemia usually have specific epigenetic modifications that cause the activation of oncogenes and, in particular, the formation of abnormal fusion genes such as aml1-eto (22, 23), a fusion protein resulting from t(8,21) translocation that commonly occurs in aml . Another typical example is the fact that bcr - abl is a constitutively active, cytoplasmic tyrosine kinase that is generated by t(9;22) translocation in more than 95% of cml (24). The central role of epigenetic modification of these genes in leukemia development promoted them as reasonable targets for understanding the differential mechanism between acute and chronic leukemia . In the present study, id4, sfrp1, and ship1 these results suggest that the epigenetic modification of these genes may play specific roles in the development of aml rather than cml and, thus, promoted them as ideal targets for drug development to treat aml . In the future, it is clearly imperative to understand the patho - physiological differential mechanisms of these genes in aml, and to finally explore novel therapeutic strategies . In present study, methylation frequencies of shp1 gene were the high detected in cml as well as aml . Shp1 is a member of the shp family of proteins, cytoplasmic protein tyrosine phosphatase (ptp), and has been known as a candidate tumor suppressor gene in lymphoma, leukemia and other cancers (25). Also, the hypermethylation of shp1 gene was frequently detected in several human cancers and the reduced expression of the shp1 gene in various types of leukemias and lymphomas mainly occurred by promoter methylation (26 - 28). Therefore, these results indicate that aberrant dna methylation of shp1 gene may be related to the tumorigenicity of myeloid leukemia . The methylation frequencies of the dab2ip, dlc-1, and runx3 genes were detected at different levels in acute and chronic leukemia patients; however, the difference was negligible . In some genes like integrin 4 gene, however, hypermethylation of these genes was frequent event in esophageal squamous cell carcinomas, lung cancers, and gastric cancers (11, 12, 15, 16). Thus, dna methylation of these genes may be not related to tumorigenicity of aml / cml in contrast with other cancers . In summary, we have identified that aberrant dna methylation of shp1 is a frequent event in aml and cml . Also, the frequencies of dna methylation of several methylation - controlled genes, including id4, sfrp1, and shp1, were higher in aml patients compared to those in cml patients . Although these results should be confirmed, more comprehensive studies are necessary involving various known risk factors such as smoking and occupational carcinogens, and genetic susceptibilities . Our results suggest that aberrant dna methylation of shp1 may be related to the tumorigenicity of aml and cml and hypermethylation of id4, sfrp1, and shp1 genes may contribute to the pathogenesis mechanism of aml specifically.
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Acute patellar dislocation is a common injury usually associated with a significant traumatic mechanism resulting in lateral displacement . Vertical axis rotation following dislocation is a rare variant of this type of injury and can prevent closed reduction in the acute setting . A 32-year old gentleman presented with an irreducible patella dislocation following an unusual atraumatic mechanism . Following attempts at closed reduction under sedation and regional nerve block, eventual open reduction and soft tissue reconstruction was required under general anesthetic . During the open reduction procedure, it was noted that the patella had dislocated into a lateral extra - articular position and rotated around its vertical axis . A review of the literature suggests dislocations such as the current presentation, are extremely rare and although have been described to occur with minor trauma, have never been described to occur following a largely atraumatic event . In such cases, closed reduction may be impossible even with adequate analgesia due to patella position and soft tissue obstruction . Acute patellar dislocation is defined as the abrupt disruption in the relationship of the patella within the femoral groove . Patellar dislocation is a relatively common occurrence and is usually managed in the emergency department with closed manipulation under sedation . However, when the patella itself rotates around its vertical axis during dislocation, surgical intervention is usually required . A 32-year old gentleman was admitted to our trauma department with a painful, immobile right knee . He sustained the injury externally rotating his knee whilst in slight flexion while he was moving from the driver s seat to the passenger s seat of his car . The first event was 15 years prior, when his knee twisted in flexed position while pushing a car . The second event was four years prior, when he sustained an injury while pushing a van, this time with the right knee fully extended . On this occasion, our patient had no other significant past medical or surgical history and explained his right knee was completely asymptomatic prior to this current event . On examination, there was an obvious deformity suggesting lateral displacement of the patella with a sulcus in the skin evident over the femoral groove . Furthermore, the knee was held fixed in 15 degrees of flexion . Due to patient positioning difficulties we were unable to obtain true ap and lateral views on x - ray . The radiographs demonstrated a laterally displaced and a mal - rotated patella in the vertical plane (see figs . 1 and 2). Lateral radiograph of the right knee demonstrating rotation of patella our patient was subsequently taken to theatre where one final attempt of closed reduction was carried out under general anaesthetic and muscle relaxation . Ultimately, this failed and an open reduction was performed . Complete rupture of the medial patellofemoral ligament (mpfl) was identified with the patella situated lateral to the lateral femoral condyle, everted by approximately 100 degrees . The knee joint was washed out with normal saline and the medial patella retinaculum was repaired . After the repair, post - operative radiographs confirm the patella in a satisfactory position in the antero - posterior and lateral planes (see figs . 3 and 4). The patient was placed in an extension splint for comfort purposes immediately post - operatively . Early mobilization was encouraged after 5 days and the patient was referred for early physiotherapy . At 3 months follow - up, our patient had no further episodes of dislocation, full range of knee extension and flexion, and normal patella tracking . A hypermobility assessment at this stage revealed a beighton score of 2 with extension beyond 10 degrees of both elbows only . Intraoperative antero - posterior florous copy image following successful reduction lateral radiograph of the right knee following surgery in knee splint patellar dislocations can vary widely in their pattern of presentation however vertical axis rotation complicates only a small number of these cases . In 1844, cooper described the earliest case of patellar dislocation with vertical axis rotation . Since then, only a handful of case reports describing vertical axis rotation of the patella have been published with the majority describing the location of the patella within the femoral trochlea (intra - articular) [2 - 11]. Our case is rare as our patient had a less common variant of extra - articular patellar dislocation complicated by vertical axis rotation and by the fact, that the causative mechanism was largely atraumatic . Previous descriptions postulate that the prominence of the lateral femoral condyle acts as a pivot point, which can cause the patella to rotate around on its vertical axis . In this case, however, the patient denies any trauma, and instead describes externally rotating the leg whilst extended as the mechanism of injury . It is almost certain that the previous dislocations in our patient s case had resulted in significant damage of the mpfl . With the patient reporting his injury taking place with the knee slightly flexed, the function of the mpfl in this case as the primary medial restraint during the first 20 degrees of flexion was likely to be inadequate . Previous authors have described similar cases of extra - articular lateral patellar dislocation with vertical axis rotation and have noted in their subsequent reviews of the limited literature that intra - aritcular dislocations were usually related to far more significant trauma than extra - articular dislocations [3 - 10]. This was also the finding in a previous report highlighting the relative minor force resulting in an extra - articular dislocation . We feel this may be an indication of the chronic deficiency in the mpfl likely contributing to cases of extra - articular dislocation . On reviewing the previous cases of extra - articular dislocation in the literature, none had described a completely atraumatic mechanism and none had discussed previous dislocations or previous surgical history in their patients . Detailed imaging can be difficult to obtain in the acute scenario; however in these cases ct imaging may clarify patella position and demonstrate mechanical engagement of the patella on the lateral femoral condyle . In one case report, ct imaging was utilized and confirmed avulsions of the vastusmedialis muscle and medial crus of the patella tendon with resultant impaction of the medial border on the patella onto the lateral femoral condyle . In this case, imaging led to the decision for open reduction being required, minimizing further attempts at closed reduction which could in theory result in further damage . It is difficult to be absolutely sure why attempts at closed reduction had failed in our particular case, but this is likely to be multifactorial . Vertical axis rotation results in impaction of the lateral edge of the patella on the lateral femoral epicondyle which may result in a corresponding femoral defect resulting in a lodging effect . Furthermore patient factors, namely obesity and large muscle mass around the knee made manipulation technically difficult in our case . Open reduction under general anaesthetic is rarely necessary for routine patellar dislocations however there are some obvious advantages . These include; the possibility of direct visualisation of the obvious damage to the soft tissue structures contributing to or resultant from the dislocation . In our case it was likely the mpfl had been damaged previously and repair is likely to help in restoring central patella tracking and overall stability . In this case, the patient was noted as having a shallow trochlea which might have accounted for his increased tendency to dislocations . Direct visual inspection may also help determine patients who are at risk of recurrent dislocations due to factors relating to their bony morphology and allow the surgeon to plan further management accordingly . Lastly, visualisation of the articulating surfaces allows the surgeon to identify and address any osteochondral defects and corresponding loose bodies which may otherwise have contributed to long term future problems requiring eventual surgical intervention . From our experience with this case, difficulty in reducing a dislocated patella should alert surgeons to the possibility of an extra - articular dislocation with possible vertical axis rotation . In these scenarios, multiple attempts at reduction we were able to achieve adequate analgesia and sedation using local and regional anaesthesia, without successful reduction of the dislocation . Although some authors have demonstrated successful closed reduction with the use of local anaesthetic and muscle relaxants, this is not always effective . Furthermore, attempted closed reduction under general anaesthesia in our case was also unsuccessful, demonstrating how closed reduction can be futile in certain cases . We would recommend a low threshold for open reduction in such cases by a specialist knee surgeon who may assess and reconstruct damaged soft tissue damage in one operation vertical axis rotation is a rare entity complicating patella dislocations . In this rare event emergency physicians should be aware of this complication and refer to the relevant specialist for open reduction under general anaesthesia in order to minimize patient discomfort and potential articular surface damage.
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Graphene oxide (go) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction . Firstly, cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 . Then, a calculated amount of go solution and formic acid (3 ml) were added . For optimization, cu2o / rgo composites with different amounts of rgo were also synthesized, which include cu2o/0.25% rgo, cu2o/0.5% rgo, and cu2o/1% rgo, in which x% represents the calculated weight ratio of the go added to cu2o . After stirring for 2 h, the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt, the final product was collected by centrifugation, washed with water five times, and dried at 70 c . Blank cu2o was synthesized through the same procedure, except for the addition of go solution . Xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv . The morphologies of the products were characterized by field - emission scanning electron microscopy (fesem, jeol-6701f) and tem (jeol-2010f). Uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer . Pl emission spectra were measured at rt by using a fluorescence spectrophotometer (f-4500, hitachi). Photocatalyst (5 mg) and nafion solution (10 l, 5 wt%) were dispersed in a water / isopropanol mixture (1 ml, 3:1 v / v) by at least 30 min sonication to form a homogeneous catalyst colloid . For the measurements, the catalyst colloid (100 l) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode . A pt wire was used as a counter electrode, and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system . Eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz . The potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz . The co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml, which was heated at 160 for 1 h prior to measurement . To remove possible trace organic contaminants, photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air (denoted cu2o treated in ar or air). A typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor . Excess (0.7 m) sodium sulfite was added to each batch as a hole scavenger.15, 46 a 150 w xe lamp (newport) was used as a light source . The light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator . The reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc (varian gc-450) with a thermal conductivity detector (tcd, connected to a molecular sieve column) to detect h2, o2, and n2 and a flame ionization detector (fid, connected to a cp - sil 5cb capillary column) to detect hydrocarbons . A methanizer was installed to enable the fid to detect co with 1000 higher sensitivity . For the isotope - tracer experiment, after the addition of cu2o / rgo (0.5 g) into co2-saturated water (10 ml), the septum - sealed reactor was purged by ar gas for 10 min . The sample was irradiated with a 150 w xe lamp for 30 min, and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc graphene oxide (go) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction . Firstly, cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 . Then, a calculated amount of go solution and formic acid (3 ml) were added . For optimization, cu2o / rgo composites with different amounts of rgo were also synthesized, which include cu2o/0.25% rgo, cu2o/0.5% rgo, and cu2o/1% rgo, in which x% represents the calculated weight ratio of the go added to cu2o . After stirring for 2 h, the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt, the final product was collected by centrifugation, washed with water five times, and dried at 70 c . Blank cu2o was synthesized through the same procedure, except for the addition of go solution . Xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv . The morphologies of the products were characterized by field - emission scanning electron microscopy (fesem, jeol-6701f) and tem (jeol-2010f). Uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer . Pl emission spectra were measured at rt by using a fluorescence spectrophotometer (f-4500, hitachi). Photocatalyst (5 mg) and nafion solution (10 l, 5 wt%) were dispersed in a water / isopropanol mixture (1 ml, 3:1 v / v) by at least 30 min sonication to form a homogeneous catalyst colloid . For the measurements, the catalyst colloid (100 l) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode . A pt wire was used as a counter electrode, and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system . Eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz . The potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz . The co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml, which was heated at 160 for 1 h prior to measurement . To remove possible trace organic contaminants, photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air (denoted cu2o treated in ar or air). A typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor . Excess (0.7 m) sodium sulfite was added to each batch as a hole scavenger.15, 46 a 150 w xe lamp (newport) was used as a light source . The light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator . The reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc (varian gc-450) with a thermal conductivity detector (tcd, connected to a molecular sieve column) to detect h2, o2, and n2 and a flame ionization detector (fid, connected to a cp - sil 5cb capillary column) to detect hydrocarbons . A methanizer was installed to enable the fid to detect co with 1000 higher sensitivity . After the addition of cu2o / rgo (0.5 g) into co2-saturated water (10 ml), the septum - sealed reactor was purged by ar gas for 10 min . The sample was irradiated with a 150 w xe lamp for 30 min, and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc as a service to our authors and readers, this journal provides supporting information supplied by the authors . Such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset . Technical support issues arising from supporting information (other than missing files) should be addressed to the authors
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In recent years, the concept of minimal intervention (mi) has prevailed in dentistry . Therefore, the importance of diagnosing caries at an early stage has increased . In conventional procedures, the diagnosis of caries has mainly consisted of visual inspection and tactile assessment with probing . However, lussi reported that the sensitivity of detecting caries was 0.62 by visual inspection and 0.82 by probing . In addition, the pressure of probing can damage the demineralized fissure and increase the risk that caries progress [3, 4]. To promote mi, diagnosis without a probe has been recommended . The laser fluorescence - based caries detection device diagnodent (kavo, germany) has been introduced as an alternative . However, no single detection method for caries is sufficient; therefore, the combination of some detection methods has been recommended [57]. Recent improvements in the personal computer have made the process of digital imaging more efficient and convenient . If the shape of caries can be quantified, and the relationship between the numerical value and the condition of the lesion can be demonstrated, this information would be helpful to diagnose dental caries . The authors of this paper have previously shown that the fractal dimension and proportion of the area of pit - and - fissure discoloration to the area of occlusal surface obtained by digital imaging were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth . For assessment of the method as a diagnostic system, the ability of the diagnosis, such as the sensitivity, the specificity, and the accuracy, should be researched in clinical situation . The aim of this study was to assess the possibility of the clinical application of the diagnosis of occlusal caries using digital imaging by examining the sensitivity, the specificity, and the accuracy in comparison with the diagnodent values and the dentists' diagnoses . One hundred teeth (36 premolars and 64 molars) with pit - and - fissure discoloration from 19 outpatients were examined at the clinic of oral diagnosis and general dentistry, dental hospital, tokyo medical and dental university . The occlusal surface of each tooth was washed with the robinson brush to remove dental plaque without any abrasive paste . Then, pit - and - fissure discoloration was dried by air and measured three times using diagnodent . The mean scores were used as the diagnodent values of the teeth (dd). Next, the occlusal surface of each tooth was photographed as large as possible with an intraoral digital camera (penscope, morita, japan). Each image was stored in a personal computer using a video capture interface (pc - mdvd / u2, buffalo, japan). Without knowing the dd, a dentist preliminarily diagnosed each tooth using visual inspection and tactile examination to decide which treatment plan would be appropriate (preventive or operative). The clinical diagnosis of preventive treatment for teeth was classified as co. on the other hand, carious lesions requiring operative treatment were removed in a conventional clinical way . If the resulting cavity preparation was limited in the enamel, then the clinical diagnosis was classified as c1 . If the resulting cavity preparation reached the dentin, and sound tissue still remained between the cavity and the pulp chamber, then the clinical diagnosis was classified as c2 . Five dentists ranging from 3 to 15 years of professional experience examined the teeth after calibration of the criteria of the caries assessment conducted before the study; the calibration was done as follows; first, the five dentists examined 30 extracted teeth and decide which treatment plan would be appropriate (preventive or operative). At that time then, the teeth were sliced pallarel to the teeth axis and to the depth of lesion for each tooth was determined . At last, the dentists discussed to accord the treatment planning for each tooth referring the depth of the lesion . The digital photographs obtained were processed and analyzed using image analysis software (image j, nih, usa). First, each image was converted to an 8-bit gray - scale image, in which the density of grayness of each pixel was linearly scaled from min 0 (black) to max 255 (white). Then, the occlusal surface in the image was isolated from the background using a density histogram of the image, and the area was measured . Pit - and - fissure discoloration was also isolated from the occlusal surface using the density histogram, and the area was measured . The proportion of the area of pit - and - fissure discoloration to the area of the occlusal surface was calculated (pa). Next, the image of the isolated pit - and - fissure discoloration was converted into a binary image, in which the density of pit - and - fissure discoloration was 0, and its background was 255, followed by calculating the fractal dimension of pit - and - fissure discoloration (fd). Differences in fd, pa, and dd between each clinical diagnosis were analyzed using two - way anova and games - howell test to reveal the clinical diagnosis and the effect of the examining dentists . The correlation between the clinical diagnosis and each fd, pa, and dd was analyzed using spearman's correlation coefficient . Discriminant formulas were obtained using discriminant analysis with the treatment plan (preventive / operative) as the objective variable and fd, pa, and dd as explanatory variables . Sensitivity and specificity were calculated by applying fd, pa, and dd to each discriminant formula . The accuracy, ratio of the number of teeth showing accordance between the treatment plan decided by the dentists and the predictive treatment plan decided using the discriminant formula to the number of all the teeth, was also obtained . The entire process was approved by the ethics committee of the faculty of dentistry, tokyo medical and dental university (no . 317). Fd, pa, and dd values corresponding to each clinical diagnosis are shown in table 1 . The two - way anova revealed that the fd, pa, and dd were different among the clinical diagnosis (p <.01). On the other hand, the difference of the examining dentists did not affect the fd, pa, and dd . Spearman's correlation coefficients between the clinical diagnosis and each fd, pa, and dd were 0.743, 0.700, and 0.652, respectively (p <.01). There were also significant correlations among fd, pa, and dd (p <.01). Table 2 shows the discriminant formula, sensitivity, specificity, and accuracy of each explanatory variable . Based on the discriminant formula of each explanatory variable, the thresholds of fd, pa, and dd between preventive and operative treatments were 1.20, 0.012, and 28.8, respectively . The sensitivity of fd was greater than that of pa, dd, and the combination of fd and pa . The specificity of pa was greater than that of fd, dd, and the combination of fd and pa . The accuracy of the combination of fd and pa was greater than that of fd, pa, and dd . Previously, it was reported that the fractal dimension and the proportion of the area of pit - and - fissure discoloration to the area of occlusal surface were significantly correlated with the depth of the caries and the diagnodent values in extracted teeth . In this study, the fractal dimensions for c0, c1 and c2 in the former study were 0.97, 1.30, and 1.52, respectively . These results indicate that image analysis of molar pit - and - fissure discoloration was clinically useful for the diagnosis of caries . An increase of the proportion of the area of discoloration corresponded to a change of the volume of caries lesion, while an increase of the fractal dimension corresponded to a change of the shape of the lesion caused by caries progression . A fractal is a geometric shape, possessing characteristics of self - similarity or self - affinity, and widely observed in nature [10, 11]. Recently, fractals have been in the spotlight in the field of medicine, and research has been introduced regarding its use in the field of diagnosis [1214]. For example, a point is described as the zero dimension; a straight line is described as the first dimension, and a plane is described as the second dimension . Such decimal dimensions can be obtained by expanding the definition of the dimension as the rate at which the perimeter (or the surface area) of an object increases, and the measurement scale is reduced . Several ways to measure the fractal dimension have been introduced . In this study, the authors used a simple way to determine the fractal dimension called box counting . In this method, a grid of squares is placed over the object, and the number of squares through which any part of the object passes is counted . This process is repeated with different grids having different sizes . The number of squares placed over the object versus the length of the side of the square are then plotted on log - log scale . When a regression line is obtained from the plots, the degree of uneven complexity of a boundary or a coast can be quantified using this approach . In this research, the fractal dimension of discoloration increased from 0.8 to 1.6 as the depth of the caries increased, which corresponded to a change in the shape of the discolored area from a point or a line to an area based on the progression of the caries . Generally, the addition of valuables into discriminant formula is one of the ways to improve the accuracy, however, in this study, the accuracy of the combination of fd and pa was similar to that of single fd . Therefore, further study to find other valuables is needed to improve the accuracy of this method by combination of valuables . Thus, the accuracy of the diagnosis of occlusal caries using digital images of discolored areas was comparable to that of diagnodent; therefore, its clinical application as a diagnostic tool is possible . Because this study was clinical, the final diagnosis of each examined tooth was not confirmed by a histological procedure but by a dentist's clinical examination . As mentioned above,, the results of two - way anova showed that the effect of the examining dentist on the dd, pa, and fd values was not significant . Therefore, we considered that difference of the diagnosis among the dentists would be small . In the present study, to examine the possibility of digital imaging in the diagnosis of occlusal caries, diagnodent was used as a comparative pre - existing dental caries detection tool . There are other caries detection tools, such as fiber optic transillumination (foti), digital imaging fiber optic transillumination (difoti), quantitative laser or light fluorescence (qlf), and electrical conductive measurements (ecm). Foti, difoti, and qlf have been tested in vivo, however, the number of clinical studies has still been small [15, 16]. On the other hand, a comparatively long time has passed since diagnodent was introduced to the market, and many findings have been reported . Sheehy et al . Reported that diagnodent had greater sensitivity and specificity than ecm . The correlation coefficient between diagnodent readings and the depth and the volume of caries lesions was reported to be 0.47 . Several researches have pointed out that diagnodent measurements are affected by other factors, such as hypomineralization, plaque, debris, staining and wetness [1921], while a high correlation between inter and intraobserver agreements was also mentioned [22, 23]. We employed diagnodent as a comparison because the diagnosis was provided as a number, the handling was easy, and, moreover, its clinical use has been discussed in other studies . In the present study, diagnosis using digital images of pit - and - fissure discoloration depended on the statistical relationship between the shape of discoloration and the depth of caries . The shape of the discolored area in the occlusal surface is, however, possibly affected by medication history, individual history, and lifestyle . Consequently, diagnosis using digital images of pit - and - fissure discoloration is rather experimental . Additionally, the procedure cannot be applied to colorless lesions, such as acute caries . Therefore, diagnosis using digital images of pit - and - fissure discoloration should not be used for definitive diagnosis . Rather, initial diagnosis, screening such as mass examination would be suitable because of its good sensitivity of fd and, the convenience of the procedure . Actually, the core of the diagnostic system is digital imaging processing by computers . If computer programing for all procedures was achieved, then screening of hundreds of examinees would be automated after photograph taking, which would make mass examination less time - consuming with low cost . For such automated uses of the computer, the process of extracting colors from the image must be improved; in this study, the threshold for the colored area was decided one by one by an observer's visual inspection . How to calculate the fractal dimension is also open to discussion . We used the box - counting method attached in the image analysis software, image j. the box - counting method is only suitable for self - similar profiles, not for more general, self - affine cases, that is, the fractal dimension using the box - counting method might be the approximate value . A special computer program must be developed to measure the fractal dimension more accurately, for example, the minkowski method or the richardson method [8, 10]. As mentioned above, the shape of the discolored area on an occlusal surface is affected by many factors . Therefore, the thresholds or the discriminant formulas acquired from this research are not universal . Further research is needed to determine the discriminant formulas to diagnose caries using the image analysis of molar pit - and - fissure discoloration.
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Craniofacial defects are rare, disfiguring facial anomalies, with an incidence of about 1.4 to 4.9 cases per 100,000 live births . Craniofacial clefts may be caused by failure of the maxillary processes to fuse, external pressure, amniotic bands, oligohydramnios, central disorganization of the neural crest, and hematomas . In 1973, the median cleft of the upper lip, or tessier 0 class, has a variety of presentations, ranging from a minimal notching of the lip, vermilion, and nose to a wide cleft that divides craniofacial structures . Tessier 0 clefts result from failure of the two medial nasal processes to fuse at midline . Median clefts are broadly classified into true and false; false clefts are due to agenesis of the medial nasal process, while true clefts are due to failure of the medial nasal process to fuse . Treatment of median clefts depends on the clinical presentation and may vary from simple alignment of orbicularis oris and vermilion, to reconstruction of the cupid's bow and philtrum for true median clefts, and complex craniofacial procedures in case of false median clefts . Various techniques have been described to repair mild or moderate true median cleft lips . Here we report using a pfeifer incision to correct a moderate true median cleft of the upper lip . Pfeifer incision includes wavy lines, which elongate the incised tissues as the waves are straightened to close in a straight line, and also provide extra tissue for a tension free closure . A five - year - old boy with a facial cleft presented to our clinic . On examination, there was a median cleft of the upper lip involving the white roll, with no bony involvements . The highest- (points a, a), and the deepest - points (points b, b) of the white roll were marked on both sides . Subsequently, a wavy incision line was made starting from the deepest point and extending over the philtrum just above the cleft (fig . A diamond incision was made over the vermilion and the labial mucosa, intra - orally extending just beyond the cleft margin . Incision was made and the mucosal tissue covering the area medial to the incision site (sterile zone) was removed . Undermining of the orbicularis oris muscle was performed and it was approximated using 4 - 0 vicryl . The cupid's bow was properly aligned, with equal height on both sides (fig . Vermilion form was satisfactory and the fullness and continuity of the orbicularis oris were maintained . Incision marking for pfeifer technique (a, a: highest points on white roll; b, b: deepest points on white roll) postoperative result after suture removal a median cleft can also be called midline cleft or vertical cleft through the centre of the upper lip, and is a rare anomaly, the exact developmental origin of which is not clear . Tessier 0 clefts occur during the third week of gestation due to failure of the two medial nasal processes to close in midline . It can occur in isolation or be a part of a syndrome such as orofacial - digital syndrome . False clefts are associated with abnormalities of the forebrain and are categorized under the category of holoprosencephaly . In 1937, veau categorized median clefts to notch of the lip, median cleft extending to the columella, and defects due to atrophy of the midline facial structures . Median cleft face syndrome, frontonasal dysplasia, and tessier 0 clefts are various terms used to describe abnormalities associated with true median clefts that are not accompanied by forebrain abnormalities . The tessier 0 anomaly may present as a small notch in the soft tissue, or in association with hypertelorism, midline craniofacial osseous defects, and hairline abnormalities . Various treatment options are present for mild deformities which do not involve the white roll . When developing the treatment plan, reconstruction of the cupid's bow, labial philtrum, vermillion, and buccal mucosa should be kept in mind . Urata and kawamoto described using a v - y flap, while weimer et al, used a diamond incision to repair these anomalies . Da silva frietas and colleagues described a mucosal z transposition technique to treat mild cases . For moderate defects involving the philtrum, these waves are subsequently approximated in a straight line, which help expanding the length and width of the tissue . This incision has previously been used to correct other tessier clefts with a high success rate . The above - mentioned studies prove the versatility of the pfeifer incision . In our case we were able to achieve adequate symmetry of the philtrum and the cupid's bow, which is important in cases like this . The postoperative results were excellent and proper approximation of the orbicularis oris muscle and the vermilion was achieved with a symmetrical cupid's bow and philtrum . The only disadvantage of this technique is that the final closure line is placed directly over the philtrum . Since the presentation of midline cleft deformities varies widely, each case should be individually considered and treated . Pfeifer incisions can successfully provide mucosal length, vermilion fullness, and lip symmetry in patients with moderate median cleft lip.
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Pseudomonas aeruginosa is an important gram - negative opportunistic pathogen that has the capability to create variable clinical infections, including, but not limited to, wound infections, urinary tract infections (utis), and blood stream infections (bsi), particularly in hospitalized and immunocompromised patients (1, 2). Pseudomonas aeruginosa was the second most common cause of hospital - acquired pneumonia, as well as the third and fifth common cause of hospital - acquired utis in usa and europe, respectively (2 - 4). Evidently, p. aeruginosa due to low permeability of its outer membrane in combination with efflux pump overexpression is intrinsically resistant to a variety of antibiotics (5). Expression of different classes of extended - spectrum -lactamas enzymes along with aminoglycoside - modifying enzymes (aminoglycoside phosphoryltransferases, aminoglycoside acetyltransferases, and aminoglycoside nucleotidyltransferases), and mutation are other well - characterized mechanisms for antibiotic resistance in p. aeruginosa (6, 7). Data extracted from several independent studies in iran showed that p. aeruginosa was responsible for 11%-32.4% of utis, 17.2%-32.4% of pneumonia, 36.7% of bsi, and 47% of wound infections (8 - 12). Moreover, based on previous studies performed in iran, treatment of p. aeruginosa infections is a major concern for health - care setting due to its high resistant rate to different antibiotics (12, 13). Since determining bacterial isolates relatedness is essential for understanding the transmission routes, different typing methods that can be divided into two major categories, phenotypic and genotypic methods, have been established (14). Phenotypic methods such as serotyping, pyocin typing, and antimicrobial susceptibility typing depend on the environmental factors, therefore, have low discriminatory power (14, 15). Genotypic methods have been performed by different typing techniques such as restriction fragment length polymorphism (rflp), pulsed field gel electrophoresis (pfge), multilocus sequence typing (mlst), enterobacterial repetitive intergenic consensus - pcr (eric - pcr), variable number tandem repeat (vntr), dna hybridization and random amplified polymorphic dna (rapd) (16 - 21). Although mlst and pfge have more discriminatory power, they are expensive, especially in developing countries (22). Rapd - pcr has the advantage of being fast, reproducible, simple, and low - cost, thus, it is an appropriate approach for primary screening of epidemic strains in large number of isolates (23, 24). To our knowledge, limited data are available about genetic diversity of p. aeruginosa recovered from different clinical specimens in isfahan; the purpose of the current study was to determine the antibiotic susceptibility profiles and genetic relatedness in p. aeruginosa isolated from patients admitted to a referral hospital in isfahan, iran . This study was conducted at a teaching referral hospital of isfahan, iran, from february 2013 to november 2013 . During the mentioned period, a total of 150 non - duplicate samples (urine, sputum, wound, blood, and eye discharge) taken from hospitalized - patients admitted to different wards of hospital were analyzed . Primary identification of p. aeruginosa was done based on the standard conventional biochemical tests, including gram staining, catalase, oxidase, oxidative - fermentative (of) tests, pigment production and growth at 42c . Then, primary identification was further confirmed with species - specific pcr using its (16s-23s rrna internal transcribed spacer) primer (25, 26). The study was approved by the ethics committee of isfahan university of medical sciences (no . 392063). Resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi (clinical laboratory standard institute) recommendation (27). The following disks (mast, uk) were applied: ceftazidime (caz, 30 g), imipenem (imp, 10 g), meropenem (mem, 10 g), ciprofloxacin (cip, 5 g), aztreonam (atm, 30 g), polymyxin b (pb, 300 units), and amikacin (amk, 30 g). P. aeruginosa standard strain (atcc 27853) was used as the quality control . Optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer, 2.5 mm mgcl2, 300 m of dntps, 1.7 u taq dna polymerase (cinnagen, iran), 3 l genomic dna (40 ng), and 10 pm of 272- agcgggccaa primer (21) (metabion, germany) in 25 l final volume . Dna amplification was carried out using biometra thermocycler (germany) and following a two - step program, 1) denaturation 5 min at 95c, annealing 5 min at 36c, elongation 5 min at 72c, for 4 cycles, and 2) 31 cycles consisted of 94c for 1 min, 45c for 1 min, 72c for 2 min, followed by a final extension at 72c for 10 min (21). Electrophoresis was carried out using 2% agarose gel (w / v) and 0.5x tbe (tris - boric acid - edta, ph = 7.5 - 8) buffer at 7 v / cm for 3h . We also used 100 bp dna ladder (cinnagen, iran) as the standard molecular size (100 bp-3000 bp). Separated bands stained with ethidium bromide (0.5 g / ml) and visualized picture was captured on gel - documentation system (uvitec, uk). Rapd - fingerprints were recorded as present (1) or absent (0) for each band . By means of freetree and treeview softwares, dice similarity coefficient and unweighted average pair group method (upgma) were used for similarity matrix calculation and cluster analysis, respectively (28, 29). Only major reproducible bands we used cut - off value of 80% for determination of potential clonal relatedness (31 - 33). This study was conducted at a teaching referral hospital of isfahan, iran, from february 2013 to november 2013 . During the mentioned period, a total of 150 non - duplicate samples (urine, sputum, wound, blood, and eye discharge) taken from hospitalized - patients admitted to different wards of hospital were analyzed . Primary identification of p. aeruginosa was done based on the standard conventional biochemical tests, including gram staining, catalase, oxidase, oxidative - fermentative (of) tests, pigment production and growth at 42c . Then, primary identification was further confirmed with species - specific pcr using its (16s-23s rrna internal transcribed spacer) primer (25, 26). The study was approved by the ethics committee of isfahan university of medical sciences (no . 392063). Resistance to antibiotics was evaluated by kirby - bauer s disk diffusion method according to clsi (clinical laboratory standard institute) recommendation (27). The following disks (mast, uk) were applied: ceftazidime (caz, 30 g), imipenem (imp, 10 g), meropenem (mem, 10 g), ciprofloxacin (cip, 5 g), aztreonam (atm, 30 g), polymyxin b (pb, 300 units), and amikacin (amk, 30 g). Optimized rapd reactions mixtures comprised 2.5 l 10x pcr buffer, 2.5 mm mgcl2, 300 m of dntps, 1.7 u taq dna polymerase (cinnagen, iran), 3 l genomic dna (40 ng), and 10 pm of 272- agcgggccaa primer (21) (metabion, germany) in 25 l final volume . Dna amplification was carried out using biometra thermocycler (germany) and following a two - step program, 1) denaturation 5 min at 95c, annealing 5 min at 36c, elongation 5 min at 72c, for 4 cycles, and 2) 31 cycles consisted of 94c for 1 min, 45c for 1 min, 72c for 2 min, followed by a final extension at 72c for 10 min (21). Electrophoresis was carried out using 2% agarose gel (w / v) and 0.5x tbe (tris - boric acid - edta, ph = 7.5 - 8) buffer at 7 v / cm for 3h . We also used 100 bp dna ladder (cinnagen, iran) as the standard molecular size (100 bp-3000 bp). Separated bands stained with ethidium bromide (0.5 g / ml) and visualized picture was captured on gel - documentation system (uvitec, uk). Rapd - fingerprints were recorded as present (1) or absent (0) for each band . By means of freetree and treeview softwares, dice similarity coefficient and unweighted average pair group method (upgma) were used for similarity matrix calculation and cluster analysis, respectively (28, 29). Only major reproducible bands we used cut - off value of 80% for determination of potential clonal relatedness (31 - 33). Out of 150 samples, 54 (36%) were positive for p. aeruginosa culture . Distribution of the 54 isolates were as follow, urine 39 (72.2%), sputum 7 (13%), wound 5 (9.3%), blood 2 (3.7%), and eye discharge 1 (1.9%). The most frequent involved wards were icu 28 (51.9%), followed by graft 12 (22.2%), internal 5 (9.3%) and surgery 9 (16.7%). Thirty - six isolates (66.7%) belonged to male patients and 18 (33.3%) belonged to female patients . The highest percentage (55.6%) of resistance was observed against ceftazidime and imipenem with 30 of the isolates being resistant; all isolates were sensitive to polymyxin b (figure 1). Twenty - eight (51.8%) isolates tested in the study revealed resistance to all applied antibiotics except for polymyxin b. electrophoresis patterns of rapd - pcr for some isolates of studied p. aeruginosa are shown in figure 2 . Genetic relatedness of clinical isolates of the detected p. aeruginosa has been carried out using rapd - pcr assay, which showed 55% to 100% similarity (figure 3). The number of bands in rapd typing varied from 2 to 12, with the length of 150 bp to 4300 bp . Rapd fingerprinting results of p. aeruginosa based on 80% showed 39 different groups (figure 3). Although the majority of isolates had unique fingerprint, group one composed of 3 isolates (patients s48, s29, s31). Isolate s29 was fully sensitive to applied antibiotics, whereas isolates s31 and s48 were resistance to applied antibiotics (figure 3). Groups 22, 31, 35, and 36, each consisted of two isolates, were also obtained from the same ward (figure 3). Based on 65% similarity level, eight main cluster, with most of the strains belonging to cluster 1 were detected (figure 3). Line 1, 14 dna ladder (3000 - 100 bp), lines 2 - 13 showed 12 different rapd types . Abbreviations: amk, amikacin; azt, aztreonam; caz, ceftazidime; cip, ciprofloxacin; imp, imipenem; mp, meropenem; sp, sputum; uc, urine; wo, wound . Constant monitoring of antibiotic susceptibility profiles and genetic relatedness among bacterial infectious agents are essential steps for infection control (34). To improve our understanding about p. aeruginosa antibiotic resistance and their distribution, 54 non - duplicate isolates of p. aeruginosa by using kirby - bauer s disk diffusion and rapd - pcr methods p. aeruginosa involved in this study were highly resistant to different antibiotic families like, monobactams, cephalosporins, quinolones, and carbapenems (figure 1). Our isolates had the highest resistance rate to imipenem and meropenem (55.6%), which is higher than similar reports from iran (11 - 13). In addition, increased percentage of resistance to ciprofloxacin and amikacin (53.7% and 48.1%, respectively) were observed in comparison with previous study (29% and 17%) that have been performed in the same hospital (35). Although, in our investigated hospital, carbapenems (imipenem, meropenem) along with amikacin are the most currently used antibiotics for the treatment of p. aeruginosa infections, results of this study revealed that prescription of them should be limited . In order to optimize treatment of p. aeruginosa infections in our hospital (also it can be mentioned as a limitation of this study), determination of resistance to other antibiotics like piperacillin, piperacillin - clavulanic acid, ticarcillin, ticarcillin - clavulanic acid, gentamicin, and cefepime, are essential to be evaluated in future studies . The finding of present study corresponds with the data presented by haeili and associates, which showed the lowest percentage of resistance (0%) to polymyxin b (11). There are reports that pfge and mlst are more reliable methods for establishing clonal relatedness among p. aeruginosa strains, but because of the high cost, their utilization is limited, especially in developing countries (36, 37). Allegedly, rapd typing is a valuable and useful method for the evaluation of genetic diversity among p. aeruginosa isolates (21, 23, 24). Out of various primers applied for rapd - pcr, primer 272 was reliable because of its higher discriminatory power and reproducible profiles (38, 39). In the present study, all 54 isolates were typeable by rapd - pcr; moreover, rapd typing allowed us to reveal 54 unique finger prints among 54 clinical isolates of p. aeruginosa . Previous studies performed in iran showed different distribution of genotypes . For instance, in the study of salimi et al ., by using rapd typing, only 8 different groups among 129 isolates of p. aeruginosa were reported (40). In addition, in another study conducted by nanvazadeh et al . 9 groups among 50 clinical samples of p. aeruginosa were observed (41). Using limited source for recovering p. aeruginosa although in a previous study the correlation between rapd type and p. aeruginosa infection was suggested, our finding is not in agreement with these data (42). Furthermore, our data demonstrated a weak correlation between rapd and antibiotic resistance profiles; it is supported by the results of other studies (34, 43). In a number of cases, the reason for this diversity may be attributed to the involved different antibiotic resistance mechanisms (5 - 7). Moreover; results of the independent studies showed that determination of antibiotic resistance pattern as phenotypic method for p. aeruginosa typing has low discriminatory power (34, 43). According to our data, the majority of isolates probably originated from host itself, but cross infection of p. aeruginosa is possible to occur in studied hospital . For instance, isolates s48, s29, and s31 were recovered from urine of the patients in different wards or isolates 26 and 33 from the same ward and sample had genetic similarity (based on rapd fingerprinting results). Before taking any decision, we think that clonal relatedness of obtained isolates should be confirmed by more discriminatory methods, such as pfge or mlst . In conclusion, prescription of common antibiotics (imipenem and amikacin) in our hospital due to high resistance rate must be restricted . According to our data, considerable genetic diversity exists among isolated p. aeruginosa; it is possible that different sources of p. aeruginosa be involved in our hospital that can lead to host colonization . In order to accurately control the infection, other p. aeruginosa isolated from hospital environment
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Polyarginine peptides are known as one of the widely used classes of cell - penetrating peptides (cpps) and cellular delivery tools . It has been reported that the presence of the guanidine group in the side chain of arginine plays a key role for improved ability of arginine - rich peptides to cross the cell membrane . Various systematic structural investigations have been performed to determine the required number of arginine residues and the length of the peptide for the optimization of cellular uptake . Short polyarginine peptides containing less than six arginine residues did not exhibit significant cellular uptake in several previously reported investigations . Thus, the presence of more than six arginine residues in the structure of polyarginine peptides is critical for their efficient cell - penetrating functions . However, several investigations were conducted to increase the cellular uptake of polyarginines by attaching the fatty acid to the n - terminal of the peptide . It has been previously reported that the acylation of the n - terminal by fatty acids can facilitate the intracellular uptake of polyarginines . For instance, katayama et al . Synthesized acylated octa - arginines and discovered that the introduction of hydrophobic fatty acid enhanced the intracellular uptake of octa - arginine peptide and its conjugated ubiquitin . Furthermore, lee et al . Designed a class of lipopeptides carrying 715 arginine residues . Among them, myristoylated - hendeca - arginine (c14r11) was found to be the most efficient cell - penetrating peptide . However, the fatty acylated polyarginine peptides that contain 715 arginine residues can potentially cause toxicity, and they can be easily degraded by proteases . Moreover, linear peptides carrying l - form are not stable in serum and therefore have a limited application for in vivo studies (figure s2, supporting information). Replacing l - form amino acids with d - form to improve the peptide stability leads to high cost production . Thus, the synthesis and development of cyclic cpps containing short amino acid sequence with less toxicity is desired . Herein, we designed acylated cyclic polyarginine peptides (acpps) containing five arginine residues and investigated their ability as cell - penetrating peptides . We compared acpps with a corresponding acylated linear polyarginine peptide (alpp) and a nonacylated cyclic polyarginine as controls . We hypothesize that the combination of acylation and cyclization of short polyarginine peptides having less than six arginine residues will increase the intracellular uptake and can generate peptides with molecular transporter properties . For convenience, square brackets [] and parentheses () phosphopeptide ptyr - glu - glu - ile (pyeei) is an optimal peptide ligand for binding to the src tyrosine kinase sh2 domain . In this study, we used negatively charged fluorescein - labeled phosphopeptide f-gpyeei as a model cell - impermeable compound . All peptides were synthesized by fmoc / tbu solid - phase peptide synthesis strategy either manually or using rainin ps3 synthesizer (protein technologies inc . ). Manual reactions were carried out in a glass reaction vessel with a sintered glass frit by mixing under nitrogen bubbling at room temperature . Fmoc - l - amino acid building blocks, fatty acids, and preloaded h - arg(pbf)-2-chlorotrityl resin were used as starting materials . 2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (hbtu), hydroxybenzotriazole (hobt), and n, n - diisopropylethylamine (dipea) in n, n - dimethylformamide (dmf) were used as coupling and activating reagents, respectively, for manual synthesis . In the case of using peptide synthesizer, 0.4 m n - methylmorpholine in dmf was used instead of dipea . Piperidine in dmf (20%) was employed to deprotect fmoc group at each step . To cleave the linear peptide from the resin, a mixture of trifluoroacetic acid (tfa)/triisoproylsilane (tis)/water (92.5/5/2.5, however, in the synthesis of cyclic peptides, the side chain protected linear peptides were first cleaved from the resin by using a cleavage cocktail, containing 2,2,2-trifluoroethanol (tfe)/acetic acid / dichloromethane (dcm) (2:1:7, v / v / v). Cyclization reaction was performed by employing a mixture of 1-hydroxy-7-azabenzotriazole (hoat) and n, n-diisopropylcarbodiimide (dic) in anhydrous dmf / dcm for 12 h. after solvent evaporation, the peptide was deprotected and cleaved from the resin by using a cleavage cocktail reagent r, containing tfa / thioanisole/1,2-ethanedithiol (edt)/anisole (90:5:3:2, v / v / v / v) for 23 h. the crude peptides were precipitated and washed with cold diethyl ether . To purify the crude peptides, we used a reversed - phase high pressure liquid chromatography (rp - hplc) system using shimadzu lc-8a preparative liquid chromatography on a phenomenex gemini c18 column (10 m, 250 21.2 mm) with a gradient 0100% of acetonitrile (ch3cn) containing 0.1% tfa (v / v) and water containing 0.1% tfa (v / v) for 1 h with a flow rate at 15.0 ml / min at the wavelength of 214 nm . As a representative example, the synthesis of dodecanoyl-[r5] is described here . H - arg(pbf)-2-chlorotrityl resin (660 mg, 0.35 mmol, 0.53 mmol / g) was swelled in dmf for 40 min by n2 . Fmoc - arg(pbf)-oh (681 mg, 1.05 mmol, 3 equiv) was coupled to the n - terminal of the resin, using hbtu (398 mg, 1.05 mmol, 3 equiv), hobt (142 mg, 1.05 mmol, 3 equiv), and dipea (366 l, 2.1 mmol, 6 equiv) in dmf (15 ml) by agitating the resin for 1 h using n2 . After the coupling, the resin was washed with dmf, followed by fmoc - deprotection with piperidine in dmf (20%). The subsequent three fmoc - arg(pbf)-oh couplings and one dde - lys(fmoc)-oh (559 mg, 1.05 mmol, 3 equiv; dde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl) coupling was carried out in the same manner, respectively . After removing the fmoc group in side chain of lysine, dodecanoic acid (210 mg, 1.05 mmol, 3 equiv) was coupled using hbtu, hobt, and dipea . Then dde protection group at n - terminal of peptide was removed by 2% hydrazine in dmf, followed by washing with dmf and dcm . The side chain protected linear peptides were cleaved from the resin by using a cleavage cocktail, tfe / acetic acid / dcm (2:1:7, v / v / v), for 1 h. the filtrate was evaporated, and the residue was dried overnight in a vacuum . The cyclization was conducted under a dilute condition with anhydrous dmf / dcm (5:3, v / v, 250 ml), using hoat (190 mg, 1.4 mmol, 4 equiv) and dic (240 l, 1.54 mmol, 4.4 equiv), and stirred for 12 h under nitrogen atmosphere . After cyclization, the solvent was evaporated, and the side chain deprotection was carried out by the addition of reagent r for 2 h. the crude dodecanoyl-[r5] was precipitated and washed with cold diethyl ether and purified by preparative rp - hplc system as described above . Fluorescein - labeled peptides were synthesized with the same protocol before the attachment of fatty acid . We used fmoc-12-aminododecanoic acid instead of dodecanoic acid, and after removing fmoc group, 5(6)-carboxyfluorescein diisobutyrate (cfdi) was attached using 7-azabenzotriazol-1-yl - oxytris(pyrrolidino)phosphonium hexafluorophosphate (pyaop), hoat, and dipea . As an example, we started fluorescein - labeled peptide synthesis in smaller scale with h - arg(pbf)-2-chlorotrityl resin (208 mg, 0.11 mmol, 0.53 mmol / g). Fmoc - arg(pbf)-oh (214 mg, 0.33 mmol, 3 equiv), dde - lys(fmoc)-oh (176 mg, 0.33 mmol, 3 equiv), and fmoc-12-aminododecanoic acid (144 mg, 0.33 mmol, 3 equiv) were used to couple each building block to the resin using hbtu (125 mg, 0.33 mmol, 3 equiv), hobt (45 mg, 0.33 mmol, 3 equiv), and dipea (115 l, 0.66 mmol, 6 equiv) in dmf . Cfdi (170 mg, 0.33 mmol, 3 equiv) was coupled with amino group of 12-aminododecaonic chain using pyaop (172 mg, 0.33 mmol, 3 equiv), hoat (45 mg, 0.33 mmol, 3 equiv), and dipea . After removal of dde protecting group with 2% hydrazine in dmf and washing with dmf and dcm, side chain protected fluorescein linear peptides were cleaved from resin using tfe / acetic acid / dcm (2:1:7, v / v / v). The molecular weights of final products were confirmed by an axima performance matrix - assisted laser desorption / ionization - time - of - flight (maldi - tof) mass spectrometer (shimadzu corporation). Dodecanoyl-(r6): maldi - tof (m / z) c48h96n22o8 calcd . 1108.7781; found 1109.7308 [m + h]. F-dodecanoyl-(r5): maldi - tof (m / z) c69h107n23o14 calcd . 1481.8368; found 1482.7586 [m + h]. W - dodecanoyl-[r5]: maldi - tof (m / z) c61h107n25o9 calcd . Human ovarian adenocarcinoma (sk - ov-3), leukemia (ccrf - cem), and embryonic kidney (hek 293 t) cells were purchased from american type culture collection . The sk - ov-3 and hek 293 t cells were grown in eagle s minimum essential medium (emem), and rpmi-1640 medium (atcc, manassas, va) was used for ccrf - cem cells in a humidified atmosphere of 5% co2 at 37 c . Both media were supplemented with fetal bovine serum (fbs, 10%) and penicillin streptomycin solution (1%, 10,000 units of penicillin and 10 mg of streptomycin in 0.9% nacl). Cytotoxicity of peptides was examined by mts proliferation assay in two human cancer cell lines (sk - ov-3 and ccrf - cem) and one human normal cell line (hek 293 t). Cells were seeded into 96-well plates (sk - ov-3 (5 10 cells / well), ccrf - cem (1 10 cells / well), and hek 293 t (1 10 cells / well)). Then, the cells were incubated with complete media (100 l) overnight . Different concentrations (0600 m) of peptide solution (10 l) were added to cells and incubated at 37 c with 5% co2 for 24 h. then, celltiter 96 aqueous solution (20 l) was added to each well and incubated for 14 h. the absorbance was measured at 490 nm using microplate reader . Sk - ov-3 cells were grown in 6-well plates (2 10 cells / well) with complete emem media 24 h prior to the experiment . The fluorescein - labeled peptide stock solution (1 mm) was prepared in water and diluted in gibco opti - mem i reduced serum medium to obtain the final concentration of 5 m . The media were removed, and the mixture containing fluorescein - labeled peptide solution (5 m) was added . After 1 h incubation, trypsin edta solution was added to detach cells from the plate s surface and remove cell surface binding peptides . After 5 min, the complete media (2 ml) were added to neutralize the trypsin . Finally, the cells were analyzed by bd facscalibur or facsverse flow cytometer using fitc channel . 5(6)-carboxyfluorescein (fam) was used as a negative control . For examination of the cellular uptake mechanism of f-dodecanoyl-[r5] at low temperature, the assay was carried out at 4 c to inhibit the energy - dependent cellular uptake pathways . The sk - ov-3 cells were preincubated at 4 c for 15 min and incubated with the fluorescein - labeled peptide for 1 h at 4 c . Cells were collected and analyzed using flow cytometry with the previously described protocol above . The data collected at 37 c were used as the control . To perform the atp - depletion assay, cells were incubated with sodium azide (10 mm) and 2-deoxy - d - glucose (50 mm) for 1 h before adding the fluorescein - labeled peptide . During the incubation time (1 h), the fluorescein - labeled bicyclic peptide (5 m) was prepared in the opti - mem i reduced serum medium in the presence of sodium azide (10 mm) and 2-deoxy - d - glucose (50 mm). Then, the cells were incubated with this solution for 1 h. the following sample preparation and flow cytometry analysis protocol was the same as described above . Sk - ov-3 cells were seeded with complete emem on coverslips in 6-well plate (1 10 cells / well) and kept until 50% confluency . The media were removed, and cells were incubated with f-dodecanoyl-[r5] (10 m) and f-dodecanoyl-(r5) (10 m) in gibco opti - mem i reduced serum medium (life technologies, grand island, ny) for 1 h at 37 c . Then cells were washed with 1 phosphate buffered saline with calcium and magnesium (pbs) for three times . The coverslips were mounted on microscope slides, and images were obtained using carl zeiss lsm 700 system with a 488 nm argon ion laser excitation and a bp 505530 nm band - pass filter . Sk - ov-3 and ccrf - cem cells were seeded in 6-well plates (2 10 cells / well for sk - ov-3 and 1 10cells / well for ccrf - cem) and grown with complete emem media (rpmi-1640 for ccrf - cem) overnight . A mixture of fluorescein - labeled phosphopeptides f-gpyeei (5 m) and peptides (10 m) were prepared in opti - mem i reduced serum medium at room temperature and incubated for 15 min . Then the cells were incubated with the premixed solution at 37 c with 5% co2 for 1 h. the sample preparation for facs analysis was carried out by previously mentioned protocol described before . In this assay, the acylated cyclic polyarginine peptides were synthesized by fmoc / tbu solid - phase peptide synthesis method . Fmoc - l - arg(pbf)-oh was coupled on h - arg(pbf)-loaded 2-chlorotrityl resin in the presence of hbtu, hobt, and dipea in dmf . Then dde - lys(fmoc)-oh was attached, and a fatty acid was coupled to the side chain of lysine . Dde protecting group was removed by 2% hydrazine in dmf, and a cleavage cocktail containing tfe / acetic acid / dcm (2:1:7 v / v / v) was used for 1 h to cleave the side - chain protected linear peptides from the resin . Cyclization of linear peptides was carried out in the presence of a mixture of hoat and dic in anhydrous dmf / dcm for 624 h. the side - chain deprotection of cyclic peptide was carried out by a cleavage cocktail reagent r for 2 h. the crude peptides were precipitated and purified with rp - hplc as described above . As a representative example, the synthesis of dodecanoyl-[r5] is shown in scheme 1 . A corresponding acylated linear polyarginine peptide (alpp) was synthesized for comparative studies with the cyclic peptide (acpp). Moreover, a cyclic polyarginine without fatty acid [r5] was also synthesized to investigate the effect of the fatty acid on cyclic peptide and its effect on molecular transporting efficiency . To investigate whether the peptide alone can enter into cells, fluorescein - labeled f-dodecanoyl-[r5] and f-dodecanoyl-(r5), where f = fluorescein, were synthesized for facs and microscopy investigations . Chemical structures of synthetic peptides used in this study (f, fluorescein - labeled; [], cyclic peptide; (), linear peptide). The cytotoxicity of all peptides were tested in two different cancer cell lines, adherent (sk - ov-3) and nonadherent (ccrf - cem) cells, and a normal cell line (hek 293 t) using mts assay (figure 2). Cyclic polyarginine [r5] without fatty acid was used to explore the effect of n - terminal acylation on cytotoxicity and cellular uptake . Alpp (dodecanoyl-(r5)) and [r5] peptides showed consistently less cytotoxicity in all three cells compared to acpps (dodecanoyl-[r5] and dodecanoyl-[r6]). After 24 h incubation, acpps showed approximately 20% toxicity in cells at a concentration of 25 m in ccrf - cem cells . However, dodecanoyl - linear (r5) and [r5] did not exhibit significant cytotoxicity at 25 m and showed less than 20% toxicity at the concentration of 100 m . In sk - ov-3 cells, dodecanoyl-(r5) and [r5] showed more than 80% cell viability at the concentration of 25 m . In normal cells (hek 293 t), all peptides exhibited less than 5% toxicity at 25 m . This differential behavior of the peptides in normal, and cancer cells can be possibly rationalized through the interaction between polyarginine peptides and cell membranes . The membrane of cancer cells holds more negative charges compared to that in normal cells because of the presence of anionic lipids such as phosphatidylserine . Therefore, polyarginine peptides can be interacted with cancer cells more effectively compared to normal cells . These data indicated that acpps are more toxic than alpp and a nonacylated cyclic peptide [r5], especially at concentration of 25 m . Thus, a noncytotoxic concentration of 510 m was used in cell - based assays . Comparison of cytotoxicity between cyclic and linear acylated polyarginine peptides and nonacylated cyclic peptide [r5] at various concentrations against ccrf - cem, sk - ov-3, and hek 293 t after 24 h. the intracellular uptake studies of fluorescein - labeled acylated cyclic and linear pp, f-dodecanoyl-[r5] and f-dodecanoyl-(r5), was carried out in sk - ov-3 cells by using flow cytometry and confocal laser scanning microscopy (clsm) methods . Fluorescein (fam, f) alone was selected as a negative control . As it is shown in figure 3, the f-dodecanoyl-[r5] and f-dodecanoyl-(r5) showed approximately 13.7- and 10.3-fold higher cellular uptake than that of control 5,6-carboxyfluorescein (fam), respectively, in sk - ov-3 cells . The cellular uptake of f-dodecanoyl-[r5] was confirmed by clsm images (figure 4). F-dodecanoyl-[r5] showed higher fluorescence intensity compared to that of f-dodecanoyl-(r5) in sk - ov-3 cells . Therefore, acpp f-dodecanoyl-[r5] was found to be a more efficient cell - penetrating peptide compared to its linear counterpart . As shown in figure 4, the fluorescence signal is extended through the whole cells, suggesting that f-dodecanoyl-[r5] can get localized in the nucleus as well as cytoplasm . Comparative cellular uptake of f-dodecanoyl-[r5] and f-dodecanoyl-(r5) (5 m) in sk - ov-3 cells (1 h). Confocal laser scanning microscope image of (a) f-dodecanoyl-[r5] and (b) f-dodecanoyl-(r5). The peptides were incubated for 1 h in sk - ov-3 cells at 10 m concentration . The mechanism of the cellular internalization of f-dodecanoyl-[r5] was investigated by a temperature control assay at 4 c along with atp depletion assay . These two assays have been widely used to examine the energy - dependent endocytosis . Facs results showed that the intracellular uptake of f-dodecanoyl-[r5] was significantly reduced at 4 c, indicating that the mechanism of internalization was mainly dependent on the endocytosis pathways (figure 5). Furthermore, atp depletion assay was performed to investigate receptor - mediated endocytosis . To induce atp depletion, sk - ov-3 cells were preincubated with sodium azide (10 mm) and 2-deoxy - d - glucose (50 mm) for 1 h prior to the experiment, and a similar concentration was maintained during the incubation (1 h) with f-dodecanoyl-[r5]. The results showed that the cellular uptake of f-acpp was inhibited in the presence of sodium azide and 2-deoxy - d - glucose, suggesting that receptor - mediated endocytosis is involved for the cellular uptake of f-acpp . In atp depletion assay, the basic cellular uptake of fam was higher compared to that in temperature control assay . However, this is evident that the intracellular uptake of f-dodecanoyl-[r5] was inhibited, and there was no significant difference between the cells treated with fam compared to those treated with f-dodecanoyl-[r5] in temperature control and atp depletion assays, suggesting that endocytosis is the major pathway for the cellular uptake of f-dodecanoyl-[r5]. Cellular uptake of f-dodecanoyl-[r5] (5 m) in sk - ov-3 cells in temperature control assay at 37 and 4 c, and atp depletion assay with nan3 (10 mm) and 2-deoxy - d - glucose (50 mm) analyzed by flow cytometry . Cells with no treatment were used as control . The ability of acpps as a molecular transporter was evaluated and compared by selecting a fluorescein - labeled phosphopeptide, f-gpyeei, as a molecular cargo . Phosphopeptide, pyeei (ptyr - glu - glu - ile) is an optimal peptide template for the sh2 domain of src tyrosine kinase . Several analogues of this peptide have been synthesized as potent ligands for this target . Because of the presence of the negatively charged amino acid residues in the structure of the peptide including phosphorylated tyrosine moreover, the internalization of the negatively charged phosphopeptide in cancer cells by diffusion is more difficult because cancer cell membranes are composed of more negatively charged lipids . Thus, cellular delivery of cell - impermeable negatively charged phosphopeptides is significantly challenging . We have previously reported different peptide - based carriers for the intracellular delivery of negatively charged phosphopeptides as model cell - impermeable drugs in several cell lines . In this study, the intracellular uptake of f-gpyeei was monitored in the presence and absence of synthetic peptides after 1 h incubation by flow cytometry . As it is exhibited in figure 6, the acpps (dodecanoyl-[r5] and dodecanoyl-[r6]) delivered the phosphopeptide more efficiently compared to alpps, dodecanoyl-(r5) and -[r5]. The intracellular uptake of f-gpyeei in the presence of dodecanoyl-[r5] and dodecanoyl-[r6] was enhanced by 3.4- and 5.5-fold higher than the uptake in the absence of acpps . However, dodecanoyl-(r5) and -[r5] only improved 1.3- and 1.4-fold intracellular uptake, respectively . The results showed that acylated and cyclized polyarginine peptides can deliver the phosphopeptide effectively . However, the intracellular uptake of the phosphopeptide did not improve significantly in the presence of either acylated linear polyarginine peptide or cyclic [r5]. These data suggest that a combination of acylation and cyclization would improve the molecular transporting efficiency of the polyarginine - based peptide (containing less than six arginines) for the intracellular delivery of a cell - impermeable phosphopeptide . This has been previously reported that the acylated linear octa - arginine increased the cellular uptake of molecular cargoes by just adding fatty acid to the n - terminal of octa - arginine . However, we discovered that both cyclization and acylation in a short penta - arginine can significantly improve the delivery of a cell - impermeable phosphopeptide in sk - ov-3 cells . Cellular uptake of f-gpyeei (5 m) in the presence of dodecanoyl-[r5] and -[r5], dosecanoyl-(r5), and dodecanoyl-[r6] (10 m) in sk - ov-3 cell line . Phosphopeptide delivery efficiency of dodecanoyl-[r5] were compared with known cpps (r7, crrrrrrr; tat, ygrkkrrqrrr). The major driving forces for the intracellular delivery are presumed to be structural rigidity through cyclization of the peptide and the interaction of the fatty acid with the cell membrane . It has been previously reported that the cellular uptake of the peptide can be increased due to the structural rigidity by cyclization of arginine - rich peptides . They proposed that the maximal distance between guanidine groups of arginine residue can lead to an efficient transduction of arginine - rich peptides . Our investigations showed that dodecanoyl-[r6] is able to deliver more efficiently by 1.6-fold higher f-gpyeei uptake compared to that of dodecanoyl-[r5]. Increasing the number of positively charged arginine residues can enhance the cellular uptake through ionic interactions with the negatively charged phosphopeptide and/or phospholipid in the cell membrane through ionic interactions . However, the higher number of arginine residue is not the only responsible element for the efficient cellular internalization . For example, it has been reported that polyarginine containing 11 amino acids (r11) showed higher cellular uptake compared to the polyarginine containing 13 amino acids (r13). At the same time, r11 was found to be a more potent transporter compared to r9 in prostate cancer cells . These investigations showed that an optimal number of arginine residues are required for the highest degree of functionality . However, the greater number of amino acid residues in cyclic peptides can decrease the structural rigidity, which lowers the ability of the peptide to get into cells . Dodecanoyl-[r5] was also compared with several commonly cpps, such as cr7 and tat (ygrkkrrqrrr) peptides . The acpp improved the cellular uptake of the phosphopeptide by 1.4- and 1.8-fold higher than those of cr7 and tat, respectively (figure 6). These results revealed that although other peptides containing arginine can deliver the molecular cargo, acpp dodecanoyl-[r5] with a shorter peptide sequence than cr7 and tat can work as a molecular transporter with higher efficiency . Chemical structures of acpps with different length of fatty acid chains (c8, c12, and c16). To investigate the effect of the chain length on the cell penetration potency, we synthesized octanoyl-[r5], dodecanoyl-[r5], and hexadecanoyl-[r5] (figure 7). Acpps showed less than 20% toxicity in cells at the concentration of 25 m (figure 8a). The in vitro toxicity results showed that increasing the fatty acid chain length caused enhanced toxicity in cells as hexadecanoyl-[r5] was more cytotoxic than dodecanoyl-[r5] and octanoyl-[r5]. These data indicate that the fatty acid chain length could alter the interaction with the cell membrane and disturb the membrane integrity . On the basis of the cytotoxicity data, (a) cytotoxicity assay of cyclic polyarginine peptide - fatty acid conjugates against sk - ov-3 cells (24 h incubation). (b) cellular uptake of a phosphopeptide, f-gpyeei (5 m), in the presence of peptide - fatty acid conjugates (10 m) in sk - ov-3 cells . It has been previously reported that there was a relationship between the length of fatty acid in polyarginines and their cellular uptake, meaning that the optimal length of fatty acid is required for optimal functionality based on the peptide sequence and cell type . The results exhibited that the cellular uptake of f-gpyeei was improved in the order of octanoyl-[r5] <dodecanoyl-[r5] <hexadecanoyl-[r5] (figure 8b). The cellular uptake of f-gpyeei in the presence of hexadecanoyl-[r5] was 9.3- and 6.0-fold higher than that in the presence of octanoyl-[r5] and dodecanoyl-[r5], respectively . These data suggest that the length of the attached fatty acid chain in the structure significantly influences the efficiency of the peptide as a molecular transporter . Sixteen - carbon chain length (c16) was found to be an optimized length for the intracellular delivery of f-gpyeei in sk - ov-3 cells . After acpps were found to act as cpp and molecular transporter, a systematic investigation was performed to modify the fatty acid to another hydrophobic moiety . W4-[r5], the whole fatty acid chain was replaced with four tryptophan residues . The second conjugate w - dodecanoyl-[r5] had one more tryptophan at the end of the dodecanoyl fatty acid chain . W4-[r5] enhanced intracellular delivery of f-gpyeei by 4.1-fold higher compared to that of dodecanoyl-[r5] in sk - ov-3 . However, w - dodecanoyl-[r5] improved the uptake of f-gpyeei by 1.3-fold higher compared to that of w4-[r5] in ccrf - cem cells (figure 9). Thus, it is not straightforward to compare the cellular uptakes in sk - ov-3 and ccrf - cem . However, the results could be assessed indirectly by relative comparison with the cellular uptake by w4-[r5]. These data suggest that the presence of hydrophobic tryptophan moieties could enhance the molecular transporting efficiency . In other words, appropriate modification of hydrophobic moiety in cpps can increase the drug delivery ability of acpp . Thus, cyclic nature, short length of polyarginine, and hydrophobic segments were found to be critical elements to generate a peptide with an optimized molecular transporter efficiency . Cellular uptake assay of a phosphopeptide, f-gpyeei (5 m), in the presence of w4-[r5] and w - dodecanoyl-[r5] (10 m) against sk - ov-3 and ccrf - cem cell lines (1 h incubation). Cells with no treatment were used as control . To evaluate whether the presence of peptides can enhance the pharmacological effect of a molecular cargo, the antiproliferative activity of doxorubicin (dox) was examined in the presence of dodecanoy-[r6] ([r6]-c12) in mcf-7 cells in a time - dependent assay . As it is shown in figure 10, the antiproliferative potency of dox (5 m) was enhanced by 7%, 11%, and 14% in the presence of [r6]-c12 (5 m) when compared with that of the drug alone after 24, 48, and 72 h incubation, respectively . These results showed that the cell proliferation of mcf-7 cells was inhibited in a time - dependent manner suggesting a sustained drug release by peptide leading to a higher inhibitory effect . The presence of the peptide possibly improved the cellular uptake of dox and enhanced the antiproliferative activity . Time - dependent antiproliferative activity of dox (5 m) in the presence and absence of [r6]-c12 (5 m). In conclusion, acylated cyclic polyarginine peptides were synthesized and examined as cpps and potential molecular transporters . Acpps showed higher potency as molecular transporter compared to the corresponding linear counterpart and cyclic polyarginine without fatty acid . The mechanism of the peptide internalization was found to be energy - dependent endocytosis . Cyclization and acylation reactions on the structure of the peptide enhanced the intracellular uptake of polyarginine peptides although they carry a short length of sequence . This intracellular delivery property of acpps can be optimized by modifying the length of fatty acid chain . To the best of our knowledge, this is the first report of cyclic fatty acylated polyarginine peptide as molecular transporter of a cell - impermeable phosphopeptide . This study provided insights about how a combination of the cyclic nature and acylation can improve the cell internalization of polyarginines . Further investigations are undergoing to determine whether conjugation of cell - impermeable hydrophobic drugs to acylated cyclic polyarginines can be an efficient method for designing novel drug delivery systems.
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As of 2014, the worldwide prevalence of type 2 diabetes mellitus (t2 dm) was estimated to be 9% among adults aged 18 years with great impact on mortality, particularly in low- and middle - income countries (lmic) [1, 2]. Moreover, globally, approximately 25% to 75% of diabetes cases remain undiagnosed [3, 4], until further complications, especially at the macro- and micro - vascular level, manifest clinically . In latin america, an important strategy to prevent or delay t2 dm complications is the early identification of those with undiagnosed diabetes; yet, universal screening for diabetes at the population level is not practical in resource - limited settings . The american diabetes association recommends the use of glucose test as t2 dm screening in people with overweight and obesity as well as in those with other risk factors . As a result, risk assessment scores have been developed to address this problem in a simple and inexpensive way . Most of the available algorithms for diabetes screening have been developed in caucasian [79] and asian populations [1013] and very few in other ethnic groups [14, 15]. To date, one diabetes risk score has been developed and validated in latin america so far which was derived from one urban area in brazil, thus bearing limited generalizability to the wider region . Furthermore, it is well established that before adopting existing risk scores as screening tools in different populations and ethnic groups, their performance needs to be evaluated, calibrated, or validated in local settings . As the american diabetes association, the peruvian ministry of health recommends diabetes screening in general population with fasting glucose in adults aged 40 to 70 years with risk factors . However, fasting glucose is not always available in primary care settings, especially in semiurban and rural areas . As a result, a major challenge to be overcome in many countries is the implementation of a simple, fast, and laboratory - free based screening method . Consequently, we aimed to develop a simple laboratory - free risk score to identify people with undiagnosed diabetes and incident diabetes in peru, a latin american country that spans coastal, andean, and rainforest settings . In order to do so, this work benefited from two large - scale population - based surveys: the first one, representative at the national level, was used to develop the score, and the second one, a cohort study, was utilized for external validation . The national survey of nutritional and biochemical indicators for noncommunicable diseases (eninbsc in spanish), conducted by the peruvian national institute of health, was used to develop our predictive model . This was complemented with the cronicas cohort study, whose baseline and longitudinal information was used to validate the risk score . The eninbsc is a national population - based survey carried out in peru between august 2004 and april 2005, designed to estimate the prevalence of hypertension, type 2 diabetes mellitus, and other risk factors for noncommunicable diseases at the national and regional level . Potential participants were those aged 20 years, habitual residents in the study area, and able to provide consent for their participation in the study . Pregnant women and those currently breastfeeding were excluded from the study . As per design, the eninbsc sample was stratified according to peru's five major regions of the country: lima, rest of the coast, urban highlands, rural highlands, and jungle . In each stratum, cluster of blocks the cronicas cohort study is an ongoing cardiopulmonary project aimed to estimate the prevalence and incidence of hypertension, diabetes mellitus, and obesity in four different settings in peru that differ in terms of their urbanicity and altitude: pampas de san juan de miraflores, in the highly urbanized lima, puno in the altitude (3,825 meters above the sea level) contributing with rural and urban areas, and tumbes, a semiurban area in the northern coast of peru . The study started in september 2010 and a follow - up visit was completed in march 2014 . A sex- and age - stratified sample was selected at random for each of the settings and all participants aged 35 years, full time residents in the study area, and able to consent, were enrolled . Follow - up data used for this analysis was collected, on average, at 30 months after baseline . The first one lasted on average 40 minutes and was carried out to apply a face - to - face questionnaire regarding data about household characteristics, demographics, lifestyles behaviors, risk factors, and blood pressure measurements . The second visit lasted 30 minutes on average and was planned to have an appropriate period of fasting for blood sampling for glucose, total cholesterol, hdl - cholesterol, and the remaining anthropometric measures (height, weight, and waist circumference) using standard procedures . Similarly, the procedures of the cronicas study has been published elsewhere . In brief, participants responded to a face - to - face questionnaire applied by trained community health workers . Data collected comprised risk factors for cardiovascular disease based on a modified version of the who step approach questionnaire for surveillance of noncommunicable disease . A period of 8 to 12 hours of fasting was required for blood sampling to collect fasting glucose, total cholesterol, and hdl - cholesterol . Height, weight, and waist circumference were also assessed, and blood pressure was measured in triplicate after five minutes of resting using an automatic monitor (omron hem-780) previously validated in adult's population . In both studies, diabetes was defined as any of the following conditions: fasting glucose 7.0 mmol / l (126 mg / dl) and/or self - report of physician diagnosis . Fasting glucose was assessed by an enzymatic colorimetric method (glucose oxidase god - pap) in both studies . After excluding individuals without known diabetes, undiagnosed diabetes variables included in the analyses were built to guarantee similarities between both studies: sex; age (<55 and 55 years); education (in years); self - reported smoking (current versus never / former smoker); alcohol use (user versus never user); self - reported diabetes in first - degree relatives (participant's parents and/or siblings); and levels of physical activity (low versus moderate / high levels, based on the transport - related domain of the ipaq). Anthropometric measurements included in the analysis were body mass index ((bmi), <25, 2529.9, and 30 kg / m), waist circumference (<90, 9099.9, and 100 cm), waist - to - height ratio (<0.50, 0.500.59, 0.600.69, and 0.70), and hypertension (measured or previously diagnosed). A total of 4,206 participants were enrolled in the eninbsc, but only 2,472 were included in this analysis . Reasons for exclusion were 1,524 because of age <35 years to make both databases comparable, 129 because of no data about fasting plasma glucose levels being available, and 81 because of known diagnosis of diabetes . In the cronicas study, 3,601 participants were enrolled at baseline but only 2,948 records were analyzed as 465 had no data about glucose levels, and 188 were excluded because of previous diagnosis of diabetes . In addition, only data from 2,577 participants was used in the longitudinal assessment of the risk score (comparison of baseline characteristics among those included and excluded from longitudinal analysis is shown in online supplement: e - table 1; see supplementary material available online at http://dx.doi.org/10.1155/2016/8790235). Initially, population characteristics of both studies were tabulated using proportions in the case of categorical variables and means and standard deviation (sd) with numerical variables . Then, the prevalence and 95% confidence intervals (95% ci) of total diabetes and undiagnosed diabetes were estimated in each study . The risk score was derived from data of the eninbsc survey taking into account the multistage sampling strategy of the study . Each potential risk factor (i.e., sex, age, family history of diabetes, etc .) Was assessed in bivariate models using logistic regression and undiagnosed diabetes as the dependent variable . Then, risk factors with a p value <0.10 in the bivariate analysis were included in a multiple logistic regression model using stepwise backward elimination with a significance level of 5% . The hosmer - lemeshow goodness - of - fit test was used to assess how well the predicted prevalence matched the observed prevalence of undiagnosed diabetes (i.e., p values over 0.20 indicate that model fits well). As we sought for an easily applicable and implementable algorithm, the risk factors in the final model were each assigned a weighted score by rounding up all regression coefficients in the final model to the nearest integer as in a previous report . For the evaluation of the risk score, the area under the receiver operating characteristic (roc) curve, sensitivity, specificity, and positive and negative predictive values (ppv and npv) were calculated . The optimal cut - point was determined using the youden index, a single statistic that captures the performance of a diagnostic test (i.e., sensitivity + specificity 1). As one of the main aims of a nonlaboratory risk score is to identify people who warrant having a blood test (i.e., fasting glucose, glycated haemoglobin, etc . ), the cut - point with the highest sensitivity was also estimated and described . We assessed the performance of our score using bootstrap techniques as well as carrying out an external validation using the cronicas cohort study . The resulting 1,000 prediction models were then assessed to estimate the bootstrap auc using the bias - corrected version of the confidence intervals . In addition, using baseline data from the cronicas cohort study, validation measures (auc, sensitivity, specificity, predictive values, and likelihood ratios) were estimated . To evaluate the performance of our algorithm, the peruvian risk score was compared to previously published models for undiagnosed diabetes including the brazilian risk score, the qingdao score, the indian risk score, the kuwaiti risk score, the patient self - assessment score, and the rotterdam risk score using the c - statistic . Finally, using the follow - up data of the cronicas cohort study, the risk score was also evaluated to detect incident cases of t2 dm by excluding those with diabetes diagnosis at baseline . Analyses were performed using stata 13.0 (statacorp, college station, tx, usa). The protocol and informed consent forms of the eninbsc study were reviewed and approved by the instituto nacional de salud and the centro nacional de alimentacin y nutricin, both part of the ministry of health in lima, peru . In the case of the cronicas cohort study, protocol and consent forms were reviewed and approved by the institutional review boards of the universidad peruana cayetano heredia and the ngo asociacin benfica prisma in lima, peru, and the johns hopkins university in baltimore, usa . Overall, participants from the cronicas study were 5 years older, reported consuming lower levels of alcohol, and were less physically active than those from the eninbsc survey . The overall prevalence of diabetes was 5.1% (129/2538; 95% ci: 4.2%5.9%) in the eninbsc survey and 8.7% (272/3135; 95% ci: 7.7%9.7%) in the cronicas cohort study's baseline . After excluding those with known diabetes, undiagnosed diabetes was present in 2.0% (48/2457; 95% ci: 1.4%2.5%) in the eninbsc survey and in 2.9% (85/2948; 95% ci: 2.3%3.5%) in the cronicas cohort study . After stepwise backward logistic regression, age, diabetes in first - degree relatives, and waist circumference were independently associated with undiagnosed diabetes (table 2). The hosmer - lemeshow test showed that the final model fitted relatively well (p = 0.21). The peruvian diabetes risk score was constructed based on the coefficients of that final regression model . The score gave an auc of 0.73 (95% ci: 0.650.78), and the optimal cut - point for undiagnosed diabetes using the youden index was 2 (figure 1). With this cut - point, about 34.8% of participants were categorized as at high risk of diabetes: sensitivity 69.6%, specificity 65.8%, and ppv and npv of 3.9% and 99.1%, respectively . With a cut - point 1, 69.8% of participants would be at high risk of diabetes with improved sensitivity (93.5%) but lower specificity (30.6%). Table 3 shows the performance of the risk score for detecting undiagnosed diabetes at different cut - points . When bootstrap was used, the performance of our risk score was similar to the obtained in the development model (auc = 0.72; 95% ci: 0.650.78). In addition, when the risk score was evaluated by applying the score to the cronicas cohort study's population, the auc for undiagnosed diabetes was 0.68 (95% ci: 0.620.73). At the suggested cut - point of 2, 42% would be categorized as undiagnosed diabetes with sensitivity, specificity, ppv, and npv of 70.2%, 58.9%, 4.8%, and 98.5%, respectively (table 4). On the other hand, with a cut - point 1, 80% would be categorized as undiagnosed diabetes with sensitivity, specificity, ppv, and npv of 94.0%, 20.0%, 3.3%, and 99.1%, respectively . When previous published algorithms for undiagnosed diabetes were applied to the cronicas cohort study, the performance of the rotterdam score (p <0.001), indian score (p <0.001), and qingdao score (p <0.01) was poorer than our score; however, our algorithm performed similar to the other assessed models, such as the brazilian risk score (p = 0.93), the kuwaiti score (p = 0.26), and the patient self - assessment score (p = 0.74), but having only three variables . The performance of this risk score was also assessed to predict incident cases of diabetes using the longitudinal data from the cronicas cohort study . One hundred twenty - one new cases of diabetes were found accounting for 6,207 person - years at risk, with an overall incidence of 1.95 (95% ci: 1.632.33) cases per 100 person - years of risk . The auc of the score was 0.66 (95% ci: 0.610.71). With a cut - point 2, 42.5% of participants were categorized as at high risk of developing diabetes: sensitivity, specificity, ppv, and npv were 69.4%, 58.9%, 7.8%, and 97.4%, whereas, for a cut - point 1, the respective values were 79.9%, 91.9%, 20.7%, 5.5%, and 98.1% . Using a national population - based survey, a simple nonblood based risk score based on age, history of diabetes in first - degree relatives, and waist circumference was built and shown to perform moderately in detecting undiagnosed diabetes when externally validated . Moreover, the performance of the score was almost similar for detecting incident cases of diabetes in the peruvian population . A relatively recent systematic literature search found 23 different blood - free prevalent diabetes risk scores: ten from europe, nine for asian populations, two from the united states, and two from middle east . In addition, and not included in the aforementioned review, only one risk score was developed in latin america using brazilian urban population . The same systematic review reported that auc for these predictive models was greater in the development studies (range: 0.65 to 0.88) than in the validation studies (range: 0.63 to 0.80), similar to our findings . Another systematic review found that several noninvasive algorithms were created using variables such as age, gender, waist circumference and/or bmi, and family history of diabetes in the final model . As impracticality due to use of the algorithms was a common barrier to the uptake of risk scores by healthcare staff and individuals, our model, created with three of these more common variables, reached a moderate - to - high sensitivity depending on the used cut - point . Moreover, two of these variables are easily evaluable during medical appointment or through individual's self - assessment, and only a measuring tape and no calculations are required to be implemented in clinical practice or at the population level . From a cross - sectional point of view, with a cut - point 2, from 1000 participants assessed by the peruvian diabetes risk score, a total of 420 would be classified as undiagnosed diabetes with the detection of 20 cases and only 6 will be missing . On the other hand, with a cut - point 1, from 1000 screened individuals, a total of 804 would be categorized as having undiagnosed diabetes with the detection of 27 cases and only 7 will be missing . Thus, the reduction of the cut - point of the risk score would increase sensitivity but reducing the specificity and imposing the need of performing a confirmatory test (i.e., fasting glucose) to almost the double of individuals, with the benefit of having only 7 more people diagnosed . Longitudinally, the same risk score would detect an important number of participants at risk of developing diabetes: 43% of screened individuals would be classified at high risk of diabetes, and of them, 8% would develop diabetes in the next 2.5 years . According to a previous study, 17 reports described a noninvasive model to predict the development of diabetes and included a median of six risk predictors, ranging from 2 to 11 . Although our score did not perform as good as other well - known longitudinal models in the literature such as the findrisc or the aric scores [35, 36], it only included three variables and was built using cross - sectional information . In addition, some variables used in the aforementioned studies are difficult to standardize within a country as peru, that is, food portions, physical activity, or sedentarism, limiting therefore its use on a wider scale and in a simple pragmatic fashion . Our algorithm performed better than the rotterdam, the indian, and the qingdao risk scores in our population, which highlights the need of calibration and/or development of a specific score for different ethnic groups before its adoption . As there are ethnic differences in risk factors for diabetes and peru is considered a multiethnic country, it is necessary to create specific scores or recalibrate existing algorithms before applying in specific contexts . In addition, with only three variables included, the performance of our predictive model was similar to the other assessed scores included in the analyses . Taken together, the score developed has the potential to augment, in a pragmatic manner, initial rapid screening for diabetes, especially at various nonspecialized primary healthcare services . Our findings also demonstrate that approximately 35% of cases of t2 dm (39% in the eninbsc survey and 33% in the baseline of the cronicas cohort study) are not aware of their disease . Results are similar to those reported in previous studies in our context and in similar settings in latin america . As the developed risk score is simple, it does not require a blood test or laboratory services, and it might be easily implemented in clinical practice . Moreover, because our score asks for general information in the form of age and diabetes in first - degree relatives and is complemented by a simple anthropometric measure of waist, there is potential for the score to be self - administered . According to our results, any patient aged 55 years and above and having at least one first - degree relative with t2 dm has greater probability of having undiagnosed diabetes but also is at risk of developing diabetes in the future . In addition, a greater central obesity, that is, 100 cm or more, independent of the other terms of the score is alone a good predictor of diabetes as reported in previous studies . Our algorithm included waist circumference instead of body mass index as other risk scores, providing a better indicator of accumulation of visceral fat and metabolic dysfunction in our context . Recently, the peruvian ministry of health has published the guide of clinical practice for diagnosis, treatment and control of diabetes mellitus in primary care and only recommends screening in general population with plasma glucose among adults between 40 and 70 years with obesity or overweight as suggested by the american diabetes association . As in other lmic, plasma glucose is not always available in primary care, especially in semiurban and rural areas; therefore, a major challenge to be overcome in many countries is the implementation of a simple, fast, and laboratory - free based screening method . Moreover, within the peruvian context, no risk score has been proposed as part of the aforementioned guide . Thus, our algorithm might fill a gap to facilitate further specialized assessment of high risk individuals for diabetes, an approach that may be of utility to various other countries facing similar challenges . The strengths of this study include the use of a national population - based survey, including urban and rural areas across major geographical regions, to develop the peruvian diabetes risk score, as well as its validation using bootstrap but also an independent longitudinal cohort study . Additionally, it is only based on three variables ensuring its simplicity to be used and implemented . However, the study has also some limitations . First, we have utilized fasting plasma glucose as the gold standard for diagnosing diabetes instead of an oral glucose tolerance test (ogtt). Although the ogtt is more sensitive and specific than the fasting plasma glucose, more cases would have been detected with the overload of glucose; it is rarely performed as part of the routine clinical practice . Second, the cronicas cohort study did not include information from the amazon rainforest as did the eninbsc survey . When a sensitivity analysis was performed excluding individuals from the jungle from eninbsc data, in addition, the score was created using a national survey to be applicable to the entire peruvian population . Third, some variables were not assessed in our logistic regression model such as dietary intake or history of gestational diabetes as such data was not available . As a result, some caution should be made when our algorithm is compared to other risk scores . Fourth, our model is based on the idea of risk stratification instead of individualisation; for instance, variables were categorized instead of being preserved as numerical . Nevertheless, the performance of our score did not change when age and waist circumference were treated as numerical variables (data not shown). Moreover, our idea was to develop a simple and easily applicable score instead of a complex algorithm for predicting undiagnosed and incident diabetes . Finally, as other diabetes risk scores, the model warrants further scrutiny before it can be used in other populations . The peruvian diabetes risk score, built using age, self - reported diabetes in first - degree relatives, and waist circumference, proves to be a simple pragmatic screening tool for undiagnosed and incident cases of diabetes in peru . This experience in generating such simple, easy - to - use approaches for the identification of t2 dm can serve to inform other similar lmic efforts who are on early stages of diabetes prevention . This tool, due to its simplicity, can facilitate various initiatives oriented to introduce and scale up early preventative and management strategies on a wider scale.
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Cancer is one of the leading causes of death worldwide and accounted for 7.6 million cases (13% of all deaths) in 2008; in 2013 in the united states there are 12,340 estimated new cases and 4,030 deaths by cervical cancer [american cancer society: cancer facts and figures 2013]. Meanwhile in romania, based on statistical data from 2012, cervical cancer is ranked third place taking in account the women cancer incidence, after the breast and colorectal cancer . Cervical cancer develops in a multi - stage process, successively passing from a cervix with a normal epithelium to cervical intraepithelial neoplasia and finally leading to invasive cervical cancer . A relevant epidemiologic risk circumstance for the progress of the cervical cancer is represented by the presence of high risk human papillomavirus infections . Consequently, considerable importance has been given to the molecular premise which leads to the progression of cervical cancer . Rnai (rna interference) is a natural mechanism able to specific inhibit a target gene . The prospective exploitation as a therapeutic has lately been strengthened by the small interfering rna (sirna) delivery to human tumours . Rnai has been demonstrated as a relevant investigation device for particular target genes by using exogenous sirnas . Sirnas are single - stranded non - coding rnas with 2023 nucleotides length, being able to negatively modulate gene expression profile at post - transcriptional level . Sirnas represent an encouraging novel type of medicaments for cancer therapy, as a result of targeting the mutated tumour suppressor genes or activated oncogenes . Many recent investigations have shown that sirna can efficiently inhibit oncogene expression in cancer cells . Various investigations demonstrated clearly that the deactivation of p53 constitutes a crucial step in carcinogenesis . The present study was designed to investigate the potential use of p53 rnai to block p53 expression, as well as the subsequent effect on cell invasion and gene expression on the human cervical cancer cell . Hela is a human cervical carcinoma cell line; this was incubated in a 5% co2 incubator at 37c . For maintenance, cells were cultured in dmem medium, 10% fcs (foetal bovine serum), 100 u / ml penicillin and 100 mg / ml streptomycin, 2 mm l - glutamine, 1% nonessential amino acids, all purchased from sigma - aldrich, bucharest, romania . For this investigations 510 hela cells / well were used for reverse - transfection, based on a preliminary optimised protocol for p53sirna, using siport neofx as transfection agent (ambion, usa). A validated p53sirna pool (sc-29435, santa cruz) was used at a concentration of 50 nm in serum starvation condition using optimem medium (gibco, invitrogen, romania), in accordance with the producer recommendations . Siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability . We performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument (roche applied science) according to the described protocol by . The total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent (sigma - aldrich, bucharest romania) based on the producer recommended protocol for each triplicate specimen . All of the samples had a rin (rna integrity number) higher than 7.5 . The 8 genes selected were assessed using specific primer and upl based on in silico design . The total rna (500 ng) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr (roche, bucharest romania). For the gene amplification we used taqman universal pcr master mix, in a 20 l volume in a 96-well plate using the roche lightcycler 480 system . The qrt - pcr reaction amplification program was as follows: 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step . The data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . As housekeeping gene the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit (r&d, catalog no . Hela is a human cervical carcinoma cell line; this was incubated in a 5% co2 incubator at 37c . For maintenance, cells were cultured in dmem medium, 10% fcs (foetal bovine serum), 100 u / ml penicillin and 100 mg / ml streptomycin, 2 mm l - glutamine, 1% nonessential amino acids, all purchased from sigma - aldrich, bucharest, romania . For this investigations 510 hela cells / well were used for reverse - transfection, based on a preliminary optimised protocol for p53sirna, using siport neofx as transfection agent (ambion, usa). A validated p53sirna pool (sc-29435, santa cruz) was used at a concentration of 50 nm in serum starvation condition using optimem medium (gibco, invitrogen, romania), in accordance with the producer recommendations . Siport neofx 0.25% concentration was able to achieve the maximum inhibition of target gene with no effects on cell viability . We performed the invasion assays on hela cells to evaluate the impact of p53sirna treatment using the cim - plate 16 with the xcelligence rtca dp instrument (roche applied science) according to the described protocol by . The total rna for the cells treated with 50 nm p53sirna andthe control group respectively at 24 hours post treatment was extracted using trireagent (sigma - aldrich, bucharest romania) based on the producer recommended protocol for each triplicate specimen . The rna concentrations and quality were assessed by the nanodrop-1100 agilent 2100 bioanalyzer spectrophotometer . All of the samples had a rin (rna integrity number) higher than 7.5 . The 8 genes selected were assessed using specific primer and upl based on in silico design . The total rna (500 ng) from all the samples was reverse transcribed using the first strand cdna synthesis kit for rt - pcr (roche, bucharest romania). For the gene amplification we used taqman universal pcr master mix, in a 20 l volume in a 96-well plate using the roche lightcycler 480 system . The qrt - pcr reaction amplification program was as follows: 10 minutes at 95c for enzyme activation followed by 45 cycles of 15 seconds at 95c and 1 minute at 60c for the amplification step . The data analysis was carried out to compare the gene expression values for the treated and untreated groups using ct method . As housekeeping gene the evaluation of the vegf protein expression at 48 hours post treatment was done using human vegf quantikine elisa kit (r&d, catalog no . Xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time, lacking any exogenous labels . The cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation, specifying the commencement and ratio of invasion and migration of hela cells . This data can facilitate to comprehend the response to treatment in dynamic . In figure 1 we can observe a delay and a reduction of the cell migration after the p53sirna treatment . Taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis . Relative gene expression quantification using ct method leads to the downregulation of the selected gene, presented in the figure 2 . After 48h post transfection with p53 sirna inhibition in hela cell line, vegf protein was found dowregulated in the culture medium than in the control group (figure 3). Xcelligence system is an innovative device that allows the scanning of cellular response via an impedance - based technology in real time, lacking any exogenous labels . The cim - plate 16 furnishes a kinetic cell - response profile to p53sirna throughout an investigation, specifying the commencement and ratio of invasion and migration of hela cells . This data can facilitate to comprehend the response to treatment in dynamic . In figure 1 we can observe a delay and a reduction of the cell migration after the p53sirna treatment . Taqman qrt - pcr assay was used to examine the effect of p53sirna on a panel of 8 genes related to apoptosis and angiogenesis . Relative gene expression quantification using ct method leads to the downregulation of the selected gene, presented in the figure 2 . After 48h post transfection with p53 sirna inhibition in hela cell line, vegf protein was found dowregulated in the culture medium than in the control group (figure 3). Although at this moment cervical cancer is considered as a preventable disorder there is a significant risk of disease recurrence causing a persuasive necessity to research new therapeutic targets for this disease management . It is now well recognized that the tumour progression of all cancers is characterized by intensified proliferation and invasion rate and diminished in apoptosis . At the same time the apoptosis and angiogenesis are interconnected as can be observed from figure 4, using string.9 database . The idea of this study is in agreement with the previous studies which are based on the hypothesis that once mutated, p53 exercised oncogenic role . By using sirna the role of the present study is to emphasize the cooperation between oncogenic mechanisms, confirming the crosstalk between apoptotic and angiogenic mechanisms . This has a significant therapeutic relevance based on the fact that mutated p53 is related to cancer aggressiveness or to promoting metastasis . In a similar study was observed that, by using sirna targeting p53/p73, tumoral cells were sensitized to chemotherapy . In a recent study, pinx1 was displayed as a novel target gene of p53, proposing the suppression of p53/pinx1 pathway as a novel mechanism to enhance the telomerase activity in cervical cancer cells, and leading to reduction of cell proliferation . Targeting oncogenic signals such as the mutated p53 gene that gains oncogenic role, we observed a downregulation of specific genes involved in apoptosis but also in angiogenesis, connected with a reduction of the invasion rate in the case of p53sirna therapy . After 24 hours of treatment with p53sirna we can observe downregulations of important genes involved in apoptosis and angiogenesis, as displayed in figure 2 . This study confirmed our hypothesis that p53 plays a central role in carcinogenesis as displayed in figure 3, as important therapeutic target . P53 could promote the convergence of the apoptotic and angiogenic pathways in cervical cancer cells . It has been proved that p53 is able to indirectly downregulate vegf gene expression, via the retinoblastoma (rb), in a p21-dependent mode, confirming its function in cell - cycle regulation as displayed by recent studies . The complete comprehension of the cross - talk between p53 and angiogenic pathways represents a challenge in cancer therapy [2426]. Another study reveals that mutant p53 correlates with overexpression of vegf . In a similar study the inhibition of mutant p53 represents a significant therapeutic target, a fact emphasized by walerych as rebel angel . P53 gene knockdown in hela cells plays an important role not only in apoptosis but also in angiogenesis through various mechanisms that regulate tumour cell development and migration . The angiogenic phenotype related to p53 targeted by sirna in human cancers is frequently associated with resistance to conventional genotoxic anticancer agents . Therefore, p53 inhibitions become dependent and rely on angiogenesis for growth and metastasis . The results in the current research may also have an important significance outside the context of cervical cancer, by using specific inhibitors for p53 for increasing therapeutic response in a wide range of tumoral pathology.
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Atherosclerosis is a coronary heart disease (chd), and is the most common cause of death in the industrialized world . High blood pressure (bp), hypercholesterolemia, diabetes mellitus (dm) and smoking are considered as the major risk factors for the development of atherosclerosis . In addition, increasing age, male sex, family history and genetic factors predispose patients to chd.1 there is some data that have suggested the possible potential role of some infectious agents in the pathogenesis of atherosclerosis.26 the possibility of infectious agents may trigger the process of atherosclerosis by direct or indirect inflammatory effects, especially at younger ages.5 although different studies have been carried out to prove this hypothesis some infections may contribute in the pathogenesis of atherosclerosis; nothing has ever been conclusively proven . The possible role of some agents such as chlamydophila pneumoniae,6 hepatitis c. virus,7 human immunodeficiency virus,7 hepatitis b virus,8 cytomegalovirus,9 herpes virus,9 epstein - barr virus,10 mycobacterium tuberculosis,11 and helicobacter pylori,3 have been considered in the pathogenesis of atherosclerosis . Among these pathogens, c. pneumoniae is important, because it could be effectively treated by antibiotics.12 c. pneumoniae is an obligatory intracellular organism, and is responsible for at least 10% of community - acquired pneumonias . This bacterium has also been associated with pharyngitis, bronchitis, otitis, influenza - like illness, sinusitis and myocarditis.1214 conflicting results have been reported about the relationship between c. pneumonia and atherosclerosis . West et al.15 apfalter et al.,16 and zibaeenezhad et al.17 reported no association between c. pneumoniae and atherosclerosis but others found significant association between c. pneumoniae and atherosclerosis.61819 the results of jha et al.,6 dabiri et al.18 and sessa et al.19 support the idea that c. pneumoniae may have a role in the development of atherosclerosis . This controversy about the role c. pneumoniae in the pathogenesis of atherosclerosis emphasizes the importance of more research works . The aim of this study was to demonstrate whether there is any difference between two group of patients with coronary atherosclerotic plaque and subjects without coronary atherosclerosis for the presence of c. pneumoniae dna by polymerase chain reaction (pcr) method . This case - control study was carried out on 60 formalin - fixed paraffin - embedded (ffpe) coronary artery biopsies in the molecular pathology laboratory, ghaem hospital, mashhad, iran in 2010 . Coronary artery tissues were selected from another study that was performed previously by our colleagues for determining prevalence of atherosclerotic plaques in autopsy cases with non - cardiac death in northeast iran . In this study, left coronary artery (lca), right coronary artery (rca) and left circumflex artery (lcx) had been evaluated grossly and microscopically . Our materials included 30 coronary artery tissues with atherosclerotic plaques that were defined as study group, and 30 coronary artery tissues without atherosclerotic plaques that served as the control group . Archived slides of the two groups were reviewed by two pathologists for confirmation of diagnosis, tissue adequacy and selection of the best paraffin blocks for dna extraction . For all subjects, the weight and height were determined, and the body mass index (bmi) was calculated . Five to seven, 5-m - thick sectionswere cut from each ffpe specimen under sterile conditions, and then the dna was extracted by using proteinase k and non - heating dna extraction method.20 dna concentration was determined by using the thermo scientific nanodrop 2000 spectrophotometer, and specimens with low dna content (<20 ng/l) were excluded from the study . Pcr was performed for detecting c. pneumoniae dna by using the pcr kits (dna technology (jsc), pcr kit, moscow, russia). 10 l of pcr master mix and 0.5 l unit of taqpolymerase (hot start taq dna polymerase) were added into each paraffin sealed tube and were mixed, then 5 l (100 ng) of dna samples were added (except for positive and negative controls) and were spun at 1,000 rpm for 3 - 5 s. the tubes were placed into applied biosystems (abi) veriti thermal cycler and pcr was performed with the program of 180 s at 94c for the first step and then 45 cycles was run as follows: 50 s at 94c, 50 s at 64c, 50 s at 72c . Amplified pcr products were electrophoresed on a 2% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light by gel documentation instrument . Pcr was interpreted as positive when the dna band corresponds to the band of the positive control (254bp) in addition to the internal control band (ic:370 bp). Results of pcr in control and atherosclerotic groups were analyzed with spss version 11.5 by a statistician . We used fisher's exact test for comparison of categorical variables and independent sample t - test for continuous variable in patients and control groups . The age range in the control group was 20 - 46 years with a mean (standard deviation) age of 26.0 (6.41) years . In the study group, it ranges from 18 to 50 years with a mean (sd) age of 32.0 (9.46) years and the t - test showed no significant difference between the ages of patients in the study and control groups (p=0.97). Twenty - five patients (83.3%) were males and five patients (16.7%) were females . The male to female ratio was 5:1 and no significant difference was seen between study and control groups for gender (p = 1.00) by fisher's exact test . The mean bmi of the patients in the study and control groups were 24.4 (3.77) and 23.9 (2.62) kg / m respectively [table 1]. The frequency of atherosclerotic plaques in the different vessels of heart of the patients in the study is as shown in table 2 . As a result, the possibility of the appearance of advanced atherosclerotic plaques s in lca was higher than the two other vessels . Concurrent occurrences of different stages of atherosclerosis were seen in our patients . Only one vessel involvement with fibro - fatty morphology (mild) was seen in five patients (16.7%), one vessel with advanced plaque was observed in three patients (10.0%), seven patients (23.3%) had one vessel with fibro - fatty and one vessel with advanced plaque, two vessels with advanced plaque were noticed in six patients (20.0%), three vessels with advanced plaque were observed in five patients (16.7%) and four patients (13.3%) had two vessels with advanced plaque and one fibro - fatty morphology . Comparisons between patients with coronary atherosclerosis and controls for weight, height and body mass index the frequency of atherosclerotic plaques in different vessels of the heart in the study group positive pcr result for c. pneumonia was seen in one (3.3%) sample among the 30 coronary artery tissues with atherosclerosis (advanced plaque), while all the control samples were negative . Fisher's exact test showed no significant difference for detection of c. pneumoniae between samples of coronary artery with and without atherosclerotic plaque (p = 1). Ischemic heart disease could be observed in the absence of its major risk factors such as increasing age, male gender, hypertension, hyperlipidemia, smoking and dm . Inflammatory cells are seen during all steps of atherogenesis, and some infectious agents may trigger this inflammation.113 although some studies showed that c. pneumoniae can increase the risk of atherosclerosis, 61819 but we did not find any significant difference in the frequency of c. pneumoniae dna isolation in the coronary arteries with and without atherosclerosis by pcr method . We believe that at least two important factors can account for this discrepancy in the results from previous studies: methodology and epidemiology . C pneumonia infection can be diagnosed by various methods such as serology with an increase in serum antibodies against the organism, dna detectionby pcr, immunocytochemistry, electron microscopyand c. pneumonia isolation by culture.14 in other studies, the most widely used method for identifying of c. pneumoniae infection was based on antibody detection . The frequency of antibody against c. pneumoniae starts to rise in children and is observed approximately in 50% of adolescents.1214 somesero - epidemiologic studies have shown a relationship between c. pneumoniae and atherosclerosis . Saikku et al.,21 podsiady et al.22 and romano et al.23 detected a higher level of antibody against c. pneumoniae in chd compared to control group but zibaeenezhad et al.,17 romeo et al.,24 and ericson et al.,25 found that the presence of antibodies was unable to predict coronary artery events . We used pcr for detection of c. pneumonia; this assay appears to be more sensitive than cell culture.1426 our finding was in concordance with those of west et al.,15 jantos et al.27 and satpathy et al.28 similarly, reszka et al.,29 studied presence of c. pneumonia dna in aortic vessels, voorend et al.30 in cerebral vessels, and kwon et al.31 in carotid arteries and could not establish any significant difference in pcr results between the study and control groups . These findings suggest that serology could detect past as well as present infection, whereas pcr detects current c. pnuemoniae infection . Considering the study of west et al.15 and satpathy et al.28 who showed a higher c. pnuemoniae igg antibody in patients with chd compared to control group but could not detect the organism by nested pcr assay in any specimen strengthens our hypothesis . We believe that the lack of standardized methods for the detection of c. pnuemoniae infection used in the aforementioned studies is probably one of the important reasons for the discrepancies in their results . C. pnuemoniae is a common human pathogen; the majority of infected subjects have few or no symptoms . The frequency of antibodies to c. pneumoniae is approximately 50% in the northern hemisphere.1432 exposure to c. pneumoniae is probably common in iran . Moghaddam et al.33 reported 38% seropositivity for c. pneumonia antibodies (igg and igm) in healthy subjects by elisa method in tehran, iran . Studies carried out in iran on the association between c. pneumonia infection, and atherosclerosis revealed conflicting results; while some studies showed the association between c. pneumonia infection, and atherosclerosis1834 others didnt demonstrate any significant associationbetween c. pneumonia and atherosclerosis.1735 the anatomical localization of vessels may also be a factor influencing the results from previous studies . Jha et al.6 showed that c. pneumoniae may be associated with chd and coronary artery was more susceptible to c. pneumoniae as compared with carotid artery . Bahrmand et al.25 and ericson et al.34 examined the relationship of c. pneumoniae infection to the severity of coronary atherosclerosis by pcr and serology respectively . They examined coronary artery samples with two different methods, and both revealed a high rate of reactivity to this bacterium in severe atherosclerosis and a much lower rate in mild atherosclerosis; therefore, they concluded that severity (stage) of atherosclerosis can be presumed as an important factor in diagnosis of c. pneumoniae infection . Our study was performed on 30 archived ffpe coronary artery tissues of patients with atherosclerosis . This sample size may be too small for final decision; therefore, future study with a larger number of case groups and also using fresh specimens are helpful . In conclusion, we did not find any significant association between c. pneumoniae infection and coronary artery atherosclerosis by pcr method . Further studies involving a greater number of patients and also using more specific methods for detecting living c. pneumonia organisms by culture can be helpful.
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Confrontation with the consequences of diabetes causes a crisis in physical, mental, and spiritual dimensions . Sometimes the spiritual crisis can be tremendous . Since spiritual health coordinates different aspects of human life, this study aimed to identify the spiritual health of patients with defects caused by diabetes this was a qualitative - phenomenological and descriptive study and the participants were selected from rehabilitation centers in isfahan and valiasr hospital in zanjan . Outcome of this phase of the study was 173 codes and 2 groups that included hindering factors in spiritual health and the promotion of the relation with god . The concepts that patients had experienced as hindering factors of the treatment process were disappointment and hopelessness, guilt, feeling distant from god, quitting obligatory acts and knowing god as cruel . The concepts that patients had experienced as contributing factors to the healing process were resorting to imams, god s ordering the disease as a reward, fear of god s punishment, believing in miracles, being closer to god, believing in the mercy of god, returning to religious practice, feeling of enjoying life and knowing that the disease is the atonement of sins . With regard to the importance of spiritual and religious care as one of the tasks of nurses, as the key members of health team, they should respect the patients beliefs and values in addition to considering their physical and mental conditions . Although the mortality rate in diabetic patients due to ketoacidosis and infection is declining, deaths from diabetes complications including diabetic foot complications has increased dramatically . According to some reports, its rate in the first year after amputation is 1340 percent, in the third year, 3565 percent and in the fifth year it is 3980 percent, which figures are comparable to death rates in malignancies . In addition, the loss of job and the need for medical and nursing care, reduction in social and family interactions, and lifestyle changes are the major problems that influence the family and socioeconomic status of these patients . The absence of the feet and lower limbs can be extremely problematic for these patients and exerts too much energy for walking; therefore, amputation of higher limbs or development of ulcers may occur . Ultimately, these patients with amputated legs need long - term hospitalization, rehabilitation, home care and social support . Organ amputation due to diabetes causes crisis in the physical, mental, and spiritual life of the patients . In this case, the spiritual health is the unique element that can harmonize physical, mental and social aspects of the human life and is necessary for coping with the disease . As a matter of fact, when the spiritual health is seriously at risk, a person may experience emotional disturbances such as loneliness, depression and loss of meaning in life . The spirit of prayer is a deep human instinct, which is the center of humanity, and where the passion and knowledge to communicate with the world comes from . Forms and statements of people who pray are different: speaking, listening, waiting and wailing . It exposes them to severe stress and they seek different approaches to cope and adapt to life . Koenig believes that religion creates a positive attitude towards the world and makes the person powerful against unfortunate events in life such as diseases and helps to improve the life with motivation and energy . This increases tolerance and acceptance of situations that cannot be changed . In many cases of emergency in which science is unable to help a person, this issue is of vital importance especially in serious cases . The increasing interest of medical scientists to the impact of praying in the treatment of other diseases can be indicative of treatment of some diseases in case of modern medicine failure . On the other hand, medical researchers also acknowledge the importance of medical procedures of traditional and complementary therapies including prayer to treat the diseases . Even the western scientific community has recently published articles showing that doctors cannot be indifferent to the religious beliefs of patients for treatment . This study was conducted to understand the spiritual experiences of the patients with organ amputation due to diabetes . The aim of phenomenological studies is to understand the phenomena of human experience through the analysis of participants verbal explanations of experiences . The study population consisted of patients with defects, caused by diabetes, who had referred to rehabilitation centers in esfahan and zanjan provinces . Sampling was purposive and the inclusion criteria for participants were the injury caused by diabetes and the willingness to participate in the interview . There were 15 participants entered the study (8 men and 7 women at 5070 years of age). All interviews were fully recorded and all participants were given a code based on the interviews . In this study, the researcher tried to be cautious with the information collected to be free from any kind of bias and to avoid persons with poor memory and those who could not provide the correct information . In the interviews, the researcher attempted to withdraw his assumptions about the phenomenon under study and thus help to strengthen the data . They were asked that how has your life changed compared to before the disease? In response, they described their life experiences with this disability, and how their performance was changed due to the disease . During the interview, the patients focused on cases where the disease led to their functioning . When it was necessary to clarify the data in specific areas, the questions were asked in details . Finally, participants were asked if they wanted to provide alternative explanations in relationship with their disease . The researcher wrote down the participants statements through repeated listening to in - depth interviews . After the formation of concepts and ideas from each interview, the next interview was conducted . The above process was repeated for each interview (second stage of colaizzi s method). After the all interviews, the concepts were formulated in particular subjects and were classified into categories (third stage) and eventually all ideas were combined so that a complete description of the phenomenon under the study was derived and presented (the fourth, fifth and sixth stages). Finally, the concepts were returned to the participants and were analyzed (the seventh). In this study, the criteria for validity and reliability of data were lincoln and guba . In order to make the study believable, the participants and the research partners also reviewed all the data . To achieve reliability and objectivity of the research data, the account access was used . To increase the portability of the present study, the complete research project was introduced . There were 15 patients participating in this study, comprised of 7 women and 8 men . The age range of participants was between 50 - 75 years with mean age of 59.7 years . Length of diabetes was between 530 years and the length of organ amputation caused by diabetes also varied from 2 weeks to 11 years with mean of 4.2 years . The education level comprised of uneducated to junior high school . Regarding employment, 53.3 percent of the participants (8 people) lost their jobs and were unemployed due to organ amputation caused by diabetes . About 66.6 percent (10 people), a leg was amputated below the knee and 26.6 percent of the participants (4 people) both legs were amputated below the knee area . In one patient both legs below the knee and the middle finger were amputated due to diabetes . The concepts derived from patients experiences as factors interfering spiritual health in the treatment process were disappointment and hopelessness, guilt, feeling distant from god, quitting obligatory acts and considering god cruel . Factors promoting the healing process were deemed by god, fear of god punishment, belief in miracles, being closer to god, believing god to be merciful, returning to religious practice, enjoying life and indicating the disease as the atonement of sins . Most of the participants, encounteringdiabetes - related organ amputatio, said that in such circumstances, intellectual protections, religious sources, and having a strong relationship with a higher power could improve the quality of life, and support the individuals and reduce the severity of symptoms . One of the participants expressed his experiences in turning to practice religion as follows: i was deemed to cut my foot to know god, i am sure there is a wisdom behind this, i ve been weak in a prayer . I was wavering, but when my leg was cut, i celt much closer to god, i know god took my foot, but after this incident, i feel that god wanted to try me, before this, if my prayer was late, i did not become upset . But now, if i do not perform my prayer on time, i will get upset, i feel that i did not do an important job, i become worried with no reason (participant 1). Another participant stated when my uncle came to visit me, he said the disease is the expiation of sins . Whenever this is said, my heart becomes calm, but i did not commit a sin to deserve such a punishment . Even, i had not bothered anything in my life . I do not remember, i wish, everything could be finished in this world (participant 3). Since my foot (right foot) has been cut off, i rub it every day . As i cannot bend i m afraid; it may give testimony against me in the doomsday and tells god that i had been overeating because our hands and legs have rights and we must consider this (participant 5). Our tenant is a believer who always reads the quran . When she saw that i was confused, she said, let me read the koran for you to be calm . Although i am illiterate and i do not understand anything from the quran, when i hear the peaceful verses of the quran, i forget my sorrows i participate in the middle of moharram mourning ceremony every year and in mourning ceremonies every thursday, but when my leg was cut, i did not go there anymore because i feel i m too far from god and as a result, these ceremonies cannot help when the wounds of the foot were fine after surgery, at fast; i performed my prayer in sitting position, but i realized that i do not feel good in just sitting and praying, i just hurt myself (participant 15). The prayer and vow are likely to enhance the human s tolerance against diseases and problems communication with the source of existence and asking him to help would repair the physical and mental powersof man and relieves a lot of disorders and diseases . Therefore, the appeal to the imams in patients with organ amputation due to diabetes is high . Several studies have also shown that when the spiritual health is at a serious risk, prayers in acute cases may face difficulty . In these cases, praying for the patient can be an important spiritual intervention . One of the most useful sentences in the form of prayers could be the short ones, asking god for fulfillment of the needs, melting away the fears and giving hopes to the patient and ultimately remembering that god is able to meet the needs of the patients . If the patient is depressed, angry or upset or suffering from extreme pain, the expression of praying sentences is like the salt to the wound . Nurses should pay close attention in the implementation of these provisions, and if the patients are upset, they should leave them alone . In this regard, a research by tracy et al . Was conducted on two thousand nurses in critical care units in which 55% of nurses reported that patients and families were asking them to pray . Another study which studied the effects of prayer on blood infection, randomly employed 1691 experimental and 1702 control patients . Parameters studied included duration of fever in patients, duration of hospitalization and mortality in the hospital . The results of this study showed a significant difference between the duration of fever and length of patients stay that was lower in the intervention group compared to the control group . Nowadays, some of the organizations that evaluate health care systems and are responsible for granting accreditation to them, suggest that the spiritual needs of the patients in health care should be also evaluated . Finally, the importance of spiritual and religious care, which is today considered as one of nurses tasks, urges them as the key members of health teams to respect patients beliefs and values as well as their physical and mental aspects.
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Vitamin d deficiency was once thought to exclusively affect bone metabolism, but now there is ample evidence of its role in many other conditions including metabolic syndrome, autoimmune diseases, and cancer . Vitamin d receptors are recognized to be in numerous extraskeletal tissues, such as pancreas and muscle . A systematic review by pittas and colleagues reported that vitamin d may have a beneficial effect on the action of insulin, either directly or indirectly . Several observational studies in adults, including the framingham heart study, have reported an inverse association between vitamin d status and insulin resistance . However, data from children and adolescents do not consistently report this inverse relationship [510]. Even in studies reporting this association in adolescents and children, further, none of the studies in adolescents have studied the effect of puberty on this association . A fall in insulin sensitivity with compensatory increase in insulin secretion has been reported in puberty [11, 12]. High prevalence of cardiometabolic risk factors, including insulin resistance (64.8% of normal weight children had at least one cardiometabolic abnormality), even in healthy young children of normal weight, has been reported in indian children and adolescents . South - asian adolescents, including those with normal bmi, have a higher prevalence of insulin resistance when compared with age and bmi matched european adolescents [14, 15]. This ethnic difference in insulin sensitivity was attributed to the higher body fat percentage in south - asian children . An alternative hypothesis to explain the ethnic differences in insulin sensitivity is vitamin d deficiency . Despite plenty of sunlight available throughout the year, a high prevalence of vitamin d deficiency has been reported in all age groups including toddlers, school children, pregnant women and their neonates, and adult males and females residing in rural and urban india . Vitamin d deficiency has been reported in 8492% children in the age group of 1018 . In view of high prevalence of both, insulin resistance and vitamin d deficiency in asian - indian children and adolescents, we sought to examine the relationship between vitamin d status and insulin resistance . We also sought to determine if puberty affected the relationship of vitamin d status and parameters of insulin resistance and sensitivity in asian - indian children and adolescents . The study was conducted as a prospective observational study after approval from the ethics committee of the all india institute of medical sciences (aiims), new delhi . Subjects were selected from the children and adolescents attending the endocrinology outdoor services at aiims for evaluation of obesity . The inclusion criteria included children between the ages of 617 years with obesity as per international obesity taskforce (iotf) criteria . The exclusion criteria included subjects with diagnosed diabetes mellitus or taking metformin or any weight reducing drugs, subjects with any known systemic illness or endocrine or metabolic disorders, known to be associated with obesity, or subjects with symptoms to suggest hypothalamic obesity were excluded from the study . Blood samples were collected in fasting state (minimum 9 hours fasting), followed by oral glucose tolerance test (anhydrous glucose = 1.75 gm / kg bodyweight, maximum of 75 gram, dissolved in 250 ml of water, ingested over 5 minutes) with sample collection at 60 and 120 minutes after glucose ingestion . Height was measured with holtain's stadiometer (holtain inc ., crymych, pembs . Waist circumference was measured (nonelastic measuring tape) at the end of normal expiration at the midpoint between the iliac crest and the lower edge of ribs in the midaxillary line . Blood pressure (bp) was measured in the right upper limb in sitting position with a mercury sphygmomanometer after 5-minute rest with an appropriate size cuff . Total body fat was measured with dual energy x - ray absorptiometry (dxa, hologic qdr 4500 with pediatric software, hologic inc ., fat mass index (fmi, fat mass in kg / height in meter) and fat - free mass index (ffmi, lean body mass+ bmc)/(height in meter) were calculated . Bone mineral area, content (bmc) and areal density (abmd) at the femoral neck, lumbar spine (l1 - 5), and forearm were measured, using a hologic qdr 4500a fan beam dxa machine . Hologic spine phantom (hologic spine phantom number 21373) was scanned daily before subject evaluation . The measured phantom bone mineral density was stable throughout the study period at 0.9150.945 gm / cm . Similarly, whole body phantom (hologic wb number 1252) was also scanned before subject evaluation and remained stable during study period . The in vivo precision error for adults was 0.62% for femoral neck abmd, 0.65% for lumbar spine abmd, and 0.77% for forearm abmd . However, in view of additional radiation exposure, the reproducibility of these scans was not assessed among children . Pubertal stage was assessed by a single endocrinologist, based on breast stage and pubic hair development in girls and genitalia development in boys . B - mode ultrasonography was used to measure cimt (7.510 mhz probe, philips envisor ultrasound machine) using standard protocol . Insulin resistance (ir) was calculated by computer - based model called homeostasis assessment model (homa - ir) utilizing fasting blood glucose and fasting serum insulin levels .the whole body insulin sensitivity index (wbisi) or matsuda index was calculated by the formula suggested by matsuda and defronzo .area under curve (auc) was calculated for blood glucose (0, 60, and 120 minutes) and serum insulin (0, 60, and 120 minutes) by using trapezoidal rule . Insulin resistance (ir) was calculated by computer - based model called homeostasis assessment model (homa - ir) utilizing fasting blood glucose and fasting serum insulin levels . The whole body insulin sensitivity index (wbisi) or matsuda index was calculated by the formula suggested by matsuda and defronzo . Area under curve (auc) was calculated for blood glucose (0, 60, and 120 minutes) and serum insulin (0, 60, and 120 minutes) by using trapezoidal rule . Complete blood counts, liver function tests, renal function test, serum calcium (corrected calcium with serum albumin), phosphate, alkaline phosphatase, uric acid, and blood glucose were measured in all subjects with an automated chemistry analyzer (roche hitachi 912 chemistry analyzer, gmi inc . Glycosylated hemoglobin (hba1c) was measured in whole blood using ion - exchange high performance liquid chromatography (bio - rad laboratories inc ., ca, us). Serum insulin was measured on an autoanalyzer (roche elecsys 2010) using electrochemiluminometric assay . Serum total cholesterol (tc), triglyceride (tg), and hdl cholesterol (hdl - c) levels were estimated directly with automated analyzer, while ldl cholesterol (ldl - c) was estimated by using the friedewald equation . Vitamin d deficiency was defined as a serum 25(oh)d levels less than 20 ng / ml . This was further subdivided to severe, moderate, and mild vitamin d deficiency if serum 25(oh)d levels were <5 ng / ml, 5<10 ng / ml, and 10<20 ng / ml respectively . Statistical analysis was carried out using spss (spss version 11.5; spss inc ., continuous variables not showing normal distribution were treated with appropriate log transformations before any parametric analysis . Student's t - test was used to determine statistical difference between subjects with serum 25(oh)d <10 ng / ml or more . Pearson's correlation was used to assess association between continuous variables and partial correlations were used to determine relationship after adjusting for relevant covariates . Stepwise linear regression analysis was used with serum 25(oh)d as dependent variable in the model . Values for serum 25(oh)d, serum insulin, homa - ir, and matsuda isi were not normally distributed; therefore, they were logarithmically transformed which yielded normal distribution . A total of 62 subjects participated in this cross - sectional study from june 2008 to may 2009 . The mean age of subjects was 13.0 3 years (35 boys; 27 girls). Assessment of pubertal status by tanner's method showed 19 subjects in stage 1, 11 in stage 2, 6 in stage 3, 3 in stage 4, and 23 subjects in puberty stage 5 . No significant difference was observed between boys and girls except higher whr in boys than girls (0.93 0.05 versus 0.89 0.05; p = 0.013). All study subjects were classified as vitamin d deficient (mean sd 8.5 4.2 ng / ml; maximum minimum: 3.919.2 ng / ml, median = 6.9 ng / ml). Severe vitamin d deficiency (<5 ng / ml) was seen in 11 subjects (17.7%) while 30 subjects had serum 25(oh)d levels between 5<10 ng / ml (48.3%). Serum 25(oh)d equal to or more than 10 but less than 20 ng / ml was present in 21 subjects (33.8%). Pearson correlation coefficient analyses showed significant inverse relationship between serum 25(oh)d and total body fat percentage (r = 0.31, p = 0.01) and serum 25(oh)d and fmi (r = 0.33, p = 0.01, figure 1). Partial correlation analysis showed persistence of significant correlation between serum 25(oh)d and total body fat percentage (r = 0.37, p = 0.005) and between serum 25(oh)d and fmi (r = 0.36, p = 0.006) even after adjustment for age, sex, pubertal stage, and bmi . Although blood glucose and serum insulin levels showed inverse relationship trends with serum 25(oh)d, but these were not statistically significant (data not shown). Similarly, no statistically significant correlation was seen between serum 25(oh)d and parameters of insulin sensitivity or resistance . Fmi showed positive correlation with homa - ir (r = 0.26; p = 0.04), auc insulin (r = 0.41; p = 0.001), auc glucose (r = 0.31; p = 0.018), and negative correlation with wbisi (r = 0.42; r = 0.001). However, total body fat did not show any correlation with parameters of glucose and insulin parameters . On the basis of serum 25(oh)d, subjects were divided into two groups, group a with serum 25(oh)d level <10 ng / ml (n = 41) and group b with serum 25(oh)d level 10 ng / ml (n = 21) (an arbitrary cutoff to compare subjects with mild vitamin d deficiency with moderate and severe vitamin d deficiency, 25). No statistical significant difference was seen in blood glucose, serum insulin, and auc for both, glucose and insulin between group 1 and group 2 . Trends for higher insulin resistance (homa - ir) and lower insulin sensitivity (wbisi) were seen in subjects with lower serum 25(oh)d concentrations but not statistically significant . Total body fat percentage and fmi were significantly higher in group 1 in comparison to group 2 (p = 0.015 and 0.007, resp . ). However, no significant difference was seen in waist and hip circumference and waist hip ratio . Similarly, ffmi, lipid parameters, or thyroid profile were also not different between the two groups . Group 1 (prepubertal group; tanner stage 1) had 19 subjects, group 2 (peripubertal group; tanner stage 2, 3, and 4) had 20, while group 3 (postpubertal group; tanner stage 5) had 23 subjects . No significant correlation was seen between vitamin d status and any other parameters including total body fat percentage, fmi and parameters of insulin resistance and sensitivity in the prepubertal (group 1) or peripubertal groups (group 2). Partial correlations after adjusting for age, sex, and bmi were also nonsignificant for both groups (data not shown). In postpubertal subjects (group 3, n = 23), a significant inverse correlation was seen between serum 25(oh)d and homa - ir (r = 0.41, p = 0.03, figure 2). This association between serum 25(oh)d and homa - ir persisted even after control for age, sex, and bmi (r = 0.505, p = 0.023). Positive trends of association between serum 25(oh)d and wbisi were also seen, but this was not significant (r = 0.29, p = 0.17), even after adjusting for age, sex, and bmi (r = 0.35, p = 0.13). Serum 25(oh)d showed significant inverse correlation with total body fat percentage (r = 0.58, p = 0.003) and fmi (r = 0.49, p = 0.016) which improved after adjusting for age sex and bmi (r = 0.63, p = 0.002 for total body fat percentage; r = 0.582, p = 0.007 for fmi). Serum 25(oh)d also showed significant inverse relationship with fasting serum insulin levels (r = 0.44, p = 0.034) which improved after adjusting for age, sex, and bmi (r = 0.53, p = 0.016). When prepubertal subjects (group 1) were compared with the prepubescent subjects (group 2), no significant difference was seen in any of the parameters including blood glucose, serum insulin, homa - ir, and wbisi . Similarly, when prepubertal subjects were compared with all other subjects (subjects in group 2 and 3 combined), significantly higher insulin sensitivity (wbisi, mean sd 6.07 5.7 versus 3.42 2.7, p <0.01) and lower insulin resistance (homa - ir, mean sd 3.68 2.7 versus 5.22 4.0, p = 0.04) were seen in prepubertal subjects . This is the first study examining the relationship between serum 25(oh)d levels and parameters of insulin resistance in obese asian - indian children . Our study confirms a significant inverse relationship between serum 25(oh)d levels and body fat indices as reported in other studies in children [6, 10]. We demonstrated trends for that higher serum 25(oh)d levels are associated with better insulin sensitivity and less insulin resistance though this correlation did not reach statistical significance . One of the main reasons for the absence of significance could be the universal presence of vitamin d deficiency (<20 ng / ml) resulting in very narrow range of serum 25(oh)d . We also show, for the first time, that the association between serum 25(oh)d and insulin resistance and sensitivity is influenced by pubertal staging . When subjects were stratified by pubertal stage, only subjects in tanner stage 5 exhibited a significant correlation between serum 25(oh)d and homa - ir, while prepubertal subjects did not show statistically significant correlation . High prevalence of vitamin d deficiency has been reported across the age range in pediatric, adolescent, and adult in obese individuals [5, 6, 2631]. This may be just reflection of very high prevalence of vitamin d deficiency in asian - indians [16, 17]. However, high prevalence of vitamin d deficiency has been reported in obese children and adolescents from populations where vitamin d deficiency is not that prevalent [5, 6, 10]. Reis et al ., in a study of 3577 adolescents in the age group of 1219 years, found significantly lower serum 25(oh)d levels in adolescents with bmi> 95th centile in comparison to subjects with bmi <95th centile (p <0.001). It has been postulated that increased sequestration of vitamin d in fat tissues, leading to decreased vitamin d bioavailability, low dietary vitamin d intake due to poor nutritional habits, and minimal sun exposure due to sedentary indoor lifestyle are important factors associated with high prevalence in the obese population [29, 32]. It has also been suggested that vitamin d deficiency is a cause of common obesity and can account for the secular trends in the prevalence of obesity and for individual differences in its onset and severity . The inverse relationship between serum 25(oh)d and insulin resistance in adults have been reported in most studies, though some have failed to find this association . In comparison, reports in children and adolescents have shown conflicting results . Delvin et al ., in a study of 1745 french - canadian children, reported modest but significant negative associations between serum 25(oh)d and homa - ir . Each 10-nmol / l (4 ng / ml) increase in serum 25(oh)d was associated with lower glycemia and homa - ir . Did not find significant correlation between serum 25(oh)d levels and insulin, homa - ir and homa - b% . In another study based on nhanes data of 3577 adolescents from 20012004 showed that serum 25(oh)d levels were inversely associated with plasma glucose concentration (p = 0.01). Lenders et al ., in a study of 58 obese adolescents, did not found any correlation between insulin indices and serum 25(oh)d in both, adjusted and unadjusted models . In our study, we did not find a statistically significant association between serum 25(oh)d and parameters of insulin sensitivity and resistance . However, trends for higher blood glucose and serum insulin levels were seen in subjects with low serum 25(oh)d level . No significant difference was seen in insulin resistance and sensitivity between group 1 and group 2 . This is in contrast to the observations made by ashraf et al . Who reported in 51 african - american obese female adolescents that higher serum 25(oh)d concentrations were associated with significantly higher wbisi (p = 0.018) but no difference in homa - ir . These differences may be due to marked difference in severity of obesity as mean bmi of study subjects was 43.3 9.9 in study by ashraf et al ., while it was 29.3 9.8 kg / sqm in our study subjects . Studies in adult population have shown that parameters of insulin sensitivity and insulin resistance as calculated by hyperinsulinemic euglycemic clamp study (m value) correlated better with serum 25(oh)d levels than indirect parameters like homa - ir . Studies with direct measures of insulin sensitivity like clamp studies are required to further investigate these issues in pediatric and adolescent population . The phenomenon of pubertal insulin resistance has been well described in cross - sectional as well as longitudinal studies . A longitudinal study of insulin resistance in american children showed that during puberty, insulin sensitivity decreased by 50%, with a compensatory increase in plasma insulin which is independent of the changes in body fat . These changes are thought to be mediated by interaction of various hormones during puberty including increased growth hormone secretion . No studies have yet reported the relationship between serum 25(oh)d concentrations and insulin sensitivity and resistance in pediatric and adolescent population and the potential influence of puberty except one . Lenders et al . Reported no significant association between serum 25(oh)d and insulin indices, even after adjusting for tanner stage . However, our study, for the first time, shows that the relationship between serum 25(oh)d concentrations and insulin resistance is influenced by puberty . When subjects with all stages of puberty were analyzed, no significant correlation was found, but when subjects in tanner stage 5 were analyzed alone, significant inverse correlation was seen between serum 25(oh)d concentrations and homa - ir (r = 0.41, p = 0.03). This association persisted / improved after controlling for age, sex, and bmi (r = 0.505, p = 0.023) but disappeared when subjects were controlled for fmi (r = 0.27, p = 0.24). Most of the evidence of an association between adiposity and vitamin d status comes from adult studies [4, 33]. A recently published study of 382 healthy subjects aged 621 years with dxa scans showed that serum 25(oh)d concentrations were more likely to be lower in those with greater bmi z - scores and fm, but not ffm . When this association was adjusted for possible covariates in the model, it became nonsignificant . . Showed that obese adolescents with and without vitamin d deficiency differed according to fm and fm percentage, but not lean mass and this relation of fm percentage to serum 25(oh)d remained significant after potential confounding variables were adjusted for . Showed that vitamin d deficiency was associated with higher visceral adipose tissue in white and greater subcutaneous adipose tissue in black american children and adolescents . In agreement with these studies, our findings indicate that body fat indices (total body fat percentage and fmi) are inversely correlated with serum 25(oh)d level . This correlation persisted even after adjustment for possible covariates like age, sex, and bmi . The differences in study findings may be explained in part by differences in study design and underlying sample characteristics like bmi and variation in serum 25(oh)d concentrations . The major strengths of our study are the use of robust measures of insulin sensitivity and resistance based on ogtt rather than fasting sample and all our study subjects belonged to one ethnic group . We have used dxa for assessment of body fat as opposed to several studies which have used bmi as a surrogate for body fat . The limitations of our study included, small sample size, presence of vitamin d deficiency in all study subjects which gave us very narrow range of serum 25(oh)d concentrations thereby limiting comparisons . Absence of pth measurement was another important limitation, since it has been implicated in pathogenesis of insulin resistance and metabolic syndrome . Another important limitation was inability of dxa technique to differentiate between subcutaneous and visceral adipose tissue . In conclusion, our study demonstrates a significant inverse relationship in obese asian - indian children between body fat indices and serum 25(oh)d concentrations which persists even after adjustment of covariates . The association between serum 25(oh)d and parameters of insulin sensitivity and resistance is influenced by puberty as a significant association was found between these two variables in postpubertal subjects (tanner stage 5) only which remained significant even after adjustment for covariates . As association is not equal to causation, future longitudinal research studies are required in determining role of puberty on association between serum 25(oh)d and insulin resistance in both, obese as well as subjects with normal bmi.
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A 21-year - old man (height = 180 cm, weight = 95 kg, body mass index = 29.3 kg / m) presented to the outpatient department with a long history of frequent and severe snoring . On physical exam, after a diagnosis of obstructive sleep apnea was made, a bilateral tonsillectomy and uvulopalatopharyngoplasty was planned for treatment . Prior to surgery, the patient had normal laboratory results with hemoglobin (hb) of 15.8 g / dl and no systemic or hematological diseases or history of bleeding disorders . On the day of the surgery, the patient received no premedication due to the history of sleep apnea . Intraoperative monitors included electrocardiogram, non - invasive blood pressure (bp), pulse oximetry (spo2), and end - tidal co2 . Pre - anesthetic vital signs were bp 120/82 mmhg, hr 68 bpm, and spo2 96% . General anesthesia was induced with remifentanil 0.5 g / kg / min and propofol 180 mg iv at 10:10 am . At loss of eyelash reflex, cisatracurium 16 mg was administered, and the trachea was intubated after a short period of facemask ventilation . Anesthesia was maintained with sevoflurane 2 vol% in 50% oxygen - enriched air and remifentanil 0.2 g / kg / min . The neuromuscular block was antagonized with glycopyrrolate 0.6 mg and pyridostigmine 15 mg iv without neuromuscular monitoring upon the completion of the surgical procedure at 11:25 am . Postoperatively, a physical examination confirmed the presence of acute bleeding in the left inferior pole of the tonsillectomy site in the post anesthetic care unit . The surgeon requested an emergent re - operation under general anesthesia for the control of bleeding . A rsi for a second round of general anesthesia was performed with propofol 120 mg, midazolam 5 mg, and rocuronium 100 mg at 11:55 am . The surgeon confirmed active bleeding due to an aberrant arterial blood supply in the left inferior pole of the tonsillectomy site . After the completion of the surgical procedure, no twitch was shown after train - of - four (tof) stimulation (tof = 0) with neuro - stim (model ns-3cc, houston, tx, usa) at 12:40 pm . The neuromuscular block was then antagonized with sugammadex 200 mg iv and the rocuronium - induced nmb was completely reversed within 2 min . The patient was transferred to the recovery room, and there were no specific findings on physical exam . A post - operative laboratory assessment showed hb of 14.6 g / dl (table 1). After 6 h and 20 min, the surgeon again requested emergent anesthesia and re - operation for control of bleeding . The surgeon attempted to control bleeding at the ambulatory otolaryngology clinic but was unable to do so . When the patient arrived at the operating room (or), vital signs were within normal limits and hb was found to be 13.2 g / dl . A third round of general anesthesia was induced with propofol 120 mg and rocuronium 40 mg by the on - call resident at 19:00 pm . The neuromuscular block was antagonized with sugammadex 200 mg iv and a tof = 0 at 20:00 pm . The patient was again extubated successfully and transferred to the recovery room with no specific findings (table 1). After 2 h and 35 min, when the patient arrived at the or, vital signs were stable and hb was 11.6 g / dl . A fourth induction of general anesthesia was performed with propofol 120 mg and rocuronium 40 mg by the same resident at 22:35 pm . During tof stimulation, 1 twitch was seen, and an additional dose of rocuronium 10 mg was given due to bucking 20 min after the induction of anesthesia . At the end of the procedure, the neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof = 1 at the end of the surgical procedure at 0:20 am . Extubation was carried out after confirming the recovery of consciousness, regular respiration, and the absence of fade on tof monitoring . After extubation, the patient vomited a large amount of blood on the operating table (approximated to be more than 200 ml). However, the patient was alert and aware of his surroundings and had regular respirations (table 1). After 20 min, the surgeon requested a re - induction of general anesthesia for additional bleeding control . An rsi for a fifth round of general anesthesia was performed with propofol 120 mg and rocuronium 100 mg at 0:40 am . The neuromuscular block was antagonized with glycopyrrolate 0.4 mg and pyridostigmine 10 mg iv with a tof = 1 after the surgical procedure was completed at 1:30 am . Approximately 15 min passed after the nmb was antagonized as above, but no evidence of recovery from the rocuronium - induced nmb was seen . Therefore, we injected a bolus of iv sugammadex 200 mg to reverse the residual nmb . Once again, the nmb was completely reversed within 2 min, and tracheal extubation was performed in the or uneventfully (table 1). A decision by surgeon was made to perform angiography of the left external carotid artery with subsequent embolization of the bleeding vessel . Embolization of the left ascending pharyngeal artery was performed with polyvinyl alcohol particle (350 m) and tornado embolization microcoil (cook, bloomington, usa) (3/2 mm) (fig . The patient's postoperative course was uneventful, and hb was found to be 10.5 g / dl . Pth occurs in approximately 1 in 20 adults (5.1%), and more than half of patients who bleed are likely to require a procedure of some type to control their hemorrhage . Aberrant arterial blood supply to the tonsillar region derived from the internal carotid artery or the carotid bulb may be present . To control bleeding, electrocauterization, packing of the pharynx, or angiographic embolization of the feeding artery may become necessary . The endovascular embolization due to refractory bleeding after tonsillectomy proved to be a valuable treatment method as an effective alternative to surgical intervention . It is a safe and permanent treatment option in this potentially life - threatening complication . In a case such as this, the aberrant arterial blood supply in the left inferior pole of tonsillectomy site caused continuous bleeding . The anesthesiologist has experienced difficulties, because an experienced surgeon has embarrassed due to repetitive bleeding . In the first induction of anesthesia, no problems were encountered with conventional anesthesia, although neuromuscular monitoring was not performed . The most common anesthetic used for rsi during the management of children with pth is succinylcholine . Since the introduction of sugammadex, rsi with rocuronium followed by reversal with sugammadex allowed for earlier establishment of spontaneous ventilation than with succinylcholine, which has many adverse side events . During the second round of anesthesia, rocuronium 100 mg (1 mg / kg) was injected for rsi, followed by reversal with sugammadex 200 mg (2 mg / kg) at deep nmb (tof = 0). During the operation, nmb monitoring was difficult due to problems attaching leads to the arms during the operation . Tof stimulation was applied at the ulnar nerve using conventional instrumental monitoring rather than quantitative monitoring due to convenience . Deep nmb was maintained for bleeding control in an attempt to not disturb the procedure . During nmb reversal, we found an absence of fade on tof monitoring just 2 min after sugammadex was administered . Re - administration of 0.6 mg / kg rocuronium could then be given after sugammadex at the recommended waiting time of 6 h . During the third anesthesia induction, an on - call resident used rocuronium 40 mg for muscle relaxation during intubation followed by reversal with sugammadex 200 mg at deep nmb (tof = 0). An absence of fade on tof monitoring was shown 2 min after sugammadex was given . For the fourth round of anesthesia, the same resident used rocuronium 40 mg for intubation 2 h and 35 min after the previous nmb reversal with sugammadex . However, we believe this decision was made without considering the recent sugammadex use . When rocuronium is used within a short time interval (less than 6 h) after sugammadex administration, the onset time of rocuronium may be prolonged and may also have an unpredictable duration of action . We suspect that the additional dose of rocuronium 10 mg due to bucking may be related to the short time interval between sugammadex and rocuronium administration . In situations such as these, the benzylisoquinoline class of medications are recommended instead of re - administering rocuronium within 6 h after sugammadex . When considering this case, cisatracurium for muscle relaxation and intubation may have been a better choice for the fourth round anesthesia . However, one drawback of cisatracurium is the slower onset time than rocuronium . Reversal of the rocuronium - induced nmb with pyridostigmine at a moderate nmb (tof = 1) was uneventful . After extubation, the patient vomited more than 200 ml of blood on the operating table; however, he was alert and showed regular respirations . Postoperative nausea and vomiting (ponv) after an operation for pth is common due to blood swallowed during the procedure . Therefore, patient awareness and complete reflexes are important, but aspiration must be avoided and monitored closely during the recovery period . However, ponv increases the risk of primary hemorrhage and unexpected postoperative hospital admissions . In this case the patient did not aspirate, but did have an increased risk of pth due to ponv . For the fifth round of anesthesia, rocuronium 100 mg (1 mg / kg) was injected for rsi after a time interval from sugammadex administration of 4 h and 40 min . It has been reported that re - administration of 1.2 mg / kg rocuronium could have been given after sugammadex 2 mg / kg without a waiting time . However, neuromuscular monitoring did not show the expected recovery, and the patient had residual paralysis after 15 min . It has been shown previously that the reversal of nmb with anticholinesterase drugs may not be complete after a large dose of a neuromuscular blocking agent . Sugammadex resolved the residual nmb within 2 min, even after reversal with anticholinesterase agents, and is a safe alternative to reversal of nmb induced by steroidal non - depolarizing agents . In this case, deep rocuronium - induced nmb can be reversed successfully with sugammadex at doses of 2 mg / kg and in as little as 2 min . Re - administration of rocuronium within a short time interval after sugammadex may result in unpredictable effects of the nmb agent . Sugammadex made it possible to perform a rapid, complete reverse when the residual block was maintained by an incomplete reversal of anticholinesterase.
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Infertility has been recognized as a public health issue worldwide (1) leading to an increasing need to the use of assisted reproductive technologies (art), including in vitro fertilization (ivf). After the first reported case of ivf in 1978(2) art enabled millions of people to have their own children in cases when pregnancy did not occur under natural circumstances . 700,000 cycles a year in the usa and europe together (3,4). Despite of evolving intracytoplasmatic sperm injection (icsi) technique a success rate of 25% and 28% has been reported in 2005(7) and 2008(8), respectively . Nowadays, this rate went up to 32% (9), which cannot be considered as a significant development . Earlier clinical protocols preferred multiple embryo transfer, but multiple gestations can result in the increased risk of preterm delivery (10 - 16). Other studies report that multiple gestations also increased the risk of low birth weight cerebral palsy (17). In the us alone, preterm births resulting from multiple pregnancies during ivf cause a 1 billion usd extra cost to the social insurance (18). In order to exclude the discussed risk factors, it is imperative, however, that accurate and economical methods should be developed to ensure that the most viable euploid embryo is selected for transfer . Ideally, such tests would be noninvasive, lessening the risks to the embryo and reducing costs and workload in the embryology laboratory (19). The biggest issue with pre - implantation viability assessment is that due to ethical reasons any assay should be completely non - invasive because no one can predict what kind of interference would be the unwanted result in the later embryonic development . The most apparent and routinely applied - way of the assessment of viability is the morphological evaluation of in vitro fertilized embryos using microscopy . There are several morphological features described which could be used for viability assessment purposes, these are dependent on the time spent after fertilization . Right after fertilization in the 1-cell embryo the size and symmetry of the two pronuclei can be examined . The time of the first cell division is also a good predictor of later implantation potential, as zygotes that divide early tend to develop more frequently to the blastocyst stage . Criteria as cleavage rate and blastomere shape and symmetry, an adequate trophectoderm layer (te) and an inner cell mass (icm) is a morphological marker of the later stages (5,20). Not only can the morphology of the fertilized embryo be used for further prediction of implantation potential, but morphological defects of the retrieved oocyte as well . Fertilization and pregnancy rate correlates with the grade of cumulus - oocyte complexes, and embryos originating from dysmorphic oocytes show a larger grade of pregnancy loss (21 - 23). The cleavage stages of morulae and blastocysts or the symmetry and patterns of cell division are also notable and frequently used aspects, and are often examined during the prediction of embryo viability (23). The biggest issue of morphological assessment is that it is still a highly subjective method (20). The reason is partly due to the fact that the final decision is made by a clinician, and not by an objective test result, and secondly it is does matter how important are the individual morphological features in the final conclusion (24 - 26). To overcome the different practice of laboratories worldwide in 2011 an international consensus (istanbul consensus) has been reached on embryo viability assessment (27). The selected morphological markers of respective stage embryos, the weighing of individual features and a scoring system has been set up . The limitations due to static time - point observation, is now solved with the use of time - lapse microscopy (28,29). Time - lapse microscopy also enables the observation of dynamics of cytoplasmic movements and cytokinesis, reflecting the functionality of microtubule and actin cytoskeleton, which is critical for proper development . In our laboratory, we aimed to improve the success rate of implantation by adopting and further optimizing the istanbul consensus . This score has been called as the optimized criteria system (ocs). According to this scoring, 3-day old embryos were divided into two subgroups: the subgroup with low blastomere number (less than 7) and with high blastomere number (7 or more). Symmetric position of blastomeres indicates the rate of symmetry of holoblastic cleavage along the embryo axis . It was classified as good (full symmetry); fair (light asymmetry); or poor (evident asymmetry). The percent values of fragmentation are based on the ratio of fragmented to total cell numbers . As a further modification to the istanbul consensus, embryos were considered as good if the fragmentation rate was <15% (instead of the original 10%). This shift from 10 to 15% was the result of our observation that a fairly high proportion of the embryos between 10 - 15% appeared to be viable . In summary the optimized criteria system (ocs) highlights 3 modified or new parameters: fragmentation (with a more permissive criterion of <15% in the good category); symmetry and the blastomere number . In addition, the blastomere size was evaluated according to the original istanbul consensus . A scoring - map was created to facilitate the evaluation (table 1) as far as the 5-day old embryos are concerned, we modified the original istanbul consensus for blastocysts by leaving out the hatched stage from the evaluation . The istanbul consensus for the 5-day old embryos has a shortcoming, i.e. It does not express the viability of embryos with a single category (good, fair, poor). We tried to overcome this by using a scoring map (table 2). In conclusion, we constructed a composite score for day-3, as well as day-5 old embryos, based on morphological parameters . As it is evident from the results, this composite score is sensitive to evaluate viability (figure 1) another possibility for non - invasive embryo viability assessment is the metabolomic examination of the culture medium surrounding the in vitro fertilized embryo . Metabolomic, (proteomic) profiling of spent embryo culture (sec) offers an exceptional, non - invasive opportunity for the assessment of embryo viability (30,31). The metabolomic profiling (32,33) of early embryo development might mean the analysis of the total metabolome by following the changes of several selected compounds, metabolomic analysis using unidentified, but significantly differing metabolomic changes, or by the analysis of a limited population of nutrients or end products . The common feature in all three concepts is that they are concentrating on the metabolomic alterations caused by differently developing embryos in the culture medium . Very simple idea is the monitoring of glucose consumption or pyruvate formation, since this would directly indicate the metabolism of the developing embryo and it is an obvious conclusion that a metabolically active embryo would have higher implantation potential . Some authors reported that the identification of these parameters resulted in successful prediction of embryo implantation potential, but other research groups describe contradictory results (34, 35). The amino acid profile of culture media is also used in the prediction of implantation potential, though not exclusively as an independent parameter, rather in combination with morphological features (36). The detection of unidentified metabolomic changes using near infra - red (nir) or raman spectroscopy (37, 38) is a very interesting and challenging possibility . More complicated is the concept when unknown, new biomarker molecules of embryo viability are searched for, assuming that these biomarkers were secreted by the embryo . The difficulty of the concept is that only 4 - 8 cells are present in the culture medium; thus a very sensitive analytical tool is required . Mass spectrometry (ms) has the potential of specific and sensitive quantification in a wide spectrum of molecular mass ranges and therefore suites well the needs of metabolomic or proteomic fingerprinting and quantification . In parallel to the spreading of mass spectrometry, proteomics is also an emerging field in the understanding of embryo development (39,40). The analysis of the embryonic secretome (41,42) provides information of the total transcriptome of the developing embryos . Mass spectrometry can be used both in targeted and discovery analysis with accurate quantification of identified biomarkers after molecular identification by bottom - up or top - down proteomics using tandem or multiple ms (43 - 46). In a recent publication from our laboratory (47) using liquid chromatography coupled mass spectrometry (lc - ms), a fragment of the human haptoglobin molecule was identified in the culture medium . Rather than analyzing the embryonic secretome, the aim this experiment was to use preexisting molecules present in the cell culture media as biomarkers . Haptoglobin - which was detected in the culture medium - is not a product of the developing embryo; the polypeptide is a contaminant of the human serum albumin standard used to supplement the culture medium (47,48). During the first three days of embryo development the formation of a subunit (alfa-1) of the human haptoglobin molecule was observed . This subunit similar to the total haptoglobin molecule was detectable in the blank control medium samples as well . The differentiation of the viable and non - viable embryos was done using the observation that compared to blank controls the samples of embryos which later did not resulted in pregnancy contained the alpha-1 subunit in a much larger quantity than the samples of embryos which did (figure 2). Clinical statistical analysis of the results revealed that the specificity of the diagnostic test was 64%, while the sensitivity was 100% . It is more informative that the positive predictive value of the assay was 51% and maybe more importantly the negative predictive value was 100% . Receiver operating characteristic (roc) analysis provides tools to select possibly optimal models and to discard suboptimal ones . Roc analysis is related in a direct and natural way to cost / benefit analysis of diagnostic decision - making . The roc curve of the morphological versus metabolomic approach in relation to the correct prediction of pregnancy outcome is illustrated in figure 3 . It is obvious that our biochemical investigation method enables a selection of the embryos by sorting out the non - viable ones . The test selected with 100% potential the embryos, which did not lead to successful implantation at all . One of the areas of collaboration between clinicians, the clinical laboratory and the research laboratory at the university of pcs is related to the research of infertility . Since the clinical background gives the beauty and the medical importance of laboratory research, it was of outstanding importance for us to receive the eflm - abbott diagnostics award for excellence in outcomes research in laboratory medicine (paris, 2015), the award given to the best published paper (47), as judged by an independent panel of experts, which demonstrates improved outcomes arising out of the application or improved utilization of an in - vitro diagnostics test.
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This study is part of a larger project examining the possible health effects of lifetime exposure to marine food and pollutants . The faroe islands constitute a unique setting at northern latitude in which the residents have an increased exposure to methylmercury and persistent organic pollutants, including polychlorinated biphenyls (pcb), because of their intake of traditional marine food, including pilot whale meat and blubber . To examine subjects aged 7074 years all 1,131 faroese citizens born between 2 january 1934 and 31 august 1937 received a letter of invitation, 713 of whom gave consent and were examined, corresponding to a participation rate of 64%, taking into account 14 deceased subjects . Furthermore, sufficient serum for determination of vitamin d status was not available from 44 subjects; thus, 668 subjects are included in the current study . The participants were asked to fast and arrive before breakfast to undergo blood sampling and a thorough clinical examination . All participants traveled to the same examination location and were examined by the same research nurses and a single physician . The participants completed current health, past medical history, and lifestyle questionnaires, including summary information on the frequency of fish dinners during the last year . A fasting capillary blood sample was used to determine blood glucose concentration using a direct - reading glucose biosensor (precision xceed and abbott precision xtra plus strips; abbott diabetes care, copenhagen, denmark). Because plasma has higher water content than whole blood, the blood values were transformed to plasma glucose concentration by multiplying by 1.12 as recommended by the manufacturer . Fasting venous blood samples were collected by phlebotomy to perform hba1c analyses by high - pressure liquid chromatography . All hba1c values were given as relative concentration,% (diabetes control and complications trial, dcct, aligned results). Hba1c% values were converted to international federation of clinical chemistry and laboratory medicine (ifcc) units (millimoles per moles) by the following equation: hba1c (dcct)% = 0.0915 hba1c (ifcc) mmol / mol + 2.15, provided by the laboratory . Edta - plasma was used for determination of fasting plasma insulin concentration by time - resolved fluoroimmunoassay (autodelfia; perkinelmer life and analytical sciences, walla oy, turku, finland). Serum hdl and triacylglycerides were measured by an enzymatic colorimetric reaction, using a modular p analyzer (roche diagnostics, indianapolis, in). Diabetic subjects were identified from self - reported doctor s diagnosis and the reported use of hypoglycemic medication; subjects were also classified as diabetic if they reported use of diabetes medication without reporting a diagnosis of diabetes . In participants not previously diagnosed, type 2 diabetes was considered to be present if the fasting plasma glucose was 7.0 insulin resistance was estimated by the homeostasis model assessment (homa - ir) and calculated as fpi fpg / 22.5 (10), where fpi is fasting plasma insulin and fpg is fasting plasma glucose . -cell dysfunction was determined by calculating the homa -index of (20 fpi)/(fpg 3.5) (10). After storage at 80c, serum 25(oh)d3 concentrations were assessed using liquid chromatography - tandem mass spectrometry (lc - ms / ms) as described previously (11). Nmol / l, and the coefficient of variation was 9.6% at 20 nmol / l and 6.7% at 60 nmol / l . Means (sd) were calculated for variables showing normal distribution and median and 2575th percentiles (interquartile range [iqr]) for those deviating from it . When transformations were insufficient to obtain normal distribution, we applied nonparametric spearman s correlation coefficient . When appropriate, multiple regression analysis was carried out . A logistic regression model was used to assess the association between serum 25(oh)d3 concentration and the risk of having type 2 diabetes . Vitamin d status was used as a binary exposure variable, i.e., dichotomized at above (sufficient) or below 50 nmol / l (deficient), and in the simple model, sex was used as an obligate covariate . In the adjusted model, additional covariates were included, i.e., bmi, serum triacylglycerides, serum hdl, pcb exposure (sum of major pcb congeners cb-138, cb-153, and cb-180 multiplied by 2), smoking, and month of blood sampling . In addition, we examined the association between vitamin d status and risk of having 1) newly diagnosed type 2 diabetes or 2) a known diagnosis of type 2 diabetes using the same models separately . Finally, the same approach was used to estimate the influence of fish intake on risk of having type 2 diabetes using subjects reporting fish intake less than or equal to two times per week as a reference group . In the models, we further tested whether there was evidence of an interaction effect between vitamin d status and sex and vitamin d status and obesity . A value of p <0.05 (two - tailed) was taken to indicate statistical significance . Stata version 11.0 (stata corporation, college station, tx) was used for statistical analyses . A fasting capillary blood sample was used to determine blood glucose concentration using a direct - reading glucose biosensor (precision xceed and abbott precision xtra plus strips; abbott diabetes care, copenhagen, denmark). Because plasma has higher water content than whole blood, the blood values were transformed to plasma glucose concentration by multiplying by 1.12 as recommended by the manufacturer . Fasting venous blood samples were collected by phlebotomy to perform hba1c analyses by high - pressure liquid chromatography . All hba1c values were given as relative concentration,% (diabetes control and complications trial, dcct, aligned results). Hba1c% values were converted to international federation of clinical chemistry and laboratory medicine (ifcc) units (millimoles per moles) by the following equation: hba1c (dcct)% = 0.0915 hba1c (ifcc) mmol / mol + 2.15, provided by the laboratory . Edta - plasma was used for determination of fasting plasma insulin concentration by time - resolved fluoroimmunoassay (autodelfia; perkinelmer life and analytical sciences, walla oy, turku, finland). Serum hdl and triacylglycerides were measured by an enzymatic colorimetric reaction, using a modular p analyzer (roche diagnostics, indianapolis, in). Diabetic subjects were identified from self - reported doctor s diagnosis and the reported use of hypoglycemic medication; subjects were also classified as diabetic if they reported use of diabetes medication without reporting a diagnosis of diabetes . In participants not previously diagnosed, type 2 diabetes was considered to be present if the fasting plasma glucose was 7.0 insulin resistance was estimated by the homeostasis model assessment (homa - ir) and calculated as fpi fpg / 22.5 (10), where fpi is fasting plasma insulin and fpg is fasting plasma glucose . -cell dysfunction was determined by calculating the homa -index of (20 fpi)/(fpg 3.5) (10). After storage at 80c, serum 25(oh)d3 concentrations were assessed using liquid chromatography - tandem mass spectrometry (lc - ms / ms) as described previously (11). Nmol / l, and the coefficient of variation was 9.6% at 20 nmol / l and 6.7% at 60 nmol / l . Means (sd) were calculated for variables showing normal distribution and median and 2575th percentiles (interquartile range [iqr]) for those deviating from it . When transformations were insufficient to obtain normal distribution, we applied nonparametric spearman s correlation coefficient . When appropriate, multiple regression analysis was carried out . A logistic regression model was used to assess the association between serum 25(oh)d3 concentration and the risk of having type 2 diabetes . Control subjects were nondiabetic subjects, i.e., subjects with fasting blood glucose 6 mmol / l . Vitamin d status was used as a binary exposure variable, i.e., dichotomized at above (sufficient) or below 50 nmol / l (deficient), and in the simple model, sex was used as an obligate covariate . In the adjusted model, additional covariates were included, i.e., bmi, serum triacylglycerides, serum hdl, pcb exposure (sum of major pcb congeners cb-138, cb-153, and cb-180 multiplied by 2), smoking, and month of blood sampling . In addition, we examined the association between vitamin d status and risk of having 1) newly diagnosed type 2 diabetes or 2) a known diagnosis of type 2 diabetes using the same models separately . Finally, the same approach was used to estimate the influence of fish intake on risk of having type 2 diabetes using subjects reporting fish intake less than or equal to two times per week as a reference group . In the models, we further tested whether there was evidence of an interaction effect between vitamin d status and sex and vitamin d status and obesity . A value of p <0.05 (two - tailed) was taken to indicate statistical significance . Stata version 11.0 (stata corporation, college station, tx) was used for statistical analyses . In the current study, the cohort consisted of 327 women and 341 men with a mean age of 72.5 years . A total of 158 septuagenarians (24%) had type 2 diabetes, of whom 88 (13%) had been previously diagnosed with the disease and 70 new subjects (11%) were identified in the current study . More than half of the subjects had vitamin d deficiency, as suggested by a serum 25(oh)d3 concentration below 50 nmol / l (median, 47.6 nmol / l; iqr, 29.864.8 nmol / l). Baseline clinical and biochemical characteristics of 668 faroese septuagenarians by vitamin d status data are means (sd) or median (2575th percentile). * hba1c (ifcc) mmol / mol can be converted to hba1c (dcct)% by the following equation: hba1c (dcct)% = 0.0915 hba1c (ifcc) mmol / mol + 2.15; homa - ir = (fasting plasma insulin fasting plasma glucose)/22.5; homa-% = (20 fasting plasma insulin)/(fasting plasma glucose 3.5); sum of major pcb congeners cb-138, cb-153, and cb-180 multiplied by 2 (12). Nmol / l, was associated with an 80% increase in the sex - adjusted odds of having diabetes (newly diagnosed and previously diagnosed subjects) compared with sufficient vitamin d status (odds ratio [or] 1.80 [95% ci 1.232.64]; p = 0.002). After further adjustment for bmi, serum triacylglycerides, serum hdl, pcb exposure, smoking, and month of blood sampling, the association between vitamin d deficiency and type 2 diabetes remained, and the or was only marginally affected (or 1.67 [1.112.50]; p = 0.013). In the separate analyses of previously undiagnosed and diagnosed subjects, we found that the sex - adjusted or for having newly diagnosed type 2 diabetes in the presence of vitamin d deficiency had more than doubled (or 2.25 [95% ci 1.313.85]; p = 0.003). After further adjustment for bmi, serum triacylglycerides, serum hdl, pcb exposure, smoking, and month of blood sampling, the association between vitamin d deficiency and newly diagnosed type 2 diabetes remained unchanged (or 1.99 [1.143.48]; p = 0.022), whereas the adjusted or for pre - existing diabetes in vitamin d deficiency was reduced considerably by similar adjustment (or 1.48; p = 0.132) (table 2). We performed logistic regression analysis on the impact of fish dinner frequencies on the risk of type 2 diabetes . Frequent fish intake (more than twice a week) did not appear to alter the odds of having type 2 diabetes (or 1.09 [95% ci 0.641.86]; p = 0.744). Obesity is a risk factor for vitamin d deficiency, and obese subjects had a lower serum 25(oh)d3 concentration than nonobese subjects (11). Furthermore, we have previously demonstrated in this cohort (11) that women have higher serum 25(oh)d3 concentration and tend to have lower bmi . However, we did not find any interaction between vitamin d status and sex (p = 0.19) or vitamin d status and obesity (p = 0.84) on risk of diabetes . Statistically significant bivariate correlations were observed between serum 25(oh)d3 concentration and several indicators of glucose metabolism, including hba1c (rs = 0.10; p = 0.01), fasting plasma insulin (rs = 0.10; p = 0.01), homa - ir (rs = 0.10; p = 0.01), and homa - b (r = 0.10; p = 0.01), whereas the plasma glucose concentration (rs = 0.01; p = 0.78) was not correlated with 25(oh)d3 concentration . However, because bmi was strongly associated with vitamin d status (rs = 0.17; p <0.0001), the observed associations of 25(oh)d3 with fasting plasma insulin and the homa indexes were substantially reduced after adjustment for bmi, sex, smoking status, and serum triacylglyceride concentration, where none of them were statistically significant (table 3). However, hba1c decreased at higher serum 25(oh)d3 concentrations after adjustment (= 0.026, p = 0.026). Excluding subjects with physician - diagnosed type 2 diabetes resulted in a smaller effect that was no longer significant, probably because of reduced variation in hba1c concentration and fewer observations (= 0.012, p = 0.112) (table 2). Finally, multiple regression analysis did not reveal any association between frequency of fish dinners and the hba1c concentration (results not shown). Unadjusted and adjusted or for type 2 diabetes in elderly faroese aged 7074 years data are or (95% ci) expressed for binary traits . Mmol / l; the crude model is adjusted for sex and compares the odds of either overall type 2 diabetes (all type 2 diabetic subjects), newly diagnosed type 2 diabetes (new subjects), or known type 2 diabetes in subjects (previously known subjects) with 25(oh)d <50 nmol / l (reference group); the adjusted model includes sex, bmi, total pcb, month of blood sampling, serum triacylglyceride concentration, serum hdl concentration, and smoking status . In this cross - sectional analysis of a population - based sample of septuagenarians, we found that the 25(oh)d3 concentration was inversely associated with the risk of newly diagnosed type 2 diabetes after adjustment for bmi and other established risk factors for type 2 diabetes . Subjects with 25(oh)d3 concentration below 50 nmol / l had the doubled risk of newly diagnosed diabetes compared with those having higher serum concentrations . Therefore, our data suggest an inverse association between the serum concentration of 25(oh)d3 and the odds of newly diagnosed type 2 diabetes, thereby suggesting a protective role of vitamin d against the development of the disease, as suggested previously (13). In further support of this association, we observed an increasing concentration of hba1c with decreasing 25(oh)d3 concentration, and this association was independent of bmi, serum triacylglycerides, smoking status, and sex . Vitamin d deficiency showed a much weaker or for type 2 diabetes previously diagnosed, as could perhaps have been anticipated, given the vigorous clinical management of the disease and the longer time interval between disease development and the assessment of vitamin d status . The confidence interval for the or for the two groups of type 2 diabetes continues to overlap considerably, and therefore, the slight difference should not be overinterpreted (table 2). Linear regression coefficients for the association between 25(oh)d3 and markers of glucose metabolism in elderly faroese including or excluding subjects with known diabetes * numbers vary because of varying missing sample material for each biochemical analysis . Is considered an indicator of average blood glucose concentrations during the preceding 2 to 3 months and, thus, a long - term marker of glucose homeostasis (14). Abnormalities may be a result of changes in insulin secretion and insulin - stimulated uptake of glucose in muscle and fat tissue . In vitro studies and laboratory animal studies suggest possible mechanisms for the effects of the active form of vitamin d, i.e., 1,25(oh)2d, on both insulin secretion and insulin sensitivity as reviewed by pittas and dawson - hughes (15). For instance, in mice expressing a functionally inactive mutant vitamin d receptor, blood glucose concentrations were increased and serum insulin was reduced, as was insulin mrna . Pancreatic -cells express the vitamin d receptor and the enzyme 1--hydroxylase, thus suggesting that the active form of vitamin d has a functional role in these cells . Even though the current study cannot elucidate the likely mechanisms involved, our results appear highly plausible given the findings in experimental studies . Increased fat mass adversely influences glucose metabolism, in part by increasing insulin resistance, and is also linked with lower 25(oh)d concentrations in serum (1618). The results suggest that the negative impact of vitamin d deficiency on hba1c and newly diagnosed type 2 diabetes risk is independent of increased fat mass . However, because bmi does not distinguish visceral from subcutaneous fat, some misclassification of adiposity may have occurred, thereby possibly causing some uncertainty in regard to the relative influence of serum 25(oh)d3 and adiposity in diabetes development . Our study results are comparable with studies on vitamin d status and type 2 diabetes in younger populations (4,16,19,20), and the current study extends this association to 70 years of age and older . Vitamin d status is known to be lower in the elderly compared with the young as the subcutaneous synthesis of vitamin d decreases with increasing age, due to a reduced concentration of 7-dehydrocholesterol in the skin (1) and reduced absorption of oral vitamin d (8). Hence, if the relationship between vitamin d status and glucose homeostasis is causal, the age - related decline in circulating concentration of 25(oh)d may augment the increase in risk of type 2 diabetes with age . In addition, our results suggest that this relationship is not the result of increased fish intake, in agreement with the existing, somewhat equivocal evidence in regard to this effect (21,22). These differences may be a result of variations in fish intake to inherent differences in the populations examined and the study designs . Among the strengths of our study, we examined a general population sample with a high prevalence of both vitamin d deficiency (11) and type 2 diabetes . However, the current study is limited by its cross - sectional design and its single measurement of vitamin d status . Nonetheless, a recent study demonstrated excellent tracking and that the correlation of serum 25(oh)d3 concentrations sampled 14 years apart was similar to that seen for other cardiovascular risk factors and that most subjects with vitamin d deficiency were still deficient 14 years later (23). Patients with type 2 diabetes and elevated hba1c concentrations are at increased risk of cardiovascular disease and total mortality compared with patients with lower hba1c levels (24). Thus, clinical management focuses on glycemic control, including treatment of other modifiable cardiovascular risk factors to reduce the macro- as well as microvascular complications . The inverse association between serum 25(oh)d3 and hba1c suggests that vitamin d supplementation could be considered a possible means for supporting glycemic control of type 2 diabetes . However, some intervention studies have shown inconclusive results on the effect of vitamin d on hba1c and type 2 diabetes (15). For example, the women health s initiative demonstrated that daily supplementation with 10 g of vitamin d3 for a median follow - up time of 7 years did not reduce the risk of type 2 diabetes in postmenopausal women (25). However, the doses used were probably too low to bring about the necessary concentrations of circulating 25(oh)d3 . Therefore, further randomized clinical intervention studies are needed to elucidate the impact of vitamin d on the risk of type 2 diabetes . In summary, our study extends previous findings that a high vitamin d status protects against type 2 diabetes in younger subjects to subjects older than 70 years . Likewise, our findings suggest an inverse association between hba1c and 25(oh)d3 also in the elderly . Because our study has a cross - sectional design, formal confirmation requires intervention studies with appropriate doses to elucidate whether vitamin d supplementation could be used to counter the worldwide epidemic of type 2 diabetes . In this regard, the need to identify the necessary daily intake of vitamin d should include the possible effect on reducing the risk of type 2 diabetes, with special emphasis on the dietary needs of the elderly.
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Leaf senescence is the last phase of plant development and a highly coordinated process regulated by a large number of senescence associated genes (sags) (12). Leaf senescence can either be naturally induced during development stages, or stimulated by environmental factors including darkness, nutritional deficiency and various stresses (13). Premature senescence is an important factor leading to the decrease of crop yield and quality, which becomes an increasing concern due to the global climate change in the recent years . Many advances in the understanding of leaf senescence at the molecular level had been achieved through the identification and characterization of hundreds of sags and senescence - related mutants in arabidopsis thaliana, lycopersicon esculentum and nicotiana tabaccum (14). Microarray expression profiling in arabidopsis revealed that more than 800 genes are up - regulated during the course of leaf senescence (5). Among them, more than 200 transcription factors, including wrky, nac, mads, myb, bzip and bhlh family members, are involved in the regulation of leaf senescence, indicating that leaf senescence is governed by complex transcriptional regulatory networks . Molecular and genetic studies of leaf senescence in recent years led to the accumulation of a large volume of scattered information related to sags . The construction of a leaf senescence related database with wide - spread collection and systematic annotation of sags may provide a useful resource and a good starting point for the further study of the molecular aspects of leaf senescence . Some initial efforts to this end had been made; including the online website of plant senescence network (sennet) constructed by thomas and his colleagues (http://www.sidthomas.net/plant_senescence/) and the corresponding senwiki web pages (http://www.sidthomas.net/senwiki/). Sennet brings together various community information including meetings, laboratories, websites and useful links related to plant senescence, while senwiki provides general information and knowledge of senescence with texts and images . However, a wide - ranging compilation and detailed annotation of known sags that would be of great help and demand to the systemic study of leaf senescence at the molecular level has yet to be done . In this regard, we have developed a leaf senescence database (lsd) (http://www.eplantsenescence.org/) by retrieving and integrating information from research papers and public databases . At present 1145 sags from 21 species were manually curated and categorized into several groups according to their function . Users can browse the entries in the database to obtain information including literatures, mutants, phenotypes, expression profiles, mirna interactions, orthologs in other plants and cross links to protein domain and family databases . Users can also search the database easily through the text search interface with locus names, keywords and author names, etc . We have implemented the blast tool kit for sequence similarity search against nucleotide or protein sequences of these sags in the lsd . A major feature of the lsd is the integration of a bioinformatics platform weblab we had developed previously (6), which allows users to perform extensive sequence analysis of the sags they are interested in . All the sag sequences are freely available for downloading . Help information including user guides, a tutorials and faqs are also available online . We plan to identify putative sags from completely sequenced genomes and add them into the database in the near future, and improve the user interface based on user feedback, provide more help documents with case studies, add more links to other useful sites, and update the database in time with more leaf senescence related data available . We hope that lsd could be a useful resource for the leaf senescence research community, as well as a gateway for the collaborative project we are working with both domestic and international colleagues . We made an extensive literature survey and collected approximately 200 leaf senescence related papers published before october 2010 on major plant biology journals including plant cell, plant physiology, plant journal, new phytologist, journal of experimental botany, among others . A total of 1145 sags from 21 species including arabidopsis, rice and so on, were identified and manually verified based on genetic, genomic, proteomic, physiological and other experimental evidence . Among them, 96 and 58 genes were supported by mutational investigation or transgenic over - expression study, respectively (table 1). Table 1.number of sags and mutants in 21 speciesspeciescommon namesagsmutantstransgenicarabidopsis thalianathale cress9499035oryza sativarice10439medicago truncatulabarrel clover3100brassica napusrape1500lycopersicon esculentumtomato803nicotiana tabacumtobacco503brassica oleraceabroccoli400pisum sativumpea410glycine maxsoybean401sorghum bicolorsorghum400solanum tuberosumpotato303zea maysmaize300hordeum vulgarebarley300astragalus sinicuschinese milk vetch101chenopodium rubrumred goosefoot110festuca pratensis huds.fescue110ipomoea niljapanese morning glory101medicago sativaalfalfa101rosa hybridrose100triticum turgidumwheat101triticum aestivumwheat100total2111459658 number of sags and mutants in 21 species the information of 154 leaf senescence related mutants such as name, ecotype and mutagenesis method were also retrieved from literatures . Expression profiling data were acquired from a classical and systemic research paper (5). In addition to manual curation, computational approaches were also employed to annotate these sags . We predicted the potential mirna targets for the sags using the rnahybrid method (7). The orthologs of each sag in other plants were retrieved from the online database orthomcl - db (8). Finally, putative function domains of sags - encoding proteins were identified with interproscan (9). To meet the general requirement of data analysis, we integrated the sequence similarity search tool blast (10) and the sequence analysis platform weblab (6) into our leaf senescence database . Users can either retrieve the sequence from lsd, or upload their own sequences to search homologs against different divisions (gene, mrna, cds and protein) in the lsd . Weblab is a web - based bioinformatics platform developed by our center and publicly available worldwide (http://www.weblab.org.cn/) (6). A user space is provided to save input data and analysis results for registered users . Users may retrieve sequence data and submit to weblab directly to perform extensive analysis for dna, mrna and protein sequences of the sags they are interested in . The lsd database enables users to retrieve and analyze sags through the browse or search page . Users may browse the entries by clicking the buttons of species, mutants or phenotypes at the main page . A tree - like structure was designed for both species and phenotypes, and a table was created for mutants . Currently, the major source of sags was from the two model organisms a. thaliana (949 entries) and oryza sativa (104 entries). The phenotypes of all sags were divided into the following groups: (i) natural senescence, (ii) dark induced senescence, (iii) nutrition deficiency induced senescence, (iv) stress induced senescence and (v) others . The text search interface allows users to make queries with three types of data: (i) locus name, genbank i d, alias, species and description of genes; (ii) name, type and ecotype of mutants; and (iii) title, author, journal and date of literature papers . Figure 1 shows the annotation for a typical lsd entry ntl9, a member of the nac transcription factor family and a membrane - associated gene that mediates osmotic stress signaling in leaf senescence (11). General information such as locus name, alias, organism, taxonomy was retrieved from the literature . Functional category, effect and evidence of senescence, as well as a brief description of this gene were manually annotated (figure 1a). Expression profiles generated from microarray data can be found for most arabidopsis sags (figure 1c). We predicted potential mirna targets for some sags and added links to mirbase (12) for these mirnas (figure 2a). Orthologs from other plants are listed with links to the orthomcl database (figure 2b). Putative functional domains of proteins encoded by sags were identified and annotated using the interproscan program (9), and matches were displayed with cross links to several protein domain and family databases such as prosite and pfam (figure 2c). (a) basic information, (b) mutant information and (c) expression profile . Figure 2.computational annotations for the arabidopsis nac transcription factor . (a) mirna targets, (b) ortholog groups and (c) cross links to other databases . A typical entry in lsd, the arabidopsis nac transcription factor . (a) basic information, (b) mutant information and (c) expression profile . (a) mirna targets, (b) ortholog groups and (c) cross links to other databases . In addition to sequence similarity search with the blast tool kit implanted in the database, users may also perform extensive analysis for sequence data retrieved from the lsd . For each entry, links to different sequence types (genomic, mrna, cds and protein) are provided . Users can click these links to bring up the corresponding sequence and submit it to weblab for further analysis, such as predicting gene structures, making pairwise or multiple sequence alignment, generating sequence logos, constructing phylogenetic trees, finding sequence motifs, etc . We will update the database regularly with more leaf senescence related data available, and predict putative sags from completely sequenced plant genomes in the near future . We will improve the user interface with comments and suggestions from the user community and add more documents including case studies to help user to make thorough analysis of sags . We hope that lsd can be a platform not only for the domestic and international collaborators we are working with, but also for the research community of leaf senescence worldwide . National natural science foundation of china (31071160 to j.l ., 30625003 and 30730011 to h.g . ); ministry of science and technology of china (2009cb119101 to h.g . ); ministry of education of china (ngi2008 - 108 - 3 to j.l ., ed20060047 to h.g . ). Funding for open access charge: national laboratory of protein engineering and plant genetic engineering.
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Female study participants were recruited from participants in a cross - sectional study assessing bmd in young women with type 1 diabetes and age - matched community control subjects (7). Cases for the cross - sectional study (baseline) were recruited from a regional tertiary hospital pediatric diabetes center and from endocrinology practices in western new york . At the time of the initial study, the diabetes center followed over 600 patients aged 221 years with type 1 diabetes . Female patients aged 1321 years in that clinical center were offered the opportunity to participate in the baseline study (n = 138). Recruitment of older women involved contacting former center patients (aged> 21 years), patients from regional endocrinology practices, and affected relatives of subjects screened by our diabetes center for the diabetes prevention trial . The comparison group included young women recruited from the same region as the case subjects; most were classmates or acquaintances of the case subjects or were volunteers who learned of the study through flyers posted at the university and hospital . The baseline study enrolled 72 case subjects (diabetes duration> 2 years) and 91 control subjects aged 1337 years who were at least 2 years postmenarchal (7). All type 1 diabetic case subjects and nondiabetic control subjects who participated in the baseline study were recruited 2 years later to assess change in bmd over time . Contact was maintained with study participants between the baseline and follow - up exam via phone and mail . Inclusion criteria for the follow - up study were participation in the baseline study, current negative pregnancy test, and signed informed consent . For individuals younger than 18 years of age, exclusion criteria for both the baseline and follow - up study included systemic illness affecting bmd (other than diabetes), other endocrine disorders (except autoimmune thyroiditis), and diagnosis of juvenile osteoporosis or other bone disease . The study was approved by the institutional review boards of the women and children's hospital of buffalo and the university at buffalo . At baseline, study participants completed questionnaires relating to personal and family health, lifestyle habits, dietary intake (food frequency questionnaire), and medication intake including use of calcium supplements . Participants also reported prior fracture history, menstrual history, and demographic information . For participants with type 1 diabetes, information on disease duration, insulin dose schedule (injections versus continuous subcutaneous insulin infusion), and diabetes complications were reported . Bmi was calculated as weight in kilograms divided by the square of height in meters . Dexa (hologic qdr-4500a; hologic, waltham, ma) was used to measure bmd in the anterior / posterior (l14) spine, total hip, femoral neck, total forearm, and whole body . A single technician performed all baseline scans and 65% of the follow - up scans; two additional technicians performed the remainder of the follow - up scans (27% and 7%, respectively). Coefficients of variation (cvs) were determined for measures of all sites for all technicians throughout the study and were all <1% throughout the study . The following biomarkers were measured in nontimed blood samples: serum osteocalcin, igf-1, igf binding protein (igfbp)-3 (by radioimmunoassay), and a1c (by high - performance liquid chromatography with bio - rad variant; bio - rad, richmond, ca). Random urinary n - teleopeptide levels were measured by enzyme - linked immuno - absorbent assay . Serum for hormonal assays was frozen at 80c and stored until sent for analysis in batches to esoterix laboratory (calabasas hills, ca). A1c (fresh plasma) was assayed in a sequential fashion at the women and children's hospital of buffalo laboratory (kaleida health). Intra - assay cv was <3% for n - telopeptide, 6% for igf-1 and osteocalcin, and 13% for igfbp-3 . Interassay cv was <7% for n - telopeptide, <9.7% for igf-1, 13% for osteocalcin, and <17% for igfbp-3 (esoterix laboratories). Data from study participants were analyzed in stratum based on age at initial enrollment (7). Demographic, lifestyle, metabolic characteristics, and bmd were compared between diabetic case subjects and nondiabetic control subjects, stratified by age . Comparisons were made for baseline differences, differences at follow - up, and percent change between the two time points . Results were presented as both unadjusted comparisons and comparisons of adjusted means (adjusted for age, bmi, and oral contraceptive [oc] use). Unadjusted differences for continuous variables were examined using student's t test and for categorical variables using the test . Data analyses were performed using sas version 8 (sas, cary, nc). Post hoc power analysis of follow - up total hip bmd (adjusted for age, bmi, and oc use) indicated that our dataset of 63 case subjects (n = 37, <20 years of age) and 85 control subjects (n = 36, <20 years of age) gave our study 80% power to detect a difference between case and control subjects with an of 0.05 and a target effect size of 0.45 in women <20 years of age and 0.63 in those> 20 years of age . Table 1 includes baseline and follow - up characteristics of women who participated in this follow - up study . For comparative purposes, the follow - up group included 63 women with type 1 diabetes (58.7% <20 years of age) and 85 control subjects without diabetes (42.4% <20 years of age) at ages 1539 years at enrollment . Of the participants in the baseline study with diabetes, 87.5% (63 of 72) participated in this follow - up study, and 93.4% (85 of 91) of control subjects participated in both examinations . More subjects from the baseline cohort 20 years of age were lost to follow - up (13.8 vs. 3.94%, cohort <20 years of age), with the highest attrition being from individuals with diabetes 20 years of age (21.2%). Participants in this follow - up study were predominantly non - hispanic caucasian (95%) and were similar in age at menarche and years since menarche . Participants 20 years of age with diabetes used oc therapy longer than control subjects (p 0.05). Although few participants were smokers, more women with diabetes reported current smoking; there were no smokers among the control subjects <20 years of age . At follow - up, women with diabetes, particularly the older women, continued to have higher bmi than control subjects . Weight change (in percent) was not significantly different over the follow - up period . As reported previously, more patients in the older cohort were using continuous subcutaneous insulin infusion (p <0.01, data not shown). While a trend toward lower daily insulin requirement was present in the older cohort (0.86 0.42 vs. 0.75 0.22 units kg day), that difference was not statistically significant . Subjects 20 years of age were more likely to have diabetes - related complications, although no statistical differences were found . Table 2 presents bmd values at baseline and follow - up for each body site measured . Unadjusted means and means adjusted for age, bmi, and oc use are shown stratified by age - group and diabetes diagnosis . In those <20 years of age, both case and control subjects had an increase in bmd over the 2-year interval . The percent increase in bmd from baseline to follow - up was not statistically different between younger subjects and control subjects . For all participants in the population 20 years of age, the bmd from baseline to follow - up remained stable . Significant differences in bmd were identified at the total hip, femoral neck, and whole body in the case subjects 20 years of age compared with the control subjects . As in the previous report, no differences in bmd were seen between groups in the younger cohort . In the adjusted model (for age, bmi, and oc use), bmd values at the total hip (baseline, p = 0.003; follow - up, p = 0.007) and femoral neck (baseline and follow - up, p <0.001) were significantly lower in older women with diabetes compared with control subjects . After adjustment, the whole - body bmd was no longer significantly different between case and control subjects; however, both age - groups showed a trend toward lower whole - body bmd in the case subjects . Further adjustment for dietary intake of calcium and vitamin d as well as physical activity did not alter the bmd results (data not shown). When the analysis was restricted to nonsmokers, the adjusted means changed very little . For the older cohort, however, the p values were attenuated for the total hip (p = 0.017) and whole body (p = 0.215). In nonsmokers> 20 years of age, the percent change difference at the total hip became more pronounced between case and control subjects and reached statistical significance (p = 0.008). Overall, igf-1 levels were lower for all groups compared with baseline (table 3). This is consistent with a physiologic decrease in igf-1 in postteenage years (esoterix normative data 11). At the 2-year follow - up, however, the igf-1 level was significantly lower in the younger diabetic subjects than in the control subjects, although still within the normal range . Igf-1 levels were not statistically different between diabetic participants and control subjects in the older cohort . Within the older age - group, igfbp-3 levels were lower in participants with diabetes than in control subjects (p <0.05). Igfbp-3 levels were comparable for control and diabetic subjects in the cohort <20 years of age . Serum osteocalcin levels were lower at follow - up for all groups, even with increased bone mineral accrual in the group <20 years of age (table 3). There was no difference in osteocalcin or n - teleopeptide levels between case and control subjects <20 years of age (table 3). After adjusting for age and bmi, case subjects had significantly lower osteocalcin levels than control subjects in the population 20 years of age (p <0.04; table 3). In addition, the percent change in osteocalcin level from baseline to follow - up was significantly different in diabetic women 20 years of age compared with control subjects after adjustment (p <0.03). Markers of bone formation did not correlate with bmd in our subjects . Metabolic control, as measured by a1c, was poorer in the younger diabetic cohort, as we reported previously (7). Overall metabolic control was stable within a given age stratum between the baseline and follow - up studies . No correlation was found between a1c and any of the bmd measurements (data not presented). When evaluating for associations between metabolic control and bmd measurements that were statistically lower in case than in control subjects at follow - up (table 2), the pearson correlation coefficient (r value) for a1c and bmd was between 0.121 (total hip) and 0.334 (femoral neck) (p> 0.095, data not shown). As in our previous study, there was no association found between bmd and diabetes duration, baseline a1c, or years since menarche . While longitudinal studies have examined the effect of insulin therapy on bmd in patients with long - standing type 1 diabetes (12), few have reported on the natural history of metabolic bone disease in younger women . Here we present bmd data for a well - characterized cohort of young women with type 1 diabetes at baseline and 2 years later . The baseline study demonstrated that the older cohort had significantly lower bmd at the femoral neck and lateral spine than age - matched control subjects (7). The data presented here demonstrate that case subjects in the older cohort had persistently lower bmd than age - matched healthy control subjects . In this cohort, bmd at the total hip and femoral neck remained lower than in control subjects after adjusting for age, bmi, and oc use, even with no significant decrease in bmd from baseline to follow - up (table 2). Similar to our initial study, there was no difference in bmd between women with diabetes aged <20 years and control subjects at any of the sites measured . However, there was a trend toward lower bmd at the total hip and whole body in women with diabetes . This difference occurred in the face of bmd increases (percent change) at both the total hip and whole body in this population, suggesting that bone mineral accrual is altered in a subtle manner during late adolescence in women with type 1 diabetes . Individuals with type 2 diabetes have higher than average bmd compared with control subjects (13), in part due to the mechanical load of obesity . In our study, women with type 1 diabetes had higher bmi than control subjects . However, bmd continued to be lower in older women with type 1 diabetes, even after adjusting for bmi; thus, overweight status does not confer protection against poor bone mineralization in these young women . Although the younger cohort had poorer diabetes control than the older cohort, we were again unable to demonstrate an association between bmd measures and metabolic control, as measured by a1c . Others have reported similar findings with respect to bmd in children and adolescents with diabetes (14). A1c measures short - term diabetes control; perhaps cumulative life - time glycemic control is a better indicator of osteoporotic risk . This hypothesis is supported by studies demonstrating that decreased lumbar spine and femoral neck bmd in adults with type 1 diabetes is associated with retinopathy, nephropathy, and peripheral neuropathy, all of which are long - term complications of poor metabolic control (1517). In addition, our data did not find a correlation between diabetes duration and bmd, further supporting a possible relation to lifetime diabetes control . The adverse effects of chronic hyperglycemia related to osteoblast function, osteoclast activity, and formation of advanced glycosylated end products that may impact bone quality as well as bone quantity (18) are difficult to measure in a systematic fashion . The role of insulin as a direct anabolic agent in bone metabolism is unclear (19). Animal models of spontaneous and pharmacologically induced diabetes demonstrate that as insulinopenia develops, there is a suppression of osteoblast markers (20). It has been postulated that in addition to insulinopenia, relative igf-1 deficiency, whether systemic or local, contributes to low bmd in diabetes . Igf-1 levels are lower in individuals with type 1 diabetes, and poor glycemic control negatively impacts igf-1 production by the liver . In adults with type 1 diabetes, igf-1 levels were lower in individuals with osteopenia at the femoral neck (1). Additionally, in a study of 127 children (aged 620 years) with diabetes, low bone mineral content correlated with low igf-1 levels (9). In our study, igf-1 levels of case subjects were significantly lower than those of control subjects only for the younger cohort . However, igf-1 levels of subjects with diabetes were lower in the group 20 years of age compared with the group <20 years of age, and igfbp-3 levels in the older cohort were significantly lower compared with control subjects . Given that no biochemical markers of bone metabolism correlated with bmd at the hip or femoral neck in our study, it could be hypothesized that a cumulative history of insulinopenia and low igf-1 levels are better correlates for low bone density . Our data demonstrate significant differences in bmd at the femoral neck and total hip that are likely to be clinically significant and may explain why women with type 1 diabetes have an increased risk of hip fractures later in life (4,5). Our data also indicate that alterations in bone mineral accrual occur within years of achievement of peak bone mass and that there is not a later period of catch - up bone mineralization . The most clinically relevant factor is whether a decrease in bmd correlates with fracture risk, which cannot be determined in this study . Besides bmd, multiple factors impact fracture risk including, but not limited to, age, nutrition status, smoking history, degree of frailty, and fracture history (21). The well - characterized population, the large percentage of women who participated in the follow - up study, and the standard procedures used in this longitudinal study all represent significant strengths . Yet, we acknowledge certain limitations . The study population is small, limiting its ability to detect true differences that may still exist . Study control subjects were community based and may not represent the general population of women . Finally, the results presented should be interpreted keeping in mind that multiple comparisons were made . We are aware of the lack of unanimity of opinion regarding the statistical approach when adjusting the statistical testing for many comparisons (22). Because many of our hypotheses are nested, and not independent, we feel that using a bonferroni approach is overly conservative . However, we have interpreted our results with caution and believe they are stronger when considered as a whole rather than as independent statistical tests . Our findings demonstrate lower bmd in young women with type 1 diabetes compared with control subjects . Although bone density testing is not routinely performed in young women, these data suggest that screening may be important in young women with type 1 diabetes . In addition, these women should be counseled regarding lifestyle interventions that may improve bone health, including adequate intake of calcium and vitamin d, and exercise.
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Many studies have shown that bracket debonding can cause enamel loss, particularly when the fracture occurs at the enamel - adhesive interface . Enamel damage may be in the form of enamel cracks, which may propagate during debonding [13]. Acid - etching, rinsing, drying and applying resin and bonding agents are the routine steps of conventional orthodontic bracket bonding . Concurrent with studies on the efficacy of the conventional technique, many studies, aimed at reducing the number of steps necessary for adhesion, have examined new bonding techniques with clinically adequate bond strength but less etching depth [56]. Traditional methods of bonding orthodontic brackets to teeth rely on acid etching to achieve adequate retention . However, maintaining a sound enamel surface after bracket removal is of primary concern to clinicians . Bond failure at the bracket - adhesive interface or within the adhesive is generally considered safer than a fracture at the enamel - adhesive interface; this is because studies have demonstrated that enamel fracture can occur during debonding . On the other hand, if a considerable amount of adhesive remains on the tooth surface after debonding, more chair time is required to remove the residual adhesive . In addition, the process of removing the residual adhesive results in further enamel loss . All - purpose or multi - purpose, self - adhesive resin cements are now commercially available in the market . They are capable of bonding to different substrates such as enamel, dentin, amalgam, metal and porcelain [910]. These cements require no surface pre - treatment due to their monomer and filler content and initiation technology . The phosphoric acid methacrylates react with basic fillers in the luting cement and the hydroxyapatite of the tooth structure [1112]. These self - adhesive cements do not require an etch and rinse phase as they are capable of conditioning the tooth surface and simultaneously preparing it for adhesion . This not only shortens the clinical application time, but also significantly reduces technique - sensitivity and risk of errors during application or manipulation . The aim of the current study was to assess the enamel cracks after debonding of brackets bonded with two different adhesive systems and also to evaluate enamel cracks after removing adhesive remnants and enamel polishing . Sixty upper central incisors, extracted due to periodontal disease, were selected and stored in normal saline at room temperature until required . The study design was approved by the ethics committee of mashhad university of medical sciences . Using a stereomicroscope (blue light industry, waltham, ma, usa) and digital camera (exwave had, sony corporation, japan) a magnified image (23.9x) the number of enamel cracks and the length and direction of each crack were assessed with the aid of adobe photoshop cs software (adobe systems incorporate, usa) (figure 1). Enamel cracks before bracket bonding (a), after deboning (b) and after polishing (c). New cracks are hatched . The number of visible cracks (i.e. Cracks visible to the naked eye) was also recorded . The teeth were then randomly divided into two equal groups . In the first group, the buccal surface of each central incisor a standard edgewise twin metal bracket (dentaurum, pforzheim, germany) was bonded with transbond xt (3 m, usa) to the center of the labial surface of each tooth . In the second group, the same bracket was bonded with self - adhesive composite cement (maxcem elite, kerr, usa). In both groups, the adhesive was cured for 40 seconds (10 seconds on each side of the bracket). The teeth were then incubated in distilled water at 37c for 24 hours to allow the material to set . All the brackets were debonded with rmo i-546 remover pliers (rmo, germany). The blades of the pliers were placed at the bracket - adhesive interface and a gentle squeezing force was applied to the pliers by a zwick / z250 at a speed of 0.5 mm / min until bond failure occurred . The present method of force application was modified from a diametric compression test for tension that indirectly measures tensile strength . This approach was used because it simulates the usual clinical method for applying debonding forces . The debonding force recorded by the zwick is not equal to the actual force applied at the bracket adhesive interface because the force was applied at a predetermined distance on the pliers beaks . These forces, however, are proportional and can be expressed in the following ratio: the actual force (f) equals the measured force (f) multiplied by (a) divided by (b): f= f (a / b). In this ratio, (a) is the distance from the point of force application to the fulcrum of the pliers and (b) is the distance from the point of actual force application to the fulcrum of the pliers . Deboning force was measured in newton and was then converted to megapascal (mpa). After debonding, the buccal surfaces of the teeth in each group were examined by the same stereomicroscope and the magnified images (23.9 x) were taken with the same digital camera . The number of enamel cracks and the length and the direction of each crack were assessed with the aid of the same software . A: the distance from the point of force application to the fulcrum of the pliers . B: the distance from the point of actual force application to the fulcrum of the pliers the number of visible cracks in each tooth was also recorded (figure 1). The adhesive remnants on each tooth were then assessed with the ari in a 03 scale: 0 indciates no composite left on the tooth; 1 indicates less than half of the composite left on the tooth, 2 indicates that more than half of the composite is left on the tooth surface and 3 shows that all the composite is left on the tooth surface with a distinct impression of the bracket base . The buccal surface of each tooth was polished with 12-blade tungsten carbide bur (g&h, usa) under water coolant . The enamel surface was examined for the third time in the same way (figure 1). The amount of shear bond strength in each group was compared with independent t - test . The enamel cracks in each group were compared before bonding, after debonding and after polishing using repeated measures anova . Friedman and mann - whitney tests were applied in case of non - normal distribution of data . The result of the independent t - test indicated that the mean shear bond strength of maxcem elite (the self - adhesive composite cement) was significantly lower than that of transbond xt (the adhesive composite) (p<0.001). The mean sbs of maxcem elite and transbond xt was 10.291.14 and 11.481.09, respectively . There were no significant inter - group differences in the number of enamel cracks before debonding . There were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . In the maxcem elite group, the difference in the number of enamel cracks before and after debonding was statistically significant (p<0.001). The difference, however, was not significant after debonding and after polishing (p=0.06). The number of visible enamel cracks followed a similar pattern . In each group, before and after debonding, a significant intra sd: standard deviation significant difference significant difference between a and b (wilcoxon test) however, before and after polishing, the difference was not significant (table 2). Sd: standard deviation significant difference significant difference between a and b (wilcoxon test) there was no significant difference in the length of enamel cracks between the two groups . In each group, however, a significant increase in the length of enamel cracks was obvious after debonding . Repeated measures test sd: standard deviation significant difference significant difference between a and b (wilcoxon test) the mann - whitney - u test showed that the differences were statistically significant among the groups (p=0.0001) (table 4). The adhesive remnant index (ari) (0, no composite left on the tooth; 1, less than half of the composite left on the tooth; 2, more than half of the composite left on the tooth; 3, all the composite left on the tooth with a distinct impression of the bracket base) the result of the independent t - test indicated that the mean shear bond strength of maxcem elite (the self - adhesive composite cement) was significantly lower than that of transbond xt (the adhesive composite) (p<0.001). The mean sbs of maxcem elite and transbond xt was 10.291.14 and 11.481.09, respectively . There were no significant inter - group differences in the number of enamel cracks before debonding . There were significantly more enamel cracks in the transbond xt group after debonding and polishing compared to the maxcem elite group . In the maxcem elite group, the difference in the number of enamel cracks before and after debonding was statistically significant (p<0.001). The difference, however, was not significant after debonding and after polishing (p=0.06). The number of visible enamel cracks followed a similar pattern . In each group, before and after debonding, a significant intra sd: standard deviation significant difference significant difference between a and b (wilcoxon test) however, before and after polishing, the difference was not significant (table 2). Sd: standard deviation significant difference significant difference between a and b (wilcoxon test) there was no significant difference in the length of enamel cracks between the two groups . In each group, however, a significant increase in the length of enamel cracks was obvious after debonding . Repeated measures test sd: standard deviation significant difference significant difference between a and b (wilcoxon test) the mann - whitney - u test showed that the differences were statistically significant among the groups (p=0.0001) (table 4). The adhesive remnant index (ari) (0, no composite left on the tooth; 1, less than half of the composite left on the tooth; 2, more than half of the composite left on the tooth; 3, all the composite left on the tooth with a distinct impression of the bracket base) the main objective of this study was to evaluate the enamel cracks after debonding brackets, bonded with two different bonding systems followed by polishing . The sbs of maxcem elite, a self - adhesive cement, and transbond xt, a conventional adhesive composite, was also compared . The number and length of enamel cracks before bonding, after debonding and after polishing were also compared as well as the ari following the removal of orthodontic brackets . Before bonding zacchrison et al . Showed that debonding of brackets created enamel cracks, which were different in size and direction . Reported a significant increase in the number and length of enamel cracks after debonding with different pliers . Stated that 82% of the teeth studied did not show significant increase in the number of enamel cracks after debonding and the teeth showing increased number of cracks had significantly greater mean bond strength . After polishing, the number and length of enamel cracks increased in all groups, although there were no differences among the groups . According to zarrinnia et al ., debonding pliers cause resin fracture at the adhesive - bracket interface and using a tungsten carbide bur, accompanied by water coolant, effectively removes all adhesive remnants . They showed that the smoothest surface was obtained with the gloss polishing paste . A direct correlation between ari and bond strength the current study found significant differences among the ari scores and concluded that, in the transbond xt group, debonding was mostly of cohesive type, in contrast to the adhesive mode in the maxcem elite group . Reported that self - adhesive cements had satisfactory bond strength to dentin and restorative materials, but that their bond strength to enamel was weak . Vicente et al . Showed that relyxunicem (a self - adhesive resin cement) possessed a significantly lower bond strength than transbond xt . Reported low bond strength for relyxunicem and suggested that the sbs of this self - adhesive universal cement needs to be increased for its successful use in bonding orthodontic brackets . . Showed that one - component adhesives (relyxunicem and maxcem) had significantly lower sbs than two- or three - component adhesives . Bishara et al . Reported that maxcem had significantly lower bond strength than transbond xt . In the current study, maxcem elite (a new self - adhesive system) the mean bond strength in the transbond xt group was significantly greater than that in the maxcem elite group, although the sbs of the latter was 10.291.14 mpa, which is clinically acceptable . Debonding brackets bonded with maxcem elite resulted in less enamel cracks . Removing remnants of adhesives and polishing with a 12-blade tungsten carbide bur did not increase enamel cracks . Maxcem elite has clinically acceptable bond strength and can be used as a routine adhesive for orthodontic purposes.
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Sensitive skin syndrome is a common and challenging condition, yet little is known about its underlying pathophysiology . Sensitive skin is a common term used by patients and clinicians, as well as the cosmetic industry, and represents a complex clinical challenge faced by dermatologists and other skin care professionals . Patients with sensitive skin often present with subjective complaints that are out of proportion to the objective clinical findings . The patients complain of severe facial irritation, burning, and/or stinging after application of cosmetic products and toiletries such as sunscreens and soaps, yet they do not demonstrate the clinical stigmata of scaling, induration, and/or erythema that would be expected in known inflammatory or allergic processes . The condition is often worsened by specific climatic conditions and may affect areas other than the face . Maibach described the cosmetic intolerance syndrome (cis), which probably covers the bulk of the clinical presentation of the sensitive skin syndrome (sss). Fisher is credited with coining the term status cosmeticus to describe one extreme of the natural history of the cis in which the patient gradually becomes completely intolerant to the application of any cosmetic product . Fundamentally, sss is a state of hyperreactivity to environmental stimuli, presenting with a clear clinical picture and resulting from a single underlying pathology or a combination of pathologies . Defined as a self - diagnosed condition, sss is by definition difficult to quantify . Some of the contradictions between investigators could be explained by flawed methodologies since the scientific community has yet to identify an acceptable objective screening test for sensitive skin . However, it is not only the subjective nature of the complaints that make sss difficult to diagnose . Robinson, by performing patch testing with sodium dodecyl sulfate (sds) and looking for intra - individual response patterns, found that there is significant positive correlation between sds and other irritants but noted overall low correlation coefficients . He therefore deduced that it is inappropriate to define a subject's reaction to a chemical based on his or her response to another irritant . She skin - tested 58 subjects with history of strong positive sds or lactic acid response . Sds- or lactic acid - positive group, a reaction to one irritant could not predict a reaction with another . To further complicate matters there is evidence to suggest that no correlation exists between the lactic acid stinging test and the response to sds . This data resonate the idea that even the reference irritants commonly used in studies of this topic do not correlate with each other and with hyperreactivity tendencies . Finally, judge tested 22 nonatopic adults with varying doses of sds and found marked inter - individual variation in the response threshold . This complexityreinforces the need for a thorough diagnostic algorithm and, specifically, the need to test patients with multiple, repeated and complete (i.e. All cosmetics applied by the patient at home) patch testing before making a diagnosis . In order to formulate a systematic clinical approach, the medical and cosmetic communities have attempted to characterize the condition . Lacking an objective screening test, investigators resorted to epidemiological studies using patient surveys . In a large epidemiological study in the uk (n = 2316), a staggering 51.4% of women and 38.2% of men self - reported themselves as having sensitive skin . Of note, interestingly, atopy did not appear to predict self - perceived sensitivity in the participating women . In addition, in the same study self - reports of sss symptoms were statistically over - represented in the self - reported sensitive cohort compared to the self - reported nonsensitive cohort . This finding validates the link between self - perception of sensitive skin and neurosensory discomfort . Two recent studies in the us and europe made similar observations . In the us, females were significantly more concerned about sensitive skin than men; however, no age or ethnic differences were found . The european study reported an overall sensitive skin prevalence of 38.4% and found no ethnic differences . The european study, once again, demonstrated that people who reported having sensitive skin were significantly more likely to experience sss symptoms . Of note, both the american and european studies used large samples (n = 994 and n = 4506, respectively); however, they were both limited by a phone survey methodology and the lack of any objective assessment . In light of the increased incidence of self - reported hypersensitivity in females, he compared the patch test responses of 384 patients to sds as the positive control and found increased reactivity in males compared to females . Lammintausta tested seven males and seven females with sodium lauryl sulfate (sls) and then performed visual inspections, transepidermal water loss measurements, and dielectric water content measurements but like other investigators found no reactivity differences between males and females, adding to the overall controversy . Female self - perception of sensitivity is consistently increased compared to that of males, yet when put through objective reactivity testing the trend is unclear . Ethnic differences in skin reactivity have been explored through the years, leading to the clinical hypothesis that black skin is less reactive than caucasian skin, which is in turn less reactive than asian skin . However, an epidemiological telephone survey performed in the us (n = 811) revealed similar incidence of self - reported sensitive skin across four major ethnic groups (afro - americans, asians, euro- american, and hispanics). The incidence rate of 52% is comparable to the incidence found in the uk (see above). The investigators did, however, find minimally statistically significant ethnic differences in the self - reported triggers and symptoms of sensitivity: afro - americans reacted less to environmental and alcoholic triggers; hispanics reacted less to alcohol; asians reacted more to wind, spices, and alcohol; and euro - american reacted more to wind . In terms of symptomology, statistically significant increase in recurrent itching on the face was noted in the asian population, and fewer euro - american avoided certain cosmetics due to skin reactivity . These findings were supported in a critical literature review, which showed that the evidence supporting the ethnic difference hypothesis using objective tests rarely reaches statistical significance and is often biased by subjective endpoints . Recent data looking into age - related differences in hyperreactivity are showing more consistent results . Whorl performed patch testing with 34 irritants on 2776 patients and found that young patients were more reactive than old patients . Robinson's findings also corroboratedthe trend towards increased reactivity in the young subjects compared to the old subjects . The mechanism leading to the sss has been debated in the literature, but the leading hypothesis relates to increased stratum corneum permeability . There is a known inverse relationship between corneocyte size and stratum corneum thickness and the permeability of the skin, and changes in this mechanical barrier result in abnormal skin penetration by irritants . In addition, low levels of ceramides in the stratum corneum have been linked to increased severity of sls - induced irritant contact dermatitis, pointing toward barrier compromise in sensitive skin . Seidenari linked self - reported hyperreactivity to decreased skin capacitance (indicating decreased water content), objectively suggesting increased absorption of irritants by the sensitive - skin population . In the same study, increased erythema was objectively measured in hyperreacting subjects suggesting involvement of some baseline vascular component, and leading more recent investigators to propose the involvement of cutaneus sensory innervation in the pathogenesis of sss . As with many syndromes, the management scheme presented here emphasizes the diagnostic framework [table 1]. The clinical approach to the sss demands a thorough process of eliminating obvious as well as more subtle diagnoses . Once the diagnosis is made, deriving the appropriate treatment becomes a relatively simple task . Note that since much of the mechanisms of sss are unknown, the clinician must be comfortable with a certain degree of ambiguity and uncertainty . Underlying mechanisms of sensitive skin syndrome it is reasonable to organize the differential diagnosis by dividing sss into visible and invisible sss . Some dermatoses that most clinicians find simple to diagnose may have atypical presentation and as a result lack the expected morphology . Eczema, atopic dermatitis, and rosacea are likely the three most common causes of sss related to barrier defect . Full discussion of these conditions is beyond the scope of this overview but we will highlight some common clinical features . Careful history, including family history and occupational history, combined with a detailed physical exam will often reveal the diagnosis . In the history particular consideration should be given to culprits such as the masking effect of other topical agents applied by the patient . The physical exam should include scrutiny of the face and scalp for subtle signs of minor inflammation, which are often masked in sss with underlying endogenous dermatoses . In all of these conditions the patients are likely over - exfoliating their skin and thus exacerbating the barrier dysfunction . Therefore, following specific treatment to halt the acute pathological process, preventing recurrence with a proper skin care regimen is indicated . In eczema and atopic dermatitis, the careful clinician may resort to short - term (2-week) use of corticosteroids to stop the inflammatory process . Calcineurin inhibitors are alternatively indicated for delicate areas on the face . In an effort to control histamine release in atopic dermatitis, antihistamines can be added and a relatively allergen - free environment should be created . In rosacea, the mainstay of treatment is oral and topical antibiotics . In seborrhoeic dermatitis, azoles are the mainstay of treatment and low potency corticosteroids and emollients can be added acutely to treat the inflammatory process . Contact dermatitis (cd) and photocontact dermatitis (pcd), as well as nonimmune contact urticaria (nicu) and immune contact urticaria (icu), are all conditions that may elicit sss symptomology, with transient objective findings on physical exam . Allergen - free products are now available and should be included as part of further skin care regimen in these patients . The main reason why nicu is often a missed diagnosis in patients presenting with the sss is the transient nature of the reaction . Thus, after testing with small amounts of product on the skin, the patient should be carefully examined using minimal magnification: the lesions will be typically revealed within 20 minutes . Common trigger agents are fragrances (e.g. Cinnamic aldehyde) and preservatives such as sorbic and benzoic acids . Once triggers icu can be demonstrated with open and occluded testing, followed by prick testing with appropriate positive and negative controls if no response is elicited . Various foods, latex, parabens, and other chemicals have been implicated in the causation of icu . However, there are cases in which no clinical clue is provided by the physical exam . In these cases, a 2-week trial of appropriate - strength corticosteroid may solve the mystery by pointing to an eczematous process in the case of symptoms resolution . Nonetheless, the careful clinician should consider avoiding prolonged use of topical corticosteroids since there is anecdotal clinical experience of topical corticosteroids inducing sss and, at least in the case of barrier dysfunction, earlier and complete steroid tachyphylaxis has been demonstrated . If no symptomatic relief is observed, the patient's sss is likely due to an invisible underlying cause . Both of these cases defined by maibach, no signs are present or can be elicited on the skin . In objective irritation the mechanism is unknown and this diagnosis likely bundles more than one pathological process in it . Interestingly (and clinically frustratingly), maibach considers subjective irritation to be the most common cause of cis . Finally, body dysmorphic disorder (bdd) should always be considered by the dermatologist when assessing skin complaints without objective findings . A recent cross - sectional study found a prevalence of 14% among patients in a cosmetic dermatology clinic . In the context of sss, dermatologists should be aware that facial irritation complaints without any findings on exam are a common presentation in the dysmorphic patients and therefore extra caution is warranted . Referral to a mental health professional is indicated since these patients are at risk of suicidal behavior . Maibach notes that patients suspected of having bdd often require a detailed diagnostic process in order to build the trust needed to safely refer them to the mental health professional . Table 2 summarizes the steps in the evaluation of a patient suspected to have sss . Specific algorithms have been developed for the management of sss but they are the result of accumulated clinical experience and lack experimental evidence to support their use . Nonetheless, algorithmic thinking may aid in ensuring complete assessment . These algorithms include four basic steps: discontinuation, assessment, testing, and slow re - introduction . Discontinuation of all topical medication and/or products, as well as elimination of activities and clothing resulting in skin friction, should be initiated and may need to last up to 12 months . The patient should then be assessed (as described above) for the occurrence of any visible dermatoses . If no clear pathology has emerged, rigorous testing is the next step . Testing should include patch and photopatch testing of routine allergens as well as of all of the patient's skin care items . Specific testing for contact urticaria should be a part of the testing protocol . Finally, the next step in management is slow re - introduction of the minimally necessary skin care products . For females, recommend adding low - allergenic- potential cosmetic products one at a time in the following order: lipstick, face powder, and blush . For all products re- introduced, recommend testing by applying the product to a 2-cm area lateral to the eye for five consecutive nights and documenting the positive and/or negative result . This exhausting process is not only a rational solution from a clinical management standpoint but may also aid in diagnosing the specific culprit in cases of invisible sss . Understanding the underlying mechanisms may lead to the development of a well - standardized screening test . This will allow clinical studies to rely on objective findings rather than self - reports and this, in turn, may result in more comprehensive and efficient management strategies . Female self - perception of sensitivity is consistently increased compared to that of males, yet when put through objective reactivity testing the trend is unclear . It is reasonable to organize the differential diagnosis by dividing sss into visible and invisible sss.
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Spontaneous pneumothorax (sp) comprises the largest group and is classified into primary spontaneous (psp) and secondary spontaneous pneumothorax . Psp usually occurs in young, tall, thin men, especially smokers, in the absence of an underlying lung disease . Although, sp is a relatively common condition, bilateral primary spontaneous pneumothorax (bpsp) is a very rare clinical condition with an occurrence ranging from 1.3 to 1.9% of all cases of sp . Herein, we present a case of bpsp in an overweight young male patient, which is an exceptionally rare event . An otherwise - healthy, non - smoker, 20-year - old, overweight (bmi 29.8 kg / m) greek male patient was presented to the emergency department complaining of low - intensity chest pain and breathlessness of progressive worsening over the preceding 7 days . On admission, blood pressure was 125/70 mmhg, heart rate 100 beats / min, sao2 97% on air and axillary temperature 36.8c . Initial plain chest x - ray demonstrated bilateral pneumothoraces (more prominent on the left side) and no deviation of the trachea (fig . 1). Plain chest x - rays on admission revealing bilateral pneumothorax . On emergency basis, the patient was managed with bilateral tube thoracostomy through the fifth intercostal spaces . During hospitalization, both lungs were gradually expanded; the use of continuous aspiration was necessary for a couple of days, in order to achieve complete expansion of the left lung (fig . 2). Next day the plain x - ray revealed recurrence of pneumothorax on the left side (fig . The tube was removed after 5 days; no recurrence took place at this time . During the next 3 days the patient remained under observation; serial physical examinations and chest x - rays were normal and the patient was discharged home . Recurrence of pneumothorax on the left side after removing the chest tube . During hospitalization a chest computed tomography (ct) was performed and revealed the presence of blebs at the apices of both upper lungs (fig . The patient was referred to a tertiary center for an elective video - assisted thoracoscopic surgery intervention (vats). Three years after, no recurrence of pneumothorax took place and the patient remains healthy . Psp usually occurs in the absence of any obvious underlying lung disease; however, blebs and bullae apparently play a role in the pathogenesis of psp, as they are found on ct or during thoracoscopy or thoracotomy in 4879% of these patients . Diagnosis of bilateral pneumothorax presents difficulties; while unilateral pneumothorax is relatively easily suspected from the patient's medical history, physical and radiological findings, the diagnosis of bilateral pneumothorax is usually delayed . The presence of equally diminished breath sounds in both sides and the presence of a not deviated trachea often mislead clinicians and delay chest drain insertion which is usually performed after the transaction of chest ct . Although sp is a relatively common condition, bpsp is a very rare clinical condition . Lee et al ., in their study of the 616 patients with 807 episodes of psp, found that the incidence of bpsp was 1.6% (13 patients). In univariate regression analysis, patients with psp compared with patients with bpsp had significantly lower body weight, bmi, higher body height / body weight ratio and higher incidence of bilateral blebs / bullae seen in hrct of the lung . However, in multiple regression analysis, only bmi and the presence of bilateral blebs / bullae retained statistically significant importance . Huang et al . Found that only lower bmi and smoking were significantly associated with the formation of bpsp . In their study, the proportion of bilateral blebs / bullae seen in hrct was higher in the bpsp group than that in the psp group (63 vs 53%, respectively), but the difference was not statistically significant (p = 0.724). The present report describes a case of bpsp in an overweight patient showing that psp is an existing condition in patients with bmi value higher than the normal . Generally, if an sp affects <20% of one lung, observation is efficient; the absorption rate of the air is 1.25% (5075 ml / day). If pneumothorax affects> 20% of the lung or if it increases during observation, chest tube drainage may be required . Air leakage can be diminished in 5 h and in 48 h in 52 and 82% of patients with tube thoracostomies, respectively . In patients with bpsp, one side should always be drained regardless of the extent of pneumothorax, whereas the other side can be managed by simple observation depending on the extent of the air in the pleura space . Prolonged air leakage is the most common indication for operation in the first episode of pneumothorax . Today compared with open thoracotomy, this procedure provides visualization of the entire thoracic cavity by video and causes less postoperative pain . Single - stage bilateral vats procedure for bpsp has been advocated as an elective procedure to avoid subsequent anesthetic and operative procedures and longer hospital stays . If any underlying pulmonary disease is detected during surgical treatment, the existence of pleural communications should be investigated and mediastinal pleura should be examined carefully . In addition, apical pleurodesis further reduces the risk of recurrence . In our case, the patient was successfully managed initially with a bilateral tube thoracostomy and finally with an elective bilateral vats procedure . In conclusion, bpsp is a rare clinical condition and usually develops in patients with low bmi and bilateral blebs / bullae . Bpsp in overweight - obese patients is an existing condition and its early diagnosis requires high suspicion index . Bpsp needs urgent assessment and management, followed by bilateral single - stage vats treatment as a safe and effective procedure.
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Familial adenomatous polyposis account for 1% of colorectal cancers, and provides a model of apc inactivation as an early genetic event for the approximately 80%85% of cancers that develop from sporadic polyps . Colorectal cancers arising in patients with familial adenomatous polyposis can be largely prevented by polyp surveillance and prophylactic colectomy . Total proctocolectomy with construction of a conventional ileostomy or ileoanal anastomosis with preparation of an ileal pouch, has various effects on the function of the terminal ileum and the intestinal bacterial flora . This may deteriorate cholesterol metabolism, as absorption of cholesterol in duodenum and jejunum requires micellar solubilization with bile acids, fatty acids, monoglycerides, and phospholipids . Hypothetically, ileal - pouch - anal anastomosis and ileostomy patients might differ with regard to the presence of various fatty acids in feces and their relationship to other reflections of lipoprotein metabolism, but we found no previous study focusing upon this issue . Dietary fatty acids are incorporated into blood and tissues, and the fatty acid composition in these tissues are often used as biomarkers of fat intake . Furthermore, the fecal amount and composition of fatty acids reflect fat ingestion, intestinal fatty acid absorption, and the activity of colonic bacteria . Although some of the discrepancies between studies may be due to the use of different methods to analyze fatty acids, differences in diet, or the fact that assessments have been performed in different body compartments, modifications in the metabolism of fatty acids have been suggested in cancer patients [5, 6]. It is not clear at what steps in the multistage carcinogenesis process a possible distorted fatty acid metabolism occurs . Notably, if such alterations occur in the development of carcinogenesis, this may affect the biological functions of essential fatty acids and their derivates . Very little is known about fatty acid metabolism in familial adenomatous polyposis, although chemoprevention affecting the fatty acid derivates and the cyclooxygenase enzymes is often administered to familial adenomatous polyposis patients . Deregulation of the cyclooxygenase-2 pathway appears to affect tumorigenesis via a number of distinct mechanisms: promoting tumour maintenance and progression, encouraging metastatic spread, and perhaps even participating in tumour initiation . Cyclooxygenase-1 and -2 are the rate limiting enzymes in the synthesis of prostaglandins and thromboxanes . Arachidonic acid is the main substrate for these enzymes, leading to the synthesis of prostaglandins which have growth promoting effects . Substituting arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins . Since cyclooxygenase-2 is a fatty acid metabolising enzyme, the relationships between cyclooxygenase-2 and fatty acid composition of different tissues is of interest . Colectomized familial adenomatous polyposis patients had a deviant fatty acid profile with high levels of arachidonic acid and docosahexaenoic acid and low levels of linoleic acid and alpha - linolenic acid in serum phospholipids, which is in accordance with studies in patients with other types of cancers [5, 1013]. In a previous familial adenomatous polyposis study, comparable treatment effects of a cyclooxygenase-2 inhibitor were observed on the fatty acid composition in serum phospholipids and duodenal lesions, presumably and most importantly the nonbeneficial effects involving essential fatty acids . The results describe the content of fatty acids in feces, and relate this to the proportions of fatty acids and levels of cyclooxygenase mrna expression in duodenal biopsies, diet, and levels of serum lipoproteins . Because the effects of ileostomy construction may differ from that of ileal - pouch - anal anastomosis because of scarcity of the bacterial flora and different surface area of the terminal ileum, we did separate analyses for these two groups . Previous familial adenomatous polyposis studies have focused upon faecal sterols and bile acids [1517], but none has to the best of our knowledge analysed the content of fatty acids in feces, including very long chained fatty acids . Data from the present study are taken from a randomized double - blind placebo - controlled intervention study with a cyclooxygenase-2 inhibitor [12, 14, 18]. The main aim was to compare the effect of rofecoxib treatment on duodenal lesions (data in preparation). The present study comprises data from baseline for only the patients operated with ileal - pouch - anal anastomosis and ileostomy (77% of total study group). All of them had been colectomized, and duodenal lesions had been verified by endoscopy and histology . Rikshospitalet university hospital is a highly specialized university hospital with national responsibilities in the area of complicated treatments, such as follow - ups on the norwegian familial adenomatous polyposis patients . Inclusion criteria were verified familial adenomatous polyposis, colectomy, 1870 years of age, and documented duodenal lesions graded as spigelman i, ii, or iii and the largest adenoma10 mm . Exclusion criteria were indications for surgical treatment, suspected or documented intestinal obstruction or stenosis, patients unwilling or unable to adhere to protocol, known cardiac failure requiring medical treatment, and pregnancy . As for the adenomas sampled, they were all assessed histologically as mild / moderate dysplasia, all below 10 mm and all flat, located in the descending part of the duodenum . Samples of feces were transferred to tubes and mixed to make a homogeneous mass of which 100200 ul were used for lipid extraction by adding 50 ul butanol, 100 l h2o, 1 ml methanol, and 1 ml chloroform . After 10 min under n2, the mixture was filtered and the filter washed with 1 ml chloroform + 1 ml chloroform / methanol (2: 1) + 0.8 ml 0.73% nacl . After mixing for 15 s, the tubes were centrifuged for 30 min at 1000 g. the upper phase was discarded and the lower phase transferred to new tubes and evaporated under n2 at 65c using a heat block . Then 0.4 ml 0.5 n naoh in methanol was added and the solution kept for 7 min at 100c under nitrogen . After cooling to room temperature, 0.5 ml 12% bf3 was added and the solution kept for 10 min at 100c under nitrogen . After cooling to room temperature, 1 ml heptan was added under nitrogen, and mixing for 30 s, 1 ml of saturated nacl solution was added . After mixing, the solution was centrifuged for 10 min at 1000 g, and the upper phase transferred to new tubes . Finally, the heptan was evaporated and 300 l hexan added before continuing with the gas chromatography procedure referred to earlier . Dietary intake was assessed by a validated food frequency questionnaire, designed to cover as much of the total habitual diet as possible . Questions were related to habitual frequency of consumption and the amount of foods eaten during the last year . They were asked to identify their habitual choices of edible fats by pointing at specially prepared pictures, in order to increase the validity of the estimate of dietary fat . Each biopsy was put into a tube containing 600 l trizol and immediately mixed on a mixermill (retsch gmbh, germany). Gene expression analyses were carried out on a 7900 ht real - time pcr machine from applied biosystems . Based on the results from running a housekeeping gene test (taqman human endogenous control plate, applied biosystems) with rna from isolated human leucocytes (data not shown). The primers and probes were initially designed as three assays per gene and validated for efficiency and specificity . The primers and probes for the cox-1 assay were: forward primer: 5-cttccaggagctcgtagga-3, probe: 5-agaaggagatggcagcagagttggag-3, and reverse primer: 5-acgcatcaatgtctccatacaat-3. Cox-2 forward primer: 5-tggaacatggaattacccagt-3, probe: 5-tgttgaatcattcaccaggcaaattgct-3, and reverse primer: 5-tcctaccaccagcaaccct-3. Gus forward primer: 5-gaaaatatgtggttggagagctcatt-3, probe: 5-ccagcactctcgtcggtgactgttca-3, and reverse primer: 5-ccgagtgaagatccccttttta-3. Tbp forward primer: 5-ctggaaaagttgtattaacaggtgc-3, probe: 5-agcagaaatttatgaagcatttgaaaacatctaccctatt-3, and reverse primer: 5-cattacgtcgtcttcctgaatc-3. Taqman universal pcr master mix (applied biosystems) was added as reaction mix . The reaction conditions were initiated by a step of 2 min . At 50c and 10 min . At 95c, followed by 40 cycles of denaturation at 95c for 15 sec . And annealing at 60c for 1 min . A combination of cdna from several samples were made and diluted in order to make a dilution curve that was included on each plate . The average of the three values for each gene was divided by the average of the corresponding tbp values, generating a normalized value of the gene expression which is a unit less value used to compare the relative amount of mrna for each gene in the different samples . Patients were informed by a physician, and in addition thoroughly written information was given to all patients . The protocol was explained to the subjects that had to give their consent before inclusion . Was approved by the norwegian health authorities and the regional committee of medical ethics 20/06/2002 (reference: s-02127). Nonparametrical statistical methods were chosen, as some of the variables were skewed and the number of observations limited . Age and fecal content of palmitolic acid, linoleic acid, and arachidonic acid differed between the ileostomy and the ileal - pouch - anal anastomosis patients (table 1). The content of linoleic acid in feces correlated negatively to the proportion of eicosaenoic acid in duodenal biopsies (r = 0.6, p <.05). The content of arachidonic acid in feces was correlated negatively to the proportions of eicosapentaenoic acid (r = 0.7, p = .02) and docosahexaenoic acid (r = 0.7, p = .008) in duodenal biopsies . Total cholesterol were negatively correlated to the content of palmitic acid (r = 0.7, p = .03), palmitoleic acid (r = 0.7, p = .02), stearic acid (r = 0.7, p = .02), oleic acid (r = 0.7, p = .02), linoleic acid (r = 0.8, p = .002), and monounsaturated fatty acids (r = 0.7, p = .02) in feces . The levels of triglycerides was negatively correlated to the content of linoleic acid (r = 0.8, p = .003) and eicosaenoic acid (r = 0.7, p = .03) in feces . No significant correlations were found between the content of different fatty acids in feces and the fatty acid dietary intake (data not shown). With the exception of the palmitoleic acid / palmitic acid ratio and levels of cyclooxygenase-2 expression in duodenal biopsies (r = 0.8, p = .003), no relationships were found between fatty acids in feces and the levels of cyclooxygenase mrna expression (figure 1). Significant correlations were found between the content of oleic acid in feces and the proportion of myristic acid (r = 0.8, p <.001), oleic acid (r = 0.5, p <.05), and eicosaenoic acid (r = 0.5, p = .03) in duodenal biopsies . Moreover, the content of linoleic acid in feces correlated significantly to the proportion of myristic acid (r = 0.8, p <.001) in duodenal biopsies, whereas the content of alpha - linolenic acid in feces correlated inversely to eicosaenoic acid (r = 0.6, p <the content of palmitic acid in feces was positively correlated to levels of ldl - cholesterol (r = 0.05, p = .05). No significant correlations were found between the content of different fatty acids in feces and the levels of cyclooxygenase mrna expression in duodenal biopsies (data not shown). Several different fatty acids in feces correlated positively to dietary intake of fatty acids, most predominantly monounsaturated fatty acids (table 2). As expected, we found a difference between ileal - pouch - anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism . Possibly, our results relate to metabolic differences caused by the different intestinal reconstructions, but the data are not suitable to explain the findings . We found no significant relationships between the levels of cyclooxygenase mrna expression in duodenal biopsies and the content of fatty acids in feces, except for the estimates of the content of delta-9-desaturase (stearoyl - coa desaturase, scd) activity, namely the ratio of palmitolic acid / palmitic acid [19, 20] in the ileostomy group . Stearoyl - coa desaturase is the central lipogenic enzyme catalyzing in vivo reactions in the synthesis of monounsaturated fatty acids, particularly oleic acid and palmitoleic acid, which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides, wax esters, and cholesteryl esters . Both delta-9-desaurase and cyclooxygenase-2 are ppar alpha regulated, although this possible link is only a speculation . A study in healthy subjects showed that human fecal water contains components can affect both the cyclooxygenase-2 protein level and enzymatic activity . One recent study claims that it is possible to detect cyclooxygenase-2 mrna in feces of colorectal cancer patients irrespective of clinical stage . Notably, increased cyclooxygenase-2 expression in duodenal compared with colonic tissues in familial adenomatous polyposis was recently reported . The significant negative correlation between content of arachidonic acid in the feces and amounts of omega-3 fatty acids in duodenal biopsies which was found in the ileostomy group is interesting, since substitution of arachidonic acid with omega-3 fatty acids has been shown to lead to the production of less potent prostaglandins . A previous familial adenomatous polyposis study established a positive correlation between the reduction in tissue prostanoid levels and clinical response, as measured by the reduction in size and number of adenomas, when patients were treated with sulindac . However, this correlation only became consistently significant when prostanoids were assayed after tissue homogenates were first incubated with arachidonic acid . We may suspect transformation of linoleic acid to arachidonic acid in feces, but data are limited as with regard to feces from colectomized familial adenomatous polyposis subjects . However, the dietary assessment showed that the intake of such foods was normal among these familial adenomatous polyposis patients . Dietary controlled intervention studies on ileostomy subjects have shown that dietary manipulation with fat and fiber may modify cholesterol absorption and sterol excretion . In the ileostomy group, levels of total cholesterol were negatively correlated to the content of palmitic acid, palmitolic acid, stearic acid, oleic acid, linoleic acid, and monounsaturated fatty acids (<18 c atoms) in feces, which is interesting since the present levels of total cholesterol are lower than those among a healthy reference group . In the ileal - pouch - anal anastomosis group separately, the content of monounsaturated fatty acid in feces was positively correlated to dietary intake of monounsaturated fatty acids in particular . Moreover, positive correlations were found between the content of oleic acid in feces and the proportion of myristic acid, oleic acid, and eicosaenoic acid in duodenal biopsies . The precise role of monounsaturated fatty acids synthesis in cell proliferation and programmed cell death remains unknown . The strong correlation of high levels of monounsaturated fatty acids and neoplastic phenotype may suggest that the regulation of stearoyl - coa desaturase must play a role in cancer development . This was an observational study, and not a metabolic balance study that was designed to investigate the metabolism of fatty acids in familial adenomatous polyposis patients . Although some studies have found that the absorption rate of fatty acids are unaffected by age [26, 27], we cannot exclude the possibility that some of the observed differences between the ileal - pouch - anal anastomosis and ileostomy patients might be due to age, rather than type of surgery, since lipoprotein clearance has been shown to be altered by age, and that might affect the relationship between dietary fatty acids and the fatty acid profiles found in tissues . Moreover, no conclusions can be drawn from the present study because this is an observational study . . However, the study addresses the need to learn more about the dietary and lifestyle habits of this subset of at - risk patients . The present study includes a number of patients close to the maximum possible number of eligible norwegian familial adenomatous polyposis patients . We may, however, suspect that the low sample size and use of a food frequency questionnaire, may have weakened any associations between the content of fatty acids in feces and dietary intake . Notably, these data do not contradict that the nutrient intake among these patients should at least meet the recommendations for healthy subjects . Nevertheless, we suggest that these data are compelling enough to suggest that future familial adenomatous polyposis studies should investigate overall fatty acid metabolism, molecular mechanisms relevant to fatty acid metabolism, inflammation, and angiogenesis, in addition to nutrition requirements . In conclusion, we found a difference between ileal - pouch - anal anastomosis and ileostomy patients concerning associations between fecal fatty acid composition and other variables involved in lipoprotein metabolism . We may suggest that fatty acid content in feces is related to dietary intake, serum lipids, and fatty acid composition in duodenal biopsies, even in colectomized familial adenomatous polyposis patients . This observational study may represent hypothesis - generating suggestions for future familial adenomatous polyposis studies.
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