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parkinson's disease (pd) is a common, progressive neurodegenerative disorder, the incidence of which rises with age, with the life-time risk of developing the disease standing at 1.5% (lees et al., 2009). the two classical hallmarks are a progressive loss of dopaminergic neurons from the substantia nigra pars compacta (snpc), and the presence of aggregates of -synuclein, called lewy bodies, that are present in many regions of the central and peripheral nervous systems (lees et al., 2009). the progressive loss of dopaminergic neurons in the snpc leads to the motor features of the disorder which are characterised by bradykinesia, akinesia and a resting tremor. there are also many non-motor symptoms such as cognitive dysfunction, orthostatic hypotension and gastrointestinal disturbances (lees et al., 2009). though administration of the dopamine precursor drug, levodopa, is a successful symptomatic treatment, its effectiveness wears off over time and levodopa-induced dyskinesias develop with prolonged use. there is therefore a critical need to develop new drugs and drugs targets to protect dopaminergic neurons and their axons from degeneration in pd. it has become increasingly recognized that epigenetic disturbances are found in patients with pd, and that these may play a role in parkinsonian pathology. one of the most intensively studied modes of epigenetic regulation is dna methylation (figure 1). this refers to the covalent methylation of residues in cpg dinucleotides within the dna sequence, by enzymes called dna methyltranferases (dnmts), and most commonly results in gene repression by blocking access to dna by transcription factors (labbe et al., 2016). evidence implicating global changes in the methylome is supported by observations of genome-wide changes in dna methylation in brain and blood samples from pd patients (masliah et al., 2013). a distinctive pattern of methylation (both increased (hyper) and decreased (hypo)) was observed in both brain and peripheral blood leukocytes from these patients, involving many genes previously associated with pd (masliah et al., such findings highlight the fact that methylation patterns have the potential to be employed as epigenetic biomarkers for pd. in support of these findings, a recent study examining methylation at the cellular level using induced pluripotent stem cell (ipsc)-derived dopaminergic neurons from patients with genetic (monogenic lrrk2-associated pd) and sporadic forms of pd showed extensive differences in dna methylation, and subsequent gene expression, in ipsc-derived dopaminergic neurons from these individuals compared to controls (fernandez-santiago et al., intriguingly, these differences were not seen in parental skin cells, undifferentiated ipscs or ipscs not enriched in dopaminergic neurons (fernandez-santiago et al., 2015), suggesting that there may be something unique about the dopaminergic methylome in pd. for example, administration of methionine (which increases global levels of dna methylation) was found to decrease levodopa-induced dyskinesias, whereas administration of rg-108 (which reduces global levels of dna methylation) exacerbated levodopa-induced dyskinesias (figge et al., 2016). this highlights the importance of the dna methylome in pd-associated motor function, and raises the potential for pharmacological manipulation of the methylome as a viable therapeutic strategy for pd. histone acetylation occurs when enzymes called histone acetyltransferases acetylate lysine residues in the n-terminal of histone proteins. this neutralizes the positive charge on the histone tail, decreasing their interaction with negatively-charged dna. this results in loosening of the dna around the histones, rendering the dna more accessible to transcription factor binding and enhanced gene transcription. these densely-methylated islands can directly prevent transcription factor binding and therefore reduce gene expression. ac: acetyl group; cpg: cytosine-phosphoric acid-guanine motif; dnmt: dna methyltranferase; hat: histone acetyltransferase(s); hdac: histone deacetylase(s); mbp: myelin basic protein. a second intensively-studied mechanism of epigenetic regulation is post-translational modification of the n-terminal tails of histone proteins, around which dna is normally coiled (figure 1). although there are many types of post-translational histone modifications, including methylation, acetylation, phosphorylation, ubiquitination and sumoylation, histone acetylation at lysine residues is particularly important (labbe et al., 2016). histone acetylation is regulated by a balance between histone acetyltransferases (hats) and histone deacetylases (hdacs). hats add acetyl groups to histones, resulting in a less condensed chromatin structure, thereby facilitating transcriptional activation, whereas hdacs remove acetyl groups from histones, exerting the opposite effect (labbe et al., 2016). as dysregulation in histone acetylation has been implicated in the pathogenesis of pd, drugs which alter levels of histone acetylation may have therapeutic potential (harrison and dexter, 2013). a recent study has shown that the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (mpp) increases the levels of acetylated histones in experimental models of pd, and that there are increased levels of acetylated histones in the brains of pd patients (park et al., 2016). this suggests that changes in the levels of histone acetylation may either 1) play a causative role in the neurodegenerative process in pd, or 2) contribute to an endogenous compensatory response in an attempt to counteract the neurodegeneration. a number of studies have shown that pan- and class-specific hdac inhibitors have neuroprotective effects in cellular models (harrison and dexter, 2013). however, it is important to note that not all hdac inhibitors have been shown to be neuroprotective, with a recent study showing that a hdac1/2 inhibitor exacerbates mpp-induced toxicity in a sh-sy5y cell model of pd (park et al., 2016). conversely, another study showed that an alternative hdac1/2 inhibitor was neuroprotective against mpp-induced dopaminergic toxicity both in vitro and in vivo (choong et al., 2016). moreover, it has also been recently shown that other hdac inhibitors can protect other neuronal cell types affected by pd, as it was shown that hdac inhibition protected both dopaminergic and sympathetic neurons from mpp-induced cytotoxicity (collins et al., 2015). these contrasting findings are not easy to reconcile, and may reflect subtle compositional and functional differences in the hdac inhibitor molecules. furthermore, the precise phenotypic outcomes of hdac inhibitors may also be concentration dependent, as highlighted by our recent work on the p300/cbp hat described below. the potential of hdac inhibitors for clinical translation is highlighted by an on-going phase i clinical trial of the fda-approved drug glycerol phenylbutyrate (an hdac inhibitor), which is exploring the potential of this drug to increase the removal of -synuclein from the brain (nct02046434). in addition to hdac inhibition, an alternative approach to increase histone acetylation is the induction of hat activity using hat activators. however, there has been limited research into the potential of hat activators as potential drug therapies for pd. to begin to address this potential, we employed a selective and potent small molecular activator of p300/cbp known as ctpb (n-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide) (balasubramanyam et al., 2003). ctpb is a benzamide that activates p300/cbp hat activity and induces p300/cbp hat-dependent transcriptional activation, but has no effect on p300/cbp-associated factor (pcaf) or histone deacetylase activity (balasubramanyam et al., 2003). to investigate the neurotrophic potential of ctpb in pd, we examined the survival- and growth-promoting effects of ctpb in the sh-sy5y neuronal cell line, a widely used model of human dopaminergic and sympathetic neurons (hegarty et al., 2016). we found that ctpb-induced p300/cbp hat activation dose-dependently promoted the survival and neurite growth of sh-sy5y cells, and that ctpb significantly increased histone acetylation in these cells, most likely through induction of p300/cbp hat activity. moreover, this study found that ctpb was capable of protecting sh-sy5y cells from the cell death induced by the dopaminergic neurotoxin 6-hydroxydopamine (6-ohda) (hegarty et al., 2016). collectively these data suggest that increasing the levels of histone acetylation either through hdac inhibition or hat activation may be neuroprotective. however in contrast to this, another recent study found that garcinol-mediated inhibition of p300/cbp and pcaf hats protected sh-sy5y cells against mpp+-induced cell death (park et al., 2016). again, it is difficult to rationalize why both activation and inhibition of p300/cbp hats could be neuroprotective in these cellular models of pd, but it could reflect intrinsic differences between ctbp and garcinol, with garcinol (but not ctpb) targeting pcaf hat activity. moreover, unpublished observations from our laboratory have shown that garcinol induces cell death in sh-sy5y cells, and that it exacerbates the toxic effects of 6-ohda (figure 2). further research is required to determine the basis of these contrasting findings, but again such discrepancies may reflect differences between the concentrations of garcinol used in these studies. a key challenge for future research is how to optimize the delivery of hat-targetting molecules to the brain. interestingly, carbon nanosphere-conjugated ctpb has the ability to cross the blood-brain barrier, localize to specific nuclei in the brain and induce hyperacetlyation in vivo (selvi et al., 2008). taken together, these studies demonstrate that small molecule-mediated p300/cbp hat activation may be an avenue to explore for neurotrophic effects in pd. garcinol dose-dependently induces cell death in sh-sy5y cells, and significantly exacerbates the neurotoxic effects of 6-ohda. (a) standardised thiazolyl blue tetrazolium bromide (mtt) assay of sh-sy5y cells treated daily for 4div with either control or garcinol (0.5100 m), as indicated (**p<0.01,***p<0.001, vs. control; anova with post-hoc tukey's test. standardized mtt assay (b) and lactate dehydrogenase (ldh) assay (c) on sh-sy5y cells treated with 15 m 6-ohda in the presence or absence of 1 m garcinol for 24 hours (**p<0.01,***p<0.001, vs. control;+p<0.05,+++p<0.001, vs. 6-ohda alone; one-way anova with post-hoc tukey's test; n=6). in summary, small molecule epigenetic modulators, such as those targeting dnmts, hdacs and hats, hold much promise as pharmacological modifiers of the epigenetic status of the cns, especially considering their ability to cross the blood-brain barrier. these drugs therefore have the ability to act both centrally and peripherally in the nervous system, and have the potential to protect all neurons affected by pd. however, further research is required to elucidate the precise mechanisms leading to the chronic epigenetic dysregulation observed in neurodegenerative diseases, such as pd. in addition to this, much more work is needed in translational animal models of pd for rationalizing the use of small molecule epigenetic modulators as a potential neuroprotective therapy for this disorder, including exploring strategies to deliver these drugs to the brain. as shown for ctpb, carbon nanosphere-conjugation offers a method to deliver such molecules to the brain. although the beneficial effects of epigenetic modulators, such as hdac inhibitors, are yet to be reported in clinical trials for pd, there is much evidence to support the continued study of molecules that target the epigenome as novel neuroprotective therapies for pd.

parkinson's disease (pd) is a common, progressive neurodegenerative disease characterised by degeneration of nigrostriatal dopaminergic neurons, aggregation of -synuclein and motor symptoms. current dopamine-replacement strategies provide symptomatic relief, however their effectiveness wear off over time and their prolonged use leads to disabling side-effects in pd patients. there is therefore a critical need to develop new drugs and drug targets to protect dopaminergic neurons and their axons from degeneration in pd. over recent years, there has been robust evidence generated showing that epigenetic dysregulation occurs in pd patients, and that epigenetic modulation is a promising therapeutic approach for pd. this article first discusses the present evidence implicating global, and dopaminergic neuron-specific, alterations in the methylome in pd, and the therapeutic potential of pharmacologically targeting the methylome. it then focuses on another mechanism of epigenetic regulation, histone acetylation, and describes how the histone acetyltransferase (hat) and histone deacetylase (hdac) enzymes that mediate this process are attractive therapeutic targets for pd. it discusses the use of activators and/or inhibitors of hdacs and hats in models of pd, and how these approaches for the selective modulation of histone acetylation elicit neuroprotective effects. finally, it outlines the potential of employing small molecule epigenetic modulators as neuroprotective therapies for pd, and the future research that will be required to determine and realise this therapeutic potential.

PMC5204215

pubmed-1302

a cornerstone of ligand optimization in drug discovery research is the comparison of activity and property data for a collection of molecules which are related by similar structural cores.(1) in order to rationalize the relationship between structure and activity, it is often beneficial to organize the structures in the form of a hierarchical tree. structures with a common core fragment are arranged in branches, in which each parent fragment is a smaller, pared-down substructure that is common to each of the children. if the tree is well constructed, considerable insight can be gained regarding which core fragments and which peripheral substituents are responsible for the properties of interest, such as binding affinity against some number of protein targets, toxicity, and relevant physical properties. given a collection of arbitrary molecular structures, there is typically no single unambiguous way to arrange them in a tree such that each parent node is a substructure of all its children. if the molecules happened to be synthesized in a particular sequence, such as by introducing a variety of substituents in a stepwise fashion to some number of similar core fragments, it may be sensible to produce a fragmentation tree which is based on the synthetic procedures. or, if a set of common scaffolds is already known, it may be sensible to start with these scaffolds as the root fragments, and from these, construct the descendency hierarchy. if the collection of molecules has significant structural similarity, but no specific information about common substructures is available, then algorithms exist for estimating which parts of a structure are most scaffold-like. these can be used to generate a fragmentation sequence which can then be expressed as a tree.(2) while the fragmentation techniques have been well developed, one part of the process remains conspicuously undocumented. a fragmentation tree of molecules with similar common scaffolds is significantly less informative if the 2d coordinates of the molecular diagrams are not arranged such that common fragments are drawn with a common layout and uniform orientation. molecular structures are often sketched by chemists showing a standard orientation when structures are added to a database, but this is not always the case. for a small group of structures, or for a rare showcase example, it is typically not a herculean task to manually ensure that all of the fragments are properly drawn and aligned. for larger collections, or if charged with the task of regularly regenerating this data, one would at least begin to feel like sisyphus. unfortunately, even if a good 2d depiction algorithm is already available, the procedure of generating 2d layout coordinates that honor common fragment ancestry, with group layout decisions made in the context of the global optimum, is not trivial. in this work, a method will be described for obtaining 2d coordinates suitable for structure diagrams, which are chosen for both independent aesthetic appeal and for clearly showing the common substructure patterns by means of layout and orientation. also discussed are higher level presentation methods for making use of the structureactivity information, which is contained in the fragmentation hierarchy. in the following section, methods will be described for generating a fragment tree, mapping sibling fragments onto each other in an optimal way, then using this mapping to guide the 2d depiction process. the results section will describe methods of presenting this information in the context of examining structureactivity relationships. for each molecule in the data set, it is necessary to propose a scheme whereby the molecule is peeled away in some number of steps, such that the last remaining fragments are the most scaffold-like. the definition of scaffolds, and the fragmentation sequences which relate them to the whole molecules, may be adapted to suit the data. the method published by schuffenhauer and ertl et al. has been found to consistently produce agreeable results for pharmacologically relevant molecules.(3) these decomposition rules are such that the last remaining fragments are typically those that have been used as scaffolds and tend to be common substructures for a particular drug discovery campaign. one modification is applied, which is to collapse sequential breaking of fused aromatic ring systems into a single fragmentation step. this fragmentation method is used exclusively in this work, but the layout methods described are general and apply equally well to any scheme which is appropriate to the data. the fragments generated for each molecule need to be organized in the form of a tree. this can be accomplished by first producing canonical string representations for each fragment (e.g., smiles).(4) the canonical strings are arranged from smallest to largest for each molecule, and sorted by alphabetical order. in this way, for a given row and column, the fragments above or below are considered to be part of the same tree node if all the fragments up to and including that column are equal, which is illustrated in figure 1. (a) shows the fragmentation sequence for four molecules which share a common root, which are grouped according to common fragments, while (b) shows the corresponding tree representation, which subsumes adjacent, analogous fragments into individual nodes. in the following section, it will be necessary to know the correspondence between each fragment and its parent fragment. this can be accomplished by assigning an arbitrary label to each atom in the original molecule, and retaining these labels as the fragmentation proceeds, for example for any node position in the tree which has more than one child molecule, the objective is ultimately to obtain an ideal 2d depiction layout and orientation such that analogous atoms in related molecules are placed in the same location, in order to make visual perception of the common features as easy as possible. before this can be accomplished, it is first necessary to find an atom-to-atom mapping scheme that relates each of the equivalent substructure fragments to each other. when all of the fragments are nonsymmetrical and nondegenerate, this process is not especially complicated, since there will only be one correct mapping between any pair of fragments. when the potential mappings are degenerate, it is desirable to consider an ensemble of possible solutions, and find a combination that leads to the most compatible topological overlap of whole molecules. consider the example shown in figure 1, where four molecules share a common root fragment, the pyridine core. pyridine itself has permutational symmetry, which means that when two such fragments are mapped onto each other, it is necessary to choose a mapping from multiple pairwise combinations. an arbitrary numbering scheme is chosen for the pyridine root fragment of the first molecule. as shown for the first option, the descendent fragment also has a benzyl substituent at atom number 6, for the second option, the benzyl fragment is attached to atom number 2. for the pyridine root fragment, there are multiple ways to map the branches to each other, which are not of equal quality. for this simple example, it is clear that a suboptimal atom-to-atom mapping scheme would eventually lead to molecules being superposed on top of each other in a manner that misleads rather than elucidates the common structural features. unfortunately, the number of possible solutions grows combinatorially, which means that it is not viable to rely on an algorithm which examines every combination to find the best topological overlap. while adequate results could be achieved by using a greedy algorithm, that is, assign the possible graph automorphisms for each fragment sequentially and pick the case that best matches the previous assignments, the method described here uses the unary quadratic optimization (uqo) method,(5) which has previously been applied to the problem of common scaffold detection.(6) each fragment can have one possible assigned permutation, and the quality of the ensemble can be reasonably expressed as the sum of compatibility scores between each pair of assignments. the interaction energy between each pair of states not belonging to the same fragment is expressed in terms of the compatibility between the substituents which are not part of the shared substructure. figure 3 shows two molecules that share a commonly assigned benzene fragment, with the tentative mapping numbers shown. the similarity of the substitution patterns is obtained by a breadth first search of the molecular graph, starting from each direct substitution point. for the first shell, the molecule in 3a is described as [2-n, 6-c], for a singly bonded nitrogen atom adjacent to position 2 and a singly bonded carbon atom at position 6. the molecule in 3b is described as [2-o, 6-c]. for the second shell, the patterns are [2-c, 2-c, 6=o, 6-c] and [2-c, 6-o, 6-c], while the third shell is [2-c, 2-c] and [6-c, 6-c]. substitution points are assigned as being the topologically closest mapped atom, while the highest bond order is taken if there is a choice of pathways. similarity between two fragments is determined by examining one shell at a time, and matching up pairs of atoms that are assigned to the same mapped atom. the score is computed as follows: 1 point for each pair, 1 point for both being carbon or both being heteroatoms, and 1 point for having the same bond order. when there are multiple mapping permutations, the best matched set of pairs is selected. the score for each shell is divided by n, which is the shell number, which starts at 1 and is incremented with each step in the breadth first search. this method of comparing substituents is simple and fast, and emphasizes the value of sharing any substition at analogous positions, with those having similar topology and heterosubstitution being further favored. the assignment of a common numbering system is done recursively. for each unique root fragment, the uqo equation is formulated and solved, which yields a unique solution for each member of the group. once the atom equivalency is established for the group of substructures which share a tree node, these assignments are stored in the fragments themselves, and also percolated through down the tree. the descendent nodes therefore share the common mapping within their partial substructures. the process is continued for each child fragment. figure 4 illustrates the mapping procedure in a stepwise fashion. in figure 4a, the assignment has been completed for the pyridine root fragment, which has been mapped for all four molecules. as can be seen, the mapping is such that the substitution is always at the atom labeled as number 6, which is an optimal solution. in figure 4b and c, the process has been recursively applied to each of the distinct children of the root fragments, and a large proportion of the structures have been mapped. these two steps are carried out separately, so the numbering systems used for the benzyl and the methylfuran substituents are not related. figure 4d shows the completed mapping, where tree singletons are assigned arbitrary numbers for the remaining atoms. stepwise assignment of a common numbering system, for a fragmentation tree consisting of 4 molecules. the method for 2d layout of atom coordinates which we use in this work normally operates by searching for a globally optimum aesthetic ideal, which is the appropriate goal for depiction of individual molecules.(7) when examining a set of molecules, such as the fragmentation tree we have described thus far, it is important to be able to represent common fragments with a common depiction motif. while this is often the case for unconstrained depiction, it can not be relied upon, since arbitrary substituents may influence overall layout decisions in conflicting ways. consider the following simple fragment, which has a prominent degree of freedom in the form of a methylene linker: two different derivatives of this fragment clearly indicate that a free-for-all depiction is not the most suitable way to produce a common layout, since the common substructure fragments which correspond to the overall aesthetic ideal are no longer superimposable: to solve this problem, we make use of the tree structure and the mapping numbering scheme, to build up the fragment depictions sequentially. while the results must honor the requirement of common layout for common fragments, degrees of freedom within the constituent fragments are dealt with in a way that emphasizes overall layout aesthetics. as a first step, we submit each of the input molecules to the depiction layout algorithm, without constraints, and store the new coordinates within the molecule datastructure. while many of the structures will have their coordinates further modified by subsequent depiction layout procedures, the coordinates obtained by unconstrained layout are used during the following procedure for clustering purposes. the tree is analyzed recursively, starting with each root fragment. for each sibling node, the 2d coordinates of the fragments comprising the node are extracted, and clustered in a greedy fashion. starting with the first set of coordinates as the first cluster, each subsequent set of coordinates is added to the same cluster as any fragment for which its coordinates can be superposed with an rmsd of less than 0.1 or to a new cluster if none. the common atom mapping scheme derived in the previous step if there is more than one cluster, which can occur when fragments have layout degrees of freedom such as rotational symmetry or aliphatic chains, the cluster with the largest number of members is selected. the first set of coordinates in this cluster is used as the definitive reference. for fragments that were found in the same cluster, this transformation is applied simultaneously to all fragments belonging to a particular molecule, not just the fragment under consideration. fragments which are not in the reference cluster require more sophisticated treatment. in each case, the whole molecule for the fragment is resubmitted to the depiction layout algorithm, with a special constraint: the coordinates for the fragment are submitted as a preblock, which forces the layout algorithm to construct the rest of the 2d molecule around the predefined fragment, whose coordinates are taken from the reference fragment. this process has been previously described for drawing molecules with common scaffolds, and is used to obtain coordinates which are optimal for the collection of molecules, if not necessarily each individual molecule.(6) the coordinates from all of the fragments corresponding to the whole molecule are updated accordingly. the fragmentation patterns for 3 molecules are shown, each of which has been subjected to unconstrained depiction layout. consider the fragments in column (b), which form a single node in the resulting tree, and are directly descended from the common root fragment shown in (a). the fragments (1,b) and (2,b) can be superposed onto each other, and so they are part of the same cluster of size 2. fragment (3,b) can not be superposed, and so it is assigned to its own cluster of size 1. the coordinates of (1,b) are used as the reference point. for the fragment (2,b), the coordinates are superposed onto the reference. for fragment (3,b), the coordinates are obtained by redepiction, using the reference coordinates as the preblock. same fragmentation tree as shown in figure 5, after the colligative depiction layout procedure has been applied. at the completion of the previous step, each of the root branches has been arranged and oriented to show the common features within the branch. there is as yet no frame of reference for comparing the structures within different root branches, because there is no common ancestor and hence not even a partial common mapping system. there is, however, quite a high likelihood that the root branches are structurally related, and so it is useful to devise a scheme to orient them in a common way by means of translation/rotation/inversion. to do this, we take advantage of the fact that the constituent fragments of the root branches are depicted in a very constrained way. their 2d shape now encodes a significant amount of information, which is generally not the case for unconstrained depiction layout. therefore, it is quite viable to search for a single transformation for each whole branch which maximizes the overall shape overlap of the 2d structures. since the orientation is a relatively imprecise step, it is sufficient to use a greedy algorithm, rather than a more rigorous clustering method. one begins by first defining the reference set to be the root branch with the largest number of constituent molecules. the subject set is the root branch with the second highest molecule count. for the subject set, an orientation is selected such that its combined 2d shape is most similar to that of the reference set. the orientation is applied to the subject set, and then it is merged into the reference set. a new subject set is selected, and the algorithm proceeds until all of the root branches have been processed. to compare the shapes of two sets, each of the molecules in each set is first translated so that the center of the root fragment is at the origin. a grid is defined, which is large enough to capture the bounds of each set as it is rotated around the origin.(8) for each set, grid values are defined by addition of a gaussian function, for each atom in each molecule: where r is the distance from the grid point to the center of the corresponding atom and n is the number of molecules in the set. the two grids are now directly comparable, and their similarity can be computed: where i and j iterate over each of the grid points, r is the grid for the reference set and s is the grid for the subject set. lower values indicate more similar grids. to find the most similar orientation, the subject set is varied by rotating about the origin in increments of 5; inverting along one of the axes; and translating by [dx, dy] {2, 1, 0, 1, 2 }, which makes for a total of 3600 evaluations. figure 7 shows the resulting orientations and the corresponding grid pattern for each of 9 root fragments from a database of dhfr inhibitors, which contains 397 structures.(9) as can be seen, at the completion of the orientation step, the layout coordinates of molecules with different root fragments are approximately comparable, and the 2d coordinates now encode significant information about shared structural features. once the fragment tree has been generated and the appropriate steps have been taken to ensure that the layout and orientation of the molecules shows the common fragmentation patterns, there are a number of ways to display this information visually. the most direct method is to display the fragmentation tree as a hierarchical structure, with each root node being a distinct entity. to make examination of the tree practical from a user-interface perspective, it makes sense to allow nodes to be interactively opened or closed, so that parts of the tree can be displayed as necessary. because molecules are usually grouped together according to common fragmentation patterns this elicits information about the activities of the molecules which are represented by each node, which can be used to identify trends in activity data. figure 8 shows several subsets of the fragmentation hierarchy for a data set compiled to show activity against various species of dhfr, using the data set described in the previous section.(9) figure 8a shows a single root fragment, and several of the immediately descended fragments. each of the nodes provides an indication of the activity values against pneumocystis carinii dhfr of the constituent molecules by means of a colored pie chart display at the top left, which is divided up into a number of slices, one for each constituent compound. the color scheme used is green for highly active (< 0.1 m), red for inactive (> 10 m) and yellow-orange for moderately active (1 m). as can be seen from the color pattern of the root fragment itself, the constituent molecules exhibit a wide spectrum of activity. (b and c) selected child of the root fragment and a selection of their descendents. by traversing the hierarchy from the root downward, it is possible to reveal groups of structurally related molecules, which show distinctive inhibition patterns. the branch shown in figure 8b shows a high degree of consistency, which suggests that the inhibition capabilities can be ascribed to the core fragments. the branch shown in figure 8c, on the other hand, shows a mixture of active and inactive constituents, which suggests that the inhibition properties are determined to a large extent by the substituents, rather than just the scaffolds. while the hierarchy display is appropriate for detailed analysis of the fragments themselves, it is impractical to examine more than several dozen structures at once with a typical display device. an alternate approach is to display the fragmentation hierarchy of all of the input structures in the form of a dendrogram, where all of the root branches are considered to be descended from a point in the center of a circle, and the branches extend outward from the center.(10) each of the leaf nodes is located at a position about the rim. to ascribe significant structural information to this layout, the ordering of the descendency pattern is chosen to maximize the similarity of any two adjacent leaf nodes. in preparation, fingerprints are calculated for each leaf node structure, using the gpidaph3 scheme.(11) a reference node is selected, which is initially set to the leaf node with the highest average tanimoto similarity to all other nodes. the tree is then traversed recursively, according to the following procedure: (1) for each of the immediate child nodes, assemble a list of all of the leaf nodes of which it is an ancestor. (2) select next-in-sequence child node to be that whose descendent leaf nodes has the highest average tanimoto similarity to the reference node. (3) if the selected node is a leaf node, set the new reference node to this. once the ordering of the tree is determined, the actual layout of the dendrogram is straightforward. the final position of each node is obtained by evenly spacing about the rim of a disk, in order of tree traversal. the parent of all root nodes is considered to be the center, and the remaining nodes are arranged in between. if the background is chosen so that each pixel within the enclosing circle is assigned a color derived from the value of the average activity of the molecules belonging to the nearest fragment node, it is possible to gain considerable insight into structureactivity relationships. this is shown in figure 9, which plots activity data against p. carinii, using the red/yellow/green activity scheme described in the previous section. it is possible to quickly identify which regions of the fragmentation tree have consistently high or low activity, suggesting that the core fragment is responsible for these properties, as well as branches whose activity is quite mixed, suggesting that the substitution patterns are primarily responsible. figure 10 shows a cutout section of the dendrogram, where several of the structures of leaf nodes are indicated. the leaf node clustering is evident by comparison of structures (a) and (b), which are descended from a different root fragment, but are almost identical structures, differing only by an additional chlorine atom on (a) and an additional nitrogen into the fused heterocycle scaffold of (b). the structures (c) and (d) have the same core fragmentation sequence, and denote the two ends of a block of very similar compounds clustered together, sharing high activity against p. carinii dhfr. combined with an interactive user interface which allows facile examination of the structures, which are represented by the fragment nodes, a significant amount of structureactivity information can be inferred from the layout and color-coding. a collection of 467 structures with corresponding activity against the cox-2 enzyme(9) was submitted to the fragmentation and depiction sequence described within. each of the structures was embedded in 3d, and its atom ordering scrambled randomly to ensure that there was no residual bias from the input sketch. figure 11 shows a selection of substructure fragments, upon which most of the structures are based. in each case, a certain motif is observed: central 5-membered ring, usually a heterocycle, is decorated by two adjacent substituents, which are either 5- or 6-membered rings, a mixture of phenyl, pyridyl, or a 5-membered heterocycle. some of the fragments show an additional substituent on the opposite side of the central ring, which is a consequence of the fragmentation ordering. the figure shows each of the fragments with a common orientation, which is chemically intuitive. in all cases, the central heterocycle bears the two adjacent substituents at approximately the 10 oclock and 12 oclock positions. the common orientation is a consequence of the layout methods described in this work: each of the branches of the fragmentation tree are depicted in such a way as to trade off aesthetic depiction against showing common substitution patterns in analogous positions, taking degeneracy into account. because this data set has a high degree of structural similarity within the core fragments and the substitution patterns, the final spatial orientation using 2d structures reveals the alignment shown. figure 12 shows a portion of a radial dendrogram, composed from the hierarchy of cox-2 inhibitors, using the same algorithm as described in the previous section. one sub-branch is shown in its entirety, and the structures of six of these compounds are shown. all of these compounds are based on the same 1,2-diphenylimidazole core, with a trifluoromethyl substituent in the 4-position. the activity values vary from 68 to 0.03 m. compounds a and b, as well as all of the compounds that are arranged in between them in the linear cluster, feature a sulfonamide substituent in the same position on the phenyl ring at the 1-position, and all of them exhibit high activity, as can be seen by the bright green color coding. the remainder of the compounds in this sub-branch have a methylsulfonyl substituent in place of sulfonamide, and their activity is much more varied and is determined by the substitution pattern on the phenyl ring at the 2-position. for example, compounds c and d, which have a small substituent in the meta position, exhibit strong activity, while compounds e and f, which have larger substituents, exhibit weaker activity. each of the compounds shown has a meta-substituted phenyl ring at the 2-position, and in each case, one of the meta substituents is depicted so that it is oriented at approximately the 2 oclock position. while an unconstrained depiction algorithm will be likely to make these layout choices on the grounds of congestion, the symmetrical phenyl substituents are explicitly mapped to each other on an atom-by-atom basis, in order to maximize the topological similarity of the interstructure mapping. a single depiction is chosen for the precursor fragment, and applied to all descendents, which ensures that the most similar substituents are placed at a consistent position. all algorithms described in this work have been implemented in svl (scientific vector language), running in moe (molecular operating environment).(12) the total processing time for the data set of dhfr inhibitors described in part 1 (397 compounds) is approximately 16 s, measured on an intel 2.0 ghz 32-bit processor. the time taken for each step breaks down roughly as follows: initial depiction (4 s), fragment generation (2 s), deriving common mapping (4 s), colligative redepiction (2 s), and branch orientation (4 s). for the cox-2 inhibitors described in part 2 (467 compounds), the total time taken was approximately 50 s, of which 33 s were devoted to deriving the common mapping scheme. the rate limiting steps scale linearly in proportion to the number of input molecules, with the exception of the mapping step, in the presence of symmetry-containing fragments. an abundance of degenerate partial fragments causes the uqo portion of the common mapping algorithm to dedicate additional computational resources to finding an optimal mapping. this is the principal reason why the cox-2 data set takes longer to analyze than does the dhfr data set, since the dhfr inhibitors are largely based on heterocyclic ring blocks with low symmetry, while the core fragments of the cox-2 inhibitors contain an abundance of substituents, such as phenyl rings, that have rotational symmetry. a method has been demonstrated for treating scaffold-like fragmentation trees so that common ancestor fragments are depicted and oriented in a consistent way that makes common structural features readily evident to the observing chemist. the algorithm operates without supervision and produces aesthetically desirable results using a combination of new and preexisting techniques. with the molecule layout and orientation method established, software applications that present fragmentation trees become significantly more valuable to medicinal chemists. there is no longer a requirement to sketch the input molecules in any particular way, nor is it necessary to postprocess the structures to elicit insight into the structureactivity information that is encoded in such trees. the value of presenting structural data in this way has been demonstrated, by using high-level and medium-level hierarchical tree views, which combine constrained clustering with activity color-coding, and the ability to examine individual structures that have a common depiction and orientation. such views are effective ways to examine structureactivity data, and the algorithms that have been described essentially remove the manual effort required to produce them. use of such visualization and grouping tools can allow the user to make intuitive selections of compounds for further development. for example, if a set of compounds which has balanced activity and diverse structure is necessary for producing a qsar validation set, either the tree or dendrogram presentation methods could be used to identify such a subset, either by visual inspection or automated selection. alternatively, when considering hypothetical new compounds to add to the collection, it may be informative to depict them under the same conditions as for the existing compounds, and insert them into their appropriate locations within the hierarchy according to the ordering scheme used by the visualization method. examination of the patterns exhibited by neighboring structures may aid in qualitatively assessing the expected properties of the compound. future research will involve developing new ways to present information-rich, easily comprehensible graphics in a concise area, for example, single computer screen, projected slide or printed page, as well as developing interactive user interfaces for arranging and querying content data. the hierarchical atom-to-atom mapping system developed in this work has been used primarily for depiction purposes, but the assignment can also be used to differentiate scaffolds vs substituents at any level within the hierarchy, which opens up possibilities for fragment-based qsar and clustering studies.

drug discovery projects often involve organizing compounds in the form of a hierarchical tree, where each node is a substructure fragment shared by all of its descendent nodes. a method is described for producing 2d depiction layout coordinates for each of the nodes in such a tree, ensuring that common fragments within molecular structures are drawn in an identical way, and arranged with a consistent orientation. this is achieved by first deriving a common numbering scheme for common fragments, then using this scheme to redepict each of the molecules, one fragment at a time, so that common fragments have common depiction motifs. once complete, the distinct root branches can be overlaid onto each other, after which all of the fragments and whole molecules have a common layout and orientation. several methods are described for preparing visual representations of molecular structure hierarchies alongside activity information. combining high level tree display and structure depiction showing common features readily facilitates insight into structureactivity relationships.

PMC2810838

pubmed-1303

successful replacement of metal-ceramic dental restorations by all ceramic core-veneer restorations has long been one of the goals of scientific dental research. the durability and effective long term service of all ceramic systems especially in load bearing situations have yet to be proven.234 few long term clinical studies56789 are available to fully evaluate the performance and life span of newer all ceramic systems for both single and multiunit dental restorations. the interfacial integrity and the reliability of the bond between the high strength core and the weaker veneering material are directly related to the mode of failure of all ceramic restorations.101112131415 the physical, chemical, and mechanical properties of core and veneer materials can significantly affect the interfacial bonding.10121617 previous researches have mainly focused on the strength and failure modes of ceramic bilayers utilizing macro-mechanical testing.161819202122 nanoindentation is a research tool with numerous applications in the dental field for both natural dental hard tissues and restorative dental materials.23242526 quantitative data from the resin-dentin interface2327 suggests that nanoindentation is a potentially useful tool for the evaluation of the bond acquired. nanoindentation derived properties from the interfaces of restorative dental materials can also be utilized in numerical models to aid in the understanding of the load transfer within a restoration.252628 high strength yttria stabilized zirconia (ytzp) core systems are commercially available for manufacturing dental crowns and bridges after veneering with sintered or heat pressed dental glass-ceramics. core-veneer matching of modulus of elasticity (e) plays a major role in the success of dental ceramic bilayers, as abrupt e and toughness changes can cast adverse effects on the survival of such restorations.1029 depending on the veneering material and/ or on the ytzp core system used, an interlayer is usually used to modify the appearance of the core material and to theoretically improve the bonding between the core and the veneering material. it has, however, been suggested that such interlayers may not enhance the resultant bond.131430 wang et al.30 found that the interface toughness was reduced in ytzp specimens lined with an interlayer prior to veneering. nanoindentation across such multi-layered ceramics and e mapping at and across these interfaces could provide further important information on the efficacy of these systems. the aim of this study was therefore to test the modulus of elasticity (e) across the interfaces of veneered dental ytzp ceramics using nanoindentation. all-ceramic veneered specimens (ytzp core plus veneer) were produced as follows. rectangular specimens (10 mm length 5 mm width 1 mm depth) were sectioned from an yttria-stabilized (5 wt% y2o3) zirconia powder blank (zs blank, lot no. la100691445, kavo everest, biberach, germany) using a diamond disc (cutman 100, no 1575202, girbach dental, germany). the sections were sintered in a pre-programmed furnace (kavo everest therm, kavo, biberach, germany) overnight at 1500 according to manufacturer's instructions. one surface of the sintered ytzp sections was manually wet lapped in a special steel mould to 1000 grit silicon carbide paper mounted on a grinder-polisher (struers knuth-rotor 3, struers, ballerup, denmark), to achieve a depth of 0.5 mm. the specimen depth was selected to reflect a realistic dental restoration core thickness. at the end of the lapping procedure, the specimens were ultrasonically cleaned (transsonic 310 elma, singen kn, singen, germany) for 2 minutes and steam cleaned. a ceramic interlayer material (ips e.max zirliner powder, lot no. h29042, ivoclar-vivadent, schaan, liechtenstein) was mixed with ips e.max ceram all-round build up liquid (lot no. h32800, ivoclar-vivadent) and was used to coat the unlapped sintered ytzp surfaces of two ytzp specimens (type-1 specimens). the type-1 specimens were then vacuum fired according to manufacturer's instructions (table 1a) in a porcelain furnace (multimat mcii, dentsply, weybridge, uk). an even layer of wax (s-u modelling wax, schuler-dental gmbh&co, germany) was applied to the surface of the sintered ceramic interlayer (ips e.max zirliner) of the type-1 specimens and the as-sintered surface of the type-2 (no interlayer) ytzp specimen using a special steel mould to a total specimen thickness of 1.65 mm. a wax sprue (3 mm in diameter) was attached with one end to each specimen at a 45 angle and the other end to a large investment ring base fitted with a surrounding silicone cylinder. the specimens were then invested using manufacturer's recommendations for crowns: 200 g of investment material (ips press vest speed powder, ivoclar-vivadent) mixed using 32 ml of investment liquid (ips press vest speed liquid, ivoclar-vivadent) and 22 ml of distilled water in a vacuum mixer (multivac compact, degudent, hanau, germany) for 2.5 minutes. after setting for 40 minutes the refractory investment cylinder was transferred to a preheated burnout furnace (5365, kavo ewl, biberach, germany) at 850 and held for 1 hour. the preheated cylinder was removed from the furnace and two room temperature glass-ceramic ingots (ips e.max zirpress ingots, lot no. h21335, ivoclar-vivadent) and an alumina plunger (23) were inserted into the refractory muffle. the refractory was next transferred to a preheated pressing furnace (cerampress ney, jeneric pentron, wallington, cn, usa), and the glass-ceramic ingots were extruded into the refractory using the manufacturer's heat pressing settings (table 1b). following heat pressing, the refractory was removed from the pressing furnace and left to cool to room temperature. the pressed components were divested using 50 m glass beads at 2 bar in a grit-blasting machine (renfert basic quattro is, renfert gmbh, hilzingen germany). the specimens were subsequently immersed in hydrofluoric acid solution (ips e-max press invex liquid, ivoclar-vivadent) in an ultrasonic bath (transsonic 310 elma) for 5 minutes to remove any reaction layer formed during heat pressing. after water rinsing for 1 minute, the specimens were separated from the sprues via a diamond disc with the low speed (15000 rpm) under water lubrication. the glass-ceramic surface of all specimens was lapped to 1000 grit silicon carbide paper to a specimen thickness of 1.5 mm and ultrasonically cleaned for 2 minutes. the specimens were then subjected to two stain firing cycles according to manufacturer's instructions (table 1a). the veneer of the specimens was glazed using ips e.max ceram glaze paste (lot h24056, ivoclar-vivadent) and the firing parameters in table 1a. the layered specimens were mounted in epoxy resin separately (epofix, struers, copenhagen, denmark) as follows: a) 1 type-1 specimen (for preliminary nanoindentation test); b) a pair of 1 type-1 and 1 type-2 specimens (for final nanoindentation test). the specimens ' core-veneer interfaces were exposed and polished on wet 500, 800, 1000 and 2400 grit silicon carbide papers mounted on a grinder-polisher (struers knuth-rotor 3). final finish was achieved on a napless cloth impregnated with a 1 m fine particle diamond suspension (hyprez liquid diamond type k standard concentration, batch 2028, engis corporation, wheeling, il, usa) under lubrication (hyprez fluid, engis corporation, wheeling, il, usa) on a polishing unit (kent 3 automatic polishing unit, engis ltd., the specimens were washed with detergent under running water and ultrasonically cleaned in water for 5 minutes. preliminary testing took place across the interface of a type-1 specimen to determine the load for the final mappings. an ultra-micro indentation system (umis) csiro 2000 (asi, canberra, australia) was used. prior to indentation, alignment between the indenter tip and the on-stage microscope was performed using the 20 objective lens to accurately target the area of indentation. the mounted type-1 specimen was bonded to a metal base using a cyanoacrylate adhesive and transferred to the magnetic stage of the umis. the umis applied a force on the tested material through a diamond indenter and recorded its penetration depth. measurements were performed using a spherical diamond indenter with nominal tip radius of 5 m. the multiple point load-partial unload method was used313233 in which, for each indentation site, the maximum force was reached in 40 increments. an e value was measured from the load-partial unload stress-strain curve of each increment. forty e values were thus recorded for each indentation site. the partial unloading fraction in this study was set to 75% for every force increment. as explained in detail by field and swain,31 the indentation modulus (e *) is calculated using: (1)e *=0.75 pmax/he where pmax is the maximum load applied, is the radius of contact, and he is the elastic penetration depth. the material modulus (em) is then calculated using the relationship between indentation modulus (e *), indenter modulus (ei), and material modulus:32,33 (2)1/e *=(1 m)/em+(1 i)/ei which solved for em gives: (3)em=(1 vm)/[(1/e *) (1 vi)/ei] where m and em are the poisson's ratio and the e of the material, respectively, and i and ei are the poisson's ratio and the e of the indenter, respectively. a common value of 0.22 for the poisson's ratio was used for the tested materials.29 the ei and i of the diamond indenter used were taken as 1150 gpa and 0.07.34 an indentation layout file was created, the schematic of which can be seen in fig. 1a to accurately map the desired indentation sites across the interface. the data retrieved were corrected for variations in the diamond indenter tip radius using a tip function previously obtained by calibration using four reference materials (glassy carbon, sapphire, silicon, and fused silica).35 the corrected data were further processed to plot graphs of e versus depth below contact for every indentation site. the test was then repeated for the type-1 and type-2 pair of specimens using the optimal load (50 mn) determined previously. the data retrieved were again corrected for tip function and further processed to plot graphs of e versus distance from the core-veneer interface. a one-way anova test (sigmastat, ver. 2.03, spss inc., san jose, ca, usa) was used to statistically compare the near-interface changes in e. a compound optical microscope (olympus bx 60, olympus optical co., ltd., suitable specimens for all the materials used in the study were fabricated and prepared for scanning electron microscopy (sem). zirliner (ips e.max zirliner, ivoclar-vivadent) powder was used to fabricate a disc specimen by moistening 1 g of powder with ips e.max ceram alround build up liquid (ivoclar-vivadent) and compacting in a hollow cylinder mould with a plunger. the zirliner disc was then sintered by receiving one zirliner firing, followed by two stain and one glaze simulated firings (table 1a). a ytzp section (zs blank, kavo everest) was cut and sintered as previously described. a zirpress specimen (ips e.max zirpress, ivoclar-vivadent) was produced by investing a 3 mm diameter wax sprue, followed by the heat pressing and divesting route described previously. the zirpress specimen then received the same firing cycles as the test specimens (two stain and one glaze firings, table 1a). all 3 specimens were embedded in epoxy moulds and polished sequentially, as previously described, to a final finish of 1 m. the ytzp specimen was then separated from the epoxy in order to be thermally etched. this specimen was placed at room temperature in a furnace (severn furnace 1800, severn thermal solutions, bristol, uk) and heated to 1450 at a rate of 10/min, where it was held for 30 min. it was then quenched to room temperature in air.36 the ips e.max zirliner and the ips e.max zirpress specimens were etched with a 1% hydrofluoric acid solution for 60 seconds and subsequently washed under running water for 1 minutes and dried. all three specimens were gold coated in a sputter coater (balzers scdo5o bal-tec, liechtenstein) at 40 ma for 100 seconds. a field emission scanning electron microscope was used (jeol jsm-6300f, hertfordshire, uk) in the secondary electron imaging mode (10 kv) and images were acquired from all 3 specimens. x-ray powder diffraction (xrd) was carried out on all the specimens produced for the sem. the ips e.max zirliner and the ips e.max zirpress materials were ground into fine powders. the sintered ytzp specimen was examined as a solid specimen to avoid potential phase transformation from the grinding process. bragg-brentano flat plate geometry/and ni-filtered cu k radiation (1=1.54059 and 2=1.54444) was used in an x'pert-pro diffractometer (panalytical b.v., almelo, the netherlands). data were continuously collected with an x'celerator solid state multistrip detector from 5 to 120 (2 range), with a step time of 200 seconds and a step size of 0.0334. phase analysis was carried out with panalytical x'pert highscore plus software using the international centre for diffraction pdf-4 database. the graphs in fig. 2 are the selected e versus depth of penetration graphs from the preliminary testing of the ytzp side only of the type-1 specimen. each e data point has been derived from each of the 40 incremental load-partial unload steps. the representative ytzp surface indentation sites tested at 10, 30, and 50 mn loads are presented here, showing how increasing the indenter maximum load influences the scatter of the acquired e values as the indenter proceeds deeper into the tested material. the results of the final e tests across the interfaces of the type-1 and 2 specimens are presented in table 2. the mean and standard deviation (sd) of e values of the last 20 data points (partial unloadings) from each indentation site (3 indentation sites per distance) were used to plot graphs of e changes across the interface for both type-1 and 2 specimens (fig. e data were analyzed using a oneway analyses of variance (anova) (sigmastat, ver. 2.03, spss inc., san jose, ca, usa) and the differences found were significant (f test; p<.001). the values were grouped according to distance from the interface for both type-1 and type-2 specimens giving a total of 60 values per distance. the distance groups were compared using tukey's multiple comparison tests (p<.05). the highest mean e values were all the values on the ytzp, which were significantly different (p<.05) to the interface and veneer e values of the type-1 specimen (table 2). there was a statistical difference between e values of the interface and the veneering materials (p<.05). veneer e values at 120 m (type-1) were found to have no statistical difference (p>.05) from the 80 m veneer values but were statistically different with all other type-1 e values (p<.05) in table 2. the type-2 specimen (no ips e.max zirliner) produced the highest mean e at the 40, 80, and 120 m values on the ytzp, which were significantly different (p<.05) to all the other type-2 e values (table 2). there was no significant difference amongst the veneer e values (p>.05). the interface value was significantly different (p<.05) when compared to all other values of type-2 specimen. the 40 m e veneer values between type-1 and type-2 specimens were found to be significantly different (p<.05). the mean (sd in parenthesis) thickness of the ips e.max zirliner layer (interlayer) at the area of interest was measured as 48 (4.8) m. the ytzp specimen showed a high area fraction of fine zirconia grains (fig. the ips e.max zirpress specimen showed nanoscale fine whiskers densely dispersed in a glassy matrix (fig. 5d). the xrd pattern of the ytzp specimen revealed a bulk tetragonal zirconia phase (fig. 6). the xrd pattern of the ips e.max zirpress specimens showed a bulk amorphous phase and some very small peaks that could not be clearly identified (fig. the xrd pattern of the ips e.max zirliner showed a bulk amorphous phase (fig. specimens were produced so that they would reflect clinical practice in terms of the core-veneer thickness ratio, and the multiple firings were done to simulate the thermal history.10 the ytzp core material to be veneered was left as-sintered for the purposes of this study in order to replicate the surface finish determined by its machining and sintering.37 the ytzp surface to be veneered can be greatly affected by heat treatments and surface finishing38 due to transformation toughening,39 resulting in unconventional interfacial properties.38 while these transformed grains may be restored to their tetragonal configuration after veneering heat treatments, the transformation associated grain volume changes can produce a flawed interface.40 one important advantage of spherical indentation is the absence of significant geometrical variations at the indenter tip. this property particularly facilitates e determination as it renders the initial surface contact elastic.41 e can quite accurately be determined using the single point unload method.32 this method requires relatively low indentation loads and penetration depths and preferably large radii indenters (> 20 m) so that the contact is purely elastic.33 using a large radius indenter in this study would have necessitated wider indentation spacings,26 potentially resulting in incomplete mapping of the interfacial compositional gradient. on this basis, a smaller radius spherical indenter combined with the multiple point load-partial unload method was chosen. this method also provides reliable modulus results,3135 by taking advantage of the elastic nature of the unloadings. in order for this to apply, the unloading force fraction has to be set to a value equal to or less than 75%.35 furthermore, as multiple values are acquired from each indentation site, statistical analysis is greatly facilitated.35 according to fischer-cripps,32 spherical indenters are generally sensitive to surface roughness effects that increase the scatter in small penetration depths, and therefore higher loads are recommended. the representative selected e versus depth of penetration graphs from the ytzp side of the preliminary type-1 tested specimen (fig. the 20 last partial unloadings using the highest tested load (50 mn) were therefore considered ample for the determination of the e values for the final testing. accuracy of nanoindentation derived data is a matter that depends on various parameters and error sources, which are dealt extensively in the study of fischer-cripps.42 in accordance to this study, corrections for machine compliance, initial penetration depth, and tip function were applied in this study. potential material pile up or sink in should also be evaluated as this can affect measured modulus values.42 the e value reported for kavo everest zirconia (scientific documentation, kavo, germany) was 210 gpa, very similar to the values obtained in this study (table 2). similarly, guazzato et al.43 obtained values of 220 gpa for a dental ytzp core material (5 wt% y2o3) using the standard astm methodology (by impulse excitation of vibration). a similar range of values with the e values in table 2 has also been reported for other dental glass-ceramics from the studies by lawn et al.44 (between 68-69 gpa) and he and swain26 [65.52 (2.89) gpa], with the latter being measured using a pointed indenter (bercovich) on a umis 2000 nanoindenter. nanoindentation using spherical indentation and the multiple point load-partial unload method may therefore be a useful predictor of e in this category of materials. in terms of precision, while low standard deviations were obtained for all e values, relatively higher standard deviations were obtained for the ytzp e values than for those of the veneering materials (table 2). we believe that this potentially may be correlated with the relationship of the sizes of the tip,26 the hertzian stress field, and the zirconia grains, as well as the orientations of the latter, and thus should be investigated further. conversely, the low standard deviations obtained for both the interlayer (ips e.max zirliner) and the heat pressed glass-ceramic (ips e. max zirpress) materials in all indentation sites (table 2) suggest that the testing parameters used in this study may be useful for similar applications and materials. the optical microscopy post evaluation of the indentation areas of the type-1 specimen (fig. 4) showed that the 40 m indents were located within the interlayer (ips e.max zirliner). this finding, coupled with the presence of a statistically significant e difference (p<.05) between type-1 and 2 specimens at this distance, suggests that the interlayer has a lower e value than the heat pressed veneer. as this does not favor the e gradation across the interface and thus modulus matching,1029 the interlayer may potentially act as a weak link between the ytzp core and the heat pressed veneer. the nano-phase whiskers 5c) found in the heat pressed veneering glass-ceramic (ips e.max zirpress) were not categorically matched to a specific crystal type due to the very small, ambiguous x-ray diffraction peaks (fig 6). the crystallite size and volume fraction may therefore be the influencing factors in this outcome. the growth of fluorapatite crystals has been reported to follow an oswald ripening mechanism, where larger crystals grow at the expense of smaller crystals,4647 and this may be responsible for the larger particulates found within the microstructure (fig. 5c). variations in the morphology and volume fraction of these crystals can be expected to affect the optical, mechanical, and thermal expansion characteristics of these materials.46 in this study (type-1, table 2) the heat pressed glass-ceramic material produced consistent e values with low standard deviations illustrating material consistency and the reliability of this testing method. the interlayer (ips e.max zirliner) had similar nano-phase whiskers sparsely distributed in the glassy matrix with no large micro-particulates observed (fig. therefore further characterization would aid in identifying and quantifying the whisker like phases present in both ips e.max zirpress and ips e.max zirliner materials. links between the resultant difference in the e (table 2) and the structural characteristics of the two materials may then be possible. the value on the interface for both type-1 and 2 specimens shows a significantly lower value (p<.05) than the one obtained for zirconia from the 40 m distance and a significantly higher value (p<.05) than the one obtained for the veneer from the 40 m distance (table 2). optical microscopy post indentation evaluation revealed possible interfacial line fluctuation, as a result of the nature of the substrate and its ' processing (fig. the values obtained can be attributed theoretically to the fact that the hertzian stress field, underneath the indents close to the interface, is influenced by both the materials that constitute it, thus giving a mixed modulus reading,2333 and the glass penetration into the zirconia grain boundaries.1448 the relatively larger sd obtained for the interfacial e values in contrast to all the other e values in table 2 may support these assumptions. according to aboushelib et al.,14 an inter-diffusion zone exists in bilayer ceramics, where elements (silica, sodium, aluminum, and potassium) migrate into the zirconia substrate extending to a maximum depth of 10 m of decreasing concentration (fig. 4). it would therefore be interesting to see whether the potential glass penetration could have yielded this reduction in the e value compared to the bulk ytzp (table 2). transmission electron microscopy and energy dispersive x-ray spectroscopy (tem-edx) may be useful in the further characterization of these areas. the width of the hertzian stress field and the subsurface morphology of the interface are however unknown, and need further investigation. the effect of the interlayer (ips e.max zirliner) in the e reduction seems to stop somewhere between the 40 and 80 m distances in the veneer of the type-1 specimen (fig. 3a), since there is no statistically significant difference (p>.05) between the 80 and the 120 m veneer e values (table 2). for the type 2 specimen, (table 2) there is no statistical difference in e values (p>.05) between 40, 80, and 120 m distances on the veneer, indicating consistent e values in the absence of the interlayer (ips e.max zirliner). on the ytzp side of both type-1 and 2 specimens, 40, 80, and 120 m e values were also not statistically different (p>.05), and thus no influence of the interface was evident at these distances. therefore the interesting zone for further work would be between 40 m or less in the ytzp and a distance equal to a maximum of double the thickness of the interlayer (ips e.max zirliner) in the veneer, since the result in these distances can be technique dependant. in order to sample this zone, where the interlayer material seems to affect e matching, narrower horizontal indent spacings (diagonally arranged in respect to the interface line) could be used, limited by the potential interaction of the stress fields.26 the size of the hertzian stress field can theoretically be minimized using a smaller radius indenter, but this would be limited by the grain size of the zirconia polycrystals to avoid sampling single crystals. the effect of water storage49 and thermocycling on the degradation of the core and thus on the e matching should also be investigated as it would be more clinically relevant. a good control for these experiments would be a metal-ceramic interface of known successful bond strength. modulus mappings at this small scale could then be utilized for finite element analysis models2833 to simulate the behavior of dental ceramic multilayers giving insight to the potential failure modes in service. the coefficient of thermal expansion (cte) of ips e.max zirpress (9.75 0.25 10 k) (scientific documentation, ivoclar vivadent) may be matched with that of the kavo everest yttrium-stabilized core material (11.5 0.5 10 k) (scientific documentation, kavo). the interlayer (ips e.max zirliner) material presents an intermediate cte (9.8 0.25 10 k) and thus should act as a favorable parameter in the gradation of the interfacial compressive stresses preventing interfacial crack initiation and thus failure. it has however been suggested that the application of the interlayer between the ytzp core and the heat pressed veneer in crown/bridge ceramic systems should be avoided as it reduces the bond strength14 and interfacial toughness.30 our findings also associate a significantly (p<.05) lower e value with this interlayer, which does not favor the gradation of e across the interface, therefore potentially affecting its reliability due to poor modulus matching.1029 all these properties and characteristics, although potentially having a significant effect on the survival of dental ceramic multilayer crown/bridge structures, can not solely determine their long term in vivo behavior. many other interfacial characteristics, including wetting,48 microstructure, core-veneer thickness ratio, geometry, processing, thermal history, and porosity,101617 may synergistically affect it. consequently nanoindentation derived data (e, hardness, fracture toughness50) may render nanoindentation a powerful complementary tool for the evaluation of dental ceramic interfaces. the conclusion of this study associates a significantly (p<.05) lower e value with the presence of an interlayer between the ytzp core material and glass-ceramic veneering material in a multi-layered all ceramic dental restorative system. the present study has shown nanoindentation using spherical indentation and the multiple point load-partial unload method to be a reliable predictor of e and a useful evaluation tool for layered dental ceramic interfaces .

purposethe aim of this study was to test the modulus of elasticity (e) across the interfaces of yttria stabilized zirconia (ytzp)/veneer multilayers using nanoindentation. materials and methodsytzp core material (kavo-everest, germany) specimens were either coated with a liner (ips e.max zirliner, ivoclar-vivadent) (type-1) or left as-sintered (type-2) and subsequently veneered with a pressable glass-ceramic (ips e.max zirpress, ivoclar-vivadent). a 5 m (nominal tip diameter) spherical indenter was used with a umis csiro 2000 (asi, canberra, australia) nanoindenter system to test e across the exposed and polished interfaces of both specimen types. the multiple point load partial unload method was used for e determination. all materials used were characterized using scanning electron microscopy (sem) and x ray powder diffraction (xrd). e mappings of the areas tested were produced from the nanoindentation data. resultsa significantly (p<.05) lower e value between type-1 and type-2 specimens at a distance of 40 m in the veneer material was associated with the liner. xrd and sem characterization of the zirconia sample showed a fine grained bulk tetragonal phase. ips e-max zirpress and ips e-max zirliner materials were characterized as amorphous. conclusionthe liner between the ytzp core and the heat pressed veneer may act as a weak link in this dental multilayer due to its significantly (p<.05) lower e. the present study has shown nanoindentation using spherical indentation and the multiple point load-partial unload method to be reliable predictors of e and useful evaluation tools for layered dental ceramic interfaces.

PMC5179487

pubmed-1304

clinical diagnosis of occlusal caries is challenging due to the complex morphology of pits and fissures and presence of staining. early diagnosis of incipient and non-cavitated carious lesions is crucial for performing preventive treatments. visual examination is more efficient for diagnosis of cavitated rather than non-cavitated and incipient lesions. furthermore, this method is subjective and its reproducibility is low, since it involves the clinical experience and scientific knowledge of the clinician. on the other hand, radiographs have high specificity and low sensitivity for the diagnosis of non-cavitated lesions and underestimate the actual depth of caries. the ekstrand index scores resemble the clinical situations and are based on signs found on the enamel surface such as opacities, white spots, brown spots, presence of cavities or micro-cavities and a combination of these conditions. new devices using new technologies are applied for quantitative and qualitative diagnosis of incipient demineralized lesions. fluorescence-based methods are among these modalities, in which the sound and decayed surfaces produce different fluorescence when exposed to a certain light or wavelength. laser fluorescence diagnodent measures the emitted fluorescent infra-red light and shows the result in whole numbers between 099. the results of in vitro studies show the ability of diagnodent in exploring relatively advanced carious lesions. these findings are consistent with histological evidence, but they have no relationship with the depth of the lesions in dentin. the result of diagnodent is affected by different variables such as dehydration of the lesion, dental plaque and stains in the grooves of the occlusal surface. evaluated the efficacy of visual examination, radiographic assessment and diagnodent for the diagnosis of occlusal caries. kouchaji evaluated 156 permanent molars and revealed that the combined use of visual examination and diagnodent laser had the highest sensitivity and specificity. additionally, rando-meirelles and de sousa applied the fluorescence laser method, radiography and visual examination to assess the extension of occlusal non-cavitated lesions in permanent molars. the aim of this study was to compare the efficacy of bitewing radiography, fluorescence laser and visual examination in the diagnosis of incipient occlusal caries in permanent first molars. this descriptive cross-sectional diagnostic study was conducted on patients aged 7 to 13 years, referred to the department of pediatric dentistry (school of dentistry, shahid sadoughi university of medical sciences, yazd, iran). patients teeth were visually examined by a dentist and 31 patients who had signs of pit and fissure caries in at least one molar tooth were selected. the proposal of this study was approved by the ethics committee of the university (p.17.1.188736). clinical examination of each tooth was performed under adequate lighting after cleaning the tooth surfaces by two examiners calibrated in a pilot study. one-hundred-fifteen teeth of 31 patients that were intact or had incipient and inconspicuous caries with or without color change were selected. the teeth with occlusal restorations, enamel hypoplasia, hypomineralization or structural defects, cavitated lesions, fissure sealant, orthodontic bands or brackets or pulp necrosis were excluded. six of the 115 teeth were also excluded due to patient dropout; therefore, the three diagnostic methods were performed for 109 teeth. occlusal surfaces of the teeth were cleaned from plaque and debris using water spray and cotton pellets if necessary. occlusal caries were scored (v0v4) using ekstrand s visual scoring system (table 1). then, bitewing radiographs were taken (planmeca prostyle, helsinki, finland) at 70kv, 8 ma and 0.36s exposure settings using e speed 2235 mm dental films (kodak, rochester, usa) in xcp film holder and processed using a dental film processor (velopex-extra x, london, england). criteria used in visual examination, radiographic examination and fissure opening images were initially examined and scored (r0r2) by a pedodontist on a negatoscope and then confirmed by a radiologist (table 1). at this time, the teeth were examined by laser fluorescence pen (diagnodent, kavo, biberach, germany) followed by cleaning with a rubber cup and pumice powder, isolation with cotton rolls, and drying. the laser fluorescence pen was placed parallel to the long axis of the tooth on incipient or suspicious dental caries and moved around following calibration of the device on ceramic and intact enamel surfaces. cavity preparation, or fissurotomy (fissurotomy miro ntf, ss white, lakewood, nj, usa) was performed in cases with obvious or ambiguous dental caries, respectively to assess and score the actual depth of lesions (table 1). then, the cavities were restored by composite or amalgam, and the fissures were sealed. roc curve was used to define the best cut-off point for diagnodent and to compare it with the gold standard (figs. 1 and 2) and the area under the roc curve (az) was calculated; then the values of sensitivity, specificity, and accuracy of this method were determined by calculating the area under the roc curve. roc curve for dentin threshold roc curve for enamel threshold these values were also calculated for visual and radiographic examinations, and compared with the gold standard (fissure opening). the agreement coefficient for radiography and visual method was measured using a kappa test and inter-examiner reliability was evaluated using icc for diagnodent. altman s paired samples method for calculating a 95% confidence interval was used to compare the accuracy of the methods. this study was conducted on 109 teeth of 31 children aged 7 to 13 (mean 11.111.34) years. after assessment of information compared to the standard method, seven surfaces were sound, 64 showed enamel caries and 38 showed dentin caries. table 2 compares the visual method scores with those of the standard method (fissure opening) in enamel and dentin, separately. in table 3, comparison of ekstrand s scoring system and the standard fissure opening method by examiners 1 and 2 we did not find score 4 in any of the teeth. frequency of radiographic scores compared to the standard fissure opening scores according to examiners 1 and 2 all surfaces showing radiolucency in dentine (score 2 based on table 1) were found to have dentin caries (dentin threshold). the best cutoff point for diagnodent in this study included: sound surfaces: 07, enamel decay: 810, dentin decay: 11. the az value for laser method was 0.83 and 0.84 for the first and second examiners, respectively, which shows high efficacy of this method. the sensitivity of diagnodent method was higher than that of the other two methods, although its specificity in enamel was lower than the other methods (table 4). sensitivity, specificity and accuracy of caries detection by each examiner (1 and 2) for all methods (visual inspection, diagnodent and radiography) the numbers inside the parentheses are calculated with 95% confidence interval. totally, all three methods had high sensitivity. the highest accuracy for detection of enamel occlusal caries belonged to diagnodent, but with no significant difference with the other two methods (table 4). the occlusal surface of the tooth is susceptible to caries. however, making a reliable diagnosis is difficult in some cases. diagnodent for caries detection has been shown in some studies; however, controversial results have been reported regarding its efficacy of caries detection [8,1214]. in this study, surface of the teeth was dried with oil-free air spray before the examination in order to decrease the refractive index between crystals from 1.33 for demineralized wet to 1 for demineralized dried surfaces. probes and explorers were not used in this study, because they would not increase the diagnostic power. furthermore, use of explorer may damage the dental tissues and impair the remineralization potential. more recent studies have used ekstrand index for standardization of the stages to detect caries using the visual method. our results also showed high sensitivity and specificity using this index for detection of enamel occlusal caries. while in our study the sensitivity of radiographic diagnosis was similar to that of visual inspection for occlusal enamel caries. this difference may be related to the quality of films and the examiner s effects. in our study, each of the radiographic images was examined by an expert radiologist followed by a pedodontist. unlike the findings of this study, another study found that diagnodent was not suitable for diagnosing incipient enamel caries. regarding our results, the specificity of diagnodent in the enamel was lower than that of other methods (table 4). inter-examiner agreement for visual examination was moderately acceptable, which was consistent with the results of earlier studies; it may be attributed to the conduction of a pilot study before the main study. in our study, the sensitivity of radiographic examination was relatively high, which is inconsistent with the results of some studies. souza et al. concluded that bitewing radiography was not suitable for caries detection because of its low sensitivity. in a study conducted by neuhaus et al, radiographic diagnosis of occlusal caries of deciduous teeth was not as accurate as laser fluorescence pen, laser fluorescence and icads methods, bader and shugars in a review study reported that diagnodent had higher sensitivity, but lower specificity compared to visual examination, and ricketts found that diagnodent led to a greater possibility of false-positive diagnosis than the visual method. in our study, the diagnostic power of diagnodent was found to be higher in dentin compared to the enamel, which is similar to a study by hasani-tabatabaee et al. they found that as the depth of carious lesions increased, diagnodent showed higher values of sensitivity and specificity. diagnodent compared to visual and radiographic methods is more sensitive and accurate for the diagnosis of enamel caries (table 4). the specificity of radiography and visual methods for the diagnosis of enamel caries was greater than that of diagnodent. visual method has a lower cost, is faster and has acceptable sensitivity; therefore, it can still be used as an appropriate method for clinical caries detection. in complicated cases, they stated that although diagnodent was more accurate, the visual method was preferred because it had no significant difference with diagnodent and it required shorter time. one limitation in using laser fluorescence pen is receiving fluorescent waves from stains, highly mineralized structures or malformed teeth. this may cause bias, lead to increased sensitivity and cause false-positive results. exclusion of these teeth in some studies (unlike ours) such as the study by kouchaji, may lead to superior results with regard to the performance of diagnodent compared to studies that included stained teeth. several studies have pointed to the reliability of laser diagnodent for occlusal caries detection in permanent teeth. in our study, the icc was used for comparison of the values read by diagnodent and showed a strong agreement between observers. in contrast, rodrigus et al, in their in vitro study reported a low agreement in values read by diagnodent between observers. their study was a histological study and the samples were stored in thymol; this antimicrobial agent destroys porphyrins and consequently weakens the signals received by diagnodent. the surface below the roc curve in our study was 84% at the cut-off point of eight and 94% at the cut-off point of 11. this was similar to the rate (92% at the cut-off point of 12) obtained by huth et al. risk factors such as age, history of dental caries, diet, attitude, and topical fluoride application must be considered before treatment of suspicious cases. considering the recent emphasis on preventive interventions and avoidance of surgical techniques, applying new technologies to detect even the slightest demineralization (incipient caries) seems necessary. one of the limitations of the current study was lack of recording of the depth of lesions, because the calibration and reliability of the measurement of the depth of lesions are difficult to achieve. additionally, in diagnodent, the qualitative property, i.e., determining the presence or absence of dentinal caries is superior to the quantitative characteristic, i.e., recording the depth of the dentin lesion. in our study, simple radiographic films were used. future research is needed to compare the validity of digital radiographs (ccd/cmos) with that of conventional radiography regarding their better contrast and resolution for detection of occlusal caries. although the visual method was not as accurate as lf, considering the insignificant differences and affordability of visual method, the latter can be the first choice for detection of incipient caries. in suspicious cases, radiography and diagnodent

objectives: early diagnosis of incipient and non-cavitated carious lesions is crucial for performing preventive treatments. the aim of this study was to compare the efficacy of three diagnostic methods of bitewing radiography, diagnodent, and visual examination in diagnosing incipient occlusal caries of permanent first molars. materials and methods: in this diagnostic cross-sectional study, 109 permanent first molar teeth of 31 patients aged 713 years were examined visually, on bitewing radiographs, and using diagnodent. scoring of visual and radiographic examinations was based on ekstrand s classification. visual examination after pit and fissure opening served as the gold standard. receiver operating characteristic (roc) curve was used to define the best cutoff point for diagnodent compared with the gold standard. inter-examiner reproducibility of visual and radiographic examinations was assessed using kappa test and intraclass correlation coefficient (icc) was calculated for diagnodent values. results:the sensitivity of detecting caries that had extended into the enamel was 81.4%, 86.3%, and 81.4% for visual examination, diagnodent and radiography, respectively. moreover, the specificity was 100%, 71.4%, and 100% for visual observation, diagnodent and radiography, respectively in the enamel. the kappa index for inter-examiner reliability was 0.7 and 0.8 for visual examination and radiography, respectively. the icc was 0.98 for the values read by diagnodent. conclusion:visual examination is the first choice for diagnosis of incipient caries. in suspicious cases, radiography and laser diagnodent can be used as adjunct procedures.

PMC4749096

pubmed-1305

salivary gland neoplasms constitute an important area in the field of pathology of head and neck region. they are mainly classified as either hodgkin's or non-hodgkin's lymphoma (nhl). nhl comprises a heterogeneous group of lymphoid neoplasms with a spectrum of behaviour ranging from relatively indolent to highly aggressive and potentially fatal. the vast majority of nhl represent b cell and less commonly t cell lineage neoplasm. nhls of b cell origin are known to be relatively common in extra nodal sites and many are thought to originate from mucosa associated lymphoid tissue (malt). this is a low to intermediate grade lymphoma that show a follicular architecture and represents the neoplastic counterpart of germinal center b lymphocytes. follicular lymphoma is very uncommon under the age-group of 40 years and is extremely rare in children. the t (14;18) (q32;q21) chromosome translocation has been purported to be the cytogenetic hallmark of follicular lymphoma. the molecular consequence is deregulation of bcl2 expression leading to over expression of bcl2 proteins in neoplastic follicles. a 40-year-old female patient reported to our institute with a complaint of swelling in the left side of lower jaw since 2 months [figure 1]. post its onset, the swelling gradually increased in size over a period of 2 months. the patient is a known diabetic since 5 years and is under oral anti diabetic medication. concurrent with the history of swelling, the patient gives a history of weight loss in past 2 months. no history of cold, cough or fever. clinical picture showing the swelling in the left submandibular region examination revealed a swelling of 9 4 cm in size which extended anteriorly 1 cm in close proximity to chin on left side and posteriorly in line with the ear lobe [figure 2]. margins were well-defined and the swelling was firm, fixed to underlying tissues and non tender on palpation. no local rise of temperature was seen in the concerned area. left submandibular lymph node was 0.5 cm in size and mildly tender on palpation. few differential diagnoses such as sarcoidosis, sjogren's syndrome, lymphomas and mikulicz disease were considered. sjogren's syndrome was ruled out as there was no history of dryness. in both sarcoidosis and sjogren's syndrome mikulicz disease is an autoimmune disease and thus was ruled out. swelling seen 2 cm below the ear lobe the following investigations were done: fnac gave an impression of chronic non specific lymphadenitis. no evidence of cytological atypia seenultra sonography report showed a circumscribed mixed area of 3 1.5 cm in the anterior region of left submandibular gland. no evidence of cytological atypia seen ultra sonography report showed a circumscribed mixed area of 3 1.5 cm in the anterior region of left submandibular gland. the patient was further subjected to ct scan of head and neck and the impression was of submandibular salivary gland tumourmultiple submental and submandibular lymphadenopthy., a diagnosis of benign tumour of the left submandibular gland was given. during excision of the tumour, it was found that whole of the gland was involved. so, total submandibular gland excision was done and the specimen was sent for histopathological analysis. gross finding revealed a single bit of soft tissue measuring 4 6 2 cm, yellowish red in color, firm in consistency. [figure 3] cut sections revealed yellowish to whitish central core surrounded by whitish brown to brownish areas [figure 4]. specimen showing 2 lymph nodes attached to the soft tissue specimen grossed specimen showing yellowish to whitish central core surrounded by whitish brown to brownish areas histopathological examination revealed hematoxylin and eosin (h and e) stained sections revealed destruction of salivary gland architecture. photomicrograph showing neoplastic lymphocytes in a follicular pattern.(h&e stain, 100) photomicrograph showing neoplastic lymphocytes with hyperchromatic nuclei and scanty cytoplasm. (h&e stain, 200) based on the histopathological appearances, a provisional diagnosis of low grade lymphoma was made and immunohistochemical profiling was done to confirm the type of lymphoma. the tissue was stained for bcl2 an anti apoptotic marker, cd20 b lymphocyte marker, cd3 which is exclusively a t lymphocyte marker, cytokeratins and ema. immunohistochemistry revealed that the cells were strongly positive for bcl-2 [figure 7] and cd 20 [figure 8] which are both cytoplasmic markers and negative for cd3 [figure 9]. (ihc stain, 200) photomicrograph showing neoplastic cells expressing strong positivity for cd20. (ihc stain, 100) photomicrograph showing neoplastic cells with negative expression for cd3. (ihc stain, 100) based on the results of immunohistochemical profiling, a diagnosis of follicular b cell lymphoma was reached. patient was referred to higher center where full body mri did not reveal any foci of disease. nevertheless, considering the indolent course of this disease, she has been on regular follow up and has not reported with any clinically suspicious changes. lymphomas also involve in the descending order of frequency, the submandibular gland (30%), minor salivary glands and the sub lingual glands. the finding of a lymphoma in a major salivary gland could mean one of two things: either it is part of a disseminated process, or it is the first clinicopathologic evidence of lymphoma. in the latter instance, whether the disease originated in the glandular stroma itself or in a para glandular lymph node and then invaded the stroma, the lymphoma is defined as primary, as long as there is no detectable disease outside the salivary gland. hyman and wolf have given few criterions for diagnosis of primary lymphoma which are: involvement of gland should be the first clinical manifestation of the diseasehistologically, the disease should involve the gland parenchyma and not the adjacent node or soft tissue alonethere should be confirmation of malignant nature of lymphoid infiltrate. involvement of gland should be the first clinical manifestation of the disease histologically, the disease should involve the gland parenchyma and not the adjacent node or soft tissue alone there should be confirmation of malignant nature of lymphoid infiltrate. lichenfield et al., reserved the diagnosis of primary salivary gland lymphoma, when lymphoma has been ruled out elsewhere in the body. the diagnosis of lymphoma in case of a salivary gland swelling is rarely done preoperatively. even though the diagnosis of lymphomas, in the algorithm of diagnosis of salivary gland swelling is rare, nevertheless, it should be considered. follicular lymphoma, although unpredictable and often associated with a prolonged course shows indolent behavior. overtime, there is a tendency to transform to a higher grade of lymphoma, usually diffuse large cell lymphoma. although various treatment modalities exist, none are curative and very few have been shown to prolong survival.

lymphomas are neoplastic diseases of lymph nodes. lymphoma of the salivary gland is rare accounting for less than 5% of lymphomas overall. furthermore, lymphomas arising in the submandibular gland are reported to comprise 916% of all salivary gland lymphomas. among lymphomas originating from salivary glands, the ratio of follicular lymphoma is very low. they can also be seen in the lymph nodes of the salivary glands which is an uncommon presentation. here, we present a case follicular lymphoma which presented as a salivary gland tumour.

PMC4211230

pubmed-1306

animals: six crossbred dogs with naturally occurring tvt (three males and three females, 25 years of age and weighing 1129 kg) were obtained from a community that promotes the welfare of dogs and dogs family planning (dog chance, ratchaburi province, thailand). the dogs were individually housed in cages at the laboratory animal facility of the faculty of veterinary medicine at kasetsart university and were acclimatized to the environment for one week. the animals were allowed access to a standard diet and drinking water ad libitum throughout the experimental period. a cytological examination and a subsequent histological examination of the biopsies the processes used to monitor the health status of all of the dogs, which included a physical examination, a complete blood cell count (cbc) and a serum biochemistry profile related to renal and hepatic function, were performed weekly throughout the studies. the tumor sizes were also measured using a vernier caliper at the beginning of the experiment and weekly before the administration of vincristine. this study was performed according to the guidelines for animal experiments and approved by the animal ethics research committee of the faculty of veterinary medicine at kasetsart university. drug administration and sample collection: the dogs were treated with vincristine sulfate at a dosage of 0.7 mg/m of body surface area through intravenous administration. blood samples for single-dose pharmacokinetic analysis were collected from the cephalic vein through iv catheter using edta tubes at 0, 5, 10, 15, 30 and 45 min and 1, 2, 4, 6, 8, 12, 24 and 48 hr after drug administration. the samples were centrifuged, and the plasma was separated and stored at 20c until analysis. the treatments were performed weekly until the tumor had visibly disappeared up to a maximum of eight treatments. the response to treatment and toxicity chemicals and reagents: the reference standards of vincristine sulfate and vinblastine sulfate (internal standard, is) were purchased from sigma-aldrich (st. louis, mo, u.s.a.). vincristine sulfate (v.c.s.; 1 mg/ml) for treatment was purchased from boryung pharmaceutical co., ltd. sample preparation: the samples were prepared according to a published method with slight modifications. briefly, 500 l of plasma was diluted with 500 l of 4% phosphoric acid, and 20 l of 500 ng/ml is solution was then added to the mixture. after thorough mixing, the mixture was applied to an oasis hlb cartridge (water, milford, ma, u.s.a.). the cartridge was washed with 5% methanol in water and then with 1 ml of methanol: water (50:50, v/v). the eluate was evaporated and reconstituted in 100 l of a mixture of mobile phase a (5 mm ammonium acetate in water titrated to ph 3.5 with acetic acid and mobile phase b (acetonitrile) (50:50, v/v). the supernatant was transferred to micro insert vials, and 10 l was injected into the lc-ms/ms system for analysis. chromatographic system: chromatographic separation through an lc system was performed using a poroshell 120 ec-c18, 3.0 50 mm, 2.7-m column (agilent technologies, palo alto, ca, u.s.a.). the mobile phase a consisted of 5 mm ammonium acetate in water titrated to ph 3.5 with acetic acid, and mobile phase b was 100% acetonitrile. the gradient elution was performed as follows: 0 to 0.5 min, isocratic 90% mobile phase a; 0.5 to 3.0 min, 90% to 5% mobile phase a; 3.0 to 5.0 min, isocratic 5% mobile phase a. the flow rate was 0.5 ml/min. the mass spectrometric analysis was performed using a 6460 triple quadrupole mass spectrometer (agilent technologies) and programmed using the agilent masshunter b.06.00 software (agilent technologies). the ionization source parameters were optimized as follows: capillary voltage, 3500 v; gas temperature, 330c; gas flow rate, 8 l/min; nebulizer, 50 psi. the fragment energy was set to 230 v. the multiple reaction monitoring (mrm) transitions were selected to be m/z 825.4 precursor ion to m/z 765.4 production ion for vincristine and m/z 811.4 precursor ion to m/z 751.3 product ion for vinblastine. validation procedures: in this study, the lc-ms/ms method was developed and validated for the identification and quantification of vincristine in the plasma of dogs with tvt. the calibration standard concentrations were prepared by spiking the working standard solution into blank plasma to yield final concentrations of 0.5, 1, 2.5, 5, 10, 50, 100 and 150 ng/ml. five duplicates of the quality control (qc) sample at concentrations of 2, 50 and 100 ng/ml were prepared and used to determine the recoveries, intra-day and inter-day precision, and accuracy of the method. the procedure was repeated five times within the same day to gain an intra-day run precision and accuracy and five times of each concentration over five different days to obtain an inter-day run precision and accuracy. the precision of the assay was assessed by calculating the relative standard deviation (r.s.d.) for each concentration level. the accuracy was calculated by comparing the average measurements with the nominal values and is expressed as a percentage. a blank plasma sample, eight calibrated standard concentrations and five duplicates of the qc samples were included in each run. the quantification was obtained using the multiple reaction monitoring (mrm) transitions for vincristine (m/z 825.4 765.4) and is (m/z 811.4 751.3). the calibration curve exhibited linearity (r=0.990) over the concentration range of 0.5 to 150 ng/ml. the lower limit of quantification (lloq), defined as the lowest concentration yielding a signal-to-noise ratio (s/n) higher than 10, was 0.5 ng/ml. pharmacokinetic study: the determination of the pharmacokinetic parameters of vincristine was performed using a two-compartmental pharmacokinetic model with pk solution 2.0(summit research services, montrose, co, u.s.a.). the cp term was the extrapolated plasma concentration time curve at zero time, k12 and k21 were micro-rate constants, auc was the area under the curve, t1/2 was the distribution half-life, t1/2 was the elimination half-life, vdarea was the volume of distribution, cl was the plasma clearance, and mrt was the mean residence time. method validation: the results of intra-day and inter-day precision and accuracy of the assay are presented in table 1table 1.intra-day and inter-day accuracy and precision of vincristine sulfate in dog plasma (n=5)qc sample concentration (ng/ml)mean sd(ng/ml)accuracy(%)precision(% r.s.d)intra-day (5 days)2.02.05 0.201026.4650.049.9 0.8199.71.59100.099.9 0.9999.90.92inter-day (5 days)2.02.22 the intra-day precision and accuracy ranged from 0.92% to 6.46% and from 99.7% to 102%, respectively. the inter-day precision and accuracy ranged from 0.902% to 4.39% and from 98.9% to 108%, respectively. the extraction recoveries were 86.3 10.54, 77.3 9.88 and 88.9 5.49% for low, medium and high qc levels, respectively (table 2table 2.recovery of vincristine following extraction process (n=5)qc sample concentration(ng/ml)mean extraction recovery(%)coefficient of variation(%)2.0 86.3 10.5412.350.0 77.3 9.8811.8100.0 88.9 5.496.18). pharmacokinetic analysis: the level of vincristine sulfate in plasma was detectable up to 120 min after drug administration. 1.time profile of vincristine plasma concentration after its intravenous administration to dogs with tvt at a dose of 0.7 mg/m. shows the mean plasma concentration of vincristine as a function of time after a single intravenous administration at a dose of 0.7 mg/m to dogs with tvt. the pharmacokinetic parameters for vincristine in dogs with tvt are presented in table 3table 3.pharmacokinetic parameters (mean sd) of vincristine sulfate after its intravenous administration at a dose of 0.7 mg/mto dogs with tvt (n=6)pharmacokinetic parameters (units)average sdk12 (min)0.094 0.035k21 (min)0.029 0.012cp (ng/ml)119 18.0t (min)21.5 6.90t (min)47.6 14.2cl (l/min/kg)0.010 0.001vd(area) (l/kg)0.660 0.210mrt (min)55.9 19.3auc (ngmin/ml)2,349 317k12, k21= micro-rate constants; cp= plasma concentration at initial time; t1/2= distribution half-life; t1/2=elimination half-life; cl=clearance; vd(area)= volume of distribution; mrt=mean residence time; auc=area under the plasma concentration-time curve .. the plasma vincristine concentration immediately after administration (cp) was 119 18.0 ng/ml. the apparent volume of distribution (vdarea) and mean residence time (mrt) were 0.660 0.210 l/kg and 55.9 19.3 min, respectively. the distribution half-life (t1/2), elimination half-life (t1/2) and plasma clearance were 21.5 6.90 min, 47.6 14.2 min and 0.010 0.001 l/min/kg, respectively. time profile of vincristine plasma concentration after its intravenous administration to dogs with tvt at a dose of 0.7 mg/m. k12, k21= micro-rate constants; cp= plasma concentration at initial time; t1/2= distribution half-life; t1/2=elimination half-life; cl=clearance; vd(area)= volume of distribution; mrt=mean residence time; auc=area under the plasma concentration-time curve. clinical responses: tumor regression was determined at weekly intervals by a physical examination and histopathological analysis. complete remission or complete response means that all of the tumors completely disappeared, and partial remission means that the tumors regressed more than 50%, but less than 100%. in our study, three to eight administrations of vincristine at a dose of 0.7 mg/m were able to induce complete remission in five dogs (83.33%). in addition, these five dogs exhibited more than 50% remission after the first week of drug administration. in contrast, one dog exhibited only a partially response after the drug was administered eight times. the tumor mass (approximately 1 cm in diameter) in this dog continued to appear on the posterior portion of the gland penis. gastro-intestinal side effects, such as vomiting, anorexia and diarrhea, were observed after the first drug administration in three dogs. hematological side effects, such as leucopenia, neutropenia and thrombocytopenia, were observed in two dogs after the third drug administration, and anemia was observed in one dog after the fourth drug administration. a high incidence of tvt is associated with a large population of free-roaming dogs with uncontrolled sexual activity. chemotherapy, particularly vincristine sulfate, has been shown to be the most effective treatment and is thus frequently used. several analytical methods for the determination of vincristine sulfate in biological samples derived from both humans and animals have been reported. currently, lc-ms/ms is the method that provides the most sensitive and reliable qualitative and quantitative analyses. the present study was conducted to evaluate the pharmacokinetic parameters of vincristine sulfate in dogs with tvt and to determine their correlation with the clinical effects of the drug using the well-suited lc-ms/ms method to determine the concentration of vincristine sulfate in dog plasma. the results from our study show that three to eight weekly intravenous administrations of vincristine at a dose of 0.7 mg/m produced a good response. this response confirmed the efficacy of vincristine sulfate as a single agent for tvt treatment, which is in agreement with previous reports [1, 16, 20]. for the pharmacokinetic evaluation, the plasma concentration as a function of time was described by a two-compartment model. the t1/2 indicates the overall rate of elimination and allows the prediction of vincristine accumulation with a value for vincristine of 47.6 min after i.v. administration in dogs with tvt, whereas the value of t1/2 was 22.8 hr in healthy dogs. thus, about 99% of the vincristine from plasma was cleared within 5 hr after i.v. the distribution half-life (t1/2, 21.5 min) of vincristine obtained in dogs with tvt was longer than in healthy dogs (0.14 hr). this may indicate that vincristine is distributed to the peripheral tissues of dogs with tvt, and this finding is further supported by the high uptake of the drug by tubulin-rich tissues [3, 21]. the above-described characteristics of the penetration of vincristine sulfate exhibited a good correlation with the clinical tumor response, which showed that more than 50% remission was obtained after the first drug injection. the volume of distribution obtained in this study is in agreement with the results from previous investigations in mice, tasmanian devils, children humans and adult humans. in previous studies, the accumulative concentration of the drug was found to be high in many organs, including the pancreas, spleen, thyroid, adrenal, intestinal mucosa, lung, liver, kidney and bone marrow, and the spleen was found to be the major organ in which the drug was accumulated. to the best of our knowledge, this study provides the first demonstration of the clinical pharmacokinetics of vincristine sulfate in dogs with tvt using the lc-ms/ms method. in conclusion, although pharmacokinetic parameters of vincristine sulfate in dogs with tvt showed rapid elimination and therefore no accumulation, it resulted in good clinical effects by dosing once a week. in addition, it is well recognized that auc is one of the most important pk parameters that have a relationship with an efficacy of antitumor drugs. based on the value of auc of vincristine in this study, it might indicate the penetration of the drug into the tumor mass, which increases the efficacy and antitumor activity of the drug. however, further investigation is needed to clarify the pharmacodynamics of the drug in tvt .

this study was conducted to evaluate the pharmacokinetic characteristics of vincristine and their correlation with its clinical effects in dogs with transmissible venereal tumor (tvt). dogs with tvt were intravenously administered vincristine sulfate at a dose of 0.7 mg/m2 of body surface area. blood samples were collected starting from 5 min to 48 hr after drug administration. the plasma concentration of vincristine was determined using liquid chromatography-tandem mass spectrometry (lc-ms/ms). the pharmacokinetic parameters of vincristine were characterized using a two-compartmental pharmacokinetic model. the volume of distribution, distribution half-life, elimination half-life and plasma clearance were 0.660 0.210 l/kg, 21.5 6.90 min, 47.6 14.2 min and 0.010 0.001 l/min/kg, respectively. tumor regression was determined at weekly interval by a physical examination and histopathological analysis. in our study, three to eight administrations of vincristine at a dose of 0.7 mg/m2 were able to induce a complete tumor regression without any evidence of gross lesion of disease. therefore, this investigation provides the pharmacokinetic characteristics of vincristine in dogs with tvt, which may be used as an integration tool to gain a better understanding of the disposition properties of the drug and the correlation of these properties with the drug s clinical effects. in addition, we validated the lc-ms/ms method and found that it is suitable for the pharmacokinetic study of vincristine in dog plasma.

PMC4300367

pubmed-1307

an overarching societal goal is to understand animal and human intelligence and translate that knowledge into technology for prosthetic, assistive, and decision support applications. traditional research in this field considers the brain to be a specially adapted information-processing system, which can be modeled using mathematical optimization or production rule artificial intelligence systems. despite many decades of investment in such learning and classification systems one proposed remedy comes from research in social robotics, which attempts to augment the understanding of intelligent behavior by capturing the important dynamics of cognition using robotic interaction with humans (dautenhahn, 2007; scheutz et al., 2007). however, almost all social robotics systems to date continue to incorporate some mixture of existing machine learning and production rule cognitive systems. for this reason, investigators are now asking whether critical neural dynamics have indeed been left out of the traditional models. fortunately, the past two decades of neuroscience research has yielded an abundance of quantitative parameters that characterize the brain's interdependent electrophysiological (markram et al., 1997; schindler et al., 2006), genomic (toledo-rodriguez et al., 2004), proteomic (toledo-rodriguez et al., 2005), researchers now have access to over a hundred neuroscience databases (society for neuroscience, 2007), including automated warehousing collections such as the allen brain atlas (allen institute, 2007) and a new data-sharing website sponsored jointly by the u.s. national science foundation and national institutes for health, called the collaborative research in computational neuroscience (teeters et al., 2008). a previous limitation to the use of biologically realistic models has been the computational overhead. fortunately, the past decade has witnessed an order of magnitude increase in computation power of individual computers and cluster configurations with a tremendous drop in cost for system components. a few groups have already reported simulations on the order of one million simplified neural elements (izhikevich et al., 2004; ripplinger et al., growth in computational technology has also encouraged nontechnical persons to participate across the internet using avatars in complex virtual reality games and social networking communities (e.g., second life), which may include not only other human participants but also programmed robots. thus, taken together, advances in computer technology and interactive 3-d software have set the stage not only to facilitate supercomputer modeling of realistic brains, but also to promote acceptance by humans that virtual reality projections may be capable of meaningful cognitive interaction. developing tenable models to capture the essence of natural intelligence for real-time application requires that we discriminate features underlying information processing and intrinsic motivation from those reflecting biological constraints (such as maintaining structural integrity and transporting metabolic products). furthermore, despite the large and increasing number of physiological parameters provided by experimental inquiry, most of the data relates either to the very small scale of individual or small groups of neurons (e.g., intracellular, 2-photon, or unit recordings at discrete recording sites), or at the other extreme, the joint effect of thousands or millions of neurons over millimeter (optical imaging) or centimeter fields (fmri and pet). thus the architecture and response patterns at the middle scale, or mesocircuit, remain largely uncharacterized, requiring that the brain modeler proposes and systematically tests plausible connection patterns and learning dynamics. another challenge in designing neuromorphic systems is that they must in some way be driven intrinsically by a motivational influence such that the dynamics that subserve information processing are themselves affected by a drive to accomplish the tasks (with neural learning that reinforces successful behavioral adaptation) (oudeyer and kaplan, 2007; oudeyer et al., 2007; samejima and doya, 2007; schweighofer et al., 2007). the motivational system must capture the aboutness of its own relationship to other behaving entities (and vice versa) in its environment (i.e., intentionality). considered together, physiological responsiveness to intrinsic motivation with intentionality should reflect behaviors consistent with emotional drive rather than by rules or objectives specified under the traditional information-processing paradigm. this suggests that intelligence has evolved most directly as a way to better serve emotional drive (rather than in spite of it). we therefore hypothesize that the development of truly intelligent systems can not occur outside the real-time, emotional interaction of humans with an intentionality-capable neuromorphic system. this does not exclude the possibility that intelligent systems, once refined, could ultimately be cloned at a point in development where they are ready to learn advanced tasks. hence, to grow intelligent systems we must start with minimalist brain architectures that are capable of being driven by intrinsic motivation and intentionality in scenarios requiring intelligent behavior in a real-world context. one approach to growing human-like intelligence is to recapitulate the way in which children develop cognitive functions over the first several years of social experience. in testing our hypothesis, it would be relevant not only to grow such intelligent systems but also to comprehend, at each step, the differential changes in architecture giving rise to novel and intelligent cognition. to address these objectives, in a recent publication, goodman et al. (2007) proposed a hybridization of neuromorphic brain modeling validation using virtually projected robots interacting with human actors, which we call virtual neurorobotics (vnr). our proposed definition, open to future collaborative revision, is defined in table 1. the definition expands upon the definition of neurorobotics, which alone would imply a biologically representative robotic control system (criterion 3), and to test our hypothesis, we additionally require that the robotic system demonstrate sufficient physical and cognitive realism that the human accepts the robot as deserving of emotional reward (criteria 1 and 4) in a real-time interactive loop (criterion 2), with a cognitive architecture potentially extensible to larger cognitive scale (criterion 5). the robot is sufficiently embodied for the human to tentatively accept the robot as a social, emotional partner the human-robot interaction loop operates in real time, with no pre-specified parcellation into receptive and responsive time windows the cognitive control is a neuromorphic brain emulation incorporating realistic neuronal dynamics with time constants that reflect synaptic activation and learning, established membrane and circuitry properties the neuromorphic architecture can potentially provide circuitry underlying intrinsic motivation and intentionality, using emotional rather than rule-based learning&reinforcement the neuromorphic architecture is expandable to progressively larger scale and complexity to support brain model development and validation the components of the real-time loop are further delineated in table 2. here, we emphasize that the interaction between human and virtual robot be unscripted, of a spontaneous, action-reaction nature. that is, there is no segmentation of behavioral time into periods wherein the robot is receptive, waiting, analyzing, and/or taking action. the action-reaction requirement implies also that the system operate nearly in real time. in our experiences, human actors readily accept delays of up to 3 or 4 s without becoming frustrated about unrealistic robotic response and loosing cognitive and emotional linkage. of course, the range of behaviors is indirectly constrained by the sensory and motor capabilities of the robot, the types of behaviors exhibited by the human, and the context (e.g., background activity). temporal sequencing (timing)computation and communication provide nearly real-time robot response, to maintain cognitive and emotional linkagetime is not segmented a priori for robot or actor receptiveness or reaction computation and communication provide nearly real-time robot response, to maintain cognitive and emotional linkage time is not segmented a priori for robot or actor receptiveness or reaction environment (scene)realistic contents, including sights, sounds, and objectsmay be affected by the actions of robot or actormay include other robots or multiple actors realistic contents, including sights, sounds, and objects may be affected by the actions of robot or actor may include other robots or multiple actors live participant (actor)human, child or adult, depending on type of target intelligencewillingness to accept scene as realistic situationwillingness to assume robot has ability to perceive and respond meaningfullywillingness to attribute intentionality to robot human, child or adult, depending on type of target intelligence willingness to accept scene as realistic situation willingness to assume robot has ability to perceive and respond meaningfully willingness to attribute intentionality to robot neuromorphic system (robot)central nervous subsystem (brain) may include neocortex, hippocampus, basal ganglia, and/or other limbic regions relating to attention, reward, and fearcomputational architecture must incorporate biologically plausible learning algorithms and support for expansion to progressively larger scale and complexityrepertoire of sensors, expressions, and behaviors are commensurate with its physical and brain complexity central nervous subsystem (brain) may include neocortex, hippocampus, basal ganglia, and/or other limbic regions relating to attention, reward, and fear computational architecture must incorporate biologically plausible learning algorithms and support for expansion to progressively larger scale and complexity repertoire of sensors, expressions, and behaviors are commensurate with its physical and brain complexity to-date there is a paucity of literature meeting our criteria (see related work, below). thus we focus here on our own research (goodman et al., 2007) as an example of the vnr principles. in that work, we chose an instinctual friend vs. foe response wherein a resting dog responds to movement in its visual field with either (1) a cautious growl while remaining in a lying position, (2) threatening bark while sitting up, or (3) happy breathing and tail-wagging while fully standing. a human actor was told that he/she is visiting a home with a dog unknown to him/her. as shown in figure 1, a robotic dog was projected in pseudo-3d onto the forward screen, with external sensors that enable its simulated brain to see and respond to the actor's movements, in the context of a background scene projected onto the rear screen (for this demonstration, we used a static image of a suburban neighborhood). the robot's eyes (a tracking pan-tilt-zoom camera) and ears (monaural or spaced stereo microphones) capture the actor's movements and voice in the context of the background scene, which is projected independently (and may contain moving elements, including other animals or actors). the brainstem is a supercomputer running threads that synchronously (1) capture and preprocess video images, sound, and touch, (2) convert preprocessed sensory images into probabilities of spiking for each primary neocortical region, (3) upload spike probability vectors to the brain simulator, (4) then from the brain simulator accept motor neuron region output spike density vectors and trigger corresponding dominant motor sequences (e.g., for the virtual dog robot: sitting, lying, barking, walking) via the robotic simulator program (webots/urbi), which makes the corresponding changes in behavior of the projected robot (and incorporates internal sensation such as proprioception and balance). the brain simulator is a neuromorphic modeling program running on a supercomputer, executing a pre-specified spiking brain architecture, which can adapt as a result of learning (using reward stimuli offered by the actor's voice or stroking of the touch pad). based on successful performance, researchers iteratively plug in alternative or more complex brain architectures. a proposed enhancement would be to couple live in vivo or in vitro neural tissue (brain slice) to the brain simulation using multielectrode arrays and optical imaging, in order to continuously calibrate and constrain synthetic brain dynamics. the simple neuromorphic brain consisted of 64 single-compartment neurons divided into four columns representing pre-motor regions (precursors to coordinated behavioral sequences), each connected to one of the visual field preferences based on gabor filter configurations. according to the probability vector received from brainstem, ncs injected short (1 ms) step current (3 na) pulses sufficient to reach the threshold of 50 mv and the actor in this scenario was told in advance that moving vertically-oriented objects (including body parts) will pose a threat to the robot, whereas moving horizontally-oriented objects will be perceived as friendly gestures; the actor was free to choose any sequence of movements in response to the perceived intent of the robot. robot behavioral sequences are triggered when the neuromorphic brain output to brainstem has 50 ms of consistent spiking in one pre-motor region compared with another. periods without domination of one pre-motor region over another trigger the robot to lie down and growl. in cell rasters, each row represents the timing of action potentials (spikes) of a single neuron; darker gray markers indicate clustered bursts of spikes. figure 2 shows pre-motor action potential spike rasters from a typical 10-s vnr interaction with a human actor. spike rasters from a 10-s behavior scenario indicating timing of actor (upper row) and robot (lower row) events. social embeddedness is a key characteristic of the proposed vnr approach, with an emphasis similar to that received initially in the stepwise, ontological development of robotic cognition (breazeal and scassellati, 2000; brooks et al., 1998) and more recently in epigenetic robotics research focused on the interaction between cognitive and perceptual brain systems (lungarella and berthouze, 2002; schlesinger, 2003). in order to map behavior to robotic cognition, almost all of these models rely on combinations of psychological production rules, fitness functions, and machine learning algorithms. notably, this includes models aimed at capturing neuronal epiphenomena such as mirror neuronal activity (triesch et al., 2007). first, we focus on understanding brain physiology at the mesocircuit level, relying on social-emotional robotics to reduce the multitude of potential architectures that could bridge the measurements at the cellular level (e.g., patch clamp and unit recordings) with those at the scales of millions of cells (e.g., optical and fmr imaging). second, because the stipulation of neuromorphic architecture excludes the use of production rules or hierarchical algorithms as psychological models, any assumptions on motivation, intentionality and behavioral triggering must emerge from the tissue models themselves, and learning from behavioral reinforcement must manifest as synaptic change. third, since realistic social interaction requires temporal coherence between the simulated robotic brain and that of the human actor, the simulation must incorporate the actual distribution of physiological time constants that characterize membranes, channels, and synapses. due to the distinguishing characteristics of the proposed vnr approach some groups have reported success in navigational tasks using neuromorphic architectures (banquet et al. 2005; cuperlier et al., 2005; krichmar et al., 2005; notable endeavors that share similarities with our work include identification of challenges and opportunities in robot-mediated neurorehabilitation (harwin et al., 2006), development of prototypes that combine robotics and virtual reality to assist in the rehabilitation of brain-injured patients and support motor control research (patton et al., 2006), and generation of artificial brains for virtual robots using a new paradigm based on the epigenetic approach (pasquier, 2004, 2005). the rationale for the virtual paradigm in vnr is rooted fundamentally on engineering and human-computer interface considerations, and is similar to that put forward by krichmar and edelman (2005) for robotic instantiation of brain-based devices. certainly, a closed-loop system could incorporate either real or virtual robots. in our vnr framework, however, we emphasize virtuality for the following reasons: (1) the human actor must find the robotic behavior believable; it is our impression that refined neurorobotic avatars are more readily accepted (perhaps due to the popularity of online virtual reality networking) than clumsy, unreliable physical robotic prototypes; (2) as investigators design and grow more complex neuromorphic brains, robotic behaviors will require additional sensory, motoric, and emotional sophistication, which in turn may entail major changes in the robot's body parts and dimensions, and degrees of freedom of joints and face all of which can be accelerated using software (often in just hours) without the delays and costs of added hardware and its engineering; and, (3) at stages of neurorobotic development at which it would be important to demonstrate the functionality of a physical robot, the software api can be compiled and transferred to a prototype of the hardware robotic system (provided that vnr simulator used a realistic control api). the use of human actors in the vnr approach might be seen as an obstacle in terms of time, resources, and variability. however, there is no other gold standard for realistic, spontaneous, emotionally intelligent interaction. moreover, it is human-level cognition that we explore and seek to elucidate in our modeling and applications. in addition, the parameters for neuronal membranes, channels, and synapses are given as time constants on the order of milliseconds to seconds, as co-optimized by evolution. this means that, for example, a system that emulates connected neurons but operates at the temporal scale of microseconds can not interact with the slower responses of humans. therefore, both the joint distribution of known biological time constants and the need for emotionally intelligent responses require the use of a closed-loop interaction of the brain prototype with an actor. as an alternative, one might consider using animals in place of humans; however, animals rely on many subtle biological sensory cues such as smell, so will readily accept neither embodied nor virtual robots as socially interactive partners. for example, within the webots/urbi environment we are currently developing a social-emotional humanoid robot with functional capabilities motivated by the mds (mobile, dexterous, social) robot under development by the personal robotics group of the mit media lab (http://robotic.media.mit.edu). our robot will incorporate language understanding and production using corresponding neocortical models based on praise and curiosity. we also plan to calibrate and constrain synthetic brain dynamics by coupling live in vitro (acute slice or sustained culture) or in vivo neural recordings to the brain simulation using multi-electrode arrays and optical stimulation and imaging. first, neuroscience research is expected to directly benefit from vnr in terms of development and validation of new, expandable brain models and architectures as well as study and exploration of various brain disorders and injuries, including strokes and genetic disorders. second, faster progress in a variety of medical applications areas will likely be enabled by vnr-based research, primary in terms of advancements in neuroprosthetics and new solutions for brain-related assistive technologies. third, a diversity of other application areas traditionally propelled by developments in artificial intelligence could take advantage of vnr method and tools. these include, but are not limited to, decision-making support driven by human-like behavior and motivation, enhanced robotics-centered navigation and security, and better understanding in the fields of neural development, neurophysiology, and neuropathology. the authors declare that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

despite decades of societal investment in artificial learning systems, truly intelligent systems have yet to be realized. these traditional models are based on input-output pattern optimization and/or cognitive production rule modeling. one response has been social robotics, using the interaction of human and robot to capture important cognitive dynamics such as cooperation and emotion; to date, these systems still incorporate traditional learning algorithms. more recently, investigators are focusing on the core assumptions of the brain algorithm itself trying to replicate uniquely neuromorphic dynamics such as action potential spiking and synaptic learning. only now are large-scale neuromorphic models becoming feasible, due to the availability of powerful supercomputers and an expanding supply of parameters derived from research into the brain's interdependent electrophysiological, metabolomic and genomic networks. personal computer technology has also led to the acceptance of computer-generated humanoid images, or avatars, to represent intelligent actors in virtual realities. in a recent paper, we proposed a method of virtual neurorobotics (vnr) in which the approaches above (social-emotional robotics, neuromorphic brain architectures, and virtual reality projection) are hybridized to rapidly forward-engineer and develop increasingly complex, intrinsically intelligent systems. in this paper, we synthesize our research and related work in the field and provide a framework for vnr, with wider implications for research and practical applications.

PMC2570068

pubmed-1308

computers now play an increasingly important role in our daily lives, and their use is associated with lower back pain. the sacroiliac joint (sij) is a widely described source of low back pain. therapeutic approaches to relieve this pain include the application of a pelvic compression belt (pcb)1. researchers have suggested that functional exercises conducted using a pcb have a beneficial effect associated with muscle strengthening2. pcb are effective for stabilizing pelvic articulation and enable exercises that address coordination and stabilization3. furthermore, evidence shows that application of a pcb can relieve pain and facilitate neuromuscular performance during rehabilitation exercises in patients with lumbopelvic problems4. in particular, pcbs effectively alter the activation patterns of hip extensor muscles in females with chronic low back pain during prone hip extension5. moreover, pcbs offer a conservative measure for the treatment of sacroiliac joint pain and are cheap and considered to be without any adverse side effects6. in addition, it has been shown that use of a pcb significantly improves health-related quality of life and possibly decreases sacroiliac joint-related pain6. hammer et al.7 suggested that pcb application is accompanied by altered rectus femoris activity when walking. furthermore, pcb improve postural steadiness7. however, it is unknown whether pcb alter trunk and lower-extremity muscle activities in healthy adults. therefore, the purpose of this study was to investigate the effects of a pcb on these muscle activities in normal healthy adults. all were given comprehensive information on the study, and all provided written informed consent according to the ethical standards of the declaration of helsinki prior to participation and agreed to participate in the study (table 1table 1.pre- and post-intervention electromyography values of subjects wearing or not wearing a pelvic compression belt (units:% mvic)without pelvic compression beltwith pelvic compression beltchange valueerector spinae*101.7 8.8 80.9 5.6 20.8 10.9oblique internus abdominis*107.8 20.4 87.0 12.420.8 11.7rectus femoris*93.7 15.977.8 8.0 15.8 17.7long head of the biceps femoris*94.7 17.383.6 11.0 11.1 13.2mean sd. their average ages, heights, and weights were 21.60 1.08 years, 171.20 6.23 cm, and 71.23 8.64 kg, respectively. significant intergroup difference between the gains achieved (p<0.05) the pelvic compression belt (the com-pressor, optp, minneapolis, mn, usa) used was an adjustable body belt with four elastic compression bands that provide stabilizing pressure and was designed to allow the amount of compression to be adjusted at targeted compression sites. the pcb was placed below the anterior superior iliac spine (asis)8, and stabilizing pressure was applied using the elastic compression bands after confirming the location of the compression site. four surface electromyography signals were processed through the mp150 system when subjects were in the bridge position and transformed into digital signals, which were filtered and processed using acqknowledge software ver. 3.7.3 (biopac systems inc., goleta, ca, usa) on a personal computer. a 1,000 hz sampling rate was used for electromyography signals, and their amplified waveform was filtered using a 60500 hz band-pass filter and a 60 hz of notch filter. for quantifying collected signals, we used root mean square values9. in addition, the signals collected from each muscle were normalized versus the maximal voluntary isometric contraction (% mvic). to measure muscle activation at maximal voluntary isometric contraction, after collection of the data for 5 seconds at maximal voluntary isometric contraction for each muscle, the average electromyographic signals as a percentage of mvic for 3 of the 5 seconds, excluding the data for 1 second each from the beginning and end, were used. muscle activation was measured using electromyogram electrodes fixed to areas of muscle fibers and by pressing on muscle parts and following the direction of muscle texture to find the appropriate positions. the locations of the surface electrodes were as follows: (1) for the elector spinae, 2 cm lateral to the spinous process at the l45 interspace10; (2) for the oblique internus abdominis (oi), in the center of the triangle formed by a horizontal line between the anterior superior iliac spine of the innominate and the umbilicus, midline, and the inguinal ligament11; (3) for the rectus femoris, the midpoint between the upper margin of the patella and asis12; and (4) for the long head of the biceps femoris, the midpoint between the gluteal fold and the knee joint13. intragroup comparisons of variables before and after the intervention were performed using the paired samples t-test. ibm spss statistics ver. 20.0 (ibm corp, armonk, ny, usa) the subjects showed a significant decrease in muscle activation in the erector spinae, oblique internus abdominis, rectus femoris, and biceps femoris while wearing the pcb (p<0.05) (table 1). this study was undertaken to determine how a pcb affects erector spinae (es), oblique internus abdominis (oi), rectus femoris (rf), and long head of the biceps femoris (bf) muscle activation in healthy adults. we observed reduced es, oi, rf, and bf activity with a pcb compared with without a pcb in the bridge position. several possible explanations exist for less muscle activity in abdominal muscles than in core muscles while wearing the pcb. stabilizing the core is a dynamic process of maintaining balance. kaushik et al.14 suggested that the transverse abdominis is the first muscle activated during lower extremity movements, indicating that it is a primary muscle linked to core stability during lower limb movements. in the present study, the decreased oi activity indicated that subjects required less effort to maintain stability when wearing the pcb. nevertheless, kim et al.15 suggested that decreasing the activation of abdominal muscles on an unstable surface using an external support, such as a pcb, is suitable for improving abdominal muscle control and lumbopelvic stability. in a recent study by hu et al.16, it was found that transverse and oblique abdominal muscles were less active with a pcb in normal subjects because these coordinated muscles are activated to press the ilia against the sacrum, creating a forced closure, and the pelvic belt may have substituted for this stabilizing activity. therefore, it is thought that the use of a pcb with external pelvic compression might have improved pelvic joint stability and altered neuromotor control of the lumbopelvic and thigh muscles. first, the small sample size may have adversely influenced certain variables and impacted results. second, the compression force of the pelvic belt was not controlled, although the belt was adjusted by a skilled physical therapist. furthermore, we recruited healthy adults without a history of low back pain or sacroiliac joint pain, and thus, our findings can not be generalized to other populations. finally, we measured emg activity of the trunk and lower extremity, but this is insufficient to represent muscle force directly. further studies are needed to investigate a more diverse sample of normal healthy subjects.

[ purpose] the purpose of this study was to investigate the effects of a pelvic belt on the activities of trunk and lower extremity muscles in normal adults. [subjects and methods] the subjects were 20 normal individuals without a history of orthopedic problems. the pelvic compression belt (the com-pressor, optp, minneapolis, mn, usa) was an adjustable body belt with four elastic compression bands that provide stabilizing pressure and was designed to adjust the amount of force applied and to alter sites of compression. the body belt was placed below the anterior superior iliac spine, and stabilizing pressure was applied to the belt using the elastic compression bands in the bridge position after confirming the site of compression. [results] the subjects showed a significant decrease in muscle activation in the erector spinae, oblique internus abdominis, rectus femoris, and biceps femoris while wearing the pelvic belt. [conclusion] the use of a pelvic compression belt with external pelvic compression might improve pelvic joint stability and alter neuromotor control of the lumbopelvic and thigh muscles.

PMC5276746

pubmed-1309

infections are common in patients with cirrhosis and have been found in up to 66% of patients with variceal hemorrhage. prophylactic treatment with third generation cephalosporins is recommended in patients with advanced cirrhosis and gastrointestinal bleeding to cover for the predominant causative gut flora organisms ,. treatment is recommended even if no causative organism is identified as up to 50% of patients with cirrhosis and clinical sepsis have negative cultures. identifying appropriate antimicrobials can be challenging without a causative organism, and the empiric use of broad spectrum antimicrobials contributes to multidrug resistant organisms. we report the first known case of paracoccus yeeii bacteremia in a patient with decompensated cirrhosis who presented with variceal hemorrhage. this rare gram negative organism may not be susceptible to third generation cephalosporins. this finding may have implications for the management of infected but culture negative patients with cirrhosis. a 42 year old gentleman with decompensated cirrhosis from genotype 1a (g1a) hepatitis c (hcv) had been treated with 24 weeks of ledipasvir and sofosbuvir which ended 4.5 months earlier. the patient presented with new onset jaundice (total bilirubin 23 mg/dl, direct bilirubin 18 mg/dl, alkaline phosphatase 99 u/l, alanine aminotransferase 31 u/l, aspartate aminotransferase 86 u/l) that peaked to total bilirubin 44 mg/dl and direct bilirubin 31 mg/dl. model for end-stage liver disease (meld) score on admission was 27. given the report of subjective fevers in the setting of recently treated k. pneumoniae bacteremia of unknown source, admission blood and urine cultures were obtained but were without growth. molecular studies revealed hcv relapse (hcv viral load=4.44 log iu/ml) without ns5b resistance (insufficient sample for ns5a) and undetectable hepatitis b (hbv) and adenovirus. there was no serologic evidence of acute infection with hepatitis e, cytomegalovirus (cmv), or epstein barr virus (ebv). liver biopsy demonstrated cirrhosis with moderate chronic inflammation, focal severe neutrophilic infiltrates with cholestasis, and ductular proliferation. magnetic resonance imaging (mri) and magnetic resonance cholangiopancreatography (mrcp) showed no cholangitis or abscess. although drug induced liver injury from ciprofloxacin was clinically suspected, two sets of blood cultures were obtained daily due to concern for infection and lack of clinical improvement. the patient elected discharge but was readmitted within 24 hours with hematemesis from variceal hemorrhage and portal hypertensive gastropathy. empiric treatment for undifferentiated septic shock including spontaneous bacterial peritonitis prophylaxis were ceftriaxone 2 g iv every 24 hours, metronidazole 500 mg iv every 8 hours, and vancomycin 2 g iv every 12 hours. diagnostic paracentesis was again attempted but the patient had minimal ascites. given the lack of clinical improvement and the finding of bowel edema and severe colitis on computer tomography, infectious disease was consulted. it was recommended to adjust antimicrobials to piperacillin-tazobactam 3.375 mg every 6 hours, metronidazole 500 mg iv every 8 hours, and vancomycin 125 mg po every 6 hours to cover for gut flora including clostridium difficile until it could be ruled out. blood cultures collected the day before discharge of the previous admission flagged as positive after 5 days of incubation in the aerobic bottle (bactec plus aerobic/f), but no organisms were seen upon gram stain. however, after two days of incubation, small mucoid colonies of oxidase positive gram negative coccobacilli were seen growing on chocolate agar only. the team was notified and antimicrobials were adjusted to only piperacillin-tazobactam 4.5 g every 6 hours. after an additional two days of incubation, an identification of paracoccus yeeii was made by matrix assisted laser desorption ionization time of flight mass spectrometry (maldi-tof ms; vitek ms) with a match of 99.9% to the vitek ms ivd unclaimed database. identification was confirmed by sequencing of the first 500 bp of the 16s gene. upon speciation, the patient was switched to ceftazidime 1 g every 12 hours to cover for p. yeeii as well as concern for a hospital acquired pneumonia. the patient completed a 2 week treatment course of gram negative antimicrobial coverage with two beta lactam agents and had clinical improvement with no other culture growth of p. yeeii. unfortunately, the patient developed end-stage liver disease and eventually succumbed to death due to septic shock from citrobacter freundii pneumonia. to our knowledge, this is the first reported case of p. yeeii in a patient with cirrhosis. patients with decompensated cirrhosis are at high risk of infection from immune dysregulation, a heightened inflammatory response, altered gut flora, and bowel translocation. however, no causative organism is found in up to 50% of patients with decompensated cirrhosis presenting with clinical sepsis. this case of bacteremia with a rare gram negative organism is instructive regarding optimal diagnosis and treatment of infection in patients with decompensated cirrhosis who have an unclear source of infection as they have an extremely high risk of morbidity and mortality. p. yeeii is an aerobic gram negative coccobacillus that has been isolated in soil and marine sediment, and has been rarely reported as infections in humans. p. yeeii has been identified in the past through commonly available biochemical tests, and this case demonstrates the utility of maldi-tof ms for the identification of this infrequently encountered organism ,. the few reported cases of p. yeeii to date have entailed peritonitis among peritoneal dialysis patients,, myocarditis in a heart transplant patient, corneal graft infection, and heart failure with bullous skin lesions. all cases identified p. yeeii through culture of affected tissues. only 2 cases, including ours, have been associated with bacteremia and ours is the first case of a patient with cirrhosis. the source of bacteremia in our case remains unclear, but it is plausible that the patient s lymphedema and bowel edema could be sources for this environmentally found organism. over the previous year, the patient had several episodes of bacteremia thought to be from bowel translocation including group g -hemolytic streptococci, streptococcus mitis/oralis, citrobacter braakii, and enterococcus faecium (the patient was not on chronic antimicrobials). maldi-tof may help with rapid diagnosis and guide appropriate treatment when initial cultures in patients with decompensated cirrhosis and clinical sepsis are negative. it is recommended that patients with advanced cirrhosis who develop gastrointestinal bleeding be treated empirically with third generation cephalosporins ,. however, this case illustrates that uncommon gram negative organisms may not necessarily be covered with third generation cephalosporins. although we did not have antimicrobial susceptibilities performed in our case, the existing literature on p. yeeii suggests that the organism is susceptible to fluoroquinolones and beta-lactams though higher minimum inhibitory concentrations (mics) were reported with third generation cephalosporins. this case reinforces the challenges in isolating rare infections in patients with cirrhosis. in the absence of obvious infectious sources in a patient with decompensated cirrhosis collaboration with infectious disease specialists and microbiologists is warranted in these cases to isolate infectious organisms and to identify appropriate antimicrobials promptly. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

highlightsno organism is found in up to 50% of infected patients with decompensated cirrhosis.in the absence of obvious infectious sources, consider rare gram negative organisms.uncommon gram negative organisms may not be covered by empiric antibiotics.maldi-tof may help with diagnosis when cultures are negative in these patients.human infections with paracoccus yeeii, a gram negative coccobacillus, is rare.

PMC5133649

pubmed-1310

coal is an important basic energy and raw material in china, and it accounts for 70% of primary energy. however, due to the complexity and particularity of the coal mine, safety accidents can not be well controlled, and it still is the key factor to restrict coal production capacity. on the other hand, the influence of coal mine safety accidents, in particular major accidents, is extremely bad, which can create serious losses to people's life and property. mine production system is a complex system, and the combined effects on various factors lead to coal mine safety accidents. many scholars and experts consider that human error or people's unsafe behavior is the main reason for coal mine safety accidents by analyzing cause of accidents, and it accounts for more than 90% in all coal mine safety accidents [35]. coal underground mining is used as the primary mining method in china, comparing to the surface mining, there are too many affecting factors in the underground, and human factors are the most important affecting factors in these factors. according to hfacs analysis methods in the coal mine accident, human factors are relatively complex and changeable, so more affecting data are unknown; it is a gray system. low prediction accuracy can be avoided due to historical data lack or inaccuracy by applying the gray scgm(1,1)c model to predict the coal mine safety accidents. at present, main methods for predicting safety accident include experience model, regression model, and the gray prediction method. xiao-fu and ya-dong had applied regression model to forecast the ship traffic accident. using the regression models and empirical models to predict accident however, accidents are mainly human error accident in the coal mine. due to human error is affect by many factors, and in a dynamic time-varying system with low accident data and the non-line random changes, so it is not suitable to use these methods for prediction. in the gray accident prediction, shan et al. had predicted mine safety accident use of unbiased gray model. meng and cefeng had applied gray correlation on human error accident prediction in the nuclear power plant.. had, respectively, applied the gm(1, 1) model and residual gm(1, 1) model to predict coal mine accidents [11, 12]. appling the gray model to predict accident, however, qualitative analysis intensity should not be impressive enough, and the prediction accuracy should be less if these gray models were individually applied. the main reason is that the model requires data sequence must be exponential distribution, and fitting will be poor when data sequence fluctuations are comparatively large. based on system cloud gray prediction model features in, combining the advantages of both gray prediction and markov theory, according to the coal mine accident deaths provided by the state administration of coal mine safety, referencing the literature [16, 17], an amended gray markov scgm(1,1)c model is proposed. the gray scgm(1,1)c model is applied to imitate the development tendency of the mine safety accident, and the amended model is to improve prediction accuracy while markov prediction is used to predict the fluctuation along the tendency, so as to further improve the prediction accuracy on random volatile accident data. according to the actual accidents situation in coal mine, human error of the coal mines is not regular; there are some characteristics which include occurrences randomly scattered, raw data samples lack, and imperfect and uncertain information. gray markov scgm(1,1)c prediction model possesses these characteristic as less information required, easy calculation, high accuracy, and so on. it does not list factors data affecting research object but finds useful information and explores the inherent laws from their own time data sequence establishing model to predict. the gray markov scgm(1,1)c prediction model is the ideal model to forecast coal mine safety incidents. taking account of randomness of human error data in mines, original time series x of coal mine safety accident deaths first, x is integrated as follows: (2)x(1)=x12,x13, ,x1n, here, x1(k)=m=2kx0 m, k=2,3, ,n. x-(0) is a close mean value generated sequence for x: (3)x(0)=x02,x03, ,x0n, here, x0k+1=x0k+1+x0k2. human error is random to lead accidents in coal mine, so majority of accidents are dynamic. given that integral sequence of safety accident deaths time series is expressed as {x-(1)(k)} that is associated with satisfaction trend of nonhomogeneous index discrete function as fr(k)=be c, thus the data of x-(1)(k) is fit to fr(k). according to gray system cloud forecast method, the system gray scgm(1,1)c prediction model can be expressed as (4)dx(1)(k)dk=ax1(k)+u, k2. its time response function can be expressed as (5)x1k=x11+uaeakua. here, (6)a=lnk=3nx(0)(k1)x(0)(k)k=3nx0k12,b=(n1)k=2nea(k1)x(1)(k)(k=2nea(k1))(k=2nx(1)(k))(n1)k=2ne2a(k1)k=2neak12,c=1n1(k=2nea(k))bk=2nx(1)(k). given x-(1)(1)=b-c, u=ac, x-(1)(k) is reverted, the system gray scgm(1,1)c prediction model of original data will be expressed as (7)x^0k=2b1ea1+eaeak1,(8)yk=x0kx^0k,(9)k=x^0kx0k, k=kx0k. y(k), (k), and k are gray fitting accuracy indicators, which reflected the degree of deviation of the predicted values to the original data. statistics data fluctuations of coal mine accidents deaths are larger, and the regularity is not very strong as uncertainty of the person's behavior. therefore, the prediction accuracy should not be too good if the scgm(1,1)c model is solely applied to predict accident deaths of coal mine. in order to improve the prediction rate and better meet the actual situation, the prediction model should be corrected to improve the accuracy. amended principle and steps are as follows.(1)the first time residuals data sequence is got in accordance with the predicted value and actual value: (10)0k=x^0kx0k, k=1,2, ,n,(11)0k=01,02, ,0n.(2)processing residuals correction sequence. the first time residuals data sequence is got in accordance with the predicted value and actual value: (10)0k=x^0kx0k, k=1,2, ,n,(11)0k=01,02, ,0n.=(1+e)(1 e)b, the scgm(1,1)c prediction will be expressed as x^(0)(k)=2ea(k-1)m. if (k) a1, m1, and b1 can be obtained in accordance with a, m, and b used method. the first time residuals corrected scgm(1,1)c model can be expressed as follows: (12)x^10k=2eak1mea1k1m1. if (k) n), the first time residuals corrected scgm(1,1)c model can be expressed as follows: (13)x^10k=2eak1m+ea1k1m1. in general, a prediction model can be repeatedly corrected residuals, and the residuals can be negative, the time dimensions are also not equal. if the first time residuals amendment can not meet the forecast accuracy required, it should do residual correction according to the above amended principles, until the accuracy meets requirements. the scgm(1,1)c prediction fitting curve is essentially an exponential curve, and the prediction result is a relatively smooth curve. because human error accidents are main part of coal mine accidents, the scgm(1,1)c model solely applied can not meet forecast accuracy requirements. markov theory has no aftereffect, that is to say, the future state of the system is only related to the current state, and has nothing to do with the past state. meanwhile, markov model is adopted to predict states trends through probability transfers, it can adapt to the randomness and variability of state. applying markov theory to correct the scgm(1,1)c prediction model of coal mine accident deaths can better solve the variability and randomness of accidents caused by human errors to improve the prediction accuracy. the annual change of the number of deaths in coal mine accidents is a dynamic nonstationary random process, and thus the prediction fitting precision indicators also are variability and randomness. because boundary and connotation of the different annual state are changeable, an adaptive state divided criterion needs to be determined, and the criterion should be consistent with basic timing trend of the coal mine accident deaths. thus, y(k) was divided into m states, and each state can be expressed as (15)ei1i,2i, i=1,2, ,m. here, 1i=y(k)+ai, 2i=y(k)+bi. in the formula, ei is expressed as i state, 1i and 2i are, respectively, expressed as the upper and lower bounds of the i state, and ai and bi are constants determined according to prediction data. because y(k) is a time function, 1i and 2i will be changed with time, so the state possesses variability. when the state is divided, the numbers of different intervals are reasonably divided according to the actual situation. if raw data are less, the interval division should be less so as to increase the number of transfers between the various states, and thus the transfer law can be more objectively reflected between states. conversely, if raw data are more, the interval division should be less in order to excavate more information from a large number of data to improve the prediction accuracy. it is suitable to adapt clustering classification method to determine class number and classification intervals due to less data and uncertain status of human error accidents in the coal mine. the original number of samples is expressed as mij(k) from the state ei transiting to the sate ej by k step, and the number of occurrences of the state ei is expressed as mi, so state transition probability is expressed as follows: (16)pijk=mijkmi, i, j=1,2, ,m. m m state transition probability matrix can be obtained as follows: (17)pk=p11kp12kp1mkp21kp22kp2mkpm1kpm2kpmmk. the state transition probability matrix p(k) reflects all statistical regularities of state transition, and the future system state steering can be predicted by investigating the matrix. in the actual analysis of the process, one step transition probability matrix p(1) given that predicted moment object is in the state ek, investigating the k row of p(1) can get the following.(1)if maxpij=pkl, the next time system should most likely shift from the state ek to the state el.(2)if there are two or more probability values identical or similar to k row in the matrix p(1), the future state steering will be difficult to determine; it needs to consider probability transition matrix p(2) or p(n) (n 3). if maxpij=pkl, the next time system should most likely shift from the state ek to the state el. if there are two or more probability values identical or similar to k row in the matrix p(1), the future state steering will be difficult to determine; it needs to consider probability transition matrix p(2) or p(n) (n 3). the system's future state will be determined by investigating state transition probability matrix, and gray change interval of relative prediction value in the future moments also will be determined; it can be expressed as [1i, 2i]. predicted value of the future moment can be expressed as the interval median as y(k): (18)yk=x0kx^0k=121i+2i=yk+12ai+bi. chinese coal mining is primarily underground mining; the main mining methods used include blasting mining, general mechanized mining, and comprehensive mechanized mining from 1990 to 2010. different methods lead to frequency of the coal mine accidents is not same. in general, blasting mining and general mechanized mining were usually applied in the town local small coal mine; their production capacity is relatively small and mining technology and management level are relatively lower compared to large coal mines, so accidents rate is high. in contrast to these small coal mines, the large state-owned coal mines mainly used comprehensive mechanized mining technology; their production capacity is relatively large, management level is relatively advanced, and the accidents rate is lower. over the last decade, due to the small and medium sized coal mines integrated and strengthened security management in china, their production capacity and mining technology were gradually improved, so the number of occurrences of accidents in the coal mine was declined. according to statistics data of 19902010 coal mine accident death provided by coal mine safety administration, the trend of accidents deaths in coal mine can be fitted by applying the gray scgm(1,1)c model and prediction accuracy can be improved by the residual modified model. finally, deaths are validly predicted by using the gray markov scgm(1,1)c model. the raw data are shown in table 1, the trend of coal mine accident deaths and death rate per million ton from 1990 to 2010 are shown in figure 1. scgm(1,1)c model of coal mine accident death toll is established by using accidents mortality data of coal mines from 1990 to 2010 in china: (19)x^0k=2b1ea1+eaeak1. in the prediction model (19), a and b are the most important parameters; they are mainly dependent on the raw data to reflect the development trend of data. here, coal mine accident deaths for 20 years in china were taken as raw data to predict, in order to find the trend of coal mine deaths. therefore, it is important to select the sample data; according to the formulas (5) and (6), a and b can be calculated. here, a=0.039436, b=236878. applying the formula (19), prediction value of coal mine safety accident deaths can be got, and then the gray fitting accuracy indicators also can be calculated by the formula (8), which reveal overall development trend of safety accident death toll in coal mine. specific forecast and actual values are shown in table 1. in accordance with table 1, according to (10), the residual initial sequence can be attained, and then one correction residual prediction model can be expressed as (20)^0k=2ea1k11ea11+ea1b1. here, according to the formula (14), a residual modification prediction value of safety accident deaths can be obtained, and the results are shown in table 2. comparing tables 1 and 2, it is shown that forecast fitting accuracy of the scgm(1,1)c model has been improved after residual modification, but volatility of the gray fitting accuracy indicators is larger from analyzing table 2. in order to solve this question, it needs to adopt markov chain prediction for further enhancing accuracy and lowering the volatility. the prediction fitting indicators data in table 2 are divided to 4 states by hierarchical clustering, and the results are shown in table 3. the corresponding states of each year can be determined according to the divided states, and applying the formula (16), the state transferred introduction matrix can be obtained, and the first step state transition matrix is shown as follows: (21)p1=1000000116161216011031035. the future coal mine accident deaths toll can be predicted based on the above state transition probability matrix. accident deaths in 2003 year be in the state e4, according to the state transition probability method, examining the state transition probability matrix p(1) line 4 can get (22)maxp4j=p44, j=1,2,3,4. after a year transition, under the control of coal mine accidents, the death toll of coal mine accidents in 2004 was most likely in the state e4. according to table 3, interval of the state e3 is [1.05,1.2], that is, 13=1.05 and 12=1.2. because 1i=y(k)+ai, 2i=y(k)+bi, and y(k)=1.19 in 2004 according to table 2, ai=13 y(k)=1.05 1.19=0.14, bi=23 y(k)=1.2 1.19=0.01. using the gray markov scgm(1,1)c model, the death toll of coal mine accidents in 2008 is most likely expressed as (23)yk=yk+12ai+bi=1.19+120.14+0.01=1.125,x02004=x^02004yk=54331.1256112. in general, in order to facilitate the calculation, similarly, according to the formula (21) and table 2, accidents deaths of coal mine from 2004 to 2013 can be predicted; prediction error comparison of the amended residuals scgm(1,1)c model and the amended residuals markov scgm(1,1)c is shown in table 4. error analysis and predictive value fitting of three methods comparing tables 1, 2, and 4, we know that table 2 was amended by using residuals prediction model on the basis of table 1, and the prediction accuracy had been appropriately improved. for table 4, comparing the residual amended model and markov prediction model can be seen that the residual amended scgm(1,1)c model can not handle abnormal events, because its prediction parameters are fixed. as china's coal enterprises are affected by the economic situation form abroad and home, their production had been in low period, and some coal was not continuously produced; they were basically in an abnormal state in the last three years, so these factors would have a big impact on mine accident forecast. national and local government paid more attention to safety management in the coal mine, and each coal mine strengthened management and prevention on human error, they would also affect the accuracy of general forecasting methods. however, the markov scgm(1,1)c prediction model is only depend on the previous state, the relationship with other states is very small, so it very better solves the abnormal accident. on the other hand, comparing accidents deaths of coal mine from 2004 to 2013 in china, the number of deaths was 6027 in 2004, and this figure became 1067 in 2013; in the recent ten years, the average decline rate of accidents deaths in coal mine is 16.8%. according to this downward trend, accidents deaths of coal mine in china will be about 889 in 2014. as figure 2 shows that the fitting degree of the markov scgm(1,1)c model is the best, the amended scgm(1,1)c is better and scgm(1,1)c model is the worst. while error change of the markov scgm(1,1)c is minimal, it illustrates that the markov scgm(1,1)c forecast model can be used as a good method to predict the accident deaths of coal mine in china comparing to the other two methods; its prediction result for accident deaths of coal mine is basically corresponded with deaths decline trend in nearly 10 years. the prediction gained by applying the amended residuals markov scgm(1,1)c model is closer to the actual value; in other words, its error is smaller, and it can better reflect the relationship between the coal mine safety accident death and the number of data series, so the prediction is reliable. the amended residuals markov scgm(1,1)c model combines the advantages of both the single-factor system cloud gray model and markov chain, using residual to amend the system gray cloud; it can take full advantage of the information given by the historical data of coal mine accidents, and overcome the data random volatile effects on prediction precision.

the prediction of mine accident is the basis of aviation safety assessment and decision making. gray prediction is suitable for such kinds of system objects with few data, short time, and little fluctuation, and markov chain theory is just suitable for forecasting stochastic fluctuating dynamic process. analyzing the coal mine accident human error cause, combining the advantages of both gray prediction and markov theory, an amended gray markov scgm(1,1)c model is proposed. the gray scgm(1,1)c model is applied to imitate the development tendency of the mine safety accident, and adopt the amended model to improve prediction accuracy, while markov prediction is used to predict the fluctuation along the tendency. finally, the new model is applied to forecast the mine safety accident deaths from 1990 to 2010 in china, and, 20112014 coal accidents deaths were predicted. the results show that the new model not only discovers the trend of the mine human error accident death toll but also overcomes the random fluctuation of data affecting precision. it possesses stronger engineering application.

PMC4897162

pubmed-1311

de ridder, vanneste, and focquaert address concerns relating to the definition of a ced and the distinction between treatment and enhancement. they raise a number of problems with the treatment-enhancement distinction and suggest that we need to ask whether we are prepared to change the definition of health used by the medical devices directive. there are in fact three questions raised here. the first is whether our proposal requires a robust characterization of the treatment-enhancement distinction, the second is whether our suggestions, as we intended them, actually involve changing the definition of heath implicit in the mdd (there is no explicit definition), and the third is whether the mere inclusion of ceds within the mdd will have the de facto effect of changing the implicit regulatory concept of health or treatment. in response to the first question, we would maintain that it is in fact an advantage of our approach that it minimizes the importance given to the treatment-enhancement distinction and thus diminishes the need to characterize it in a way that is immune to criticism. on our approach, devices intended for enhancement are regulated in a similar manner to therapeutic medical devices. thus, if some enhancement devices are misclassified as therapeutic devices, or vice versa, this will not have major implications for their regulation. what matters, from our perspective, are risks and benefits. by contrast, some alternative approaches, including the one recommended by de ridder and collaborators, would regulate ceds and therapeutic medical devices quite differently, with the result that misclassification would have more significant regulatory effects, since devices regulated under the general product safety directive alone are held to less specific and less stringent standards. in short, we agree that the treatment-enhancement distinction is problematic and some of us have elsewhere rejected it but we believe a desire to mitigate the problems it raises counts in favor of our approach, not against it. we would also dispute the suggestion that our approach requires any modification to the concept of health. our proposal was not to change the definition of health (nor treatment) but, rather, to bring some non-therapeutic devices, which do not aim to improve health problems, within the remit of the mdd. our proposals involved leaving the definition of a medical device used by the mdd unaltered. ceds would not be medical devices on the mdd definition. instead, an ancillary positive list is proposed to bring specific devices with a non-medical purpose within the remit of the mdd alongside those devices defined as medical devices according to the criteria employed in the directive. by proposing an ancillary list for specific cognition-affecting devices without a medical purpose, we in fact reinforce the concepts (ie medical and non-medical) underpinning the european commission's proposed creation of annex xv for implantable and other invasive devices for which the manufacturer claims only a non-medical purpose. we proposed that this list should be extended to include non-invasive neuromodulation for non-medical purposes. however, the third implicit question indeed requires consideration: will there be a de facto change in what is meant by health and treatment if ceds are regulated under the mdd? arguably, the mere association of ceds with therapeutic medical devices could result in an expansion of the concepts of treatment and health. the possible implications of a shift in what is seen as health-related or there, they suggest that medical device regulation might underscore the illusion that devices are beneficial. however, we suggest that the idea that tdcs and tms techniques can yield cognitive enhancement in healthy adults is not a mere illusion. further, we suggested that regulatory approval of moderately risky brain stimulation devices should be dependent on evidence of at least some degree of performance. in this respect, it would be important to notify consumers explicitly on which population the ced has shown to be beneficial while reminding that it can be ineffective or detrimental to other populations. regulation would therefore go some way towards ensuring that effects are not illusory for those devices that are approved (although individuals could believe effects to be bigger than they are). whilst we concede de ridder, vanneste, and focquaert's point that there is currently no substantive evidence that ceds produce lasting effects outside of research and clinical settings (p. 320), there is a wealth of scientific research that provides proof of concept for the cognitive enhancing effects of tdcs and tms techniques. that the ceds on the market have not been subject to objective assessment is part of the reason why regulation is needed and counts in its favor. they argue that contemporary scholarship shows no serious harms associated with the use of neurofeedback or tdcs. in response to this, we wish to point out particularly in relation to active devices used for tdcs and tms that, whilst devices have indeed been use safely in the laboratory, and the regulated devices that researchers use in such contexts are indeed conducive to safe use, these facts do nothing to ensure that the unregulated devices on sale for enhancement exhibit the same safety profile. first, the exclusion criteria for brain stimulation research are much more conservative than marketed exclusion criteria. second, while brain stimulation experiments lasts usually between a single session to a dozen (the latter is a relatively rare scenario), there is not safety data on the usage of brain stimulation over a long period of time such as months or years. essentially, regulation is needed to ensure that devices sold for enhancement are as similar as possible to those devices about which kuersten and hamilton make their safety claims. indeed and to illustrate the point from another perspective fitz and reiner raised concerns about the foc.us device in their commentary: the internal electrodes exceed the general safety guidelines for current density at all stimulation levels, the voltage limits do not behave as specified in the manual, the device behaves unpredictably when its connection to the head is lost, and under some circumstances the foc.us can generate small voltage current spikes. the internal electrodes exceed the general safety guidelines for current density at all stimulation levels, the voltage limits do not behave as specified in the manual, the device behaves unpredictably when its connection to the head is lost, and under some circumstances the foc.us can generate small voltage current spikes. accordingly, we emphasize: just because tdcs can be safe does not mean that all of the particular devices on sale are meeting the same level of safety. further, at the end of their commentary, and perhaps in tension with their assertion that the sorts of ceds we discuss present no significant risks, kuersten and hamilton say that low-risk devices should not be excluded from on-going oversight, since so much concerning the brain, what affects it, and how remains unknown (p. 347). we wish here to emphasize that low-risk devices, as we envisioned them, would typically be non-active devices such as neurofeedback equipment, which they correctly claim pose no risks qua devices (see below). given their comments on the safety of neurofeeback, especially considered simply as a device, we would be surprised if they would think that such equipment required on-going oversight. thus, even if there is room to disagree about whether ced is the right label for non-active devices (as we discuss below), we in fact seem to agree on the lack of need for the continual regulation of low-risk devices, such as those that do not transfer energy through the skull. kuersten and hamilton object to our inclusion of neurofeedback as a ced. however, in offering our definition of ceds, we were not aiming to provide the definitive view on what does and what does not fall within this category. our interest in offering a definition was only to facilitate identification of a class of non-therapeutic devices that should receive regulatory attention and, on that definition, neurofeedback plausibly does qualify as a ced. note also that doubts about whether neurofeedback equipment qualifies as a ced on a precise understanding of that term could also be raised regarding tdcs equipment, which kuersten and hamilton believe does qualify. they argue that what actually affects the brain is therapy used with [neurofeeback equipment] (p. 341). but tdcs also only enhances cognitive performance when combined with cognitive training exercises, so could be excluded from the category of ceds on similar grounds. nevertheless, kuersten and hamilton are right to highlight that tdcs directly modulates brain activity whereas neurofeedback modulates it through psychological mechanisms. we have no problem with the suggestion that this is a significant difference, and indeed it informs our suggestion that tdcs and tms should be classed as iia or iib devices, due to their active nature, and neurofeedback should be in class i, if regulated at all. kuersten and hamilton next argue that including ceds under the definition of a medical device would not result in our suggested problem of overbroadness (which we suggested would occur if the definition of a medical device were to be altered), since devices, they claim, would be limited by the definition to those that investigate, replace or modify the anatomy or a physiological process. their claim is based on the assumption that ceds could fall under the definition as it is currently articulated. however, it was the possibility that an altered definition would be overbroad that we thought counted in favor of a supplementary positive list. in setting out our proposals, we worked on the assumption that the current definition did not in fact capture ceds (due to the principal criterion that a medical device be intended to treat, prevent or diagnose) and that changing the definition to remove this principal criterion across the board would have the result that it would then be overbroad. if the principal criterion were simply that a medical device investigate, replace or modify the anatomy or a physiological process then all sorts of devices would fall under the remit of the mdd from earrings to nail extensions. indeed, the european commission used precisely this argument to support the creation of a positive list of implantable or other invasive devices without a medical purpose. according to their impact assessment on the revision of the regulatory framework for medical devices, the definition of a medical device stipulates that it be intended for treatment, prevention or diagnosis, and the removal of this criterion would result in overbroadness, making a positive list the preferred solution in the case of certain cosmetic devices. our argument for an analogous list for ceds is based on the assumption that the european commission is correct in its understanding of the mdd and the implications of the current and possible definitions employed therein. fitz and reiner broadly support our proposals and endorse our central claim, viz. that the regulatory framework for medical devices should be extended to include ceds. nevertheless, they raise a concern about the ability of ced users to evaluate the risks of ceds, saying that evidence that consumers are in a strong position to evaluate the risks associated with ced use is lacking (p. 323). whilst we agree that consumers will not be perfect rational calculators, we emphasize that our proposals would eliminate devices from the market that were manifestly dangerous or far more dangerous than needed to serve their intended function. in addition, our liberal view is committed to the contention that allowing some room for error in consumers weighing of benefits vs. risks in relation to their individual well-being can be a justifiable cost of allowing individuals greater autonomy and freedom of action. we stress also that what the risks of devices are their nature and likelihood is something that would, in line with the procedure for medical devices, be assessed by a group of experts prior to approval for the market. so, although we agree that leaving some room for consumer valuation of risk leaves open the possibility that individuals will not factor this carefully into their decision whether to use a product, we contend that this is an acceptable cost of a liberal approach to regulation. further, we would like to emphasize that the current lack of regulation implicitly signals to home users that there are no real concerns with ceds, as otherwise they would have been regulated ie the perception is likely to be that, given the lack of regulation, there are no risks for consumers to evaluate at all. thus, even if consumers are not perfect risk calculators, the current situation it likely to be more misleading than one in which risks have been identified by regulators. king, gavaghan, and mcmillan provide an interesting critique of our proposals relating to consumer assessment of ceds, and in particular their risks and benefits. they agree that the concept of medical benefit is not always appropriate for ceds (especially where designed and sold only for enhancement) and that well-being would be the theoretically appropriate construct when assessing such ceds. however, they suggest that well-being, harm and risk are difficult to assess in the pre-market approval process and raise a particular concern about the phenomenon of risk compensation. the central feature of this phenomenon is that some individuals have a propensity for taking a certain level of risk and will increase the riskiness of what they are doing until this propensity is met. they therefore challenge the assumption that pre-market approval will make the use of ceds safer overall, especially in the context of experimental home use. they suggest that, where users are determined to use devices in a risky way, they will do so despite safety standards. in response to this, we raise a point of empirical uncertainty, and emphasize what can still be achieved through regulation, despite the phenomenon of risk compensation instantiated in some individuals. it might be true that people will tend to fulfill their propensity for a particular total amount of risk in their lives, but it is unclear whether they do this by assuming superfluous risk to achieve a particular goal or seeking further risky goals. perhaps, as king and colleagues suggest, achieving one goal more safely allows one to assume additional risk in relation to other pursuits. however, a risk quota might also be met by increasing or maintaining the level of risk one exposes oneself to in pursuit of one's current primary goal. which is the case is likely to depend on the number of goals one believes to be valuable to pursue. if ceds are made safer, would users with high risk thresholds seek to obtain riskier devices, or would their risk quota be freed up to pursue additional risky activities? the question is essentially whether individuals tend to increase riskiness across all activities until their risk propensity is met, or whether they increase the riskiness of every activity to some threshold level for each activity, regardless of how superfluous this risk may be. if the former, risk reducing regulation could still have benefits in the form of facilitating pursuit of other risky, but perhaps all-things-considered valuable, activities. even if individuals with a high risk threshold are inclined to practice riskier use as the device itself becomes safer, we assume that king and colleagues would agree that it is still better that the devices available pose as low a risk as possible to achieve their effects, and that unjustifiably dangerous devices are prohibited from placement on the market. eg by using failsafe mechanisms, audible warnings, and limits on stimulation duration and strength. an individual's propensity to take risks with a product does not render consideration of that product's safety redundant. by analogy, just because some individuals routinely drive dangerously, this does not negate the need to try to make cars as safe as possible. even if those engaging in risk compensation reap no overall benefit from making ceds safer, failing to minimize risks would unfairly jeopardize those who do not so compensate. johnson, gillett, and snelling suggest that the most convincing argument in favor of our position is based on the regulatory assessment of risk, which they believe should be the sole consideration in pre-market assessment. we suggest here that our respective proposals are not as different as they may have seemed, as we proposed that risk should be the primary regulatory concern. however, we diverge from the position advocated by them in our contention that objective improvements to cognitive capacities are amenable and relevant to assessment. baseline (or, we infer, upper limit) level of risk that society feels an individual should be able to take (eg skydiving or climbing), and then require manufacturers to quantify risks only. they argue for this position by pointing out that when technologies are new, and particularly when new technologies are used for an innovative application, variation in the effects on individuals and useful endpoints my not be obvious ab initio. our first response is to note that the same applies for risks novel applications may present novel and variable risks that are as difficult to predict as the cognitive improvements one might obtain from such applications. our second response is to emphasize that the model we proposed is actually quite close to a risk-only model; at least, it is a risk-first model. we suggested using risk as the primary way of categorizing a device and that benefits should only be considered in terms of objective improvements eg demonstrated improvements in a cognitive capacity, such working memory which are precisely the sorts of effects about which manufacturers make their claims. importantly, this consideration of objective benefit is not intended to downplay the broader benefits that an individual might attain from using a device. whilst objective benefits can be considered, they must not be thought to constitute the only determinants of well-being. nonetheless, regulators should still ensure that consumers are provided with information on objective benefits. indeed, the distinction between thick and thin considerations of well-being drawn by king, gavaghan, and mcmillan is illuminating in this regard. thin well-being, they say, consists only in all-purpose goods, whereas a thick well-being incorporates the individual agent's conception of value and his particular circumstances. an objective impairment in, say, verbal working memory (that occurs as a trade-off of an objective improvement in, say, visuospatial working memory), will present the very same challenges for quantification, despite such impairment constituting a risk rather than a benefit. to the extent, as the risk only model assumes, that objective risks can be quantified (and impairment in verbal working memory indeed permits such quantification) objective benefits along the same sorts of dimensions can also be quantified). further, we suggested that room should be made to account for the difficulty of measuring effects (positive or negative) on thick well-being. our proposal was that, once the manufacturer had made it clear what the objective benefits and risks are for moderately risky devices, the consumer should have the freedom to decide whether that benefit risk ratio is acceptable for them (for example, because they expect it to result in a net gain in thick well-being). on the other hand, we proposed that high-risk devices, presenting risks that might be classed all-purpose bads (eg a high risk of seizure), should be prohibited for sale on the consumer market. whatever the individual's life plans, in the preponderance of cases of healthy adult use, a seizure is unlikely to promote these plans. where there is scope for disagreement about the value of an effect for example as to whether a small impairment in verbal working memory is a reasonable price to pay for a similarly sized improvement in visuospatial working memory level of acceptable risk therefore plays a greater role than quantification of benefits in our overall framework. as an objection to our model, king, gavaghan, and mcmillan present the example of the surgical insertion of a ventriculoperitoneal shunt to treat normal pressure hydrocephalus. they note that this would be a high-risk procedure on our proposals, as it comes with the risk of seizures, stroke, paralysis, and death. moreover, there has never been a comprehensive multicenter randomized controlled trial to demonstrate the performance of this product. this is thus a high-risk procedure for which there is no good evidence of benefit. nevertheless, they seem to suggest, the use of this procedure should be permitted. it might seem, then, that consistency requires permitting the use of some high-risk ceds as well, calling into doubt our claim that high-risk ceds should not be allowed on the market. in response, we would note that patients with normal pressure hydrocephalus typically have much more to lose from non-intervention than do healthy individuals considering whether to employ ceds. indeed, one might think that advancing dementia is amongst the worst fates that might befall a person. there is thus a case for tolerating much greater risk in devices intended to treat this condition than in ceds. king and collaborators also use the ventriculoperitoneal shunt example to suggest that it may not be practical to enforce the provision of evidence-based measures of efficacy to consumers for ceds. in response to this, we emphasize that we do not have in mind a requirement such that each manufacturer must perform a randomized controlled trial using their device. research investigating tdcs and tms in healthy adults is demonstrating efficacy, and we envisage that manufacturers should be required to show how their device complies with design parameters equivalent to those that have been shown in the scientific literature to produce enhancement effects. for example, devices with electrodes positioned in locations on the scalp that have no evidence of producing the particular effect claimed by the manufacturer should not be approved for sale alongside such claims. manufacturers must be able to identify a credible body of scientific literature that supports the claimed efficacy and safety, given the product's characteristics electrode location, stimulation intensity, duration, and so on. further, we agree with king, gavaghan, and mcmillan that manufacturers should be required to state clearly the lack of evidence for any unsubstantiated claims, if they are to be allowed to make them at all. kuersten and hamilton also comment on the risk benefit assessment of ceds, and the standard to which we proposed they should be subjected. they argue that there is a lot of room for maneuver in the mdd to assess acceptable risks when weighed against the benefits (p. 346). this is true, but we nonetheless think a framework for assessment is helpful, especially when the devices in question purport to offer non-medical benefits, a concept for which medical device regulators have little precedent. indeed, we argued against adopting the approach originally suggested by the mhra for cosmetic devices, which is very risk-averse: according to their approach such devices must present no or the minimum acceptable risk, as they confer no clinical benefit. conceding that some framework could be helpful, kuersten and hamilton suggest that, because the concept of benefit is nebulous, in the case of ceds, only risk should matter for pre-market approval. in fact, as discussed above, we are broadly in agreement with such a position benefits become increasingly harder to quantify and assess as they move beyond the sorts of benefits that enable individuals to pursue the standard range of activities most people wish to pursue. for example, not being in great pain would count as a benefit for most people, regardless of what they valued, but a small improvement to mathematical ability, for example, would not permit such evaluative consensus. as noted above, our inclusion of regulatory consideration of benefits for moderately risky devices was intended only to refer to objective improvements about which consumers should not be misled. for example, if manufacturers claim that their device improves, say, the user's linguistic fluency, this is a claim that can be tested and measured objectively. beneficial such an objective improvement is to any particular person will vary depending on their goals, but there will be a fact of the matter about whether the device is able to confer such an improvement aside from the subjective question of how valuable this improvement might be. certainly, we would advocate regulating the claims that manufacturers make in relation to what their devices can achieve. however, far from our proposals being overly paternalistic, we argued that where there was room for disagreement about how to quantify a benefit in relation to risks, regulation should err on the side of consumer freedom. further to their concerns regarding individuals abilities to weigh risks, fitz and reiner emphasize the remaining problem of do-it-yourself (diy) users who, in constructing devices from scratch, will be afforded no protection by the regulation of direct-to-consumer devices. one of their proposals, with which we agree, is to bolster our recommendations with additional attempts at active harm reduction. they suggest that members of the professional community could join together to create an inclusive online community, where information could be gathered and disseminated with professional oversight. we are broadly sympathetic with much of what they propose and would support the creation of an inclusive online community, were there to be sufficient expert interest in creating and maintaining it. indeed, we are currently exploring how expert advice could be dispensed via existing fora, such as reddit. de ridder, vanneste, and focquaert also raise the problem of unsupervised use and suggest potential solutions in the form of age limits, safety-by-design, and requiring user licenses. we agree that the unsupervised use of ceds needs consideration, but do not agree that this should lead to their apparent conclusion that we should regulate the use of ceds rather than their placement on the market. the first task must be to ensure that the devices that people use (unsupervised or otherwise) are not unnecessarily dangerous or simply defective by design. this can be achieved for example through european medical device law, which focuses on premarket requirements and reporting on post-market experience, even though it does not regulate use (apart from requirements related to the instructions the manufacturers must provide). there is no point carefully regulating use if the devices being used are not safe to begin with. further, the authors suggestion that ceds should be made safe by design requires enforcement beyond that granted by the general product safety directive, and this is precisely the sort of thing that could be achieved by a model of the sort we propose. de ridder, vanneste, and focquaert raise the concern that the societal benefits of enhancement might be lost if devices are in fact effective but regulated. in relation to this, we emphasize that the regulation we advocate is not akin to prohibition. to the extent that our proposals would prevent devices from being placed on the market, this would be limited to very dangerous devices or devices making implausible claims hardly the sorts of devices that would confer net societal benefits. the important point that many other commentators on our model appear to overlook is that the effectiveness of brain stimulation techniques as a type of intervention does not guarantee the effectiveness of particular token devices claiming to be of this effective type. moreover, as low risk, effective devices are developed, these would be available under our proposal and a vigorous market will emerge. de ridder, vanneste, and focquaert raise concerns about the misuse of resources, which assumes a position markedly different from that which we set out in our paper. they argue, persuasively, that resources should be directed to the most important causes and that enhancements might not be of a high enough priority to warrant use of our limited resources. we agree that limited resources need to be allocated carefully, but our proposal does not threaten such allocation: the devices that would be prohibited under our proposal are ones that would certainly not warrant significant investment of public resources, and our proposal leaves open how resources should be allocated among those that would be permitted. perhaps de ridder and collaborators assumed us to be advocating the inclusion of ceds under the healthcare funding arrangements, but this is not what we envisaged. the extension to the scope of the mdd, as we recommend it, has no direct implications for the distribution of healthcare resources. relatedly, the authors ask whether the regulation of ceds requires new regulations regarding who can use them. again, the answer is for the most part no: our proposals merely affect what can be placed on the market for consumers to purchase. the only exception should be restrictions governing the use of ceds on children or vulnerable adults through contraindication labeling and the criminal law. in contrast, adult consumers will not need a prescription to purchase and use ceds on themselves. indeed, king, gavaghan, and mcmillan provide a compelling argument in their commentary for why medical practitioners should not serve as gatekeepers to cognitive enhancement. also of relevance to the question of the correct place of enhancement in healthcare, king, gavaghan, and mcmillan suggest that our reference to formally trained practitioners was not developed. we used this phrase in relation to the use of ceds in children and vulnerable adults, since our proposals envisaged that healthy adults would be able to directly purchase and use ceds, with no medical intermediary. they ask whether we envisage a further tier of regulation and raise a number of interesting points that would need to be addressed if medical professionals or those offering alternative therapies were to serve as gatekeepers to the use of ceds in such populations. they are correct that the regulation of such practice would indeed introduce a further tier of regulation, and we acknowledge that this issue was not addressed in our original paper. however, we believe that the scope for the permissible use of ceds on children may be limited. further, neuromodulation offered to vulnerable adults overseen by medical professionals will primarily be governed by existing medical ethical guidelines, since such use is likely to be therapeutic and hence occurs within the clinical domain. in relation to children, king, gavaghan, and mcmillan are correct that further thought is needed to establish how use in adolescents should be controlled, and with whose oversight. however, ensuring the safety of devices that might be used for enhancement in children and placing controls on such use are separate issues and both are important. whilst our proposals were concerned with controlling which devices are sold directly to consumers, we agree that the regulation of services, especially those offered to children needs close attention. kuersten and hamilton object that we fail to cite much law on the remit of the mdd. the suggestion seems to be that this has left us underinformed. attempting to clarify the current regulatory situation, they say that instead of the definition of a medical device being the significant factor in the non-application of the mdd to ceds, ceds are instead unlikely to fall under the mdd because manufacturers targeting the general market are discouraged from intending them for a medical purpose (which they argue manufacturers have considerable latitude to do) (p. 344). manufacturers are deterred, they say, because intending that a device be used for a medical purpose mandates costlier and more time-consuming requirements. we suggest that this is precisely the problem: manufacturers should not be able to evade regulation just because they deem it to be too burdensome. we are fully aware that it is the manufacturers intentions identifiable from the claims they make in relation to their products that are instrumental in bringing a device within the definition of a medical device. we argue that, particularly in the case of brain stimulation devices, opting not to fall under the definition of a medical device should not be a possibility. however, kuersten and hamilton suggest that manufacturers intentions will in fact not matter once a new definition comes in to force, which will specifically define a medical purpose. they cite the mhra's overview of the current proposals for the current revision of medical devices legislation, proposals with which we are very familiar. they quote the document as follows, saying that the definition of medical purpose will remove this decision from manufacturers: [m]edical device means any instrument, apparatus, appliance, software, implant, reagent or other article, intended by the manufacturer to be used alone or in combination, for human beings for one or more of the specific medical purposes of: investigation, replacement or modification of the anatomy or of a physiological process or state (pp. [m]edical device means any instrument, apparatus, appliance, software, implant, reagent or other article, intended by the manufacturer to be used alone or in combination, for human beings for one or more of the specific medical purposes of: investigation, replacement or modification of the anatomy or of a physiological process or state (pp., we suggest that it can not be seen how the manufacturer's intentions become irrelevant; on the contrary, they are referred to explicitly. further, rather than the indents defining medical purpose, the scope of medical purpose is constrained to the specific instances given in the indents. indeed, the consultation documents from the european commission and the uk's medical and healthcare products agency underscore the continued relevance of the medical/non-medical purpose distinction in relation to devices that replace or modify anatomy: their proposal of a positive list (in annex xv) of implantable or other invasive devices without a medical purpose would be redundant if any replacement or modification of anatomy were to constitute a medical purpose. again, the indents in the definition of a medical device serve to constrain, rather than define, medical purpose. thus, we rely not only on the court of justice decision, but also on the interpretation of the mdd offered by the mhra and other european commission documents. kuersten and hamilton perhaps think that there should not be a gap, or may think that the definitions should be interpreted differently. but the fact is, given the way the definitions are currently being understood by the relevant parties, it will take more than the cited revised definition of a medical device to bring ceds within the scope of the mdd. we were gratified that our paper prompted so much discussion. in reading the commentaries and articulating our responses, it became apparent that the comments and critiques served foremost to highlight the modest scope of our original proposals. whilst we still believe that pre-market approval is highly important for ceds especially for those that transfer energy across the skull the further proposals made in these commentaries serve to set an agenda for continued discussion of the optimum integrated policy response to ceds and, indeed, other new and emerging technologies designed for enhancement.

our (2014) model for the regulation of cognitive enhancement devices (ceds) received a great deal of interest from those involved in european device regulation and from academic commentators. further, since the publication of our recommendations, the number of manufacturers of brain stimulation devices for non-medical purposes has increased, underscoring the need for a regulatory response. in this paper, we clarify aspects of our original proposal and address additional regulatory issues beyond our original focus on the sale of devices. we begin with theoretical points pertaining to the definition of a ced and the distinction between treatment and enhancement. we then respond to practical challenges raised by the prospect of implementing our regulatory framework. next, we address some wider societal considerations relating to users and other stakeholders. finally, we revisit the broader regulatory context within which the various discussions are situated.

PMC5034395

pubmed-1312

oral hygiene habits are instilled in childhood itself irrespective of the nationality or geographic location of an individual. the most reliable and accepted method of oral hygiene maintenance the world over are mechanical methods of tooth cleaning but adjuvants for decreasing plaque formation and maintaining oral hygiene have been sought. presently chemotherapeutic agents are used as adjuvant agents to reduce plaque formation but they have their own disadvantages.1 kavala graha or gandoosha2 are procedures recommended for oral hygiene maintenance in ayurveda. it is described as a procedure in which an individual takes a comfortable amount of oil/medicated oil and holds it or swishes it in the mouth. when the oil turns thin and milky white it is spit out without swallowing.2 dr. f. karach popularised this procedure as oil pulling.3 he claimed that oil pulling can cure several illnesses including oral diseases, but his claims were not supported by evidence. recent studies of oil pulling therapy using sunflower oil4 and sesame oil5 were found to decrease plaque induced gingivitis. even though coconut oil is used for gargling among the people in coconut farming communities, no studies have been done on the benefits of oil pulling using coconut oil, to date. coconut oil is an edible oil and is consumed as a part of the staple diet in many tropical countries. coconut oil is different from most other dietary oils because the predominant composition of coconut oil is a medium chain fatty acid, whereas in the majority of other oils the basic building blocks are almost entirely long chain fatty acids. human breast milk is the only other naturally occurring substance with such a high concentration of lauric acid. lauric acid has proven anti-inflammatory effects and antimicrobial effects.678 therefore a study was conducted to assess the effect of coconut oil on plaque formation and plaque related gingivitis. the aim of the study was to evaluate the effect of coconut oil pulling/oil swishing on plaque formation and to evaluate the effect on plaque induced gingivitis. a total of 60 age matched subjects in the age-group of 16-18 years with plaque induced gingivitis were included in the study. the use of systemic or topical antibiotics and the history of dental treatment in the past one month were set as exclusion criteria. the study was designed to compare the baseline values and the post intervention values in a single group performing coconut oil pulling in addition to their oral hygiene routine. a thorough history regarding the medical condition and the medication taken in the past 6 months was obtained from the subjects. all the chosen subjects had a habit of brushing once or twice a day with toothbrush and paste. six subjects had the habit of flossing once in the night along with brushing twice a day. the subjects were advised to routinely perform oil pulling with coconut oil every day in the morning in addition to their oral hygiene routine. five subjects discontinued from the study as they could not tolerate the taste of the oil and three subjects discontinued from the study because of antibiotic usage during the period. modified gingival index9 and plaque index10 were used as the clinical measures to assess gingival inflammation and plaque formation respectively. plaque and gingival indices were measured at baseline that is, and on days 1, 7, 15, 30 after the oil pulling routine was started. reliability of clinical examination was tested for all the days of assessment and the interexaminer reliability was found to be substantial to good. the kappa coefficient scores were in the range of 65-92 [table 1a and b]. the mean gingival index was 0.91 and the plaque index was 1.19 at baseline. in comparison to the baseline values both the gingival and the plaque indices substantially reduced during the period of assessment. there was a steady decline in both the plaque index and the gingival index values from day 7. the average gingival index score on day 30 was down to 0.401 and the plaque index score was 0.385 [figures 1 and 2]. statistical analysis using the paired t test showed that the decrease was statistically significant [tables 2 and 3]. kappa scores for modified gingival index kappa scores for plaque index shows the mean and standard deviation of plaque index scores shows the mean and standard deviation of gingival index scores comparison of plaque index scores between baseline, 7, 15, and 30 days comparison of gingival index scores between baseline, 15, 30 and 45 days dental plaque is defined clinically as a structured, resilient substance that adheres to intraoral hard surfaces and is composed of bacteria in a matrix of salivary glycoprotein and extracellular polysaccharides. plaque induced gingivitis is the result of an interaction between plaque and the tissues and the inflammatory response of the host. it is associated with the subtle microbial alterations as the plaque matures.1112 oral hygiene measures using chemo mechanical procedures reduce the incidence of plaque related diseases by decreasing the plaque accumulation. our study aimed at checking the effectiveness of oil pulling with coconut oil as an adjuvant to brushing, in decreasing the plaque accumulation and plaque induced gingivitis. plaque index by sillness and loe10 and modified gingival index9 were used for clinical assessment in the study as they are the most widely used indices in trials for therapeutic agents.13 oil pulling with sunflower oil was found to significantly reduce plaque index and gingival index after 45 days.4 asokan et al., found oil pulling therapy with sesame oil was equally effective as chlorhexidine in decreasing plaque induced gingivitis.5 in our study also there was a significant decrease in the plaque and the gingival index at the end of 30 days. there are various hypotheses on the mechanisms by which oil pulling may act in decreasing the plaque and gingival index. in oil pulling, as the oil is swished in the mouth the mechanical shear forces exerted on the oil leads to its emulsification and the surface area of the oil is greatly increased. the oil film thus formed on the surface of the teeth and the gingiva can reduce plaque adhesion and bacterial co aggregation.5 it was also proposed that the alkalis in the saliva can react with the oil leading to saponification and formation of a soap like substance [figure 3] which can reduce the adhesion of plaque.514 coconut oil has a high saponification value and is one of the most commonly used oil in making soaps. the soaps produced with coconut oil can lather well and have an increased cleansing action.15 the lauric acid in the coconut oil can easily react with sodium hydroxide in saliva during oil pulling to form sodium laureate, the main constituent of soap16 which might be responsible for the cleansing action and decreased plaque accumulation. saponification reaction the significant reduction in gingivitis can be attributed to decreased plaque accumulation and the anti-inflammatory, emollient effect of coconut oil. in animal studies coconut oil was found to be an effective burn wound healing agent and this was attributed to its anti-inflammatory and antiseptic properties.17 coconut oil showed moderate anti-inflammatory effects on ethyl phenylpropiolate induced ear edema in rats, and carrageenin and arachidonic acid-induced paw edema.18 it was found be effective and safe when used as an emollient and moisturiser.19 there are many commercially available mouthwashes. listerine (phenol compound) and meridol (an amine/stannous fluoride mouthwash) were found to be less efficacious than chlorhexidine in controlling plaque induced gingivitis. after 3 weeks of rinsing, plaque indices remained the lowest in the chlorhexidine group, while subjects using listerine or meridol the score were similar but significantly lower than that of individuals rinsing with the placebo solution. the antimicrobial potential of chlorhexidine was found to be the highest followed by meridol.20 in our study there was a 50% decrease in the plaque and gingival index scores in 4 weeks which is comparable to the decrease produced by chlorhexidine. chlorhexidine on long term use alters taste sensation and produces brown staining on the teeth which is very difficult to remove. the mucous membranes and the tongue can also be affected and may be related to the precipitation of chromogenic dietary factors on to the teeth and mucous membranes.21 staining is also associated with the of long term use of phenol compound and stannous fluoride containing mouth washes.22 in the present study there were no reported alterations in the taste or noticeable staining from coconut oil at the end of 4 weeks. as an antimicrobial agent, chlorhexidine is effective against both gram positive and gram negative bacteria. its antibacterial action is due to an increase in cellular membrane permeability followed by coagulation of the cytoplasmic macromolecules.232425 it has also been shown that chlorhexidine can reduce the adherence of porphyromonas gingivalis to epithelial cells.26 pure-culture studies of 10 oral bacteria (eight genera) showed that actinomyces naeslundii, veillonella dispar, prevotella nigrescens, and the streptococci were highly susceptible to chx, while lactobacillus rhamnosus, fusobacterium nucleatum, were less susceptible.27 studies show that coconut oil also has substantial antimicrobial activity. it is shown to have significant antimicrobial activity against escherichia vulneris, enterobcater spp., including c. albicans, c. glabrata, c. tropicalis, c. parapsilosis, c. stellatoidea and c. krusei728 studies also show that coconut oil is affective against s. mutans and c. albicans in an in vitro oral biofilm model.29 the antimicrobial potency of coconut oil was not tested in our study. further studies with a have been planned to check the antimicrobial potential of coconut oil. the fact that a control group with a proven chemotherapeutic agent was not used is the major the limitation of our study. oil pulling has been proven to be an effective method in reducing plaque formation and plaque induced gingivitis. this preliminary study shows that coconut oil is an easily usable, safe and cost effective agent with minimal side effects which can be used as an adjuvant in oral hygiene maintenance. more studies on the antimicrobial potency of coconut oil on microorganisms causing oral diseases is required to authenticate the use of coconut oil as an effective oral antimicrobial agent. further studies on coconut oil with a large number of subjects and comparative studies using various chemotherapeutic agents can improve the quality of evidence.

background: oil pulling or oil swishing therapy is a traditional procedure in which the practitioners rinse or swish oil in their mouth. it is supposed to cure oral and systemic diseases but the evidence is minimal. oil pulling with sesame oil and sunflower oil was found to reduce plaque related gingivitis. coconut oil is an easily available edible oil. it is unique because it contains predominantly medium chain fatty acids of which 45-50 percent is lauric acid. lauric acid has proven anti inflammatory and antimicrobial effects. no studies have been done on the benefits of oil pulling using coconut oil to date. so a pilot study was planned to assess the effect of coconut oil pulling on plaque induced gingivitis. materials and methods: the aim of the study was to evaluate the effect of coconut oil pulling/oil swishing on plaque formation and plaque induced gingivitis. a prospective interventional study was carried out. 60 age matched adolescent boys and girls in the age-group of 16-18 years with plaque induced gingivitis were included in the study and oil pulling was included in their oral hygiene routine. the study period was 30 days. plaque and gingival indices of the subjects were assessed at baseline days 1,7,15 and 30. the data was analyzed using paired t test. results:a statistically significant decrease in the plaque and gingival indices was noticed from day 7 and the scores continued to decrease during the period of study. conclusion:oil pulling using coconut oil could be an effective adjuvant procedure in decreasing plaque formation and plaque induced gingivitis.

PMC4382606

pubmed-1313

taurodontism is defined as the enlargement of the pulp cavity of a molar tooth at the expense of root length. the name was given because of the apparent similarity between these teeth and those of ungulates, especially bulls. hence, we have the term taurodont, tauro from the latin term for bull and do nt from the greek term for tooth appears to be a continuous trait. identification of the taurodont teeth can only be made by radiographic examination as the external morphology of the teeth is within normal configurations. appearance of a taurodont tooth is very characteristic, and radiographic examination is the only way to visualize a rectangular configuration of the pulp chamber. the pulp chamber is extremely large with a greater apico-occlusal height and there is no cervical constriction of the teeth. these teeth have short roots and the bifurcation or trifurcation may be only few millimeters above the apices of the roots. it has been suggested that the anomaly represents a primitive pattern, a mutation, a specialized or retrograde character, an atavistic feature, an x-linked trait, familial or an autosomal dominant trait. although taurodontism has been reported in association with certain syndromes and some genetic defects, its true significance is still obscure. taurodontism appears most frequently as an isolated anomaly, but it has also been associated with several developmental syndromes and anomalies including amelogenesis imperfecta, down's syndrome, ectodermal dysplasia, klinefelter syndrome, tricho-dento-osseous syndrome, mohr syndrome, wolf taurodontism has also been reported to present with other rare syndromes such as smith magenis syndrome, williams syndrome, mccune most studies of its prevalence have, however, employed a categorical approach. extreme enlargement of the pulp cavity in an ancient population was first reported in neanderthal teeth from the krapina, who described it as a distinguishing feature of this middle palaeolithic human population. the prevalence of taurodontism is reported to range from 2.5% to 11.3% of the human population. this range is likely accounted for by variations in race and differences in diagnostic criteria. taurodontism is exhibited in 2.5-3.5% of the chromosomally normal caucasian population, and most of these teeth are hypotaurodontic. the aim of this retrospective study was to evaluate the frequency of the occurrence of taurodont molars in an indian dental school patient population using full-mouth periapical radiographs. in this paper, we also emphasize the diagnosis, etiology and anatomic and radiographic characteristics of taurodont teeth and also its association with various syndromes and anomalies. a total of 1000 patients retrospective full-mouth periapical radiographs, which were recorded in the department of conservative dentistry and endodontics, from january 2007 to december 2012, were screened. each of these patients had to have full-mouth periapical radiographs, had to be at least 18 years of age and had to be of indian origin. personal details including age, sex and race of all these patients were recorded to ensure that all patients were of indian origin. in order to obtain a balanced distribution between male and female patients, an equal number of (500) full-mouth radiographs of male and female patients were studied. the full-mouth radiographs were taken using kodak ultra-speed films (kodak, stuttgart, germany). the radiographs were placed on a viewing box and light surrounding the radiograph was blocked. if disagreement existed, a joint evaluation of all authors was made until a consensus was reached. shifman and chanannel proposed the following criteria for determining the presence of taurodontism, the distance between the cementoenamal junction and the floor of the pulp chamber is 2.5 mm and if the distance from the lowest point at the occlusal end of the pulp chamber (a) to the highest point at the apical end of the chamber (b) divided by the distance from a to the apex is 0.2 or greater [figure 1]. the relative incidence and the correlations regarding the location of taurodont teeth (maxillary versus mandibular and male versus female) were analyzed [table 1 and figure 2] using the chi-squared test. measurements based on the study by shifman and chanannel were used to determine the presence of taurodontism. (a) the lowest point at the occlusal end of the pulp chamber and (b) the highest point at the apical end of the pulp chamber. cej, cementoenamel junction distribution of teeth examined and taurodont teeth in the maxilla and mandible the relative incidence and the correlations regarding the location of taurodont teeth (maxillary and mandibular) in males and females one thousand patients, 500 male and 500 female, between the age of 20 and 70 years (average, 45 years) were included in this study. maxillary molars comprised 3805 teeth and mandibular molars comprised 3810 teeth [table 2]. of the 7615 molars examined, 40 (0.53%) teeth were found to have taurodontism. these teeth were detected in 28 (2.8%) of 1000 subjects, with 17 as males (60.71%) and 11 as females (39.28%). twenty-eight patients were found to have a taurodont molar teeth (24 teeth in male and 16 teeth in female, p=0.267). taurodontism was present in 17 of the 500 male patients (3.4%) and 11 of the 500 female patients (2.2%), p=0.250 [table 3 and figure 3]. distribution of right and left molars in the maxilla and mandible distribution of total subjects and subjects having taurodont teeth by gender p=0.250 (ns) distribution of total subjects and subjects having taurodont teeth by gender a cluster analysis of total taurodonts in the mandible (45%) versus the maxilla (55%) of both males and females combined showed a statistically significant difference (p<0.05; table 1). although there have been several studies reporting the prevalence of various dental anomalies, no reported study has been conducted on the prevalence of taurodontism in an indian population. in the present study, patients who reported in the opd of the department of conservative dentistry and endodontics, faculty of dental sciences, who either underwent restorative treatment or an endodontic procedure, providing an estimation of the prevalence of taurodontism in the indian sub-population were analyzed. the present data indicated that the occurrence of taurodontism in the indian population was 2.8%. there is one study in our knowledge that evaluated both maxillary and mandibular molars, reporting the prevalence of taurodontism in indians being 2.49%. in a recent study of various malocclusions, found a higher rate of 8.0% in jordanian dental patients; shifman and chanannel reported a prevalence of 5.6% in israeli patients, whereas macdonald-jankowski and li reported an even higher rate (46.4%) of taurodontism in an adult chinese population. the prevalence rates reported by previous studies range from 0.25% to 48% in different populations. in the present study, men presented a higher prevalence of taurodont teeth than women, although these differences were not statistically significant (p=0.250). however, the present observation is different from a previous study on asians that reported a higher prevalence in women. the diagnosis of taurodontism using panoramic radiographs is difficult; the anterior regions of both jaws may become distorted and this may result in an incorrect diagnosis of taurodontic molars. additionally, the differential diagnosis between taurodontic teeth and other teeth exhibiting large pulp chambers (patients with amelogenesis imperfecta seem to have large pulp chambers because of the lack of enamel) is of clinical significance. nevertheless, a reliable diagnosis of taurodontic molars has been shown to be possible using orthopantomograms. however, to ensure proper identification and the best accuracy of assessment, full-mouth periapical radiographs were used in the present study. on the basis of the external morphology, shaw proposed a classification of taurodont teeth, but later a more accurate approach was established by keen for the categorization of taurodontic teeth using internal morphology. he proposed the taurodont index as an objective method for the assessment and related the height of the pulp chamber to the length of the longest root. this index is used as a biological landmark that undergoes changes during the whole life. to overcome these changes, shifman and chananel used landmarks that are not modified by apposition of reparative dentine or morphological changes of the root anatomy. however, even this ratio can be affected by incomplete root formation or radicular resorption. in addition, root length varies by sex and ethnic groups, which may cast some doubt on the credibility of any metric method as a screening tool for taurodontism between different populations, and limits the comparability of the results of different studies. recently, a case report highlighted the use of high-end diagnostic imaging modalities such as spiral computerized tomography in making a confirmatory diagnosis of the multiple morphologic abnormalities such as taurodontism, dens invaginatus, pyramidal cusps of the premolars and dens evaginatus. from an endodontist's view, taurodontism presents a challenge during negotiation, instrumentation and obturation in root canal therapy. magnification devices such as magnifying loupes or surgical microscopes can be helpful to locate canal orifices, evaluate the pulp chamber and obturate the canals. the importance of taurodontism is felt more by endodontists while negotiating the complex internal anatomy of such teeth. shifman and buchner stressed on the importance of gaining access to the canal orifice, which is not difficult in such teeth due to the absence of reactional dentin that is present in normal teeth; however, this view was contradictory to the study by durr et al., which says that such a morphology could make the orifice location difficult, creating further difficulty during root canal preparation and obturation. hayashi reported mandibular taurodont tooth with five canals, wherein only three canals could be instrumented till the working length. he also explained the presence of extra root canals in terms of shape and number. it is therefore stressed that careful exploration of canal orifices and grooves is mandatory under the magnification the diagnosis of taurodont tooth is established. the voluminous pulp present in taurodont teeth needs to be removed completely for the success of treatment; it is here that the importance of irrigation comes into play. suggested copious irrigation with 2.5% sodium hypochlorite to remove pulpal tissue from the irregular canal walls in such teeth. grossman and meiman reported that irrigation with 5% sodium hypochlorite for 20 min to 2 h dissolves all pulpal tissue. moorer and wesselink demonstrated that one of the major factors in pulpal tissue removal was agitation with irrigation. it is highly recommended that agitation must be done with copious irrigation to get rid of the pulpal tissue in taurodont teeth. the proximity of the orifices and deeply situated opening of the canals made it difficult to obturate the canals with any single method of obturation. therefore, a combination of lateral condensation and warm vertical condensation technique can be performed to achieve the best results. within the limitations of this study, it was found that: the present study found the prevalence of taurodont molar to be 2.8%male have a higher prevalence rate then female (3.4% vs. 2.2%, respectively), with p=0.250maxillary molars are most commonly involving teeth than mandibular molars in males, while females have an equal frequency of taurodont teeth. the present study found the prevalence of taurodont molar to be 2.8% male have a higher prevalence rate then female (3.4% vs. 2.2%, respectively), with p=0.250 maxillary molars are most commonly involving teeth than mandibular molars in males, while females have an equal frequency of taurodont teeth. these variations in prevalence between different populations may be due to ethnic variations, but may also be influenced by differences in criteria used for the interpretation of taurodontism and also the specific teeth examined.

background: it is very important for dentists to be familiar with anomalies of teeth not only for the clinical complications but also their management. taurodontism also provides a valuable clue in detecting its association with various syndromes and other systemic conditions. the purpose of this study was to assess the prevalence of taurodont molars among a north indian population. materials and methods: a total of 1000 patients full-mouth periapical radiographs were screened. the radiographs were evaluated under optimal conditions using double magnifying glasses. a total of 7615 molars (including third molars) were evaluated. the relative incidence and the correlations regarding the location of taurodont teeth (right versus left side and maxillary versus mandibular) were analyzed using the chi-square test. results:twenty-eight patients were found to have a taurodont molar (11 women and 17 men [p=0.250]). the prevalence of taurodont molar was 2.8%. males had a higher prevalence rate than females (3.4% vs. 2.2%, respectively). a cluster analysis of total taurodonts in the mandible (45%) versus maxilla (55%) of both males and females combined showed a statistically significant difference (p<0.05). conclusion: the occurrence of taurodontism is basically based on racial expression in different populations. these variations in prevalence between different populations may be due to ethnic variations. the occurrence of taurodont molars among this indian population was rare.

PMC4357075

pubmed-1314

various environmental chemicals, industrial pollutants and food additives have been implicated as causing harmful effects. one such food additive is monosodium glutamate (msg) and it is sold in most open market stalls and stores in nigeria as the safety of msg's usage has generated much controversy locally and globally. in nigeria, most communities and individuals often use msg as a bleaching agent for the removal of stains from clothes. there is a growing apprehension that its bleaching properties could be harmful or injurious to the body, or worse still inducing terminal diseases in consumers when ingested as a flavour enhancer in food. despite evidence of negative consumer response to msg, reputable international organizations and nutritionist have continued to endorse msg, reiterating that it has no adverse reactions in humans. notably of such is the directorate and regulatory affairs of food and drug administration and control (fda&c) in nigeria, now nafdac has also expressed the view that msg is not injurious to health. msg improves the palatability of meals and thus influences the appetite centre positively with it resultant increase in body weight. though msg improves taste stimulation and enhances appetite, reports indicate that it is toxic to human and experimental animals. msg has a toxic effect on the testis by causing a significant oligozoospermia and increase abnormal sperm morphology in a dose-dependent fashion in male wistar rats. it has been implicated in male infertility by causing testicular hemorrhage, degeneration and alteration of sperm cell population and morphology. it has been reported that msg has neurotoxic effects resulting in brain cell damage, retinal degeneration, endocrine disorder and some pathological conditions such as addiction, stroke, epilepsy, brain trauma, neuropathic pain, schizophrenia, anxiety, depression, parkinson's disease, alzheimer's disease, huntington's disease, and amyotrophic lateral sclerosis. it can not be stated that msg is the cause of such varied conditions as epilepsy and alzheimer's disease, although there may be concerns of its involvement in its etiology. the fallopian tubes are paired, tubular, seromuscular organs whose course runs medially from the cornua of the uterus toward the ovary laterally at the upper margins of the broad ligaments between the round and utero ovarian ligaments. millions of tiny hair-like cilia which beats in waves hundreds of times a second catching the egg at ovulation and moving it through the tube to the uterine cavity, line the fimbria and interior of the fallopian tubes. other cells in the tube's inner lining or endothelium nourish the egg and lubricate its path during its stay inside the fallopian tube .. the tubal wall consists of three layers: the internal mucosa (endosalpinx), the intermediate muscular layer (myosalpinx), and the outer serosa, which is continuous with the peritoneum of the broad ligament and uterus, the upper margin of which is the mesosalpinx. the endosalpinx is thrown into longitudinal folds, called primary folds, increasing in number toward the fimbria and lined by columnar epithelium of three types: ciliated, secretory, and peg cells. in the ampullary and infundibular sections, secondary folds of the tubal mucosa also exist, markedly increasing the surface areas of these segments of the tube. the myosalpinx actually consists of an inner circular and an outer longitudinal layer to which a third layer is added in the interstitial portion of the tube. the serosa of the tube is composed of an epithelial layer histologically indistinguishable from peritoneum elsewhere in the abdominal cavity. this work was carried out to investigate some probable histological effects of msg on the fallopian tubes and its likely involvement in female infertility in nigeria. about 15% cases of female infertility, investigation will show no abnormality. in these cases abnormalities this study was given approval for the methodology and other ethical issues concerning the work by the university of benin research ethics committee. twenty four, (24) adult female wistar rats with average weight of 230 g were randomly assigned into three groups a, b and c of (n=8) in each group. groups a and b (n=16) serves as treatment groups while group c (n=8) was the control. the rats were obtained and maintained in the animal holdings of the department of anatomy, school of basic medical sciences, university of benin, benin city, nigeria. they were fed with growers mash obtained from edo feed and flour mill limited, ewu, edo state, and were given water liberally. the monosodium glutamate (3g/sachet containing 99+% of msg) was obtained from kersmond grocery stores, uselu, benin city. monosodium glutamate administration: the rats in the treatment groups (a and b) were given 0.04mg/kg and 0.08mg/kg of msg thoroughly mixed with the growers mash, respectively on a daily basis. the control group (c) received equal amount of feeds (growers mash) without msg added for fourteen days. the fallopian tubes were quickly dissected and fixed in 10% buffered formaldehyde for 24 hours and embedded in paraffin wax for routine histological techniques. the 0.04mg/kg and 0.08mg/kg msg doses were chosen and extrapolated in this experiment based on the indiscriminate use here in nigeria due to its palatability, and previous work done with this additive. the two doses were thoroughly mixed with fixed amount of feeds (550 g) in each group, daily. histological study: the fallopian tube tissues were dehydrated in an ascending grade of alcohol (ethanol), cleared in xylene and embedded in paraffin wax. ph otomicrographs of the desired results were obtained using digital research photographic microscope in the university of benin research laboratory. the fallopian tubes (ft) of the control group showed normal histological features, illustrating a well defined tubal wall which consists of three layers: the internal mucosa (endosalpinx), the intermediate muscular layer (myosalpinx), and the outer serosa (fig. the fallopian tubes of the treated groups showed some cellular hypertrophy (ch) of the columnar epithelium, distortion of the basement membrane separating the endosalpinx from the myosalpinx. there were degenerative and atrophic changes (adc) observed in some parts; these were more pronounced in those that received 0.08mg/kg of msg. there were marked vacuolations (v) and lysed red blood cells (lrbc), (0.08mg/kg treated rats) appearing in the stroma cells (figs. 2 and 3). adc: atrophic and degenerative changes; ch: cellular hypertrophy; v: vacuolation. treatment section of fallopian tubes (0.08mg/kg msg); h&e (mag. adc: atrophic and degenerative changes; v: vacuolation; ch: cellular hypertrophy; lrbc: lysed red blood cells. the results of the hematoxylin and eosin staining (h&e) reactions showed some cellular hypertrophy of the columnar epithelium, distortion of the basement membrane separating the endosalpinx from the myosalpinx. degenerative and atrophic changes were observed in some parts; these were more pronounced in those that received 0.08mg/kg of msg. there were marked vacuolations and lysed red blood cells (0.08mg/kg treated rats) appearing in the stroma cells. the increase in cellular hypertrophy of the columnar epithelium in the treatment groups as reported in this study may have been as a result of cellular proliferation caused by the improved intake of food which msg influences. the vacuolations observed in the stroma of the fallopian tubes in this experiment may be due to msg interference. degenerative and atrophic changes and lysed red blood cell which were observed were more pronounced in the groups treated with higher dose (0.08mg/kg) of msg. as a result of the distortion and disruption in the lumen of the fallopian tubes, the ciliary action and other functions of the fallopian tubes may have been highly affected as a result of probable toxic effect of msg. it may be inferred from the present results that higher dose and prolonged administration of msg resulted in degenerative and atrophic changes observed in the fallopian tubes. the actual mechanism by which msg induced cellular degeneration observed in this experiment needs further investigation. degenerative changes have been reported to result in cell death, which is of two types, namely apoptotic and necrotic cell death. pathological or accidental cell death is regarded as necrotic and could result from extrinsic insults to the cell such as osmotic, thermal, toxic and traumatic effects. in this experiment msg the process of cellular necrosis involves disruption of membrane's structural and functional integrity which was also a landmark of this experiment. cell death in response to toxins occurs as a controlled event involving a genetic programme in which caspase enzymes are activated. the results obtained in this study following the administration of 0.04mg/kg and 0.08mg/kg per day of msg to adult wistar rats caused some cellular hypertrophy of the columnar epithelium, distortion of the basement membrane separating the endosalpinx from the myosalpinx. degenerative and atrophic changes, which were more pronounced in those that received 0.08mg/kg of msg .

background: the effect of monosodium glutamate used as food additive on the fallopian tubes of adult wistar rat was investigated. material and methods: adult female wistar rats (n=24) of average weight of 230 g were randomly assigned into three groups a, b and c in each group (n=8). the treatment groups (a&b) were given 0.04mg/kg and 0.08mg/kg of monosodium glutamate thoroughly mixed with the growers mash, respectively on a daily basis. the control group (c) received equal amount of feeds (growers mash) without monosodium glutamate added for fourteen days. the growers mash was obtained from edo feeds and flour mill ltd, ewu, edo state and the rats were given water liberally. the rats were sacrificed on day fifteen of the experiment. the fallopian tubes were carefully dissected out and quickly fixed in 10% buffered formaldehyde for routine histological procedures. result:the histological findings in the treated groups showed evidence of cellular hypertrophy, degenerative and atrophic changes, and lysed red blood cells in lumen with the group that received 0.08mg/kg of monosodium glutamate more severe. conclusion:msg may have some deleterious effects on the fallopian tubes of adult female wistar rats at higher doses and by extension may contribute to the causes of female infertility. it is recommended that further studies aimed at corroborating these findings be carried out.

PMC3354428

pubmed-1315

however, oxygen also poses a potential hazard via reactive oxygen species (ros) and reactive nitrogen species (rns), with biological and functional alterations of lipids, proteins, and deoxyribonucleic acid (dna) [13]. therefore, ros/rns have been initially considered as harmful products of the normal aerobic metabolism. the control of ros/rns production plays physiological roles, especially, in regulating cell signaling to involve cell proliferation, differentiation, and apoptosis [13]. oxidative stress (os) mediated by free radicals is defined as an imbalance between the production of ros/rns and the antioxidant capacity of the cell [13]. the predominant inhibitory neurotransmitter in the brain is -aminobutyric acid (gaba), and almost all researchers have focused on gaba or the regulation of gaba receptor (gabar) in the brain [48]. currently, gaba is considered to be a multifunctional molecule with various physiological effects throughout the body [9, 10]. in the brain, many researchers have found that the regulation of gabar has preventive effects against os-induced damage [5, 7, 8]. these results in the brain were mainly explained via specific pathways against os (i.e., inhibition of the response to dna damage [5, 11, 12] and promotion of cell survival [13, 14] or the free radical scavenging system [15, 16]). however, in the liver, the effects of gabar regulation have not been reported. orthotopic liver transplantation (olt) is an accepted therapy for children and adults with end-stage liver disease, and it currently provides long-term survival and quality lifestyle. however, cold ischemia during organ storage and subsequent reperfusion severely damage the transplanted liver. during cold ischemic preservation, parenchymal cells swell and bleb, and then kupffer and endothelial cells trigger ros/rns production after warm reperfusion. this cold ischemia/warm reperfusion (ciwr) injury is still a major cause of morbidity and mortality after olt due to primary graft dysfunction or a nonfunctioning graft. reperfusion not only triggers the liver regeneration cascade but also causes fatal damage in the liver graft due to os [18, 19]. proactive strategies through pharmacological pretreatment to limit graft damage from ciwr injury have the advantage of excellent graft function after olt. a small-for-size graft (sfsg) is also an issue in deceased-donor liver transplantation (ddlt) and living-donor liver transplantation (ldlt). an sfsg is defined as a ratio of graft weight against standard liver volume<40% [20, 21]. an inevitable insufficiency of graft size can not be avoided in ldlt or split orthotopic liver transplantation (solt) for ddlt. shear stress not only triggers the liver regeneration cascade but also causes fatal damage in the sfsg by os [22, 23]. an sfsg in ldlt or solt is accompanied by ciwr injury, as well as shear stress with portal hypertension. the choice of a left-side graft is preferred from the viewpoint of greater donor safety and expanded donor candidates in ldlt [20, 24]. guaranteed solt with successful outcomes resolves a donor shortage in ddlt [24, 25]. our laboratory has focused on the effect of gabar regulation on liver damage by using rodent models [2628]. we failed to show beneficial effects in gabar regulation ex vivo and in gabar regulation by a specific antagonist [27, 28]. however, gabar regulation in vivo by a specific agonist showed a subtle reduction in liver damage in a murine hepatectomy model involving shear stress with portal hypertension and in a rat orthotopic liver transplantation model with a whole-liver graft involving ciwr injury. proactive strategies through pharmacological pretreatment to limit graft damage from ciwr injury and shear stress with portal hypertension have advantages for overcoming a current issue. as a final goal of gabar regulation in the liver, we investigated the strategic potential of graft pretreatment in vivo by a gabar agonist in the rat solt model with a 40%-sfsg, and we examined the possible pathways involved. graft donors and recipients were 812-week-old rats (approximately 250 g). the experimental protocols were approved by the ethical committee of our institution (mayo clinic, institutional animal care and use committee, no. rats were cared for in accordance with the institutional guidelines for animal welfare based on the national institutes of health guide for the care and use of laboratory animals. a dose of 43.56 nmol/g body weight of gabar agonist (gabaa receptor agonist, muscimol, 114.10 g/mol; 70015, fluka, sigma-aldrich co., st. four hours before graft harvest, the donor rat intravenously received 1.0 ml of gabar agonist into the penile vein. comprehensive details of the surgical procedures for rat solt and postoperative care in our institution have been previously described [29, 30]. briefly, the syngeneic graft had a cold ischemic time of 2 h at 4c in normal ringer's solution. the liver graft was washed twice by 10 ml of normal ringer's solution, immediately after the graft harvest and before graft implantation. the 40%-sfsg was made by the left median and lateral segments at the back table [29, 30]. to avoid any irrelevant signaling, the hepatic artery was reconstructed by ultramicrosurgery in this study [29, 30]. each rat was housed separately after surgery, and body temperature was maintained by a heating pad. postoperative observation was performed every 30 min until 6 h after solt, and 1.0 ml of warm lactate ringer's solution was routinely administered every 1 h until 6 h after solt. in this model, we previously demonstrated the importance of a shortened anhepatic phase and exclusion of unreliable samples based on autopsy findings [29, 30]. in this study, the anhepatic phase was maintained within 20 min in each solt, and no surgical complications were observed in each case at autopsy. recipient rats were divided into three groups according to donor treatments and the recipient's surgery as follows: (i) saline (normal saline, 1.0 ml, i.v.) and laparotomy, (ii) saline (normal saline, 1.0 ml, i.v.) and solt with 40%-sfsg, and (iii) gabar agonist (muscimol, 43.56 nmol (4.98 g)/g body weight, 1.0 ml, i.v.) and solt with 40%-sfsg. first, a survival study was performed (n=10 in each group). cell signalings involving cell proliferation, differentiation, and apoptosis were investigated from the early postoperative period [18, 3133], and subsequently, progressive necrosis was observed [18, 3133]. serum, plasma, and liver samples for histopathological/immunohistological assessment and western blotting analyses were then collected 6 h after solt (n=5 in each group). aspartate aminotransferase (ast), alanine aminotransferase (alt), and total bilirubin (t-bil) levels, and the international normalized ratio of prothrombin time (pt-inr) were measured. serum ast, alt, and t-bil levels were assessed (sgot, sgpt, and total bilirubin reagent, respectively, biotron, hemet, ca, usa). the pt-inr in plasma was measured by the i-stat system (abbott, princeton, nj, usa). liver tissue was fixed in 10% neutral-buffered formalin, embedded in paraffin, and sliced into 4 m sections. morphological characteristics and graft injury scores were assessed after hematoxylin-eosin (he) staining. the graft damage score (points) has previously been described elsewhere [30, 34, 35]. scores were counted in 10 fields (100) in each slide, and then these scores were averaged. induction of apoptosis was assessed by immunostaining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (tunel) (apoptag peroxidase in situ apoptosis detection kit, s7100, chemicon international, inc., billerica, ma, usa) and cysteine aspartic acid protease (caspase) 3 (cleaved caspase-3 (asp175) antibody, 9661s, cell signaling technology, inc., danvers, ma, usa). tunel-positive nuclei were stained brown, and negative nuclei were counterstained light blue. slides were scanned with an automated high-throughput scanning system (scanscope xt, aperio technologies, inc., vista, ca, usa). to quantify the immunohistological findings, positively stained nuclei were counted by aperio imagescope software (aperio technologies, inc.). all nuclei were classified into four color intensity levels, and the higher two levels were considered as positive. the ratio of positively stained nuclei to all nuclei was calculated, and the mean ratio per mm was determined. the primary antibodies for 4-hydroxynonenal (4-hne) (4 hydroxynonenal antibody, ab46545, abcam, cambridge, ma, usa), ataxia-telangiectasia mutated kinase (atm) (phospho-atm/atr substrate rabbit mab, cell signaling technology), phosphorylated histone h2ax (phospho-histone h2ax antibody, 2577, cell signaling technology), phosphatidylinositol-3 kinase (pi3k) (phospho-pi3k p85/p55 antibody, 4228, cell signaling technology), akt (phospho-akt rabbit mab, 4058, cell signaling technology), superoxide dismutase (sod) 1 (cu/zn superoxide dismutase, ls-b2907, lifespan biosciences, seattle, wa, usa), sod 2 (mn superoxide dismutase, ls-c62194, lifespan biosciences), and catalase (catalase, ls-b2554, lifespan biosciences) were used. liver samples were collected, homogenized, and centrifuged at high speed for 10 min at 4c. the supernatant was then collected and used for bicinchoninic acid protein determination (bca protein assay reagent, thermo fisher scientific, rockford, il, usa) and western blot analysis. forty micrograms of protein were run on 420% tris-glycine gels and transferred onto 0.45 m nitrocellulose membranes. the membranes were then blocked with 5% nonfat milk made up in a tris-buffered saline solution. after blocking, the next day, the membranes were washed three times for 10 min with tris-buffered saline solution and then incubated with the peroxidase-conjugated secondary antibody for 1 h, with shaking at room temperature. after incubation, the membranes were once again washed three times for 10 min with tris-buffered saline solution and then developed using chemiluminescence. signals were quantified by using imagequant 5.0 software (molecular dynamics, sunnyvale, ca, usa). the student's t-test was used for the comparison of unpaired continuous variables between groups. survival curves were constructed by the kaplan-meier method (log-rank test). solt with a 40%-sfsg clearly showed poorer survival than laparotomy (p<0.0001), and graft pretreatment by gabar agonist prolonged survival after solt (p=0.0369). inflammatory cell infiltration, vacuolization, hepatocyte ballooning, and necrosis were confirmed after solt with a 40%-sfsg. actual histopathological findings in h-e staining are shown in each group in figures 1(b)1(d). there were significant differences between laparotomy and solt with saline (0.0 0.0 versus 5.8 1.1 points, p<0.0001) and between solt with saline and solt with gabar agonist (5.8 1.1 versus 4.1 1.0 points; p=0.0280) (figure 1(e)). there were significant differences in serum ast levels between laparotomy and solt with saline (45.4 10.3 versus 387.4 36.8 u/l; p<0.0001) and between solt with saline and solt with gabar agonist (387.4 36.8 versus 296.0 32.3 there were significant differences in serum alt levels between laparotomy and solt with saline (54.2 9.2 versus 354.2 32.1 u/l; p<0.0001) and between solt with saline and solt with gabar agonist (354.2 32.1 versus 272.4 31.3 there were significant differences in serum t-bil levels between laparotomy and solt with saline (0.28 0.04 versus 1.37 0.29 mg/dl; p<0.0001) and between solt with saline and solt with gabar agonist (1.37 0.29 versus 1.02 0.15 mg/dl; p=0.0453) (figure 2(c)). there were significant differences in pt-inr between laparotomy and solt with saline (0.99 0.04 versus 1.22 0.06; p=0.0001) and between solt with saline and solt with gabar agonist (1.22 0.06 versus 1.13 0.06; p=0.0456) (figure 2(d)). the ratio of tunel-positive nuclei was significantly different between laparotomy and solt with saline (0.001 0.002 versus 0.166 0.052; p<0.0001) and between solt with saline and solt with gabar agonist (0.166 0.052 versus 0.092 0.038; p=0.0324) (figure 3(d)). the ratio of caspase 3-positive nuclei was significantly different between laparotomy and solt with saline (0.0001 0.0001 versus 0.115 0.019; p<0.0001) and between solt with saline and solt with gabar agonist (0.115 0.019 versus 0.080 0.024; p=0.0347) (figure 4(d)). normalized 4-hne showed significant differences between laparotomy and solt with saline (1.00 0.06 versus 1.38 0.22; p=0.0068) and between solt with saline and solt with gabar agonist (1.38 0.22 versus 1.05 0.15; p=0.0276) (figure 5(b)). actual intensities of atm and h2ax in each group are shown in figure 6(a). normalized atm showed significant differences between laparotomy and solt with saline (1.00 0.11 versus 1.21 0.11; p=0.0131) and between solt with saline and solt with gabar agonist (1.21 0.11 versus 0.90 0.28; p=0.0477) (figure 6(b)). normalized h2ax showed significant differences between laparotomy and solt with saline (1.00 0.10 versus 2.59 0.66; p=0.0007) and between solt with saline and solt with gabar agonist (2.59 0.66 versus 0.83 0.25; p=0.0005) (figure 6(c)). actual intensities of pi3k and akt in each group are shown in figure 7(a). normalized pi3k showed significant differences between laparotomy and solt with saline (1.00 0.11 versus 0.59 0.27; p=0.0139) and between solt with saline and solt with gabar agonist (0.59 0.27 versus 0.92 0.13; p=0.0443) (figure 7(b)). normalized akt showed significant differences between laparotomy and solt with saline (1.00 0.12 versus 0.34 0.24; p=0.0006) and between solt with saline and solt with gabar agonist (0.34 0.24 versus 1.11 0.22; p=0.0007) (figure 7(c)). actual intensities of sod 1, sod 2, and catalase in each group are shown in figure 8(a). normalized sod 1 showed significant differences between laparotomy and solt with saline (1.00 0.10 versus 0.81 0.16; p=0.0445) but not between solt with saline and solt with gabar agonist (0.81 0.16 versus 0.82 0.12; p=0.8248) (figure 8(b)). normalized sod 2 showed significant differences between laparotomy and solt with saline (1.00 0.13 versus 0.79 0.14; p=0.0361) but not between solt with saline and solt with gabar agonist (0.79 0.14 versus 0.84 0.15; p=0.5765) (figure 8(c)). normalized catalase showed no significant differences between laparotomy and solt with saline (1.00 0.14 versus 0.95 0.14; p=0.6904) and between solt with saline and solt with gabar agonist (0.95 0.14 versus 0.96 0.26; p=0.9764) (figure 8(d)). based on the current situation in the clinical field, the 40%-sfsg needs to be investigated in detail because successful solt overcomes a donor shortage in ddlt, and the shift to a left-lobe graft provides donor safety in ldlt [20, 24, 30]. however, the 40%-sfsg is prone to ischemia/reperfusion injury and shear stress with portal hypertension, and therefore, the os-induced damage after solt is more fatal [18, 3638]. in our study, a survival study, biochemical assays, and histopathological assessment showed that the 40%-sfsg received the liver injury enough. os causes dna damage and subsequent apoptosis [13], and in our study, immunohistochemistry showed that solt induced apoptosis in the 40%-sfsg. ros/rns can attack and damage a variety of critical biological molecules [13], and the products of lipid peroxidation reliably and rapidly reflect sensitive and specific signals due to os occurring in vivo [39, 40]. the fatty aldehyde 4-hne is an end product of lipoperoxidation [39, 40]. therefore, os after solt with a 40%-sfsg resulted in apoptotic induction and subsequent necrosis. os mediated by free radicals is defined as an imbalance between the production of ros/rns and antioxidant capacity [13]. ros/rns have been suggested as a major contributing factor for dna damage in the progression of os. as a sensor of dna damage responses, the protein kinase atm can be initiated through rapid intermolecular autophosphorylation induced by dna damage [12, 41]; it phosphorylates various proteins, and subsequently amplifies the responses to dna damage. h2ax is required for cell cycle arrest and dna repair following double-stranded dna breaks [5, 42]. dna damage results in the rapid phosphorylation of h2ax by atm at sites of dna damage [5, 4345]. our study showed that this response to and repair of dna damage via atm/h2ax was clearly triggered after solt with a 40%-sfsg and that this cascade is a possible pathway in the process of os-induced injury after solt with sfsg. our preliminary data in the rat olt model with whole-liver grafts (i.e., a model for only ciwr injury) suggested that gabar regulation by a specific agonist showed differences in atm/h2ax. we consider that gabar regulation may have a beneficial effect against ciwr injury via the atm/h2ax pathway in the liver. from the viewpoint of the production of ros/rns in the process of os, akt also plays a critical role in controlling apoptosis [41, 46, 47] and promotes cell survival [4750]. apoptotic machinery is inhibited by the activation of akt [46, 51, 52]. akt is a component of the antiapoptotic process related to the activation of pi3k, and pi3k is upstream from akt [47, 53]. the cell survival pathway via pi3k/akt is also considered as an important signaling pathway to control apoptotic induction in the liver [54, 55]. our study showed that this promotion of cell survival via pi3k/akt was disturbed after solt with a 40%-sfsg and that this cascade could be one of the possible pathways in the process of os-induced injury after solt with sfsg. our preliminary data in the murine hepatectomy model (i.e., a model for only shear stress with portal hypertension) suggested that gabar regulation by a specific agonist showed differences in pi3k/akt. therefore, we consider that gabar regulation may have a beneficial effect against shear stress with portal hypertension via the pi3k/akt pathway in the liver. from the viewpoint of antioxidant defenses, free radical scavenging enzymes, such as sod and catalase, also play an important role in reducing dna damage and subsequent apoptosis [2, 3, 56]. normal cells are able to defend themselves against os through this scavenging system [3, 56]. our study showed a decrease in sod 1 and sod 2 levels after solt with a 40%-sfsg, although we initially expected that antioxidant enzymes would increase. our results appear to be consistent with a previous opinion that os impairs mitochondrial importing and processing of sod. however, another possible explanation for our results may be that this scavenging system failed, and some reactive molecules evaded the detoxification process and damaged potential targets because of drastic damage after solt with a 40%-sfsg, even though these scavenging enzymes can handle large amounts of ros/rns. our results of the survival study, histopathological assessment, and biochemical assays showed that pretreatment for sfsg by gabar regulation in vivo affected graft damage after solt. moreover, immunohistochemistry showed that this pretreatment reduced apoptotic induction after solt. in the field of brain research, the effect of gabar regulation on the prevention of os has been reported [57]. although gaba was initially thought to be confined to the central nervous system, gaba is currently considered to be a multifunctional molecule with various physiological effects throughout the body [9, 10]. although the liver contains gaba and hepatic gabar [10, 17], the effects of gabar regulation in the liver are unknown. our study suggests that gabar regulation may have a strategic potential for 40%-sfsgs as a pharmacological pretreatment for reducing os-induced damage after solt, although solt with a 40%-sfsg involves fatal os due to dual damage (i.e., ciwr injury and shear stress with portal hypertension). whether gabar regulation ex vivo (i.e., a procedure during organ storage) is more suitable for ldlt is unknown. although our results showed the strategic potential of gabar regulation in vivo as a pretreatment for liver grafts, we failed to confirm a positive effect of gabar regulation ex vivo. therefore, some innovations are still required for clinical application. in previous reports on the brain, many investigators have suggested that gabar regulation by a specific agonist or antagonist affects the response to reduce os-induced injury [5, 7, 8]. their preventive effects in the brain have been mainly explained via specific pathways against os (i.e., inhibition of the response to dna damage [5, 11, 12] and promotion of cell survival [13, 14] or the free radical scavenging system [15, 16]). many previous investigators have suggested that gabar regulation in the brain has certain effects on the response to and repair of dna damage via the atm/h2ax pathway in vivo and in vitro in the process of os [5, 11, 12]. our study showed that the regulation of hepatic gabar also appeared to reduce os-induced dna damage via the atm/h2ax pathway as well as to have effects in the brain. with regard to the effects of gabar regulation on os in the brain, the pi3k/akt pathway promotes cell survival against dna damage [5, 13, 14, 46, 59]. our study showed that regulation of hepatic gabar appeared to promote cell survival via the pi3k/akt pathway against os-induced dna damage as well as to have effects in the brain. however, antioxidant enzymes reduce os-induced damage. from the viewpoint of this scavenging system, some researchers have shown that gabar regulation in the brain has preventive effects against os-induced damage via antioxidant enzymes [15, 16]. although sod 2 plays an important role in preventing dna damage in the sfsg, our results suggested that the effects of the regulation of hepatic gabar against os did not depend on this scavenging system. overall, we speculate that the regulation of hepatic gabar has a preventive effect against os, by reducing dna damage via the atm/h2ax pathway and by promoting cell survival via the pi3k/akt pathway. however, antioxidant enzymes might be important for gabar regulation in the brain [15, 16]. in conclusion, regulation of gabar by a specific agonist in vivo works well in the liver, as well as the brain. even though ciwr injury and shear stress with portal hypertension affect 40%-sfsgs after solt and results in fatal os, graft pretreatment in vivo by gabar regulation clearly improves graft damage after solt. this strategy may be advantageous for overcoming current issues in the ddlt and ldlt fields. the effects of gabar regulation on graft damage after solt with a 40%-sfsg appear to prevent os by reducing dna damage via the atm/h2ax pathway and by promoting cell survival via the pi3k/akt pathway.

background. graft pretreatment to limit postoperative damage has the advantage of overcoming a current issue in liver transplantation (lt). the strategic potential of graft pretreatment in vivo by a specific agonist for -aminobutyric acid receptor (gabar) was investigated in the rat lt model with a small-for-size graft (sfsg). methods. recipient rats were divided into three groups according to donor treatments and recipient surgeries: (i) saline and laparotomy, (ii) saline and split orthotopic liver transplantation (solt) with 40%-sfsg, and (iii) gabar agonist and solt with 40%-sfsg. survival was evaluated. blood and liver samples were collected 6 h after surgery. immunohistological assessment for apoptotic induction and western blotting for 4-hydroxynonenal, ataxia-telangiectasia mutated kinase (atm), histone h2ax, phosphatidylinositol-3 kinase (pi3k), akt, and free radical scavenging enzymes were performed. results. pretreatment by gabar showed improvement in survival, histopathological assessment, and biochemical tests. apoptotic induction and oxidative stress were observed after solt with an sfsg, and this damage was limited by gabar regulation. gabar regulation appeared to reduce dna damage via the atm/h2ax pathway and to promote cell survival via the pi3k/akt pathway. conclusions. pretreatment in vivo by gabar regulation improves graft damage after solt with an sfsg. this strategy may be advantageous in lt.

PMC3817746

pubmed-1316

we aimed to compare the international association of diabetes and pregnancy study groups (iadpsg) and the world health organization (who) criteria to diagnose gestational diabetes mellitus (gdm) in chennai, india. we reviewed the retrospective data of 1351 pregnant women who underwent screening for gdm at four selected diabetes centers at chennai (three private and one government). all women underwent an oral glucose tolerance test using 75 g glucose load and fasting, 1-h, and 2-h samples were collected. a total of 839 women had gdm by either the iadpsg or the who criteria, of whom the iadpsg criteria identified 699 and the who criteria also identified 699 women as having gdm. however, only 599/839 women (66.6%) were identified by both criteria. thus, 140/839 women (16.7%) were missed by both the iadpsg and the who criteria. 687/699 (98.2%) of the women with gdm were identified by the who criteria. in contrast, each value of iadpsg criteria i.e., fasting, 1 h, and 2 h identified only 12.5%, 14%, and 22%, respectively. a single who cut-point of 2 h>140 mg/dl appears to be suitable for large-scale screening for gdm in india and other developing countries. along with the rising tide of the current epidemic of diabetes in india, the prevalence of gestational diabetes mellitus (gdm) is also rising in india. however, the diagnosis of gdm has always been beset with problems related to differing diagnostic criteria with conflicting evidence regarding the maternal and fetal outcomes. till recently, the gdm diagnostic criteria proposed by world health organization (who) or the american diabetes association (ada) were followed in most countries. though the who recommendation was not based on studies with maternal and fetal outcomes, the 2-h cut-off value of>140 mg/dl for diagnosis of gdm was found to predict the neonatal outcomes in a fairly robust manner. recently, the international association of diabetes and pregnancy study groups (iadpsg) based on the hyperglycemia and adverse pregnancy outcome study has introduced new gdm criteria in an attempt to unify the gdm criteria throughout the world. the iadpsg criteria require three samples i.e., fasting, 1 h, and 2 h after 75 g glucose, whereas the who criteria need two samples namely the fasting and 2 h, although in practice, only the 2 h is used. in this paper, we have applied the who and the iadpsg cut-off values and compared the two criteria with respect to their impact on diagnosing gdm among pregnant women seen at four diabetic clinics in chennai city in southern india. we reviewed the retrospective data of 1351 pregnant women who underwent screening for gdm at four selected diabetes centers at chennai (three private and one government). at diabetes centers, pregnant women with a high index of suspension with elevated glucose levels hence, the prevalence of gdm at such center would be very high and hence they do not reflect the prevalence of gdm in the community. all women underwent an oral glucose tolerance test (ogtt) using 75 g glucose load and fasting, 1-h, and 2-h samples were collected. the iadpsg and who criteria were compared for diagnosis of gdm. according to the iadpsg criteria, any one of the following criteria was used for diagnosis of gdm, i.e., fasting 92 mg/dl (5.1 mmol/l), 1 h 180 mg/dl (10.0 mmol/l), or 2 h 153 mg/dl (8.5 mmol/l). according to the who criteria, either fasting 126 mg/dl (7.0 mmol/l) or 2-h value 140 mg/dl (7.8 out of a total of 1351 pregnant women who underwent screening for gdm at four diabetes clinics in chennai, 839 women were diagnosed to have gdm either by the iadpsg or by the who criteria. the iadpsg criteria identified 699/839 (83.3%) of the total number of women classified as gdm. the who criteria, again, identified 699/839 (83.3%) of the total number of women with gdm. however, as shown in the venn diagram [figure 1], only 559 women of the total 839 women with gdm (66.6%) were identified by both the iadpsg and who criteria. thus, 140/839 (16.7%) of the gdm women would have been missed if iadpsg criteria alone were used. conversely, 140/839 (16.6%) of the gdm women would have been missed if who criteria alone was used. venn diagram showing the gestational diabetes cases identified by both international association of diabetes and pregnancy study groups and world health organization criteria and by either criteria we next compared the usefulness of the who 2-h criteria alone in comparison to the full who criteria namely 2 h and fasting who criteria and the results are shown in table 1. use of the who 2-h criteria was found to identify 687/699 (98.2%) of the gdm cases identified by the full who criteria. comparison of the components of the international association of diabetes and pregnancy study groups and world health organization criteria to identify gestational diabetes mellitus table 1 also compares the percentage of gdm women identified by the fasting, 1 h and 2 h iadpsg compared to the full iadpsg criteria i.e., any 1 elevated value. it can be seen that in contrast to the 2-h who criteria where one value could pick up the majority (> 98%) of gdm cases, each value of the iadpsg criteria identified much lower percentages of gdm cases compared to using all three values. using the iadpsg criteria, the fasting value alone identified 12.5%, 1-h alone identified 14% and the 2-h alone identified 22% cases and using any two values identified only 51% of the gdm cases. hence, it is clear that all three values are needed to identify gdm cases by the iadpsg criteria. of the 88 women with fasting value alone elevated according to the iadpsg criteria, only 30 (34%) were picked up by who 2-h value. among the 98 individuals who had elevated 1-h value on iadpsg, only 47 (48%) were picked up by who 2-h value. comparing the 2-h cut-points of 140 mg/dl (who) and 153 mg/dl (iadpsg), it is seen that 113 women had a 2-h cut-point between 140 and 153 mg/dl. this study shows that the number of gdm cases identified at four selected diabetes centers in chennai is the same by who criteria and the iadpsg criteria. however, only 66.6% of the gdm cases identified by the who and iadpsg criteria are the same individuals, whereas 16.7% each of the gdm women identified by iadpsg and who criteria are different individuals. the who first proposed criteria for gdm using a 75 g ogtt in the 1980s. in its technical report published in 1994, it defined gdm as diabetes mellitus (dm) first recognized during pregnancy, and gestational impaired glucose tolerance (gigt) as impaired glucose tolerance (igt) first recognized during pregnancy. in 1998, who recommended new criteria. with regard to gdm, pregnant women who met the who criteria for dm or igt were classified as having gdm and, therefore, the term gigt disappeared. some studies have been published taking fasting plasma glucose (fpg)>126 mg/dl as the criteria for gdm. however, the more recent studies have altogether ignored the fpg criteria and have used only the 2-h>140 mg/dl criteria of the who. when the ada lowered the fpg to 100 mg/dl from the previous 110 mg/dl for diagnosis of impaired fasting glucose in non-pregnant adults, the fpg level of 126 mg/dl in pregnancy started looking too high and most people just chose to ignore the fpg level for the diagnosis of gdm. however, till date, there is no official recommendation from who to drop fpg criteria and to follow only the 2-h value of 140 mg/dl. it appears an anomaly that in the who criteria, the fasting cut-off had been set at 126 mg/dl which is diagnostic of diabetes in non-pregnant adults, whereas the 2-h cut-off was set at 140 mg/dl, which is the diagnostic cut-point for igt in non-pregnant adults. probably because of this inherent contradiction in the diagnostic criteria, the fasting values in the who criteria are not particularly useful to diagnose gdm and this might explain why the who 2-h value alone picked up over 98% of all cases diagnosed by both fasting and 2-h who criteria in this study. another point to be noted is that if a pregnant woman has a fpg 126 mg/dl, it is considered overt diabetes complicating pregnancy, and not as gdm, by the iadpsg criteria. another issue of concern is whether too many women would get diagnosed as gdm because of the low fpg cut-off in the iadpsg criteria. indeed, of the 88 women who were diagnosed as gdm by virtue of their fpg abnormality alone using iadpsg criteria, only 30 (34%) were classified as gdm by the who criteria. a similar comparison with those with gdm according to the iadpsg 1-h cut-off value showed that only 47/98 (48%) had gdm by who criteria. it is thus possible that by reducing the fpg cut-point to 92 mg/dl, we could be over-diagnosing gdm in normal pregnant women. we have earlier reported that the sensitivity of the 2-h value in the glucose tolerance test (gtt) is much higher than the fasting plasma glucose among non-pregnant indian adults. thus, it is reasonable to assume that since the iadpsg has raised the 2-h value in the iadpsg to 153 mg/dl, many cases of gdm could be missed. one of the limitations of the study is that with our present data, we can not conclude whether iadpsg or who criteria is better for indian pregnant women as we do not have data on the maternal and fetal outcomes. in the absence of the outcome data, which however, was beyond the purview of the study, it was not possible to comment on the suitability of diagnosing gdm by either of the two criteria in this population. nonetheless, this study compared the ease of use of two criteria in the population studied. future studies should compare the outcomes of the gdm cases diagnosed by iadpsg and who criteria as this would provide the final answer as to which criteria is more suitable for indians. the second limitation is that as the cases were selected from diabetes centers where women had been referred with suspected diabetes, this study can not be used to study the prevalence rates of gdm. this should be done in a maternity clinic or a general population where the entire population of pregnant women is screened to study the prevalence rates of gdm. this study however reflects the usefulness of the who and iadpsg criteria at a diabetes centers. however, the strength of the study is that it is the first to our knowledge, to directly compare the iadpsg and the who criteria, especially in an asian indian population. the who 2-h criteria of>140 mg/dl alone appears to be sufficient to diagnose gdm, as it picks up the majority of gdm cases diagnosed by both the whole who criteria as well as the same number of cases as the three sample iadpsg criteria. this could have great benefit especially in rural areas in india where obtaining three blood samples as required by the iadpsg criteria, could be a major challenge.

aim: we aimed to compare the international association of diabetes and pregnancy study groups (iadpsg) and the world health organization (who) criteria to diagnose gestational diabetes mellitus (gdm) in chennai, india. materials and methods: we reviewed the retrospective data of 1351 pregnant women who underwent screening for gdm at four selected diabetes centers at chennai (three private and one government). all women underwent an oral glucose tolerance test using 75 g glucose load and fasting, 1-h, and 2-h samples were collected. the iadpsg and who criteria were compared for diagnosis of gdm. results:a total of 839 women had gdm by either the iadpsg or the who criteria, of whom the iadpsg criteria identified 699 and the who criteria also identified 699 women as having gdm. however, only 599/839 women (66.6%) were identified by both criteria. thus, 140/839 women (16.7%) were missed by both the iadpsg and the who criteria. 687/699 (98.2%) of the women with gdm were identified by the who criteria. in contrast, each value of iadpsg criteria i.e., fasting, 1 h, and 2 h identified only 12.5%, 14%, and 22%, respectively. conclusions:a single who cut-point of 2 h>140 mg/dl appears to be suitable for large-scale screening for gdm in india and other developing countries.

PMC3784877

pubmed-1317

saudi arabia (sa) is a large country with an area of about 2.2 million square kilometers and total population exceeding 27 million, of which 69% are saudis. less than one quarter of cancer patients in sa present with localized disease. indeed, most cancer patients present with advanced disease with regional or distant extension. the crude and age standardized cancer incidence rates in 2007 were 52.3 and 82.1 per 100,000 populations, respectively. the five most common cancers, in descending order, are colorectal, non-hodgkin lymphoma, leukemia, lung, and liver in males; and breast, thyroid, colorectal, non-hodgkin lymphoma, and leukemia in females. the palliative care (pc) program at king faisal specialist hospital and research center (kfshrc), riyadh, was established more than 20 years ago as the first program of its kind in the country. the program delivers services for inpatients, outpatients, as well as for those at home through a home health care service. the program has also established a structured postgraduate fellowship for physicians pursuing subspecialty in palliative medicine. awareness of symptomatology pattern in cancer patients enables health care professionals to place appropriate emphasis on various challenges in a prioritized manner. prioritization process may include patient care plans, professional education plans, and the needs of health care services. furthermore, the quality of life of patients with advanced cancer could be significantly improved if specialized palliative care teams were involved in addressing the variety of symptoms that are common among these patients. therefore, impeccable identification and management of symptoms are two crucial aspects of palliative care. a recent report from 11 european countries suggests that there are variations between countries in the number of inadequately treated cancer-related symptoms. to the best of our knowledge, no previous report has described the prevalence and severity of nonpain symptomatology among patients with advanced cancer in saudi arabia (sa). this paper aims at determining the prevalence and severity of 10 nonpain symptoms in cancer patients visiting the pc outpatient clinic at kfshrc, riyadh, sa. this is a cross-sectional survey exploring the pattern of 10 common nonpain symptoms in an outpatient palliative care clinic. the research advisory council (rac) of kfshrc have approved the research project (rac number 2101053) based on the approval of the research ethics committee. adult cancer patients who were oriented to person, place, and time were consecutively included in the study. the 10 nonpain symptoms included tiredness, nausea/vomiting, anxiety, depression, shortness of breath, drowsiness, insomnia, dry mouth, loss of appetite, and confusion. the intensity of each symptom at the time of the encounter in the clinic was determined by each patient based on a 010 numerical scoring system where zero means the absence of respective symptom and 10 means the greatest severity a person could possibly imagine. the performance status was determined using the palliative performance scale (pps), which is a reliable and valid observer-rated tool for measuring performance status in pc patients. the pps consists of five domains (on a scale of 0%100%, with increments of 10%), namely ambulation, self-care, activity level/evidence of disease, intake, and conscious level. all interviews were conducted by one of the authors (oa) to avoid the potential of inter-rater variability. data analysis was conducted using the software package sas version 9.2 (statistical analysis system, sas institute inc., the student's t test for independent samples was used to compare means between two groups, such as those related to gender, age group, encounter type, and performance status. one-way analysis of variance was used for comparing the means among more than two groups such as in the case of grouping according to cancer type. a value of p less than 0.05 was considered significant. a total of 124 patients were interviewed, with a median age of 56 years and dominance of female gender (59%). one third of the patients were seen on follow-up basis and the rest were new to the palliative care outpatient clinic. all patients had advanced incurable cancer, with the majority (102; 82.3%) diagnosed with metastatic disease. for patients who were still receiving anticancer treatment, the most frequently encountered cancer types were female breast (27.4%), head and neck (15.3%), and genitourinary (12.9%). only one patient denied any nonpain symptom while all other patients had from 1 to 10 nonpain symptoms per patient, with a mean number of symptoms of 5.1 2.2. the majority of patients (93; 75%) had more than 3 nonpain symptoms each. the mean number of symptoms was higher in patients with pps of less than 70% (5.9 1.9) as compared with those who had better performance status (4.6 2.3) (p=0.002). however, the mean number of symptoms did not show any statistically significant difference between groups of patients based on age, gender, cancer type, or the type of outpatient encounter. the most frequently reported symptoms were tiredness (79.8%), loss of appetite (71.8%), dry mouth (69.4%), and anxiety (60.5%), whereas the least prevalent symptoms were nausea and confusion (22.6% each) [table 2]. for symptomatic patients, the mean severity scores were highest for tiredness (5.1) and loss of appetite (5) and lowest for nausea and confusion (3.4 each) as shown in table 2. older patients (above the median age, 56 years) had lower mean anxiety score compared with younger patients, 1.9 and 3.2, respectively (p=0.003). females had higher mean severity scores than males in 4 symptoms, namely, tiredness (4.6 vs 3.5; p=0.04), nausea/vomiting (1.0 vs 0.4; p=0.02), anxiety (3.2 vs 1.7; p=0.001), and loss of appetite (4.2 vs 2.7; p=0.003). patients with lower pps tended to have significantly higher severity scores for tiredness, shortness of breath, drowsiness, dry mouth, and loss of appetite, see table 2. the mean severity scores of symptoms did not significantly differ between patients according to their cancer type or the nature of outpatient visit (new vs follow-up). however, when patients with primary or secondary lung or pleural disease were pooled in one group, they had higher mean severity scores of shortness of breath compared with other patients, 2.4 and 1.2, respectively (p=0.01). symptom prevalence and severity the participation of all approached patients in our study was not unexpected because the questions were all focused on the presence and severity of symptoms known to be fairly common among cancer patients. the median age of our study population agrees with the findings of a previous report on a larger group of advanced cancer patients in sa and appears generally younger than the median age of the palliative care population in western countries.[911] this might be explained by the fact that more than 97% of the saudi population represents those younger than 65 years. the finding that metastatic or locally advanced disease is the rule in the studied population may be explained by the trend of delayed diagnosis of cancer in sa as well as the common practice of delayed referrals to palliative care in kfshrc. symptom prevalence studies on patients with advanced cancer have mostly targeted inpatients rather than outpatients. furthermore, most reports have major variations in demographics of surveyed populations and in methodologies implemented, which obviously make comparisons between the findings of various reports less meaningful. that being said, our findings have shown higher prevalence of nonpain symptoms among patients with advanced cancer compared with what other researchers have reported. for instance, grond and colleagues have reported that cancer patients attending an outpatient pain clinic were having on average 3.3 nonpain symptoms the most prevalent of which were insomnia (59%) and loss of appetite (48%). according to several reports, it seems that the more symptoms are actively assessed the more symptoms will likely be reported.[1517] similar to our results, there is a common finding among various reports that tiredness (also reported as fatigue or weakness in some studies) is often on the top of the list of prevalent nonpain symptoms among patients with advanced cancer.[1620] potter and coauthors have reported nonpain symptoms in patients seen at an outpatient pc clinic in the uk, with the most prevalent symptoms being tiredness and nausea (18% each) and loss of appetite (17%). a recent report from lebanon has shown high prevalence of fatigue (88.4%), dyspnea (65.1%), depression (53.5%), and confusion (37.2%) among inpatients with advanced cancer. alshemmari et al. from kuwait have studied cancer patients who were largely having advanced incurable disease and found the most common nonpain symptoms to be fatigue (80%), anorexia (67%), weight loss (49%), and dyspnea (42%). overall, the severity of symptoms reported by our patients was generally more than what has been reported elsewhere on a group of cancer patients hospitalized for palliative reasons. our findings have supported previous reports suggesting that poorer performance status may be associated with higher symptom burden. in addition, we have found that some symptoms might be associated with higher severity scores in patients with poor performance status. have found, our data failed to show an association between symptom prevalence and age or gender of patients. however, younger patients in our study had higher severity scores of anxiety than older patients. also, females reported higher severity scores as compared with males in few symptoms including anxiety. our finding that patients with primary or secondary lung or pleural cancer were having severe shortness of breath supports what has been suggested by kirkova et al. that some symptoms may vary in prevalence or severity (or both) based on cancer site. in the pc outpatient clinic at kfshrc, it is common for patients relatives to attend follow-up clinics on behalf of patients for the sake of medication refill, hence the lower proportion of follow-up patients as compared with new patients in our study. however, one reason could be the fact that many patients reside outside riyadh and, like most saudis, have widely extended families with good chances of having relatives living in riyadh who would be happy to visit the clinic on behalf of the patients. although air fares for health care-related travel are usually covered by the government, some patients may find it difficult to attend the clinic in person due to poor performance status. however, we are under the impression that a good number of patients who prefer to send relatives to attend the clinic on their behalf might be having symptoms that are fairly controlled. this could partially explain the high prevalence and severity of symptom burden in our sample as well as the finding that symptom prevalence and severity in our study was similar in patients new to the pc clinic and those returning to the clinic for follow-up. limitations of our study include the relatively small number of participants and the cross-sectional nature of the study design. a longitudinal design with sequential assessments of a larger group of patients from different centers could have generated more generalizable data. a much longer list of nonpain symptoms could have been better representatives of symptom burden among this group of patients. future multicenter research on the same lines may be focused on longitudinally assessing symptom burden in such patients, with emphasis on exploring the efficiency and effectiveness of symptom management strategies in pc outpatient settings. despite the wide variation in reports that explored symptom pattern and prevalence in patients with advanced cancer, it is clear that they all agree upon the fact that patients with advanced cancer are polysymptomatic. the significant prevalence and severity of nonpain symptoms among new and follow-up cancer patients seen in a pc outpatient clinic emphasizes the need for comprehensive assessment and routinely audited symptom management plans.

background: epidemiology of cancer-related nonpain symptoms receives less attention in literature as compared with cancer pain. objective:this paper aims at exploring the prevalence and severity of nonpain symptoms in cancer patients attending a palliative care (pc) outpatient clinic. materials and methods: over a 5 months period, consecutive adult cancer patients attending pc outpatient clinic at a tertiary hospital were evaluated for the presence and severity of 10 nonpain symptoms. patients were grouped to new or follow-up cases and were also grouped according to performance status and cancer type. prevalence and severity of symptoms were compared between groups using t test or analysis of variance as appropriate. results:fifty-one males and 73 females were interviewed. the most common cancer is female breast (27.4%) followed by head and neck (15.3%). majority of patients (67%) were new to pc clinic. patients had 5.1 nonpain symptoms on average, with most common symptoms being tiredness (79.8%), loss of appetite (71.8%), dry mouth (69.4%), anxiety (60.5%), and depression (50.8%). the least common symptoms were confusion and nausea (22.6% each). the median scores of severity were highest for tiredness, loss of appetite, dry mouth, and insomnia (5 points each). symptoms were fewer among patients with good performance status (p=0.002), whereas age, gender, cancer type, and encounter type were not associated with difference in symptom prevalence. younger patients, females and those with poor performance status have shown a tendency toward higher severity scores for several symptoms. conclusion:the significant prevalence and severity of nonpain symptoms among new and follow-up cancer patients seen in a pc outpatient clinic emphasizes the need for comprehensive assessment and routinely audited symptom management plans.

PMC3477372

pubmed-1318

tuberculosis (tb) is common disease in developing and developed countries caused by various strains of mycobacteria, usually mycobacterium tuberculosis (m. tuberculosis)14). the most common initial infection site is the lung, but the brain can be infected by hematogenous transmission. tb meningitis and tuberculoma are the two most common forms of tb of the central nervous system. surgical treatment is only recommended when medical therapy fails, decompression is necessary, or the diagnosis is uncertain9,15). transient worsening, appearance of new symptoms, or radiological manifestations of tb can develop after initiating of anti-tb medication4). these paradoxical responses are not uncommon in human immunodeficiency virus-negative patients and have been observed in 6-30% of patients with a tb infection6,7). these episodes of paradoxical deterioration are commonly associated with extrapulmonary tb and the central nervous system is the most common location for this presentation. however the lesion revealed indistinguishable radiological findings of a malignant intracranial tumor such as a high grade glioma after anti-tb treatment. we describe the radiological findings of the paradoxical response of a cerebral tuberculoma mimicking a malignant brain tumor. she underwent a chest computed tomography (ct), which showed minimal cardiac fluid collection with pleural effusion but without pleural enhancement or thickening. the pleural fluid was an exudate, with a white blood cell count of 907/mm and 30% lymphocytes. the quantiferon tb and immunoglobulin m for the ebstein-barr virus capsid antigen was positive. she refused anti-tb medication, and her symptoms improved after aspirin and steroid treatment. brain magnetic resonance images (mri) showed 18 mm and 6 mm sized lesions on the pons and occipital lobe, respectively. the lesions were hypointense on t1-weighted images, and iso-intense with a central hyper-signal on t2-weighted images associated with perilesional edema and rim-enhancement after contrast administration (fig. the diffusion images showed diffusion restriction in the central area but no increased cerebral blood volume on mr perfusion images (fig. a follow-up chest ct showed randomly distributed miliary nodules and mixed small centrilobular nodules, suggesting pulmonary tb with miliary dissemination. anti-tb medication was started with isoniazid, rifampicin, ethambutol, and pyridoxine. at the 1 month follow-up, the thickened, peripherally enhanced lesions were enlarged and associated with aggravated perilesional edema (fig. h mr spectroscopy revealed increased choline, lactate and lipid peak, and reduced n-acetyl aspartate and creatine, suggesting a high grade glioma rather than tuberculoma (fig. 2), and pcr for tb was positive. her medication was changed to isoniazid, rifampicin and ethambutol. after 13 months of medication, the right hemiparesis improved and the lesion had decreased in size with decreased perilesional edema (fig. she continued the anti-tb medication to treat the remaining lung and brain lesions. involvement of the central nervous system (cns) is the most severe form of tb11). a ring-enhancing lesion can be seen in intracranial infections or tumors such as tuberculomas, neurocysticercosis, bacterial cerebral abscesses, neurosarcoidosis, cerebral metastases, glioblastomas, and cns lymphomas8). a caseating tuberculoma with a solid center shows ring and central heterogeneous enhancement and is iso- to hypointense on t1- and t2-weighted mri. a caseating lesion with a liquid center shows ring enhancement and is hypointense on t1- and hyperintense on t2-weighted mri. intense focal gyral enhancement can be seen, and a secondary cerebral infarction from obliterative end arteritis can be associated with intracranial tb16). a tuberculoma with liquid necrosis shows restricted diffusion with a low apparent diffusion coefficient, whereas those with solid necrosis do not. h mr spectroscopy of the lesion reveals lipid with increased choline and reduced n-acetyl aspartate and creatine12). mr perfusion also shows increased cerebral blood volume on initial images, which gradually normalize with treatment, suggesting healing of the lesion12). the diagnosis is established based on the pathology results of a biopsy or detecting m. tuberculosis dna in a pcr study2). treatment of a tuberculoma is based on anti-tb treatment regimens, which include two important antibiotic agents10). isoniazid and rifampicin can be used for several months with combinations of other agents due to bacteria resistance. the initial treatment of extrapulmonary tb includes these two agents and the brain is not an exception. expansion of an intracerebral tuberculoma or newly detected lesions can be seen on follow up images after anti-tb medication, which is called a paradoxical response or paradoxical progression5,17). the tuberculomas increase in size at 1-7 months after starting chemotherapy. the explanation for this paradoxical response during therapy remains unclear, but it may be related with local tissue reactivity17). the brain is known as ' immunologically privileged ', meaning that immune reactivity in brain is selective and modified. foreign antigens including pathogens deposited in the brain parenchyma are not detected efficiently by the immune system in the cns. the experimental data showed that peripheral immune sensitization can result in the immune-mediated delayed-type hypersensitivity response, which can be used to explain ' paradoxical reaction ' of intracranial tuberculoma3). once the active tb is under control with chemotherapy, enhanced delayed type hypersensitivity can cause activation and accumulation of lymphocytes and macrophages at the site of bacillary deposition or toxin production1). therefore, many factors may combine to produce the paradoxical response, which might be related to a host immune response, virulence of the tubercle bacilli, the infection site, antigen load, or the effects of chemotherapy. these aggravated lesions can be misdiagnosed as treatment failure or other tumorous pathology. in our case, after 1 month of treatment, our patient developed worsening symptoms, radiological aggravation with tumoral spectrum on h mr spectroscopy, and increased blood volume on mr perfusion. these findings mimicked a high grade glioma even if this center of the lesion with liquid necrosis showed a diffusion restriction. initial improvement followed by deterioration despite adequate diagnosis and treatment is strong evidence to suspect the paradoxical response on clinical grounds. the response can be effectively managed by continuing the anti-tb drugs, and eventual clinical improvement is observed in almost all reported cases. systemic corticosteroids are probably indicated to manage symptoms and cerebral edema for most patients with a cerebral paradoxical response. however, careful consideration of corticosteroid administration must be given due to possible complicating factors such as the host's immune status or the presence of concomitant infections. surgical intervention should be considered for patients with increased intracranial pressure or an uncertain diagnosis. brain tuberculoma could be aggravated mimicking brain malignancy during administration of anti-tb medication. this paradoxical response can be effectively managed by continuing the anti-tb drugs with careful radiologic follow-up.

we report a case of a paradoxical response of a tuberculoma in the brain mimicking a brain tumor. a 76-year-old woman presented with a 2 week history of headache, dysarthia, and orthopnea. brain magnetic resonance images (mri) revealed two rim-enhancing lesions on the pons and occipital lobe, and chest computed tomography showed randomly distributed miliary nodules. the tentative diagnosis was tuberculosis (tb) of the brain and lung. she complained of right hemiparesis and worsening general weakness after taking the anti-tb medication. on the monthly follow-up images, the enhanced lesions were enlarged with increased perfusion and choline/creatinine ratio, suggesting a high grade glioma. a surgical resection was completed to diagnose the occipital lesion, and the tuberculoma was pathologically confirmed by a positive tb-polymerase chain reaction. the anti-tb medication was continued for 13 months. a follow-up mri showed decreased size of the brain lesions associated with perilesional edema, and the clinical symptoms had improved. brain tuberculoma could be aggravated mimicking brain malignancy during administration of anti-tb medication. this paradoxical response can be effectively managed by continuing the anti-tb drugs.

PMC4414779

pubmed-1319

left ventricular noncompaction (lvnc) is a rare primary congenital cardiomyopathy arising from intrauterine arrest of myocardial compaction. it results in persistent prominent ventricular trabeculations and deep intertrabecular recesses and is characterized morphologically by 2 layers i.e., thin compacted epicardial layer and thickened noncompacted endocardial layer.1) the prevalence of lvnc is approximately 0.05%-0.24% in the general population according to echocardiography, the diagnostic tool of lvnc.2) the etiology of lvnc is unidentified; however, possible linkage between the disease and various genetic background is recently suspected.3) diverse clinical manifestations previously reported range from no symptom to the development of heart failure, arrhythmias, systemic embolic events or sudden cardiac deaths.3) interestingly, several abnormal electrocardiograms (ecgs) were reported in lvnc patients but none had the feature of left atrial (la) standstill imitating atrioventricular (av) nodal block. we described a 29-year-old man who presented with a symptom of cerebrovascular accident (cva) and concomitant asymptomatic lvnc and la standstill. a 29-year-old man with the history of stroke was transferred from a local medical center to determine whether he should receive pacemaker insertion due to abnormal ecg findings. at first, he was admitted to a local clinic with dysarthria a month prior to the transfer, and was diagnosed as left middle cerebral artery territory infarction. his brain magnetic resonance imaging (mri) demonstrated multiple cortical infarcts implying embolic origin (fig. 1). magnetic resonance angiography findings showed no evidence of pathologic stenosis or atheroma of the neck and head arteries. asymptomatic bradycardia was shown on the local ecg with 2:1 av block reported on local holter monitoring (seer light, ge medical, usa). there was neither specific family history nor past history of specific diseases except the recent stoke. the initial ecg obtained after transfer showed bradycardia (55 beats/min), pr prolongation followed by av block characteristic of second degree av block, mobitz type 1 with left axis deviation and poor r wave progression (fig. the patient's vital signs were as follows: blood pressure, 120/80 mmhg; heart rate, 49 beats/min; respiratory rate, 20 breaths/min; temperature, 36.7. he had white blood cell count 802010/l (neutrophil 68.5%); hemoglobin, 15.1 g/dl; platelet count, 202000/l; and serum c-reactive protein, 0.42 mg/dl. additionally, serum electrolyte, serum creatine kinase-myocardial band, serum troponin i level and n-terminal pro b-type natriuretic peptide were within normal range. treadmill test was performed for the evaluation of a chronotrophic response; however, the patient's maximum heart rate observed during bruce protocol stage 3 was 117 beats/min without reaching the target heart rate of 163 beat/min. transthoracic echocardiography demonstrated a loss of a wave mimicking atrial fibrillation with peak e wave velocity of 59.72 cm/sec and deceleration time of 183 msec. global left ventricular (lv) ejection fraction was 67% with e/e ' of 5.24 implying normal lv systolic function and filling pressure. end diastolic diameters of each ventricle were within normal ranges i.e. 51.80 mm on the left and 33.10 mm on the right ventricle. contrast echocardiography showed flow communication between multiple intertrabecular recesses and lv cavity with noncompacted/compacted ratio of approximately 3:1 (fig. 3b). cardiac mri demonstrated prominent trabeculations and deep intertrabecular recesses with thinner compact epicardial layer of the lv characteristic of lvnc (fig. 4). mild spontaneous echo contrast in la appendage without definite thrombi and decreased mean la appendage flow velocity of 19.93 cm/sec were observed on transesophageal echocardiography with no definite atherosclerotic change from the aortic arch to thoracic aorta. eletrophysiologic (ep) test was performed to evaluate the cause of arrhythmia and to determine pacemaker insertion. 5a); interestingly, although there was 1:1 av relationship during pacing from high right atrium (ra), there was no atrial signal on coronary sinus (cs) bipole with no correlation between atrial and ventricular signal while pacing from ra appendage that implied absence of electrical activity of la on ep study (fig. for further corroboration of the results, we carried out an additional ep study using 3 dimensional voltage mapping (fig. the map showed no voltage signal of the entire la, septum and body of ra, which explained the capture failure during pacing of ra septum and la. based on the prior results, we decided to initiate warfarinzation keeping in mind permanent pacemaker insertion; however, we could not perform the procedure due to the patients ' refusal. he was discharged from hospital and remained asymptomatic without further complication until the latest follow up visit. we reported a case of a young male patient with concomitant lv noncompaction and la standstill that has not been reported before. the etiology of cva in this case can be explained by 2 conditions: firstly, the noncompaction itself as a complication; and secondly, low velocity in the la appendage. lvnc was first described as " spongy myocardium " in 1975 by dusek et al.4) and is characterized by morphological abnormality of prominent trabeculae representing " persistent sinusoids " and myocardial wall thickening; the new term " isolated non-compaction of lv myocardium " was introduced by chin et al.5) in 1990 emphasizing deep recesses communicating with the cavity of lv and absence of other cardiac deformities. although it is defined as a primary cardiomyopathy by the american heart association, it still remains as a " unclassified " cardiomyopathy according to the world health organization.6) the etiologies of isolated lvnc are yet obscure, though several genetic mechanisms that might be involved in the arrest of myocardial development during embryogenesis were recently suggested.3) the various clinical manifestations of lvnc reportedly include congestive heart failure, systemic embolic events, and arrhythmias. the degree of symptoms usually depends on the grade of noncompaction and ventricular function.3) two representative methods used for the diagnosis of lvnc are echocardiography and cardiovascular magnetic resonance (cmr). generally, the diagnosis of lvnc largely depends on 2 dimensional/3 dimensional echocardiography, with or without contrast echocardiography due to cost-effectiveness. we applied 3 different echocardiographic diagnostic criteria by chin et al.,5) jenni et al.,7) and stllberger et al.8) based on parameters of ventricular morphology, although there is no officially established diagnostic criterion; therefore, multimodal imaging approach inevitably improves diagnostic accuracy. in case of low echocardiographic image quality, cmr shows better signal to noise ratio, contrast to noise ratio, unlimited imaging planes and ability to use tissue characterization. there are 2 cmr criteria proposed by petersen et al.9) and jacquier et al.,10) either using the ratio of noncompact/compact myocardium depth (> 2.3) or trabecular mass (> 20%). management of lvnc focuses on standard medical therapy for systolic and diastolic ventricular dysfunction and prevention of systemic embolic events.3) we applied warfarin for the prevention and treatment of cva. there is controversy regarding cardioverter-defibrillator (implantable cardioverter-defibrillator) implantation in patients with lvnc but should be considered as primary prevention in high risk patients; alternatively, cardiac transplantation can be considered in these patients.11) once lvnc is diagnosed, family screening of first degree relatives is recommended.3) the prognosis of lvnc is still controversial. initial reports showed very poor prognosis in patients with lvnc; however, recent studies supported a better prognosis.6)12) one study showed 15% mortality among lvnc patients during a mean 30 month follow-up.12) previously reviewed studies and case reports indicate various ecg findings among the lvnc patients; for example, normal ecg, atrial fibrillation, bundle branch block, qt interval prolongation, left ventricular hypertrophy, av block, and repolarization abnormality.13) there were several cases of both atrial standstill in lvnc patients; however, our patient showed isolated la standstill that has not been reported previously in lvnc patients. atrial standstill is an uncommon arrthymia characterized by the absence of electrical and mechanical activity in the atrium.14) on egc, atrial standstill is distinguished by the absence of p waves with bradycardia and narrow junctional escape rhythm.14) known etiologies of atrial standstill are electrolyte imbalance, familial cause, cardiomyopathies, valvular disease, muscular dystrophy and toxicity of antiarrhythmic agents.15) several types of atrial standstill were previously described; the more frequent transient type, the rare persistent type and total or partial types.15) recently, familial atrial standstill was reported in several studies showing tendency of sudden cardiac death with higher probability of cardiomyopathies at a young age. the genetic causes of familial type are still obscure; however, cardiac sodium channel gene scn5a was identified as a related gene mutation.16) the study patient showed features of isolated la standstill demonstrating electrical silence in the la that is isolated from partially electrically active ra. isolated la standstill is extremely rare; it has previously been reported in severe mitral valvular stenosis, dilated cardiomyopathy or following la ablation.15) however, none of the possible factors was found in our patient, thus raising the question whether the abnormal ecg finding was correlated with lvnc or occurred independently. the ep study demonstrated complete electrical block between ra and la, showing no atrial conduction impulse in the cs bipole with no electrical activity in la, which supported la thrombus formation as the etiology of cva. in conclusion, both lvnc and la standstill have risks of embolic events. while the connection between these 2 conditions is still ambiguous, coagulation is a certainty. here we reported a case of a young man who had lvnc combined with la standstill with cva sequel without any cardiac symptoms.

isolated left ventricular noncompaction (lvnc) is a rare cardiomyopathy with morphologic characteristics of two distinct myocardial layers i.e., thin compacted epicardial and thick noncompacted endocardial layers. the noncompacted myocardium consists of prominent ventricular trabeculae and deep intertrabecular recesses. it can lead to arrhythmias, heart failure or systemic embolisms. electrocardiographic patterns of patients with lvnc are various and non-specific; however, the most common findings are intraventricular conduction delay, left ventricular hypertrophy, and repolarization abnormalities. we reported the first case, to the best of our knowledge, of a 29-year-old man who had recent cerebral infarction and incidental lvnc with spontaneous left atrial standstill.

PMC4580704

pubmed-1320

the deregulation of signaling pathways in tumors can lead to enhanced cancer cell growth, proliferation, survival, invasion, and metastasis or reduced apoptosis 1, 2. such pathways became the focus of the development of targeted cancer therapies during the last decades 3-5. kinases are of special interest within these systems, either as receptor molecules or downstream regulators of signaling cascades (see table 1 for an overview of targeted therapies). examples of receptor kinases to be further discussed in this review are the human epidermal growth factor receptors 1 (egfr) and 2 (her2). both kinases are targets for anticancer drugs and can be analyzed for their expression by clinically approved tests, such as immunohistochemistry (ihc) and fluorescence in situ hybridization (fish). egfr overexpression due to gene amplification is often found in human cancers; in gliomas, this deregulation is often associated with structural rearrangements leading to in-frame deletions in the extracellular domain of the receptor 6. her2 overexpression as found in 25 to 30% of human breast cancers can be mediated either by transcriptional activation or gene amplification 7-10. the her2 status of breast cancer patients does not only have a predictive value, but the receptor itself is also a target for the monoclonal anti-her2 antibody trastuzumab 8. recent evidence has demonstrated that besides being an important therapeutic target in breast cancer, her2 is a target for the treatment of metastasized gastric cancers 11, 12, and thus, the anticancer drug herceptin (trastuzumab) was approved for the treatment of advanced gastric carcinomas. her2 is currently detected by immunohistochemistry (ihc) and fluorescence in situ hybridization (fish), and protocols for the detection of her2 in gastric cancers by ihc were recently suggested by rschoff et al. 13. however, although her2 is well-established as a therapeutic target, recent evidence suggests that down-stream signaling molecules may be better predictors for a response to a her2 directed therapy than the receptor itself. this follows from the fact that the membrane-bound molecule alone does not necessarily lead to an activation of the signaling cascade. for that reason, identifying the activation status of cancer-related signaling cascades might provide a better insight into the mechanisms underlying the success and failure of targeted therapies, thus providing a useful approach to stratify patients for optimal personalized treatment regimens. to individualize cancer, biomarker identification has become even more important for the stratification of patients for special treatment regimens. urokinase-type plasminogen activator (upa) and its inhibitor plasminogen activator inhibitor1 (pai-1) are prominent examples of such biomarkers. both are used to provide a more detailed prognosis for nodal-negative breast cancer patients and have reached the highest level of evidence (loe i) for this purpose 14. however the level of these two markers can just be determined in fresh-frozen tissue. this is problematic as formalin-fixed, paraffin-embedded (ffpe) tissue is the main source of patient material world wide. therefore for clinical determination of diagnostic and therapeutic targets new methods are crucially needed to maximize the data that can be gleaned from this kind of tissue. the recently developed method to extract full-length, immunoreactive proteins from ffpe tissues (for review see 15) is a major progress for such approaches and provides the means to quantitate clinically relevant proteins like her2 and analyze cancer-related pathways by reverse phase protein microarray (rppa)16. because the field of targeted therapy is growing rapidly, it is important to emphasize the necessity of standardization for pre-analytical as well as analytical settings. in this review, we shed light on new developments for the detection of therapeutic protein targets and diagnostic biomarkers in clinical tissues and comment on suggestions for the standardization of tissue handling and analysis. formalin is the standard fixative in clinical tissue collections worldwide and the method of formalin fixation was described in detail in a previous article of the authors 17. the protein and nucleic acid cross-linking induced by formalin maintains the tissue in an excellent condition for the pathological examination of the morphological characteristics of diseased tissues 18. with the new interest in macromolecules (dna, rna, proteins) as therapeutic targets or biomarkers, scientists began to analyze these entities first in frozen material because ffpe fixation required to preserve the tissue morphology had adverse effects on macromolecules, especially proteins. by adoption of antigen retrieval first described by ikeda et al. 19 it was possible to develop an assembly of protocols enabling extraction of proteins from ffpe tissues 18-26. an overview of these methods is provided in a recent review by berg et al., compared to other recent studies 27-30, using these protocols non-degraded, full-length, and immunoreactive proteins are obtained and may then be used for protein profiling for enhanced diagnosis. however one should keep in mind that all protocols require high sds concentrations, temperature and ph, which is problematic for some down-stream applications such as elisa assays. to assure the integrity of isolated proteins we always do a quantification of the total protein obtained by extraction (bradford-assay) and additionally check for correct protein masses by control western blots. other authors suggest controlling for protein integrity by ms 26, 31-33. in the scope of tumor marker research and personalized medicine further aspects of investigation became of major interest: pre-analytical workflow and tissue quality. for molecular diagnostics protein biomarkers need to be precisely measured in high-quality tissue samples. it is known that specimens undergo numerous processing steps from the collection of patient samples to the final diagnostic analysis. this aspect of the sample history is very important with regard to the identification of disease-related biomarkers. but so far there were no detailed investigations concerning for example the influence of temperature and time during transportation, fixation method and storage on the molecular integrity of tissue samples. in order of reproducibility of subsequent diagnosis the impact of pre-analytical parameters on biomolecular integrity and expression needs to be analyzed in detail. furthermore we need guidelines for the standardized collection, handling, stabilization and storage of biosamples and quality assurance indicators for artificial, post collection changes of biological samples. two large international initiatives are working toward this goal, one funded by the eu (www.spidia.eu) and one by the us (http://biospecimens.cancer.gov). in table 2 we highlight the most important problems occurring during the pre-analytical phase of sample preparation. the issue of the evaluation of adverse and variable effects of sample preparation is discussed in more detail in a recent review by becker and taylor 34. although morphological parameters, namely tumor size, grade and staging, are the most important parameters for diagnosis, they do not address the complexity and heterogeneity of individual tumors at the molecular level 35, 36. however, since individualized cancer therapies are on gaining ground more detailed patient classifications are necessary to be able to identify the right therapy for each patient. anyway, it is important to realize that transcript profiling does not accurately reflect the complexity of cellular protein networks (e.g. protein-protein interactions, protein localization or posttranslational modifications) 37. additionally, several groups demonstrated that there is no constrained correlation between gene transcript levels and protein expression or the functional state of a protein. however especially the communication in such a protein network, which is responsible for activation and deactivation of involved proteins, is often altered in cancer and may then lead to aberrant cellular functions that result in proliferation, apoptosis, differentiation, survival, invasion and metastasis. that's why it is of great importance not only to look at the expression of genes or at the activation state of a single protein but to analyze whole protein signaling networks that then can provide fundamental information about the functional state of signaling pathways 42, 43. one method which meets all demands for such a network monitoring is the reverse phase protein microarray (rppa). this array format allows the simultaneous analysis of multiple samples for the expression of several proteins under the same experimental conditions even from small numbers of tumor cells or small specimens such as biopsies 44, 45. like this changes in protein expression levels or phosphorylation states, before and after treatment, between disease and non-disease states and between responders and non-responders the procedure to generate such an array is simple. after protein extraction from any kind of material (e.g. cells, ffpe tissue or fresh-frozen material), each sample is arrayed in triplicate on nitrocellulose-coated slides using several dilutions to ensure that each analyte/antibody combination can be analyzed in the linear dynamic range. after the spotting each slide may be detected with an antibody against the desired protein (for an overview of the rppa methodology, see figure 1). however one should keep in mind that for a successful implementation of rppa highly specific antibodies are needed as one ca n't distinguish between different molecular weights (as e.g. in western blot) but only one signal is obtained. especially for phosphorylated proteins and other posttranslationally modified proteins this seems to be one of the major limiting factors 35, 46. another advantage of the rppa is the possibility to quantify the total amount of protein in the sample via the application of purified recombinant proteins one the same slide 15, 16. this method enables to measure protein expression from ffpe tissues more precisely than it is possible by ihc (e.g., for her2) and implement the analysis of clinical markers, which until now were only measurable from fresh or frozen tissues (e.g. for upa/pai-1). this highly quantitative approach is advantageous for patient selection; however, the correlation between protein abundance and histology, as provided by ihc, is lost. nevertheless, we successfully established a set of about 50 antibodies for use in rppa analysis by stringently selecting for specificity and sensitivity by western blot 15, 16 thus providing a starting point for the analysis of major cancer-related signaling pathways from ffpe tissues of cancer patients. based on this expertise we recently developed an algorithm to validate antibodies for their simultaneous application to extract based and classical morphology based methods (schuster c, malinowsky k et al. we plan to combine the advantages of both approaches to get broad insight into all aspects of cancer formation and development. for the application of rppa generated data for such a purpose in clinical routine it is necessary to be able to compare the results of arrays generated in different hospitals and experimental settings. this can be achieved by reference samples on each slide, to which the signal may be normalized to. such a control has to be renewable, reproducible in large-scale, successful over a broad range of end points, stable over a long period of time and as closely related to the test sample as possible 47. to validate the reproducibility of the method, we performed experiments analyzing the variability between sample preparations, array slides and experimental setups (inter-sample as well as inter-assay comparisons) and found the method to be highly reliable 15. in comparison to the widely used elisa approaches rppas are more sensitive and a two-site antibody sandwich method is not used, hence there wo n't be any experimental variability introduced due to labeling yield (or) epitope masking. they are used for the rapid and comprehensive analysis of new drug candidates found by in silico approaches or by binding screens for their biological functions 48-50, as well as for biomarker screenings 51. many recent studies have demonstrated that rppa technology is a very promising tool for signaling pathway profiling of human tissues and cell lines to produce valuable information for the development of new therapeutics or patient selection. feinberg et al. was the first to utilize a microspot technique to detect antigens in serum 52. in 2001, another study used rppas to show the activation of pro-survival proteins and pathways during prostate cancer progression 45. in our group, we provided insight into the role of upa/pai-1 in cancer related signaling 16. other prominent examples of cancer-specific signaling deregulation namely the activation of the phosphatidylinositol 3-kinase (pi3k) pathway in a significant number of ovarian and colon tumors 53, 54.and, alterations in the mitogen-activated protein kinase (mapk) pathway or the overexpression of her2,8, 53-57 were summarized in an earlier review by the authors 17. these examples demonstrate the diverse signaling-based mechanisms underlying cancer progression and indicate that cancer types (e.g., breast cancer) can be subdivided e.g., into small, well-defined subgroups that express a typical protein profile. some authors go even further, suggesting that the use of a targeted therapy should not be based on the identity of the cancer but rather on the deregulation of a certain pathway 58. such an approach would challenge the design of future clinical studies and the approval of new drugs for targeted therapy but could be beneficial for patients by providing highly specific therapies that are only based on the availability of specific targets regardless of the classical characterization of the cancer type. such a specific treatment could greatly increase the quality of life for patients by minimizing adverse side effects of cancer treatment. in addition to classical biomarker screenings rppa is also an adequate tool to compare different types of samples regarding their protein expression profiles. one prominent example is the expression analysis of her2 from resected tumors versus core needle biopsies. currently, core biopsies are routinely used for diagnosis of breast cancer and they are often the only sample for providing prognostic and predictive markers before treatment. after extraction of full-length proteins from ffpe tissues, berg et al. used rppas to compare her2, estrogen receptor (eralpha), and progesterone receptor (pgr) expression levels in a series of 35 ffpe breast cancer surgical specimens and their corresponding core biopsies. we found a high concordance between protein expression in core biopsies and surgical specimens. in this study, the authors could show that her2, eralpha, and pgr expression can be assessed reliably on core biopsies of ffpe breast cancer tissues using rrpa. these results might further strengthen the implementation of rppa technology in routine clinical settings (berg et al. protein microarray-based comparison of her2, estrogen receptor and progesterone receptor status in core biopsies and surgical specimens from ffpe breast cancer tissues. gene expression profiles (e.g., genes regulating cell cycle, invasion or metastasis) have been proposed as potential biomarkers for the prognosis, prediction of treatment response and the identification of potentially new drugs 59. in addition to the well-established mrna profiling, the analysis of small non-coding rnas called mirnas has become a focus in biomarker research. in recent years, evidence has suggested that whole groups or families of mirnas are deregulated in different diseases, especially cancer 60, 61. the quantitative analysis of protein expression described in this review may enable the parallel identification of protein and rna profiles (mrna and mirna) using only one ffpe tissue sample. with this method, it is possible to simultaneously determine mutations in critical genes and relate them to expression profiles. gene mutations frequently lead to a loss of gene function, although the mutated gene is still expressed at the rna and protein levels. a classical example for such a mutation is the cell adhesion molecule e-cadherin, in which a loss of e-cadherin function is found in many diffuse-type gastric cancers, although the protein is expressed at high levels in these tumors 62. another useful application of the combined dna and protein analysis is the utilization of egfr as a therapeutic target; although egfr expression is detectable on a protein level, many patients with colorectal cancers do not respond to cetuximab therapy 63-65. the downstream target of egfr is mutated in many of the non-responders, therefore blocking the egfr does not inhibit proliferative signaling. therefore, the parallel assessment of the level of egfr protein and the mutational status of the egfr gene is one important prerequisite for successful cetuximab treatment 63-65. the remaining egfr-positive patients express the wild-type form of the protein and provide a good example for the need of additional biomarkers for the prediction of therapeutic response. many of these patients do not respond to cetuximab therapy, probably due to further alterations in pathways downstream of egfr. in recent years, several additional candidate biomarkers of egfr resistance were suggested. molecular aberrations occur in braf, pik3ca and pten, which are known downstream effectors of egfr 66-69. somatic mutations in these egfr effectors correlate with the reduced efficacy of cetuximab in patients with metastatic colorectal cancers 70, 71. an activating mutation of braf, which lies downstream of the egfr signaling cascade, was found in a proportion of patients with wild-type egfr that do not respond to cetuximab treatment 66. for that reason, braf mutational analysis is currently recommended by the national comprehensive cancer network (nccn) clinical guidelines for patients with wild type metastatic or recurrent colorectal cancers that are receiving cetuximab 72. recently, we succeeded in the parallel extraction of proteins and rna, including mirna, from the same ffpe tissue sample (malinowsky et al.; unpublished data), demonstrating the proof of principle of our vision. while ffpe samples are a large challenge of the clinical application of the combined analysis of protein and rna, new formalin-free fixatives, e.g. paxgene tissue fixation and stabilization reagents 73, hope 74-76, rcl2 77-79, finefix 80, metacarn 81 or umfix 82 show great potential to serve as novel multimodal fixatives for modern pathology, enabling extensive protein biomarker studies on clinical tissue samples. regardless of the nature of the fixative, our approach allows the integration of analysis at all three levels of gene expression (dna, rna and protein), providing insight into the whole spectrum of potential biomarkers (figure 2). in the scope of new targeted cancer therapy approaches new technologies which meet the issues of fast and precise target detection and quantification are desperately needed. it has already been implemented in several biomarker studies and becomes even more powerful when combined with the huge sample reservoir provided by ffpe tissues. it is clear now that proteins can be extracted from ffpe tissues and reliably analyzed. we believe that applying these new techniques-probably in conjunction with novel tissue fixatives-will greatly facilitate the search for new biomarkers and therapeutic targets in the near future. more importantly, the great translational potential of the new approaches are apparent as the methodologies discussed can easily combine classic histopathology and modern multiplex protein profiling. however, in depth evaluation of antibodies and the use and design of proper controls for tissue quality are essential.

in recent years, new anticancer therapies have accompanied the classical approaches of surgery and radio- and chemotherapy. these new forms of treatment aim to inhibit specific molecular targets namely altered or deregulated proteins, which offer the possibility of individualized therapies.the specificity and efficiency of these new approaches, however, bring about a number of challenges. first of all, it is essential to specifically identify and quantify protein targets in tumor tissues for the reasonable use of such targeted therapies. additionally, it has become even more obvious in recent years that the presence of a target protein is not always sufficient to predict the outcome of targeted therapies. the deregulation of downstream signaling molecules might also play an important role in the success of such therapeutic approaches. for these reasons, the analysis of tumor-specific protein expression profiles prior to therapy has been suggested as the most effective way to predict possible therapeutic results. to further elucidate signaling networks underlying cancer development and to identify new targets, it is necessary to implement tools that allow the rapid, precise, inexpensive and simultaneous analysis of many network components while requiring only a small amount of clinical material.reverse phase protein microarray (rppa) is a promising technology that meets these requirements while enabling the quantitative measurement of proteins. together with recently developed protocols for the extraction of proteins from formalin-fixed, paraffin-embedded (ffpe) tissues, rppa may provide the means to quantify therapeutic targets and diagnostic markers in the near future and reliably screen for new protein targets.with the possibility to quantitatively analyze dna, rna and protein from a single ffpe tissue sample, the methods are available for integrated patient profiling at all levels of gene expression, thus allowing optimal patient stratification for individualized therapies.

PMC3005552

pubmed-1321

collagen is a major connective tissue protein that plays an important role in the extracellular matrix in animals. as such atelocollagen is a type of soluble collagen produced from tropocollagen, the collagen molecule that makes up collagen fibrils, via the elimination of the telopeptide moieties, which are considered to account for most of collagen's antigenicity [1, 2]. thus, atelocollagen is considered to have little immunogenicity, which makes it a safe biomaterial. for example, a minipellet atelocollagen formulation has been demonstrated to sustain the release and maintain stable blood concentrations of protein drugs for more than 1 week. many kinds of protein drugs such as interferon-, interleukin-2, nerve growth factor, and bone morphogenetic protein, and so forth, have been administered using this drug delivery system, and interferon- and interleukin-2 showed strong antitumor activities in animal models when administered in this manner [3, 4]. in the past decade, as well as being used as a solid substrate, dissolved atelocollagen has been used as a drug delivery vehicle for nucleic acid-based medicines for gene conversion, inflammatory disease [8, 9], and tumor therapy. atelocollagen can be used to deliver most kinds of nucleic acid-based medicines including plasmid dna, antisense oligodeoxynucleotides (odn) [1113], short interference rna (sirna) [1420], and micro rna (mirna) [2123]. it is also capable of delivering oligonucleotides to subcutaneous xenografts and metastatic tumors after its local and/or systemic administration. many studies, including some involving in vivo tumor models, have evidenced the contribution of atelocollagen to the enhancement of drugs ' antitumor activities, and some of them described the mechanisms. for example, nucleic acids delivered by atelocollagen are protected against degradation by host nucleases [8, 14, 24], and it has also been shown to improve the delivery efficiency of oligonucleotides to tumors [15, 16]. however, the biological functions of atelocollagen and the mechanism by which it enhances delivery efficiency are still not fully understood. it is essential to reveal the biological characteristics of atelocollagen in order to be able to fully exploit its drug delivery potential. while we were studying the basic properties of atelocollagen, we discovered another of its functions: it increases endothelial permeability. here, we describe the results of a study of the effects of atelocollagen on intercellular sealing function. we measured transendothelial electrical residence (ter) in order to estimate intercellular barrier function and performed an immunohistochemical examination to see whether any cellular morphological changes were induced. rhodamine red-conjugated atelocollagen was prepared in accordance with the manufacturer's instructions (fluoreporter rhodamine red-x protein labeling kit; life technologies japan, tokyo, japan). the oligodeoxynucleotides (odn) and double stranded rna (dsrna) were synthesized by eurogentec (seraing, belgium). the sequences of the oligonucleotides are listed in table 1 [20, 25, 26]. each atelocollagen-oligonucleotide formulation (ac formulation) was prepared by gently mixing aqueous atelocollagen with a solution containing a defined concentration of oligonucleotides. the final oligonucleotide concentration was usually 5 m and that of atelocollagen was 0.1% w/v unless otherwise stated in the text, tables, and/or figures. normal human dermal microvascular endothelial cells (hmvec) were purchased from eidia (tokyo, japan). these cells were cultured in egm (endothelial growth media; eidia, tokyo, japan) until they reached confluence on 12 mm transwell filters with a 0.4 m pore size (corning glass works; corning japan, tokyo, japan) coated with rat tail collagen. porcine brain microvascular endothelial cells (bmvec) were purified and maintained according to the method described in a previous study. ter was determined using an evom voltohmmeter and an endohm-12 chamber (world precision instruments, sarasota, fl) at 37c. cell growth was monitored by measuring ter. once stable intercellular seals had formed; that is, at confluence, the medium in the inner chamber was exchanged for 400 microliters of culture medium containing 30% v/v of ac formulation. one hour after treatment with the ac formulation, which was performed as described in section 2.2, 100 l of 0.36% w/v texas red-conjugated dextran (mw: 40 kda; life technologies japan, tokyo, japan) were added to the inner chamber. one hour later, the dextran concentration of the medium in the outer chamber was analyzed by measuring its fluorescence. to investigate the enhancement of paracellular transport by atelocollagen, solute transportation was compared among the ac formulation, bovine serum albumin, and dextran. once stable intercellular seals had formed, the medium in the inner chamber was exchanged for 400 microliters of culture medium containing 30% v/v of the ac formulation, which had been produced using 0.1 or 0.3% w/v rhodamine red-conjugated atelocollagen (i.e., approximately 0.03 or 0.1% w/v atelocollagen was added; molecular weight (mw): 300 kda); 0.1% w/v of fluorescein conjugated dextran (mw: 70 kda; life technologies japan, tokyo, japan); 0.1% w/v of alexa fluor 594 conjugated bovine serum albumin (bsa; mw: 66 kda; life technologies japan, tokyo, japan). the solute concentrations of the outer chambers were analyzed by measuring their fluorescence at 1 and 2 hours after the medium exchange. after treatment for 1 hr with the ac formulation, oligonucleotide alone, atelocollagen alone, or phosphate-buffered saline (pbs) as a control, hmvec cells were fixed with 1% paraformaldehyde for 10 min and then treated with 0.2% triton x-100 for 10 min. after preincubation with 5% skimmed milk, they were incubated for 1 hr at room temperature with rabbit or mouse antibodies against vascular endothelial (ve)-cadherin (bd biosciences, san diego, ca), zonula occludens-1 (zo-1) (zymed laboratories, san francisco, ca), claudin-5 (zymed laboratories, san francisco, ca), and -tubulin (amersham, poole, uk). then, the samples were incubated for 1 hr with appropriate secondary antibodies labeled with alexa fluor-488 or alexa fluor-596 (life technologies japan, tokyo, japan). actin filaments were labeled with alexa fluor-546 phalloidin (life technologies japan, tokyo, japan). the expression of each protein was examined using a laser scanning confocal microscope (mrc 1024; bio-rad, hercules, ca). western blotting was performed according to the method described in a previous report. for western blotting of the total cell lysates, the dishes were washed with pbs, and then 300 l of sample buffer (1 mm nahco3 and 2 mm phenylmethylsulfonyl fluoride) was added to 60 mm culture dishes. the cells were scraped and collected in microcentrifuge tubes and then sonicated for 10 sec. the protein concentrations of the samples were determined using a bca (bicinchoninic acid) protein assay reagent kit (pierce chemical, rockford, il). for each sample, aliquots of protein (15 g per lane) were separated by electrophoresis in 4/20% sodium dodecyl sulfate polyacrylamide gels (cosmo bio, tokyo, japan) (sds page). after being electrophoretically transferred to nitrocellulose membranes (immobilon; millipore, billerica, ma), the membranes were saturated with blocking buffer (trisbuffered saline [tbs] supplemented with 0.1% tween 20 and 4% skimmed milk) for 30 min at room temperature and incubated with antiactin, anti-zo-1, anti-ve-cadherin (bd biosciences, san diego, ca), anticlaudin-5 (zymed laboratories, san francisco, ca), anti-p38 mitogen-activated protein kinase (map kinase or mapk) (santa cruz biotechnology, santa cruz, ca), antiphospho-p38 mapk (cell signaling, beverly, ma), anti-p42/44 mapk (promega, madison, wi), antiphospho-p42/44 mapk (cell signaling, beverly, ma), anti-rho-a, and anti-cdc42 (santa cruz biotechnology, santa cruz, ca) antibodies (1:1000) for 1 h at room temperature. the membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit or mouse igg (dako a/s, copenhagen, denmark) at room temperature for 1 h. the immunoreactive bands were detected using an ecl western blotting analysis system (ge healthcare, little chalfont, uk). paracellular flux is dependent on the function of tight junctions [30, 31]. we assessed the effects of an ac formulation on the ter of hmvec to evaluate their tight junction function. as shown in figure 1 and table 1(a), the odn containing ac formulation caused a time-dependent reduction in ter, while ter was hardly affected by treatment with odn or atelocollagen alone. as for the type of oligonucleotide in the formulation, phosphorothioate odn produced a more significant reduction in ter than phosphodiester odn, which only produced slight alterations. various formulations containing different ratios of odn and atelocollagen were examined in order to understand which parameters have the greatest effect on the change in ter. as a result, we found that the ter change was dependent on the size of the odn and the composition of the formulation, but not the odn sequence, as shown in tables 1(b), 1(c), and 1(d). specifically, odn composed of 15 or more bases were effective and those containing around 30 bases were the most effective, but 10-base-long odn were not effective. the change in tight junction function was also dependent on the concentrations of odn and atelocollagen in the formulation. to verify that the ac formulation increased paracellular flux, the amount of texas red-labeled dextran (molecular weight: 40 kd) transported across an endothelial cell layer was examined. as shown in figure 2(a), dextran transport was increased approximately twofold in the cell cultures incubated with the ac formulation. next, the paracellular transport of atelocollagen was analyzed and compared with those of bsa and dextran. only very small amounts of bsa and dextran penetrated the cell sheet during the 2-hour study period; on the other hand, much more atelocollagen passed through, even though the molecular weight of atelocollagen is 4-5 times higher than those of bsa and dextran (figure 2(b)). an examination using bmvec was performed to determine whether the effect of the ac formulation was specific to hmvec. as a result, we found that the ter value of the bmvec was also reduced by the ac formulation (and only the ac formulation), as shown in figure 3. bmvec forms the blood-brain barrier (bbb), where intercellular sealing function is strictly maintained. these results showed that the ac formulation is able to affect the paracellular flux of endothelial barriers. it is well known that increased endothelial permeability is associated with impaired intercellular contact [3235]. we carried out an immunohistochemical analysis of the cells treated with the ac formulation to clarify how their intercellular sealing was affected. as shown in figure 4(a), treatment with the ac formulation markedly reduced the degree of intercellular contact, as shown by intercellular gap formation, actin stress fiber formation, cellular contraction, and a lack of ve-cadherin. adequate expression of claudin-5, one of the key components of the endothelial barrier, was noted at the cell periphery. however, zo-1 protein expression was absent from the intercellular gaps. on the contrary, western blotting revealed that treatment with the ac formulation did not affect the expression of these proteins (figure 4(b)). although the ter value remained low as long as the ac formulation was present in the culture medium, the treatment did not cause toxicity. the cells survived well for at least 24 hrs, and both the ter and morphology of the cells could be recovered by removing the formulation (data not shown). no such morphological changes were induced by treatment with odn or atelocollagen alone (figure 4(a)). microtubules play an important role in regulating actin formation and hence, endothelial barrier function [36, 37]. as shown in figure 5, -tubulin formed a fine network in the blank control. however, treatment with the ac formulation caused the peripheral fine structure of the -tubulin network to be lost. many studies have shown that increased endothelial permeability and impaired intercellular contact can be induced by signal transduction, mainly that of rho a [32, 38] and p38 map kinase [36, 3942]. thus, we investigated the effects of the ac formulation on signal transduction. as a result, no differences were found in the expression levels of rho-a, cdc42, or p42/44 map kinases or their phosphorylated forms. regarding p38 map kinase, although no changes were noted among the control, oligonucleotide alone, or atelocollagen alone groups, the levels of phosphorylated p38 map kinase were markedly increased in the cells treated with the ac formulation (figure 6), which indicates that the impact of the ac formulation on tissue permeability is associated with the activation of p38 map kinase. collagen plays an important role in the extracellular matrix by supporting cells so that they can form tissues and organs. atelocollagen is produced from type i collagen and is widely used in its solid state as a biomaterial for medical and surgical products because of its biocompatibility and workability. however, the kinetics, dynamics, and biological functions of atelocollagen after its injection into the living body are still poorly understood, and it is essential to elucidate the characteristics of atelocollagen in order to fully exploit its potential. here, we demonstrated a novel biological function of atelocollagen. when endothelial cell sheets were treated with atelocollagen or oligonucleotides alone, the intercellular structure of the sheet was not changed. however, when the atelocollagen and oligonucleotides were administered together, intercellular gaps formed and consequently the paracellular flux of the sheet was elevated. the abovementioned changes were elicited via the activation of p38 map kinase, a signal-transduction-related molecule. in addition, the changes observed in this study are similar to those triggered by thrombin, histamine, tnf- [34, 36], and vegf (vascular endothelial growth factor), and so forth. as shown in section 3.1, the degree to which endothelial function was affected was dependent on the molecular structure of the oligonucleotides including their size and chemical modifications, suggesting that the three-dimensional structure of the oligonucleotide and atelocollagen complex stimulates a signal transduction pathway that acts as a permeability modulator, although the specific pathway that it stimulates remains unknown. to date, no severe systemic edema or side effects of the ac formulation have been noted, even after the intravenous administration of atelocollagen as an oligonucleotide drug carrier. these findings indicate that atelocollagen could be used as a permeability enhancer at local treatment sites without the adverse systemic effects that cytokines and chemokines sometimes provoke. since tight junction modulators are regarded as practical drug delivery enhancer candidates [4446], the function of atelocollagen demonstrated in the present study should be thoroughly investigated. the unique biological functions of atelocollagen have led to the development of unique antitumor therapies and products, such as surgical products; formulations that sustain the release of antitumor proteins [24]; treatments that enhance the antitumor activities of various molecules including antisense odn [1113], sirna [1420, 24], and mirna [2123]. obtaining more information about atelocollagen would allow us to develop the next generation of atelocollagen-mediated drug delivery systems.

atelocollagen is a major animal protein that is used as a highly biocompatible biomaterial. to date, atelocollagen has been used as an effective drug delivery technology to sustain the release of antitumor proteins and to enhance the antitumor activity of oligonucleotides in in vivo models. however, the biological effects of this technology are not fully understood. in the present study, we investigated the effects of atelocollagen on endothelial paracellular barrier function. an atelocollagen formulation containing oligonucleotides specifically increased the permeability of two types of endothelial cells, and the change was dependent on the molecular size, structure of the oligonucleotides used and the concentrations of the oligonucleotide and atelocollagen in the formulation. an immunohistochemical examination revealed that the formulation had effects on the cellular skeleton and intercellular structure although it did not affect the expression of adherens junction or tight junction proteins. these changes were induced through p38 map kinase signaling. it is important to elucidate the biological functions of atelocollagen in order to be able to exploit its drug delivery properties.

PMC3312293

pubmed-1322

in order to characterize the radial undulations from a simulated vesicle, we start by recasting the positions of the bilayer beads into an equi-angular discrete surface representation using a, -grid (colatitude and longitude respectively), where [0,] and [0, 2], with =0 at the northern pole. the angular resolution is defined as1 where s is the arc-length where the bilayer fluctuations transition between a continuum mode to molecular (s 4 nm), r0 is the average vesicle radius, and ns is the number of sampling points per s with ns 2 to satisfy nyquist sampling theorem. to render the surface, we define the origin at the com of the vesicle (lipids+membrane inclusions) and then transform the system into spherical coordinates (xi, yi, zi ri,i, i, where ri,i, i corresponds to radius, colatitude, and longitude for each bead i, respectively). the direct use of an equi-angular, -grid to bin bead positions introduces discontinuities at the poles. we mitigate these discontinuities by implementing an arc-length low-pass filter with filter cutoff qarc=2/s. our previous work with undulation analysis on flat patch systems identified a cutoff wavenumber, q0=1.5 nm, at which the long-wavelength undulations transition into molecular structure fluctuations. we use this q0 as a guide in defining an appropriate qarc for the vesicle systems. figure 1a shows a snapshot from an equilibrated, 34 nm dmpc vesicle, and figure 1b presents a cutaway view, emphasizing the vesicle s diameter relative to its thickness. we parse all lipids into the inner and outer monolayer using the average vector between the acyl-chain terminal beads and the phosphate bead, where the radial component defines which monolayer the lipid belongs to (i.e., r<0, inner monolayer; r>0, outer monolayer). using an arc-length filter with qarc=2.5 nm, we define the local, -membrane surface for both inner and outer monolayers, rin(,) and rout(,), respectively. figure 1c schematizes the first stages of our method, with the inner (red) and outer (blue) monolayer beads shown for numerous, -positons. on the left hemisphere of figure 1c, regions of rin(,) and rout(,) are presented for illustration. the average of the two surfaces define the undulating radial surface as2 a. snapshot of a 34 nm dmpc-lipid vesicle. c. an arc-length filter is used to define a surface for both inner and outer monolayers (blue and red selection beads respectively). d. rund(,) is determined as the average of the inner and outer monolayers (color-map indicates fluctuations about the average radius, r0, (units in nm). figure 1d shows rund(,) calculated from one frame of the dmpc trajectory. the color-map displays the deviations about the average vesicle radius, r0 (units in nm), which is typically smaller than the ideal sphere radius r0. from rund(,), we then define the normalized radial fluctuations as3 next, spectral decomposition of rund(, spherical harmonics are standing waves on a sphere. given that rund(,) is a discrete surface defined on a spherical manifold, we can represent it as a linear combination of spherical harmonics with degree (l) and order (m), corresponding to the number of waves in the, -dimensions, respectively. spha decomposes rund(,) into a series of standing waves on a sphere with degree l and order m. helfrich s formulism for spha expands the normalized radial fluctuations in spherical harmonics as4where alm are the spherical harmonic coefficients of degree l and order m (l 0,1,lmax and m l, ... ,l) with lmax defined by the number of colatitude grid points.ylm are the spherical harmonic basis functions5where plm() are the fully normalized associated legendre polynomials with the normalization factor, nlm, defined as6 because f(,) is a real-valued function alm=al, m *, the complex conjugate of alm. we thereby redefine alm such that7allowing us to write8where now m 0, ... ,l. we generate the matrix p with dimensions (2n*lmax) n for a given, -distribution, where n is the number of colatitude parallels, such that for each li 0, ... ,lmax, the corresponding 2 m+1 rows are defined as9from which the transformation matrix y follows as10 from the matrix y we can write the spherical harmonic forward transform as11where alm1 is a recasting of the spherical harmonic coefficients alm with dimension n 1 corresponding to the row construction of y, and f,1 is a matrix of positions with dimension (2n*lmax) 1. whereas the forward transform is exact, the inverse transform (i.e., spha) is overdetermined. using the factorize package in matlab, we compute the pseudoinverse of y using qr-factorization and apply a least-squares approximation to determine alm1 as12 implementation of the spha algorithm results in error propagation from numerous sources (i.e., truncation error, round-off error, and least-squares approximation). we evaluated the magnitude of these effects by calculating the root-mean-squared-difference (rmsd) between rund(,) and the transformed rund(,), where13 rmsd calculated for the dmpc system is 3.5 10 nm and for the dmpc+cholesterol system is 1.8 10 nm. these errors are 2 orders of magnitude lower than the magnitude of the radial fluctuations, o (0.5 10 nm), allaying concerns regarding the inherent propagating errors. membrane structure ((z) and al) for flat-patch membrane systems was determined using our previously published method. briefly the urs was determined using the phosphate atoms and subsequently filtered with a 1.5 nm cutoff. the filtered surface was then used to reference every atom to the local bilayer midplane. these distances were subsequently binned and volume normalized to extract the number density profile. the standard method for determining the radial membrane number density profile, rbin(r), references every bead/atom in the system relative to the com of the vesicle in spherical coordinates, averaging across all, -angles. we have previously shown that the membrane structure profile is smoothed out in systems where long-wavelength undulations develop. by isolating the long wavelength undulations and referencing every bead/atom to the local undulating reference surface following the same principles for our method developed for flat-patch systems, we apply a low-pass ideal filter to the spherical harmonic coefficients with an order cutoff lcut=qarcr0 0.5 such that14where lm1 are the filtered coefficients. for the dmpc vesicle lcut=26, whereas for the slightly larger dmpc+cholesterol vesicle, lcut=29. we next apply an the inverse spherical harmonic transform to resolve a filtered radial-undulation surface rund(,) as15 with the filtered surface we resolve the vesicle s local membrane structure, uc(r), by referencing every bead/atom i relative to it is position on the surface, such that16where rund(, )nearest corresponds to the closest equal-angular grid point on rund(,). the grid definition samples the corresponding filter wavelength at least 2 times (satisfying nyquist sampling theorem). as a default we employ 4-samples per wavelength (at qarc=2.5 nm the arc-length resolution is 0.4 nm), providing a close approximation to the unique rund, i(,). the resulting ri(,) is then binned with a bin-width of dr=0.1 nm and subsequently normalized by the differential volume shell17with r0the radius of the ideal sphere with equal volume as rund(,) defined as18and adjusted for every bin index b by the corresponding distance from the surface. in addition to number density profiles, we can identify the vesicle s area per lipid, al, both as a whole and independently for both monolayers. instead of defining r0 with all vesicle beads/atoms, we parse them independently as inner and outer monolayers. then using eq 18, we obtain two additional ideal sphere radii (three in total), r0,vesicle, r0,inner, and r0,outer. the corresponding al for each ideal radius is simply192021where nl, inner and nl, outer refer to the number of lipids in the respective monolayer. from alm1, we obtain the undulation power spectrum by binning the modulus of the spherical harmonic coefficients across degree l. the resulting profile can be interpreted according to the helfrich continuum model for undulations on a sphere with vanishing spontaneous curvature as22where t is temperature and l 2, ... ,lmax. in the flat-patch spectral method, the undulations power spectrum, |u(q)|, determined by direct fourier transformation of the bilayer selection atoms can be modeled as23where n is the number of selection atoms, a is the projected area per lipid, and s(q) is the in-plane structure factor. s(q) is subtracted from n|u(q)| to provide a broader range of modes within the q regime. kc is then determined by fitting the low-q, long-wavelength modes. using a filter to define the urs attenuates the spectral intensity of the undulations. this attenuation can span a broad frequency bandwidth, bleeding through into the desired frequencies of the signal. we explored a range of arc-length filter cutoffs to characterize the filter s frequency response, with the goal of identifying a cutoff where signal attenuation is limited to frequencies above the crossover wavenumber, q0=1.5 nm. figure 3 presents the power spectra for the dmpc vesicle system using four different arc-length filters with wavenumber cutoff, qcut, ranging from 0.5 nm to 2.5 nm. undulation spectra for a range of arc-length cutoff wavenumbers, (qcut=0.5 nm: blue; 1.5 nm: green; 2.0 nm: red; and 2.5 nm: cyan) for the dmpc vesicle system. the frequency response of the arc-length filter is far from ideal, with significant bleed-through extending below the desired cutoff wavenumber. this is most noticeable when comparing the crossover wavenumber q0=1.5 nm (black dashed-line)the wavenumber where continuum undulations transition into molecular fluctuations to the 1.5 nm filter cutoff (green triangles). there is significant loss of undulation intensity for degrees 1225, all before the 1.5 nm crossover wavenumber. this loss of intensity skews the kc-fit, resulting in a larger kc. furthermore, increasing the filter cutoff increases the number of degrees that comprise the linear region below q0, thereby improving the fit to eq 22. bending with qcut=2.5 nm, all 26 degrees (spanning the full range, q0 1.5 nm) can be used to determine kc. all simulations were run using the martini force field and using the gromacs v4.5.3 program. isobaric (npt) ensemble at constant pressure and temperature (1 bar and 303 k, respectively) using either a 25 fs time step for pure lipid or 10 fs time step for cholesterol systems. pressure coupling was isotropic for the vesicle systems and semi-isotropic for flat-patch, with independent xy- and z-barostats, resulting in a tensionless bilayer. initial equilibration for vesicle systems included 100 000 steps of steepest descent minimization followed by 500 ns of dynamics using the velocity-rescaling thermostat and a berendson barostat (flat-patch systems underwent 10 000 steps steepest descent minimization and 100 ns velocity-rescaling dynamics). hoover thermostat and the parrinello rahman barostat with a time constant of 2.5 and 250 ps, respectively. pressure coupling was applied isotropically for vesicle simulations and semi-isotropically for flat-patch systems. vesicle production simulations were 2.5 s (10 s scaled simulation time), flat-patch simulations were 5 s (20 s scaled time) sampled every 1 ns. the starting configurations for the dmpc vesicle (11 126-dmpc, 1 123 315-coarse grained water+antifreeze, high-water, 1 113 742-cg water+antifreeze low-water) and dmpc+cholesterol (12 771-dmpc+5473-cholesterol, 1 313 698-cg water+antifreeze) were constructed by randomly seeding two opposing monolayers with the appropriate number of lipids (dmpc and/or cholesterol) based on the area per lipid of each species, the surface area of the monolayer shell, and the mole fraction of the mixture. a second dmpc vesicle was simulated using the 3 s frame as a starting configuration with 10% of the internal water beads removed to explore effects of system construction on structure and bending rigidity. flat-patch bilayer systems were constructed with 3200-dmpc lipids and 2240-dmpc +960-cholesterol, randomly seeded in flat monolayers with 70,400 coarse grained water+antifreeze particles. during the early stages of equilibration, rapid pore formation occurred throughout the vesicles. these pores coalesced and closed quickly within 70 ns providing an opportunity for both lipid flip-flop and water exchange across the vesicle to equilibrate the system. for the low-water system, the vesicle collapsed into an ellipsoidal shape within 100 ns, and maintained an ellipsoidal character throughout the 3 s production run. trajectories were manipulated and processed using both the gromacs v.4.5.3 simulation package and the mdanalysis python library. further data analysis and figure rendering was performed using matlab (v.r2012a) with use of the factorize library. having established the framework to define rund(,) for vesicle simulations, it now becomes possible to extract the underlying membrane structure from the fluctuating vesicle s trajectory. figure 4 presents the number density profiles for the dmpc vesicle, rbin(r) and uc(r), and the dmpc flat-patch, uc(z), systems. vesicle profiles are illustrated as dashed lines, flat-patch profiles as filled distributions, with the component groups color-coded as described in the caption. negative radial positions correspond to the inner monolayer, positive values correspond to the outer monolayer. comparisons of component number densities for dmpc vesicle and flat-patch systems. flat-patch profiles are filled-distributions with headgroup (black/gray), carbonyl-glycerol (red/pink), and acyl-chain (blue/cyan). (a), (b) rbin for the high-water and low-water dmpc system, respectively. (c), (d) uc for high-water and low-water dmpc system. the same flat-patch profile has been included in all panels. figure 4a compares the high-water vesicle profile, rbin(r), to the flat-patch profile, uc(z). the uc(z) profile was translated by the average vesicle radius, r0, to allow direct comparison to the vesicle profiles. as expected, using a global reference frame with a fluctuating bilayer results in a broadening of the component distributions (rmsd between rbin(r) and uc(z) is 3.08 nm). this broadening results in a loss of structural resolution in the vesicle profile, most noticeably in the acyl-chain distributions, where the typically pronounced terminal methyl trough is absent. figure 4b compares rbin(r) from the ellipsoidal, low-water dmpc system to the flat-patch profile. as fluctuations increase, the low-water rbin(r) displays significant distortion (rmsd is 7.12 nm) across all lipid-component distributions, highlighting the problem with using a global com reference frame to define membrane structure. figure 4c, d present comparisons of our new method, uc(r), to the flat-patch profile for both the high- and low-water dmpc systems, respectively. in both cases, the resulting number density more closely resembles the flat-patch result (rmsd between uc(r) and uc(z) is 1.66 nm for low-water dmpc and 2.34 nm for high-water). each component distribution is tightened up relative to rbin(r), and key structural features (e.g., the terminal methyl trough) are properly characterized. in the high-water system (figure 4c), the headgroup distributions are centered at the appropriate position (i.e., the bilayer thickness matches that of the flat-patch). however, the amplitude and width of the headgroup distributions do not fully agree with the flat-patch result: the amplitudes are too high and the widths to narrow. this small discrepancy suggests that the lipids are under tension, either due to water-density or lipid density asymmetry across the bilayer. comparison of the low-water profiles (figure 4b, d) demonstrates the dramatic improvement in calculated membrane structure when undulations are isolated and characterized during structure determination. even with a dramatic ellipsoidal geometry, the uc(r) method extracts the underlying membrane structure. the low-water profile amplitudes agree with the flat-patch profile, more so than the high-water result. however, the membrane appears thicker and the component distributions are slightly broader than the flat-patch result. comparing figure 4c with 4d (uc(r) for the spherical and ellipsoidal dmpc vesicle systems) as expected, varying the number of waters inside the vesicle can lead to different membrane tensions and corresponding structures. with our new algorithm, this type of comparison can serve as a guide in the initial construction of vesicle simulations, evaluating the membrane structure for imbalances in water density or lipid density across the bilayer. analysis of the dmpc+cholesterol system highlights additional complexities that can develop due to the different system geometries. supplemental figure 1 presents rbin(r) and uc(r) for the dmpc+cholesterol system. the undulation correction still sharpens the component distributions, albeit to a lesser extent than the pure dmpc system. cholesterol s facile ability to flip-flop during the time-scale of the simulation results in an asymmetric cholesterol distribution across the bilayer in the vesicle geometry. the development of an asymmetric cholesterol distribution is not surprising for the vesicle as the spontaneous curvature difference between inner and outer monolayers will induce an asymmetric partitioning of lipids with non-neutral spontaneous curvature. close inspection of all vesicle number density profiles highlight an asymmetry (particularly in the headgroup distributions) where the inner monolayer has a higher density relative to the outer monolayer (figure 4 and supplemental figure 1). we can not know for certain whether this asymmetry is due to a redistribution of lipid components or an artifact from a poorly equilibrated vesicle. vesicle structure does not change across the last 1.5 s for both the dmpc and dmpc+cholesterol systems. however, the differences in structure between high-water and low-water dmpc vesicle systems highlight the effect of water density imbalances across the bilayer. furthermore, in all simulations, lipid flip-flop was only observed for cholesterol and never for dmpc. significantly longer simulation times (> 100s of s) of standard md would be necessary to allow for both water equilibration and sufficient lipid flip-flop events to fully equilibrate the vesicle. our focus here has been on the development of a method to calculate structure from vesicle simulations and not to perfectly refine these particular simulations. nevertheless, the problem of vesicle construction and equilibration is an important challenge that must be answered to facilitate further study of these more complex systems. risselada et al. employed an artificial pore allow for both water and lipid exchange across the vesicle prior to the production md simulation. for simple lipid compositions, this approach is preferable over an iterative refinement of the lipid and water distributions (altering them by hand on the basis of structural profile imbalance) and far more computationally efficient than extending the simulation until sufficient lipid flip-flop is observed. figure 5 presents the area per lipid (al) time-series for the dmpc vesicle systems. we compare values determined along the urs for the full vesicle (all, blue) and both inner and outer monolayers (green and red, respectively) with those of the flat-patch al (cyan). a summary of the average al is presented in table 1. in the high-water dmpc system (figure 5a) there are three distinct differences between the vesicle and flat-patch result: (1) the inner and outer monolayer have different al, with inner monolayer lipids occupying more lateral area than those in the outer leaflet; (2) all measures of al are greater than the flat-patch al; and (3) the fluctuations in al are significantly decreased in the high-water vesicle system versus the flat-patch. the imbalance between inner and outer monolayers correlates with the structural asymmetry in the headgroup uc(r). (a) al trajectory for the high-water dmpc vesicle system with total vesicle (blue), inner monolayer (green), outer monolayer (red), and flat-patch system (cyan). we presume that this reflects that the inner monolayer is under greater tension than the outer, constraining the inner headgroups in the radial dimension and resulting in a tighter position distribution (recall that the thickness is the same, figure 3b). there are two potential causes of this tension, either an imbalance in the water density or the lipid density across the vesicle. the area per unit cell, auc=2 asurface/ndmpc, for the dmpc +cholesterol system shows a similar trend as the dmpc vesicle (see supplemental figure 2). figure 5b illustrates the same al analysis for the ellipsoidal, low-water dmpc system. it is immediately evident that the water content dramatically affects the vesicle s structure and dynamics. the ideal sphere al is increased for all three al metrics, even though the average vesicle radius is smaller. there is a partial recovery in the al symmetry across the monolayers (i.e., a reduction in al=al, out the residual discrepancy between al, out and al, in likely stems from a lipid density imbalance across monolayers, which is still evident in figure 4d. although the structure from the low-water system more closely matches the flat-patch profile, it is the al of the high-water system that is in better agreement. the number density profile calculation isolates the increased fluctuations in the low-water system, whereas the al does not. figure 6 presents the power spectra and subsequent model fits for dmpc high-water (black) and dmpc+cholesterol (red) in both vesicle (figure 6a-c) and flat-patch (figure 6d) geometries. in each case the spectra was determined over the final 1.5 s of the trajectory. as expected, in both the flat-patch and vesicle simulations cholesterol reduces the magnitude of the undulation intensity, reflecting the sterol s well-know rigidifying effect.kc values were determined by fitting the linear regime across low degrees corresponding to q0 1.5 nm to the appropriate helfrich continuum model for vesicle or flat-patch geometries (l 26 for dmpc and l 29 for the slightly larger dmpc+cholesterol vesicle). as shown in table 2, we observe a similar cholesterol-induced increase in kc in the vesicle- and flat-patch geometry (the latter of which was obtained via our the previously published method). power spectra for both vesicle systems (a) with corresponding kc fit for dmpc (b) and dmpc+cholesterol (c). undulation power spectrum and kc-fits for flat-patch systems. for all panels, dmpc (black) and dmpc+cholesterol (red). the*denotes values from brandt et al.; experimental values (* *) from pan et al .. the high-water (hw) dmpc vesicle kc is in better agreement with our previously published kc from a large flat patch, where a 30 000 lipid dmpc coarse-grained flat-patch system had kc=1.5 10 j. to ensure the use of an equi-angular grid did not introduce any artifacts into the vesicle analysis, we repeated the spha on the high-water dmpc vesicle in a 90 rotated reference frame. the results were identical to the nonrotated system (see supplemental figure 3). the increased kc from our smaller 3200 lipid dmpc flat-patch highlights potential system-size effects with spectral methods to extract kc. the low-water (lw) system shows a reduced kc, relative to the high-water kc, agreeing with the increased al fluctuations (see figure 5b, table 2, and supplemental figure 4). as we discuss below, other factors may also contribute to the differences in kc across the two system geometries, including hydration levels. for example, in the low-water system, we observed a dramatic increase in fluctuations and concomitant decrease in kc. the increase in rigidity for the dmpc+cholesterol system, determined as the ratio of kc between dmpc+cholesterol and dmpc, shows excellent agreement for the high-water (hw) case, 1.8 for flat-patch versus 1.9 for vesicles. for both cases (pure lipid and+cholesterol), the vesicle s kc is smaller than the corresponding flat-patch (1.3 10 j versus 2.1 10 j for dmpc and 2.4 10 j versus 3.8 10 j for dmpc+cholesterol). in both the flat-patch and vesicle systems, the calculated kc value for dmpc is an order of magnitude greater than that determined experimentally (0.58 10 j, determined from diffuse x-ray scattering on oriented bilayer stacks). however, given that our goal is to establish a reliable and robust (i.e., force-field independent) algorithm, we consider the agreement between the established flat-patch method and our new vesicle method as the relevant measure of success, rather than close agreement between our result and experiment. that said, we do note that there is much better agreement between the kc calculated from simulation and the experimental values for both dmpc low-water (6.1 10 j versus 5.8 10 j) and the dmpc+cholesterol systems (2.4 10 j versus 2.73 10 j, respectively). achieving better agreement with experiment in these values remains a challenge for force-field development, though it remains to be seen whether any force field (even fully atomistic ones) will be able to accurately capture kc across all lipid types. because of the sensitivity of the spectrum to the arc-length filter cutoff (see figure 3), it was important to evaluate the sensitivity of the kc fit to the loss of undulation intensity. we did so by varying the number of degrees used in the data-fitting, testing both the 1.5 nm and 2.5 nm filters. supplemental figure 5 illustrates the sensitivity of kc fits and time course of kc for both dmpc and dmpc+cholesterol vesicle systems. the increased linear region for the 2.5 nm spectra results in a much broader span of converged kc fits for both vesicle systems (supplemental figure 5b). this added robustness confirms our choice of filter parameters, specifically the 2.5 nm arc-length filter (for rendering the initial surface) and the 1.5 nm ideal filter (to define the urs and truncate the sph coefficients for the kc-fit at the transition from continuum to molecular length scales). the evolution of kc shows very small changes across the duration of the simulations for both dmpc and dmpc+cholesterol systems (supplemental figure 5c). we have developed a method to determine membrane structure (number density and area per lipid) and bending rigidity from md simulations of lipid vesicles. comparisons of number density profiles determined from vesicle simulations using either a global or a local reference frame highlight the broadening effect introduced by local bilayer fluctuations, similar to what was previously observed in large flat-patch simulations. using a urs removes the fluctuation-induced broadening effect and recovers the underlying structure profile, even under extreme vesicle deformations (e.g., elliptical vesicles). our new spha method can be applied to any md simulation where the topography is such that radial lines intersect the topography only once (e.g., vesicles, bubbles, micelles). in systems where the topography is more tortuous (e.g., where radial lines intersect the topography multiple times) the spha method breaks down. nevertheless, the local structure profile can still be resolved via the urs, as long as the method used to define the urs is modified to accommodate the more complex topography. our choice of using an equi-angular grid in order to define the urs as well as in implementing the spha was done to simplify the calculation of the sph coefficients via matrix transformation (eq 12). alternative surface definitions (e.g., geodesic gridding) are equally viable; however, they may introduce increased complexity and computational cost in determining the transformation matrix. any predefined, surface reference frame requires either a real-space filter (e.g., arc-length filter) or a direct spherical harmonic transformation that requires treating every membrane bead as a radial delta function to obtain the urs. although the latter is the direct corollary to our flat-patch direct fourier method, the increased computational cost of the explicit sph transform makes that approach prohibitive. the spherical harmonics analysis of the urs allows us to determine the al for the equivalent ideal sphere that samples the same area as the undulating bilayer. we define a unique al for the vesicle as well as both monolayers. these three al metrics describe structural imbalances that exist due to initial vesicle construction (e.g., water or lipid imbalances across the bilayer). the convergence of these three metrics may serve as a simple readout for vesicle equilibration. this paper lays the groundwork for an iterative process to improve vesicle construction. with flat-patch membranes, numerous studies have been successful in matching experimental structure profiles by tuning the al through altering the periodic box dimensions. the vesicle membrane geometry raises a new and more difficult challenge: obtaining al, structure, and bending rigidity correct by varying the lipid distribution in the two monolayers and the water density inside the vesicle. our algorithm can extract these structural and mechanical properties to guide the construction and equilibration of these complex vesicle systems. comparison of these structural and mechanical results with experimental measurables is the ultimate goal. although we are currently limited by the accuracy of the simulation force fields, changes in structure that correlate with changes in rigidity may provide insight into the interpretation of experimental results .

we have developed an algorithm to determine membrane structure, area per lipid, and bending rigidity from molecular dynamics simulations of lipid vesicles. current methods to extract structure from vesicle simulations define densities relative to the global center of mass of the vesicle. this approach ignores the long-wavelength fluctuations (undulations) that develop across the sphere and broaden the underlying structure. our method establishes a local reference frame by defining a radially undulating reference surface (urs) and thereby removes the broadening effect of the undulations. using an arc-length low-pass filter, we render the urs by defining the bilayer midplane on an equi-angular, -grid (colatitude, longitude). this surface is then expanded onto a truncated series of spherical harmonics. the spherical harmonic coefficients characterize the long-wavelength fluctuations that define both the local reference frame used to determine the bilayer s structure and the area per lipid (al) along the undulating surface. additionally, the resulting power spectrum of spherical harmonic coefficients can be fit to a helfrich continuum model for membrane bending in spherical geometry to extract bending rigidity (kc). kc values determined for both dmpc and dmpc+cholesterol (30 mol %) vesicles are consistent with values from corresponding flat-patch systems determined using an independent, previously published spectral method. these new tools to accurately extract structure, al, and kc should prove invaluable in evaluating the construction and equilibration of lipid vesicle simulations.

PMC4159217

pubmed-1323

recent research indicates that at least one-third of us adults report regularly getting less than the 79 hours of sleep per night recommended by the national sleep foundation. insufficient sleep, variably defined, has been associated with health-risk behaviors, such as smoking [2, 3], alcohol use [2, 3], and with adverse health outcomes such as obesity, and frequent mental distress (fmd) (14 days/past 30 days in which respondents report their mental health was not good). noting that the average sleep duration among us adults has decreased during the past 50 years along with a concomitant increase in the prevalence of obesity, wheaton et al. found a strong positive relationship between perceived insufficient sleep and body mass index (bmi) among community-dwelling adults. given the linkage between insufficient sleep and increased bmi, the association between insufficient sleep and diabetes is not surprising. consistent with these findings, sleep restriction (defined as 5 hours/night for one week) was found to be associated with a significant decrease in insulin sensitivity. notably, the prevalence of several factors associated with insufficient sleep has been reported to vary between different race/ethnicities in large epidemiologic studies. specifically, an analysis of 20062008 bmi data from the behavioral risk factor surveillance survey (brfss) revealed prevalences of obesity of 23.7% among non-hispanic whites (nhw), 35.7% among non-hispanic blacks although ai/an were not specifically delineated in the latter survey, rates of obesity in ai/an adults have been reported to be 1.5 times greater than among nhw adults. similarly, aggregated prevalences (19992004) of diagnosed diabetes reported from the national health and nutrition examination survey (nhanes) were 5.9% among nhw, 11.1% among nhb, and 10.9% among mexican americans. an analysis of data from both the brfss and the indian health service across 19942002 revealed that the age-adjusted prevalence of diabetes among ai/an adults was more than twice that of us adults overall. despite the relatively high prevalence of factors associated with insufficient sleep in ai/an, few epidemiologic investigations of insufficient sleep have been conducted in this population. in investigations delineating race/ethnicity disparities, short sleep duration (average 6 hours/24-hour period) among workers was reported significantly more frequently by nhb (38.9%), nh other (35.3%), and nh asian (33.2%) than among nhw (28.6%) or hispanic (28.8%). in related research using nhanes 20052008 data to assess sleep-related difficulties (e.g., concentrating, remembering, driving, and working), nhb reported a greater prevalence of sleep-related difficulties in driving or taking public transportation (14.8%) than the other racial/ethnic populations studied (nhw, mexican americans, and others). similarly, reporting 30 days of insufficient sleep or rest was significantly more prevalent among nhb (13.3%) than nhw (11.2%) 2008 brfss respondents. it is worthy of note that because of small sample sizes of ai/an in national surveillance systems, ai/an are often categorized as others, thus rendering it difficult to ascertain the prevalence of sleep sufficiency and risk factors in this population. as many risk factors associated with frequent insufficient sleep appear to be of increased prevalence in ai/an, we sought to determine the prevalence of insufficient sleep in a community-based sample of this population. specifically, to examine if ai/an are more likely to experience insufficient sleep relative to nhw, as has already been reported among nhb, we aggregate data from the 2009 and 2010 brfss. furthermore, we assess the impact of recognized sleep correlates (i.e., socioeconomic indicators, lifestyle behaviors, obesity, age, and fmd) on potential race/ethnicity disparities in insufficient sleep. data were obtained from the recent brfss, a large, random-digit-dialed telephone survey conducted in all 50 states, the district of columbia, and us territories. the 2009-2010 brfss collected data on health-related behaviors, including sleep, smoking, physical inactivity, binge drinking, obesity, and frequent mental distress among the us civilian population aged 18 years living in households with landline telephones. as previously noted, we combined 2009 and 2010 brfss data in order to yield an adequate sample size of ai/an. before aggregating these data, we observed no difference between 2009 and 2010 in frequent insufficient sleep, defined as 14 days/past 30 days in which respondents reported that they did not get enough rest or sleep. additionally, the two years shared a similar median response rate to the question assessing frequent insufficient sleep (52.5% and 54.6%, respectively) (brfss 2009 and 2010 summary quality report, version 1, revised on 2/18/2011 for 2009 and on 5/2/2011 for 2011). a detailed description of the brfss survey design, data collection techniques, and the full-text questionnaire can be found at http://www.cdc.gov/brfss. data were obtained from 810,168 respondents (96.9%) who self-identified as nhw, (n=671,448), nhb (n=67,685), hispanic (n=59,528), or ai/an (n=11,507) after excluding respondents who had missing data on the insufficient sleep question (n=15,322) and other variables of interest (n=25,636). all respondents were asked, during the past 30 days, for about how many days have you felt you did not get enough rest or sleep? we defined frequent insufficient sleep as 14 days, as this cutoff has been shown to have a strong relationship with the prevalence of chronic disease and health risk behaviors. sociodemographic characteristics included sex, age in years (1824, 2534, 3544, 4554, 5564 or 65), years of education (< 12, 12, or>12), and employment status (employed for wage/self-employed, unemployed, retired, unable to work, or homemaker/student). health-related lifestyle behaviors included smoking status (current smoker, former smoker, or never smoked), binge drinking (for men, 5 alcoholic beverages on one occasion in the previous 30 days; for women, 4 alcoholic beverages on one occasion in the previous 30 days), and physical inactivity (respondent indicated no to the question, during the past month, other than your regular job, did you participate in any physical activities or exercising such as running, calisthenics, golf, gardening, or walking for exercise?). frequent mental distress (fmd) was defined as a response of 14 days to the question, now thinking about your mental health, which includes stress, depression, and problems with emotions, for how many days during the past 30 days was your mental health not good ?. assessment of obesity was based on the body mass index (bmi, kg/m), calculated from respondents ' self-reported height in inches and weight in pounds (obese: bmi 30 kg/m versus not obese: bmi<30.0 kg/m). first, the racial/ethnic-specific distributions of the selected characteristics and frequent insufficient sleep were obtained in order to examine unadjusted race/ethnicity disparities. next, the prevalence of frequent insufficient sleep by race/ethnicity and groups defined by obesity, smoking, binge drinking, physical activity, and fmd status was calculated to demonstrate race/ethnicity-specific associations between these covariates and frequent insufficient sleep. we used multivariate logistic regression modeling to assess the race/ethnicity disparity in frequent insufficient sleep in separate models controlling first for age, then in a second model with the addition of sex, education, and employment, to which obesity was added to create a third model. a fourth model added smoking status, physical inactivity, and binge drinking to the third model, with a fifth model adding fmd. all analyses were conducted using sas-callable sudaan to account for the complex sampling design. ai/an had a higher proportion of male respondents than other racial/ethnicity groups. relative to nhw, we found that nhb, hispanics, and ai/an tended to be younger, to report<12 years of education and were more likely to be unemployed or to report being unable to work (p 0.01). in addition, nhb, ai/an, and hispanics were more likely to be obese relative to nhw (p 0.01) and, concomitantly, to report they were physically inactive (p 0.001). ai/an were more likely to report being current (33.2%) or former (25.7%) smokers than any of the other race/ethnicity groups examined. binge drinking was similar in all race/ethnicity groups except among nhb who were less likely to report binge drinking. table 1 also indicates that the unadjusted prevalence of fmd was significantly greater (p 0.001) among ai/an than any other race/ethnicity group. most notably, we found significantly higher unadjusted prevalences (95% confidence interval [95% ci ]) of frequent insufficient sleep among ai/an (34.2% [32.136.4 ]) and nhb (29.4% [28.630.1 ]) and a significantly lower prevalence among hispanics (25.4% [24.726.1 ]) compared to nhw (27.4% [27.127.6]). table 2 lists the race/ethnicity-specific prevalences of frequent insufficient sleep by selected characteristics. obese respondents had a statistically higher prevalence of frequent insufficient sleep than nonobese respondents among all race/ethnicities, excepting ai/an. regardless of race/ethnicity, current smokers were more likely to report frequent insufficient sleep than former smokers or never smokers (p 0.01). a higher prevalence of frequent insufficient sleep among binge drinkers compared to nonbinge drinkers reached statistical significance only among nhw and nhb (p 0.01). a positive relationship between physical inactivity and frequent insufficient sleep was observed regardless of race/ethnicity (p 0.01). persons reporting fmd were significantly more likely to report frequent insufficient sleep than those without fmd in all race/ethnicity groups (p 0.001). results from our adjusted multivariate logistic regression models comparing the likelihood of frequent insufficient sleep among nhb, hispanics, and ai/an relative to frequent insufficient sleep in nhw are shown in table 3. after age adjustment (model 1) the disparity in frequent insufficient sleep between nhw and nhb (pr=1.02; 95% ci: 0.991.04) was no longer statistically significant. however, both nhb (pr=0.95; 95% ci: 0.920.98) and hispanics (pr=0.84; 95% ci: 0.810.86) remained significantly less likely (p 0.001) to report frequent insufficient sleep than nhw, even after controlling for all the covariates in the model (model 5). table 3 also reveals a significant age-adjusted excess prevalence of frequent insufficient sleep in ai/an relative to nhw in model 1, (pr=1.20; 95% ci: 1.121.27). this was modestly weakened by the addition of sex, education, and employment (pr=1.12; 95% ci: 1.041.19) as indicated by model 2. this relationship was further attenuated in ai/an by the separate addition of obesity in model 3 (pr=1.10; 95% ci: 1.031.18), and then by the addition of smoking, physical inactivity, and binge drinking (pr=1.08; 95% ci: 1.011.16) in model 4. finally, the disparity in frequent insufficient sleep was no longer significant after adding fmd in model 5 (pr=1.05; 95% ci: 0.991.13). this is the first investigation to document the excess prevalence of frequent insufficient sleep among ai/an relative to other race/ethnicity groups. over 34% of ai/an reported frequent insufficient sleep 14 days in the past month in contrast to 29% of nhb, 27% of nhw, and 25% of hispanics. the disparity in frequent insufficient sleep prevalence between ai/an and nhw remained even after we adjusted for recognized sleep correlates that included sociodemographic characteristics, lifestyle behaviors, and obesity. however, we also found that this disparity was attributable, in part, to the relatively high rate of fmd in the ai/an population. notably, previous sleep research in this population has largely been limited to the identification of factors associated with the increased prevalence of sudden infant death syndrome [1618] and has documented the prevalence of sleep disturbance among ai/an veterans. other research on sleep health in ai/an suggests possible implications of the results of the present investigation. specifically, noting that cardiovascular disease (cvd) is the leading cause of death among ai/an, sabanayagam et al. assessed both symptoms of insomnia including short sleep duration and self-reported cvd among 449 ai/an aged 55 years of age in the native elder care study. notably, these investigators found that relative to participants reporting a 7-hour sleep duration, the multivariable odds ratio for cvd of those with a sleep duration 5 hours was 2.89 (95% ci: 1.177.16), after adjusting for demographic, lifestyle, and clinical factors. these results suggest that the excess insufficient sleep documented in the present investigation could manifest itself in the increased risk for cvd-related morbidity and mortality in the ai/an population. in a review of the literature metabolic cost, and triggers a cascade of metabolic, neuroendocrine, and behavioral changes designed to increase food consumption and decrease energy expenditure, thereby giving rise to obesity. the excess insufficient sleep observed in this study may thus underlie findings indicating that ai/an are at increased risk of obesity relative to other race/ethnicity groups [2325]. furthermore, as diabetes is associated with obesity [2629], our findings may describe a heretofore unrecognized risk factor underlying the increased rate of diabetes among ai/an [3032]. first, our measure of frequent insufficient sleep was based on subjective self-report and is not corroborated by actigraphy or polysomnography. second, the wording of the insufficient sleep question includes both the terms rest and sleep, thereby being potentially subject to varying interpretations among respondents. third, our sample is limited to only households with landline telephones and to respondents residing in noninstitutionalized settings; thus, our findings may not be generalizable to the overall us population. our finding of excess insufficient sleep among ai/an thus appears to confirm the importance of sleep health to chronic disease prevention and health promotion in this population. specifically, assessment of sleep sufficiency appears particularly germane to healthcare providers serving this population, as well as to public health messaging targeting sleep health (including sleep correlates) in ai/an communities. available interventions designed to promote awareness of the importance of sufficient sleep include a summary for patients describing the influence of both diet and sufficient sleep on efforts to lose weight, as well as cognitive behavioral therapy for insomnia [34, 35], with the latter improving both subjective and objective sleep quality, as measured by polysomnography. finally, as fmd appears associated with frequent insufficient sleep among ai/an, mental health assessment and intervention emerge as important components in the evaluation of insufficient sleep in this population.

objective. frequent insufficient sleep, defined as 14 days/past 30 days in which an adult did not get enough rest or sleep, is associated with adverse mental and physical health outcomes. little is known about the prevalence of frequent insufficient sleep among american indians/alaska natives (ai/an). methods. we assessed racial/ethnic differences in the prevalence of frequent insufficient sleep from the combined 2009-2010 behavioral risk factor surveillance survey among 810,168 respondents who self-identified as non-hispanic white (nhw, n=671,448), non-hispanic black (nhb, n=67,685), hispanic (n=59,528), or ai/an (n=11,507). results. we found significantly higher unadjusted prevalences (95% ci) of frequent insufficient sleep among ai/an (34.2% [32.136.4 ]) compared to nhw (27.4% [27.127.6]). however, the age-adjusted excess prevalence of frequent insufficient sleep in ai/an compared to nhw was decreased but remained significant with the addition of sex, education, and employment status; this latter relationship was further attenuated by the separate additions of obesity and lifestyle indicators, but was no longer significant with the addition of frequent mental distress to the model (pr=1.05; 95% ci: 0.991.13). this is the first report of a high prevalence of frequent insufficient sleep among ai/an. these results further suggest that investigation of sleep health interventions addressing frequent mental distress may benefit ai/an populations.

PMC3595691

pubmed-1324

because steroid hormones play an important role in a wide range of physiological processes, the potential to disturb endocrine effects is a major concern in the development of novel pharmaceutical drugs such as etomidate and aminoglutethimide. the adrenal gland is the most common target for toxicity in the endocrine system in vivo, because steroid hormones are primarily synthesized through enzymatic reactions in the adrenal cortex [25]. indeed, in these studies based on chemically induced endocrine lesions observed in vivo, the most frequent site of reported effects was the adrenal gland. therefore, the prediction of human adrenal toxicity based on the mechanism of on- or off-target actions in the early stages of drug development is important. the nci-h295r human adrenocortical carcinoma cell line has been used to elucidate mechanisms of adrenal steroidogenic disrupting compounds [1, 6]. the h295r cell line was established by gazder and his collaborators in 1990, which expresses all key steroidogenic enzymes and steroidogenesis-related proteins [79]. h295r cells have the physiological characteristics of zonally undifferentiated human fetal adrenal cells and the ability to produce steroid hormones found in the adult adrenal cortex [1, 7, 9]. in vitro bioassays using the h295r human cell line have been able to evaluate the effects of chemicals on steroid hormone production [1015], steroidogenic enzyme activities [11, 16, 17], and the expression of steroidogenic genes [11, 18]. in transcriptome studies, the mechanisms of action of many steroidogenic disrupting compounds have been qualitatively assessed in terms of adrenal toxicity. furthermore, measuring a few specific steroid hormones may not be a useful approach to study the mechanisms of steroidogenic disrupting effects in complex pathways such as adrenal steroidogenesis. to systematically understand how exogenous compounds affect adrenal steroidogenesis, simultaneous determination of all detectable steroid hormones and integrative analysis of these complex data would be important. as an exploratory approach to analyze complex data, toxclust developed by zhang and colleagues in 2009 is able to visualize concentration-dependent response relationships in the characteristics of chemically induced toxicological effects. however, this exploratory approach is unable to provide a quantitative understanding of the mechanism of action of adrenal toxicants or reveal systematic information about the effect of each enzymatic reaction, interactions, and feedback in the adrenal steroidogenesis pathway. systems biology based on computational models of biological processes and the comprehensive measurement of biological molecules is the most powerful approach to quantitatively understand the influence of each factor in complex biological pathways. in recent studies by our collaborators, a computational model of adrenal steroidogenesis has been developed in nci-h295r cells, including the steroidogenic disrupting effects of metyrapone to inhibit enzymatic reactions of cyp11b1 [21, 22]. the model reproduces the dynamics of adrenal steroidogenesis in nci-h295r cells and the influence of metyrapone. a current computational model of adrenal steroidogenesis was incorporated with a reaction of oxysterol synthesis as a bypass to consume cellular cholesterol. in addition, all reactions in this model are described by a kinetic equation of the first-order reaction. it is difficult to quantitatively evaluate the influence of each protein in the complicated system of adrenal steroidogenesis using the reported models, because it is simple and any biochemical and cellular biological information is not sufficient. for example, to clearly understand the cause of the change from the differentially dynamic patterns of steroid hormones, it is necessary to consider the substrate inhibition of steroidogenic enzyme because most of steroidogenic enzymes recognize multiple steroids as the enzymatic substrate. however, the substrate inhibition of steroidogenic enzyme can not be described by the mathematical model based on kinetic equations of first-order reaction that does not consider michaelis constant km expressing the affinity of the substrate. to quantitatively estimate the mechanism of steroidogenic disrupting compounds from comprehensive experimental data of adrenal steroidogenesis in nci-h295r cells, the reported model should be improved according to the following two points. first, the kinetic equation of enzymatic reactions should be exchanged from the first-order equation to a steady-state kinetic equation based on the mechanism of the enzymatic reaction. because a mathematical model organized by first-order equations operates in a simple structure-dependent manner, it does not show complex behavior based on molecular interactions, feedback, or regulation. second, intracellular localization processes of cholesterol should be incorporated as a considerable mechanism. because intracellular cholesterol molecules are stored as cholesterol esters or widely distributed as membrane components, only a few cholesterol molecules localized on the mitochondrial inner membrane are available for the adrenal steroidogenesis pathway [23, 24] moreover, cholesterol-trafficking processes from the outer to inner mitochondrial membranes, which are regulated by steroidogenic acute regulatory (star) protein, are one of the rate-limiting steps in adrenal steroidogenesis. by overcoming these limitations in the reported steroidogenesis model, systems analysis of adrenal steroidogenesis in h295r cells may be able to quantitatively estimate the mechanism of action of steroidogenic disrupting compounds. in the present study, to quantitatively estimate the toxicological mechanism of endocrine-active compounds in adrenal steroidogenesis and to predict human adrenal toxicity of novel pharmaceutical drugs in the drug discovery phase, we developed a novel computational model of steroidogenesis in nci-h295r cells. it includes cholesterol transport into intracellular regions from the extracellular space, the cholesterol translocation system in intracellular regions, including oxysterol synthesis, the metabolic pathway of adrenal steroidogenesis, and transport of steroid hormones. global sensitivity analysis of this adrenal steroidogenesis model is able to evaluate the influence of each steroidogenic enzyme and related protein for each steroid hormone observed in an in vitro steroidogenesis assay of nci-h295r cells. furthermore, the mechanisms of action of steroidogenesis disrupting compounds for steroidogenic enzymes can be estimated by the optimization method to solve the reverse problem from the concentration changes of 12 steroid hormones measured by liquid chromatography/mass spectrometry in the steroidogenesis assay of nci-h295r cells in vitro. using this developed model of adrenal steroidogenesis and the analytical approach, the in vitro steroidogenesis assay of nci-h295r cells can assess the human adrenal toxicity of a novel pharmaceutical drug based on quantitative understanding of its toxicological mechanism in adrenal steroidogenesis. nci-h295r human adrenocortical carcinoma cells were purchased from the american type culture collection (cat #crl-2128, manassas, va) and cultured at 37c in a humidified atmosphere with 5% co2. the cells were maintained in a 1: 1 mixture of dulbecco's modified eagle's medium (dmem, gibco, life technologies, carlsbad, ca) and f-12 medium (mp biomedicals inc., irvine, ca) supplemented with 15 mm hepes (dojindo laboratories, kumamoto, japan), 0.00625 mg/ml insulin (sigma-aldrich, inc., st. louis, mo), 0.00625 mg/ml transferrin (sigma-aldrich inc., st. louis, mo), 30 nm sodium selenite (wako pure chemical industries ltd., osaka, japan), 1.25 mg/ml bovine serum albumin (bsa, sigma-aldrich inc., st. louis, mo), 0.00535 mg/ml linoleic acid (sigma-aldrich inc., st. louis, mo), 2.5% nu serum (becton, dickinson and company, franklin lakes, nj), 100 u/ml penicillin (meiji seika pharma, tokyo, japan), and 100 mg nci-h295r cells were stimulated with adrenocorticotrophic hormone (acth), forskolin, and angiotensin ii to initiate steroidogenesis. changes in steroid concentrations over time were measured after stimulation in both cells and culture medium to construct a simulation model. the cells were seeded at 6 10 cells/well in 6-well plates. after 3 days of culture, the culture medium was changed to stimulation medium consisting of dmem/f-12 (1: 1) medium supplemented with 0.00625 mg/ml insulin, 0.00625 mg/ml transferrin, 30 nm sodium selenite, 1.25 mg/ml bsa, 0.00535 mg/ml linoleic acid, 10% fetal bovine serum (gibco, life technologies, carlsbad, ca), 100 u/ml penicillin, 100 mg/l streptomycin, 50 nm acth (sigma-aldrich inc., louis, mo), 20 m forskolin (sigma-aldrich inc., st. louis, mo), and 100 nm angiotensin ii (calbiochem, merck millipore, darmstadt, germany). culture media and cells were collected at 0, 8, 24, 48, and 72 h after stimulation. the cells were collected in 100 l distilled water and sonicated to produce a cell lysate. the cultures were conducted in four wells/time point (n=4). the concentrations of 12 steroids, pregnenolone (preg), 17-hydroxypregnenolone (hpreg), dehydroepiandrosterone (dhea), progesterone (prog), 17-hydroxyprogesterone (hprog), androstenedione (dione), testosterone (testo), 11-deoxycorticosterone (dcortico), 11-deoxycortisol (dcort), corticosterone (cortico), cortisol (cort), and aldosterone (aldo), in the medium and cell lysate were measured by lc/ms. concentrations of estrone (e1) and 17-estradiol (e2) were measured by enzyme-linked immunosorbent assays (wako pure chemical industries ltd. in addition, the concentration of cholesterol was measured using a commercial kit (wako pure chemical industries ltd., osaka, japan) based on the cholesterol oxidase method. a lc-vp series (shimadzu, kyoto, japan) consisting of an sil-htc autosampler, lc-10advp pump, cto-10acvp column oven, and dgu-14am degasser was used to set the reverse-phase liquid chromatographic conditions. the column was a cadenza cd-c18 column (100 2 mm i.d., 3 m, imtakt corp., kyoto, japan) used at 45c. the mobile phase included water/acetonitrile/formic acid 95/5/0.05 (v/v/v, solvent a) and water/acetonitrile/formic acid 35/65/0.05 (v/v/v, solvent b). the gradient elution programs were 0% b (0-1 min with an isocratic gradient), 040% b (1-2 min with a linear gradient), 40% b (27 min with an isocratic gradient), 40100% b (712 min with a linear gradient), 100% b (1214 min with an isocratic gradient), 1000% b (14-15 min with a linear gradient), and 0% b (15-16 min with an isocratic gradient) at a flow rate of 0.3 ml/min. the autosampler tray was cooled to 45c and the injection volume was 5 l. a triple quadrupole mass spectrometer api4000 (applied biosystems/mds sciex, concord, canada) coupled with an electrospray ionization source was operated in the positive ion mode. the optimized ion source conditions were as follows: collision gas, 6 psi; curtain gas, 40 psi; ion source gas 1, 50 psi; ion source gas 2, 80 psi; ion source voltage, 5500 v; ion source temperature, 600c. nitrogen was used as the collision gas in the multiple reaction monitoring (mrm) mode. the conditions of declustering potential, collision energy, and collision cell exit potential were optimized by every steroid. the transitions in mrm were as follows: preg m/z 317 299, hpreg m/z 315 297, dhea m/z 289 271, prog m/z 315 109, hprog m/z 331 109, dione m/z 287 97, dcort m/z 331 123, dcortico m/z 347 161, cortico m/z 347 100, cort m/z 363 309, aldo m/z 361 343, and testo m/z 289 109. mass spectroscopic data were acquired and quantified using the analyst 1.4.2 software package (applied biosystems/mds sciex, concord, canada). cell volume was estimated from the number of cells in the well and the average diameter of the cells. cells were detached from the well using 0.025% trypsin (mp biomedicals, inc., irvine, ca) in a 0.02% edta solution (dojindo laboratories, kumamoto, japan) at the start of preculture, start of stimulation, and at 24, 48, and 72 h after stimulation. the numbers and diameters of the cells were measured by a cell counter vi-cell xr 2.01 (beckman coulter, krefeld, germany) after trypan blue staining. parameters of the cell volume and number of cells were estimated to fit experimental time-course data using exponential curves. nci-h295r cells were exposed to seven well-characterized inhibitors of steroidogenesis, and then the concentrations of the steroids in the culture medium were measured to estimate the enzyme inhibition to evaluate the performance of the simulation model. the adrenal steroidogenic inhibitors included aminoglutethimide (agt, bachem ag, bubendorf, switzerland), o, p-ddd (ddd, sigma-aldrich inc., st. louis, mo), spironolactone (sp, sigma-aldrich inc., st. louis, mo), metyrapone (mp, sigma-aldrich inc., st. louis, mo), ketoconazole (kc, wako pure chemical industries, ltd., osaka, japan), miconazole (mc, wako pure chemical industries, ltd., osaka, japan), and daidzein (dz, sigma-aldrich inc., st. the cells were also exposed to four adrenal toxicants whose adrenal toxicity is not mediated through steroidogenesis inhibition. the toxicants were acrylonitrile (an, wako pure chemical industries, ltd., osaka, japan), salinomycin (sm, sigma-aldrich, inc., st. louis, mo), thioguanine (tg, tokyo chemical industry co., ltd., tokyo, japan), and fumaronitrile (fn, wako pure chemical industries, ltd., osaka, japan) and added to the culture medium at 1: 1000 dilutions. nci-h295r cells were cultured for 3 days in 6-well plates and then stimulated with the above-mentioned compounds. upon the start of stimulation, various concentrations of test chemicals were added to the cultures. after a further 3 days of culture with the chemicals, the concentrations of 12 steroids (preg, hpreg, dhea, prog, hprog, dione, dcortico, dcort, cortico, cort, aldo, and testo) in the culture medium were measured by lc/ms/ms. the cytotoxicity assay was conducted in 96-well plates using atp content in cells as an endpoint (celltiter-glo luminescent cell viability assay, promega). the test concentrations of adrenal steroidogenesis inhibitors and other compounds are shown in table 1. comparisons were performed by the two-sample welch's t-test with bonferroni multiple testing correction for each steroid hormone species. steroid hormones secreted from human adrenal corticocarcinoma nci-h295r cells are synthesized from cholesterol through the c21-steroid hormone biosynthesis pathway. a mathematical model of adrenal steroidogenesis in nci-h295r cells was constructed with cholesterol transport and the intracellular localization pathway, the oxysterol synthesis pathway as a bypass of steroidogenesis, the c21-steroid hormone biosynthesis pathway as the main steroidogenesis pathway, passive transport of steroid hormones, and cell proliferation (figure 1). in this model, two compartments, the intracellular space and culture medium, were incorporated as the available region. equations and parameters of the cell proliferation and diffusional transport of steroid hormones have been proposed by previous studies [21, 22]. cholesterol transport and the intracellular localization pathway including the oxysterol bypass were integrated using a part of the acth-stimulated cortisol secretion model described by dempsher and colleagues. the c21-steroid hormone biosynthesis pathway includes 14 steroid hormones, preg, hpreg, dhea, prog, hprog, dione, testo, dcortico, dcort, cortico, cort, aldo, e1, and e2, and 17 enzymatic reactions catalyzed by nine steroidogenic enzymes, cholesterol side chain cleavage enzyme (cyp11a1), 17-hydroxylase (cyp17h), c17,20-lyase (cyp17l), 3-hydroxysteroid dehydrogenase (hsd3b2), 21-hydroxylase (cyp21a2), 11-hydroxylase (cyp11b1), 18-hydroxylase (cyp11b2), 17-hydroxysteroid dehydrogenase (hsd17b3), and aromatase (cyp19a1). in this mathematical model of adrenal steroidogenesis in nci-h295r cells, the flux velocities of molecular transportation and enzymatic reaction rates of steroidogenic enzymes were defined based on the first-order reaction and rapid-equilibrium enzyme kinetics, respectively. all equations in the mathematical model of adrenal steroidogenesis of nci-h295r cells were described in a supplementary document (see supplementary material available online at http://dx.doi.org/10.1155/2016/4041827). the rate constants and the maximum activities were estimated by fitting to experimental time-course data of the concentrations of cholesterol and all steroids. initial values of cholesterol and the 14 steroid concentrations were used in each experimentally measured value, and every steroid concentration was assumed to rapidly reach the equilibrium state between the culture medium and intracellular space. all fixed values of static parameters and initial values of variable parameters in this model were described in tables s1 and s2 in a supplementary document, respectively. this computational model of adrenal steroidogenesis in nci-h295r cells was developed on the simbio platform which is a general environment of biological dynamic simulation and computational model development. odes were solved by the fourth-order runge-kutta method with a variable time step. the time step (dt) was adjusted to refer to the maximum absolute value of flux velocities or enzymatic reaction rates at each time point, and the range of the time step was from 1 10 to 10. to confirm whether the range of the time step was suitable, the numerical error ratio was calculated by certain fixed time steps in the range of the time step, which was under 1 10 in every time step. the duration time of computational simulation of adrenal steroidogenesis in nci-h295r cells was set at 72 h. to reconstruct experimental time-course patterns of the concentrations of cholesterol and the 14 steroids in the culture medium and intracellular space, we optimized every rate constant and maximum velocity of the steroidogenic enzymes. this parameter optimization problem was solved by the levenberg-marquardt method which is one of the nonlinear least squares methods [4951]. the objective function of optimization was used as the following normalized least squares distance (nlsd):(1)nlsd=h i jxh, i, jexpxh, i, jsim2xh, imax2,where h is the compartment (culture medium or intracellular space), i is the molecular species (cholesterol and the 14 steroids), j is the time point (0, 8, 24, 48, and 72 h), xh, i, j is the experimentally measured concentration of molecule i in compartment h at time point j, xh, i, j is the simulated concentration of molecule i in compartment h at time point j, and xh, i is the maximum concentration of molecule i in compartment h over all time points. data points under the lower quantitation limit were excluded from the evaluation by the objective function. effects of every static model parameter for parameter optimization were calculated from differences of fitting the objective function using sensitivity analysis. metabolic steroid profiling and differential patterns of the adrenal steroid hormones by chemical perturbation were reconstructed to optimize the relative activities of the steroidogenic enzymes. the input data for the quantitative mechanistic analysis of adrenal toxic compounds was a fold change (ratio) of the measured 12 steroid concentrations induced by drug exposure for 72 h. the two-step optimization method of the real-coded genetic algorithm (rcga) was adopted as a global optimization method in the quantitative mechanistic analysis of adrenal toxic compounds. the operations of the crossover and generation alteration model in rcga were used for the real-coded ensemble crossover (rex) and just generation gap (jgg) [5255]. as the initial parameters of rcga, maximum generation, population size, selection size of parent individuals, population size of child individuals, and termination criteria were 1000, 100, 6, 25, and under 0.1 of nlsd, respectively. the search space for the relative activities of the steroidogenic enzymes was from 1/100 to 100. to evaluate the fitness of each individual, the sum of squared residuals for fold changes of measured 12 steroid concentrations was used as the objective function. nonlinear least squares optimization by the levenberg-marquardt method was used as a local search [4951]. as the estimated mechanisms of actions of the adrenal toxic compounds, the relative activities of eight steroidogenic enzymes (cyp11a1, cyp17h, cyp17l, hsd3b2, cyp21a2, cyp11b1, cyp11b2, and hsd17b3) were optimized by the above-mentioned 2-step optimization method. the property of every kinetic parameter in this computational model of steroidogenesis in nci-h295r cells was evaluated by dynamic sensitivity analysis. the sensitivity (sx, y) of kinetic parameter x for variable parameter y was defined by the following equation:(2)sx, yt=yt/ytx/x, where variable parameter y was the concentration of a steroid hormone in the cytosolic space of nci-h295r cells. the perturbation for kinetic parameters was+10% (x/x=0.1). all steroid hormones in the culture medium were significantly increased after 72 h of stimulation with 50 nm acth, 20 m forskolin, and 100 nm angiotensin ii (figure 2(a)). mass balances in steroidogenesis of nci-h295r cells under nontreatment and control (stimulated) conditions are shown in figures 2(b) and 2(c), respectively. under stimulation, the dynamics of net mass in these experiments were unchanged, and accumulated cholesterol was converted to adrenal steroids. viabilities of cells treated with each compound were expressed as a relative value to the atp level of the control. effects of an, sm tg, fn, agt, ddd, sp, mp, kc, mc, and dz on cell viability were determined to be valid under 80% of the relative atp level at 7 days after treatment. an, sm, tg, and fn showed cytotoxicity at over 100, 1, 10, and 10 m, respectively. agt, mp, and dz did not affect cell viability at up to 100 m. ddd, sp, kc, and mc induced less than 80% of cell viability at over 100, 50, 100, and 25 m, respectively. after nci-h295r cells were exposed to each test compound during three days, the concentrations of 12 steroid hormones in the culture medium were simultaneously measured by lc/ms/ms. all effects of the compounds on adrenal steroidogenesis were evaluated at the concentration without any overt cytotoxicity. the differential metabolic steroid profiles of 11 adrenal toxic compounds were classified and visualized by using hierarchical clustering analysis (figure 3). four adrenal toxicants without steroidogenic inhibition, an, sm, tg, and fn, did not change the medium concentrations of all steroid hormones by more than 2-fold. above-mentioned 4 compounds at every condition and 7 adrenal steroidogenic inhibitors at the low exposure concentration were gathered into a big cluster as nonchange group. the 7 steroidogenic inhibitors at the maximum exposure concentration showed the characteristic steroid profiles each, but 100 m dz and 10 m sp were classified as a cluster. agt drastically decreased the medium concentrations of preg, hpreg, dhea, prog, dcortico, cortico, and aldo at 100 m. ddd dose-dependently decreased the medium concentrations of prog, dcortico, cortico, cort, and aldo at>10 m and decreased preg, hpreg, dhea, prog, hprog, dione, and dcort at the maximum exposure concentration of 25 m. sp increased preg, hpreg, and dhea and decreased prog, dione, dcortico, dcort, cortico, aldo, and testo at 10 m. mp dose-dependently decreased cortico, cort, and aldo and decreased dhea, hprog, dione, and testo at the maximum exposure concentration of 100 m. kc drastically decreased the medium concentrations of preg, hpreg, dhea, hprog, dione, dcortico, dcort, cortico, corto, aldo, and testo at 10 m. mc increased the medium concentrations of prog and decreased dione, dcort, cort, and testo at 10 m. dz increased preg, hpreg, and dhea and decreased dione, dcortico, dcort, cortico, cort, aldo, and testo at 100 m. the mathematical model of adrenal steroidogenesis in nci-h295r cells was optimized for several kinetic parameters of cholesterol transport, intracellular localization, the oxysterol pathway, and maximum velocity of steroidogenic enzymes to fit the experimental time-course data. the optimized mathematical model was reconstructed with the experimental dynamic patterns of cholesterol and the 14 steroid hormones in the intracellular space and culture medium. optimized kinetic parameters were calculated sensitivities for the nlsd value as the fitting score and are shown in table s1 in a supplementary document. the highly sensitive parameters for fitting the nlsd score were the extracted nine kinetic parameters, kcholesterol transport, kf, kf, kb, vmax, km, vmaxa, vmaxa, and vmaxa, which had higher than 3.0 fitting sensitivity. effects of adrenal toxic compounds on steroidogenic enzymes were quantitatively predicted from the change in the ratio of the measured medium concentrations of the 12 steroid hormones at 72 h after drug exposure using the mathematical model of adrenal steroidogenesis in nci-h295r cells. the estimated effects of 11 adrenal toxic compounds on eight steroidogenic enzymes are shown in figure 5. the adrenal toxic compounds without steroidogenic inhibition, such as vasculotoxic agents (an, sm, tg, and fn), were not estimated for the target steroidogenic enzymes under noncytotoxic conditions. every fitness values were under 0.05 of nlsd values used as the fitting objective function (figures 5(a)5(d)). other steroidogenic inhibitors (agt, ddd, sp, mp, kc, mc, and dz) are described in detail below. the mechanism of action of agt in adrenal steroidogenesis was estimated by inhibition of cyp11a1, hsd3b2, cyp21a2, and cyp11b1 at 100 m (estimated inhibitions were 77.0%, 78.0%, 81.1%, and 59.8%, resp.) (figure 5(e)). agt has been reported to inhibit cyp11a1, cyp21a2, cyp11b1, and cyp11b2 [6, 2730]. in particular, cyp11a1 appeared to be inhibited strongly by agt. in our study, hsd3b2 inhibition of agt was shown by mechanistic analysis based on systems biology approaches as a novel mechanism of action of agt. however, the concentration of aldo in the culture medium decreased to 3.8% of the normal stimulated condition. inhibition of agt by cyp11b2 has been shown using sheep adrenal homogenates as well as a human adrenal homogenate from a patient with cushing's syndrome. the activity of 18-hydroxylase induced by cyp11b2 was determined as the conversion of corticosterone to 18-hydroxycorticosterone in the previous study. the cause of the discrepancy regarding the effect of agt on cyp11b2 was suggested to be substrate inhibition, because the intracellular concentration of cortico was increased by over 10 times of that in the culture medium to reach 50 m. another possibility was poor quantitative reliability of the experimental data, because the aldo concentration was under the lower limit of quantification at 100 m agt. hecker and colleagues reported that 3 m agt decreases preg and prog concentrations and increases the testo concentration. one possibility is that the concentration of testo was already enhanced by about 3.3-fold through stimulation with acth, forskolin, and angiotensin ii. the mechanism of action of ddd in adrenal steroidogenesis was estimated by dose-dependent inhibition of cyp11a1, hsd3b2, cyp21a2, and cyp11b1 (estimated inhibitions at 25 m were 87.0%, 86.9%, 76.9%, and 84.9%, resp.) ddd has been reported to inhibit cyp11a1, hsd3b2, cyp21a2, cyp11b1, and cyp11b2 [29, 31, 32]. inhibition of ddd by cyp11b2 was not estimated in our study. however, the concentration of aldo in the culture medium decreased to 3% of that in the normal stimulated condition. inhibition of ddd by cyp11b2 has been shown using mitochondrial and microsomal fractions prepared by standard centrifugation procedures from a bovine adrenal cortex homogenate. the cause of the discrepancy regarding the inhibition of ddd by cyp11b2 could not be explained by same effect in the case of agt. the mechanism of action of sp in adrenal steroidogenesis was estimated by inhibition of hsd3b2, cyp21a2, and hsd17b3 (estimated inhibitions at 10 m were 70.2%, 59.5%, and 59.3%, resp.) sp has been reported to inhibit cyp17h, cyp17l, cyp11b1, and cyp11b2 [6, 3335]. the inhibitory effect of sp on the hsd3b2 enzyme was a novel mechanism of action. sp has also been reported to exert some off-target effects by binding to androgen, glucocorticoid, and progesterone receptors [5658]. sp has been shown to inhibit the production of aldo and cort from preg induced by angiotensin ii in h295r cells, but the specific mr antagonist eplerenone did not show the inhibitory effects. therefore, hsd3b2 inhibition by sp is not mediated via mr, and the action might be direct inhibition of hsd3b2 enzymes or a part of known off-target effects mediated through other nuclear hormone receptors. regarding cyp17h and cyp17l, our results were consistent with previous reports [33, 34]. the fact that there were no inhibitions of cyp17h or cyp17l in our study suggests that sp might not be deacetylated to 7-thiospironolactone in nci-h295r cells. it has been shown that 30 m sp inhibits cyp11b1 and cyp11b2 in human and bovine adrenal mitochondria. the cause of cyp11b1 and cyp11b2 inhibition by sp could not be determined in our study, which might be due to the lower maximum exposure concentration of sp than that in the previous report. we could not examine sp concentrations over 10 m because these concentrations were cytotoxic in nci-h295r cells. the mechanism of action of mp in adrenal steroidogenesis was estimated by dose-dependent inhibition of cyp11b1 (estimated inhibitions at 1, 10, and 100 m were 57.1%, 82.7%, and 98.2%, resp.) mp has been reported to inhibit cyp11b1 as its major effect and cyp11a1 and cyp11b2 as a weak effect [6, 29, 3639]. the results were able to show that mp is a selective inhibitor of cyp11b1 in the previous report. however, the estimated effect of mp at a high concentration, 100 m as the maximum exposure concentration, was unclear. according to the previous report, selectivity of mp for cyp11b1/cyp11b2 is about five times. in addition, 20 m mp has a slight inhibition effect on cyp11a1 in h295r cells. the mechanism of action of kc in adrenal steroidogenesis was estimated by inhibition of cyp11a1, cyp17h, cyp17l, hsd3b2, cyp21a2, cyp11b1, and cyp11b2 (estimated inhibitions at 10 m were 92.6%, 94.3%, 51.8%, 83.0%, 88.2%, 97.4%, and 79.8%, resp.) kc has been reported to inhibit cyp11a1, cyp17h, cyp17l, hsd3b2, cyp21a2, and cyp11b1 [6, 29, 4043]. kc inhibits cyp11a1, cyp17h, cyp21a2, and cyp11b1 in nci-h295r cells at 10 m and cyp17h, cyp17l, cyp21a2, and cyp11b1 in human adrenal mitochondria and leydig cell microsomes at 25 m [60, 61]. however, kc has shown only weak inhibition of hsd3b2 and hsd17b3 in leydig cells at the millimolar level [60, 61]. regarding cyp11b2 and hsd17b3, we considered that these estimated inhibitions of kc did have sufficient reliability in terms of quantitative prediction precision, because aldo and testo concentrations were less than the lower limit of quantification at 10 m kc. the mechanism of action of mc in adrenal steroidogenesis was estimated by inhibition of cyp17h, cyp17l, cyp11b1, and hsd17b3 (estimated inhibitions at 10 m were 69.1%, 53.0%, 76.4%, and 57.1%, resp.) (figure 5(j)). mc has been reported to inhibit not only cyp17h and cyp17l but also cyp11a1, cyp21a2, and cyp11b1 [41, 44, 45]. the results in the previous reports were able to estimate that mc is a cyp17 inhibitor. however, cyp11a1 inhibition by mc, probably instead of a reduction in star expression, was not clearly detected in our study using nci-h295r cells, because there were no decreases in the concentrations of preg and prog in the culture medium. indirect inhibition of cyp11a1 via the peripheral-type benzodiazepine receptor has been reported in mouse adrenocortical y-1 cells treated with mc in the absence of stimuli by measuring preg production. on the other hand, reductions of star protein expression and/or transport activity without affecting total steroid synthesis or cyp11a1 and hsd3b2 enzyme expression or activities have been reported in (bu)2camp-stimulated ma-10 leydig tumor cells treated with mc by measuring prog production. therefore, the effect of mc on the initial reaction in adrenal steroidogenesis from cholesterol should be different according to the cell type and stimulation condition. inhibition of cyp21a2 and cyp11b1 by mc has been reported as decreases in the consumption of prog and dcortico, respectively. inhibition of cyp11b1 was estimated by the action of mc in this study, but that of cyp21a2 was not detected. in the previous experimental report, inhibitory sites by mc might have been reflected by inhibition of cyp17h activity, because cyp21a2 activity was measured as a decrease in labeled prog. the mechanism of action of dz in adrenal steroidogenesis was estimated by inhibition of cyp11a1, hsd3b2, cyp21a2, cyp11b1, and hsd17b3 (estimated inhibitions at 100 m were 58.6%, 94.1%, 96.5%, 87.2%, and 98.1%, resp.) (figure 5(k)). these estimated effects of dz on cyp11b1 and hsd17b3 were unclear, because the concentrations of aldo and testo were less than the lower limit of quantification at 100 m. in addition, these enzymes act downstream of the strong action points of dz, such as hsd3b2 and cyp21a2. to comprehensively understand the dynamics of adrenal steroidogenesis, dynamic sensitivities were calculated for steroid concentrations secreted by nci-h295r cells using our constructed mathematical model of steroidogenesis. the results of dynamic sensitivity analysis at 72 h of duration and 6 h of interval time are presented as a heat-map in figure 6. the top 10 parameters of the total area under the curve of dynamic sensitivity for cholesterol and the 14 steroids in culture medium were vmaxa, vmax, vmaxa, kf, kb, km, vmaxa, kcholesterol transport, kf, and vmaxb in order from the top. cholesterol uptake (kcholesterol transport), star protein (kf), and cyp11a1 (vmax), which are determining factors of the capacity for steroidogenesis, promoted the production of mineralocorticoids (dcortico, cortico, and aldo) and restrained the synthesis of glucocorticoids (dcort and cort) and sex steroids (dione, testo, and e1) because of the accumulation of intermediate molecules in steroidogenesis (preg, hpreg, dhea, prog, and hprog) only by self-activation. the dynamic patterns of the intermediate molecules in steroidogenesis were mainly dependent on the activity of cyp17h and hsd3b2 with preg as the substrate of these enzymes, in which the dynamic sensitivities of vmaxa for hpreg, and hprog and vmaxa for prog, hprog, and dcortico reversed the direction of sensitivity at 4966 h after stimulation. the dynamic sensitivities of the maximum activities of hsd3b2 for preg (vmaxa) and cyp21a2 for prog (vmaxa) were related to all steroids at 72 h. almost all model parameters had positive sensitivity for downstream steroids in the adrenal steroidogenic pathway and negative sensitivity for direct-binding steroids as substrates of the steroidogenic enzyme. the sensitivity of vmax in all steroidogenic enzymes was relatively higher than km for the same steroid substrate. to clearly show the property of the metabolic shift between mineralocorticoid and glucocorticoid biosynthesis, we performed two-dimensional parameter scanning of the enzymatic activities of cyp17h and hsd3b2 (figure 7). nci-h295r cells lost the ability to produce all steroid hormones when enzymatic activities of cyp17h and hsd3b2 were changed by over 60% and 30%, respectively. activation of cyp17h and/or hsd3b2 induced the metabolic shift that enhanced the glucocorticoid biosynthesis and deviated from the mineralocorticoid biosynthesis. on the other hand, inhibition of cyp17h and/or hsd3b2 induced the metabolic shift that enhanced the mineralocorticoid biosynthesis and deviated from the glucocorticoid biosynthesis. moreover, the enzymatic activity of hsd3b2 regulated the metabolic balance of sex steroids and the precursors on adrenal steroidogenesis of nci-h295r cells. e1, testo, and dione were produced by nci-h295r cells when activating the enzymatic activity of hsd3b2. conversely, e2 and dhea were produced by nci-h295r cells when suppressing the enzymatic activity of hsd3b2. the biosynthesis of downstream steroids in adrenal steroidogenesis pathway, such as mineralocorticoids and glucocorticoids, was almost completely terminated when the enzymatic activity of hsd3b2 was decreased by over 80%. our systematic analysis using the mathematical model of adrenal steroidogenesis in nci-h295r cells revealed that the enzymatic activity of 3-hsd controls the dynamics of adrenal steroidogenesis. the activity of the star protein controls the net capacity of steroidogenesis in steroidogenic cells, which is the transport of cholesterol from the outer to inner mitochondrial membranes. both the expression levels of star protein and mrna are rapidly elevated in response to stimulation by tropic hormones such as acth [62, 63]. another important factor in adrenal steroidogenesisis is the cholesterol side chain cleavage enzyme cyp11a1, the first rate-limiting and hormonally regulated step in the synthesis of all steroid hormones, which is conversion of cholesterol to pregnenolone in mitochondria. according to our results of global sensitivity analysis (supplementary table 1 and figure 6(d)), in addition to cyp11a1 and star proteins, 3-hsd was one of the key regulators in adrenal steroidogenesis of nci-h295r cells. and also, this result suggests that a significant regulatory mechanism in steroidogenesis pathway is very reasonable. star, cyp11a1, and 3-hsd (isoforms 1 or 2 in humans) proteins generally respond to the same hormones that stimulate steroid production through common pathways such as camp signaling in adrenal glands and testes [65, 66]. moreover, our data also support recent experimental evidence from clinical and in vivo studies, suggesting that the enzymatic activity of 3-hsd plays an important role in the regulation of mineralocorticoid synthesis in adrenal steroidogenesis and contributes to hypertension caused by abnormal overproduction of aldosterone [6770]. circadian clock-deficient cry-null mice show salt-sensitive hypertension due to abnormally high synthesis of aldosterone, which is caused by constitutively high expression of hsd3b6 mrna and protein in the adrenal cortex [67, 68]. recent clinical observations have revealed predominant expression of hsd3b2 mrna and protein in tumor cells of aldosterone-producing adenoma (apa), and hsd3b1 mrna significantly correlated with cyp11b2 mrna levels and plasma aldosterone concentrations in apa patients [69, 70]. however, the relationship is unclear and disputed in a small-scale clinical study indicating that genetic variation in hsd3b1 affects blood pressure and hypertension in apa patients. the results of our simulation study suggest that 3-hsd protein (human genes are hsd3b1 and hsd3b2) is one of the determination factors for the dynamic property of adrenal steroidogenesis. our results support the clinical evidence of doi and colleagues, and we believe that the hsd3b1 enzyme has a promising potential as novel drug target for endocrine hypertension. the metabolic properties of adrenal steroidogenesis in nci-h295r cells were revealed by dynamic sensitivity analysis using the mathematical model (figure 6). mineralocorticoids, such as dcortico, cortico, and aldo, and intermediate steroids upstream of the adrenal steroidogenesis pathway, such as preg, hpreg, dhea, prog, and hprog, were accelerated by reactions of cholesterol import (kcholesterol transport), star protein (kf and kf), and cyp11a1 (vmax). on the other hand, glucocorticoids, such as dcort and cort, and sex hormones, such as dione, testo, and e1, were suppressed by these model parameters. therefore, enhancement of the net adrenal steroidogenesis capacity, which supplies preg precursor to the pathway, causes a production shift from glucocorticoids to mineralocorticoids by substrate inhibitions of cyp17h, hsd3b2, and cyp21a2 caused by accumulation of initial intermediate steroids such as preg and prog. sensitivities of cyp17h (vmaxa) and hsd3b2 (vmaxa) for the products were dynamically changed and these parameters determined the metabolic balance of downstream steroids in the adrenal steroidogenesis pathway. according to these results of dynamic sensitivity analysis of star, cyp11a1, cyp17h, and hsd3b2, we suggest that the enhancement of cyp17h and hsd3b2 activity during acth stimulation was important to shift the steroidogenic output away from aldo biosynthesis towards cort biosynthesis, as well as adrenal androgen production. this suggestion partially supports a comparative animal study in which molecular and cellular variations in cyp17h activity dramatically affect acute cortisol production, resulting in distinct physiological and behavioral responses. results of two-dimensional parameter scanning of the enzymatic activities of cyp17h and hsd3b2 quantitatively showed the detail of the metabolic relationship between mineralocorticoid and glucocorticoid biosynthesis (figure 7). particularly, the results showed that the balance of these enzymatic activities was very important for the typical function of nci-h295r cells, namely, the ability to produce all steroid hormones. nci-h295r cells lost this function when enzymatic activities of cyp17h and hsd3b2 were changed by over 60% and 30%, respectively. in addition, they became mineralocorticoid (aldo) secreting cells when the enzymatic activity of cyp17h or hsd3b2 was inhibited by over 50% or glucocorticoid (dcort and cort) secreting cells when these enzymes were activated by over 50%. in particular, this analysis also showed that hsd3b2 was a key player in the adrenal steroidogenesis of nci-h295r cells, because hsd3b2 inhibition by over 80% almost completely inhibited the biosynthesis of downstream steroids. the ratio of cyp17a1 to hsd3b2 mrna expression levels has been related to several endocrine diseases with a low level in apas and high level in cortisol-producing adenomas. furthermore, the expression levels or enzymatic activities of cyp17a1 and hsd3b1 have been related to androgen production in polycystic ovary syndrome [75, 76]. these clinical studies support our simulation results indicating that the balance of enzymatic activity of cyp17h and hsd3b2 determines the shift in steroidogenic output to mineralocorticoids, glucocorticoids, or androgens. according to our results obtained using the mathematical model of steroidogenesis in nci-h295r cells, such as sensitivity analysis, comprehensive analysis based on systems biology is available to quantitatively estimate the mechanism of action of steroidogenic disrupting compounds from differential profiling of adrenal steroid hormones, because dynamic patterns of steroid hormones in adrenal steroidogenesis pathway are highly complex. our proposed method of quantitative mechanistic analysis of steroidogenic inhibitors was able to predict known action sites in the adrenal steroidogenesis pathway at only one time point (72 h after drug exposure). moreover, according to the results of sensitivity analysis (figure 6), vmax of all steroidogenic enzymes was more sensitive than the km, because the intracellular concentrations of steroid hormones were almost maintained at sufficiently high levels compared with km values of steroidogenic enzymes. these results suggested that estimation of the mechanism of action of drugs is more effective and detectable when using the influences of vmax as the searching parameters such as our proposed method. our data showed that the proposed method based on a systems biology model is a very powerful tool for exploratory screening of steroidogenic disrupting compounds. rcga as a solver of parameter estimation problems in systems biology has been applied to biological network identification of gene regulatory networks and metabolic pathways and optimization of biological processes using experimentally observed time-course data [7782]. in this study, rcga was useful to estimate the mechanism of action of novel pharmaceutical drug candidates for adrenal steroidogenesis as a new application of rcga in systems biology. we had two issues when applying rcga to the quantitative mechanistic analysis of drug actions. these issues were the vast calculation cost and multimodality of quasi-optimum solutions in solving the optimization problem, because the mathematical model in systems biology consists of many equations and parameters. a proposed optimization strategy using rcga based on rex/jgg was a highly stable and efficient calculation method for a better quasi-optimum solution than the unimodal normal distribution crossover (undx)/minimum generation gap (mgg) method that is well applied in the engineering field. in addition, we expanded the rcga optimization program based on rex/jgg to a hybrid method of ga and then applied a local search as recommended by harada and kobayashi [28, 83]. a final optimal solution was obtained with a good convergence property. because these problems are general in systems biology studies, we suggest that the proposed hybrid method based on rex/jgg is a very useful tool for quantitative mechanistic analysis of novel pharmaceutical drugs, not limited to steroidogenic disrupting compounds. the novel mathematical model of adrenal steroidogenesis was constructed in this study, including cholesterol transport and distribution, the c21-steroid hormone pathway, steroid transport, and cell proliferation, which could reproduce adrenal steroidogenesis in nci-h295r cells. according to the results of dynamic sensitivity analysis using the new model, hsd3b2 plays the most important role in the metabolic balance of adrenal steroidogenesis in nci-h295r cells. moreover, to quantitatively estimate mechanisms of action of adrenal toxic compounds, we analyzed differential metabolic profiles of 12 steroid hormones at 3 days after exposure to 11 adrenal toxic compounds, by using the new mathematical model and a hybrid optimization method of the rcga and a local search (nonlinear least squares). we could estimate which steroidogenic enzymes were affected by these compounds using the hybrid optimization method. vasculotoxic agents were estimated to have no effect according to the results obtained by our method. in terms of adrenal steroidogenic inhibitors, the predicted action sites were approximately matched to the target enzymes as reported in the literature. thus, our computer-aided method based on a systems biology approach may be useful to analyze the mechanism of action of endocrine-disrupting compounds and to assess the human adrenal toxicity of novel pharmaceutical drugs based on steroid hormone profiling.

adrenal toxicity is one of the major concerns in drug development. to quantitatively understand the effect of endocrine-active compounds on adrenal steroidogenesis and to assess the human adrenal toxicity of novel pharmaceutical drugs, we developed a mathematical model of steroidogenesis in human adrenocortical carcinoma nci-h295r cells. the model includes cellular proliferation, intracellular cholesterol translocation, diffusional transport of steroids, and metabolic pathways of adrenal steroidogenesis, which serially involve steroidogenic proteins and enzymes such as star, cyp11a1, cyp17a1, hsd3b2, cyp21a2, cyp11b1, cyp11b2, hsd17b3, and cyp19a1. it was reconstructed in an experimental dynamics of cholesterol and 14 steroids from an in vitro steroidogenesis assay using nci-h295r cells. results of dynamic sensitivity analysis suggested that hsd3b2 plays the most important role in the metabolic balance of adrenal steroidogenesis. based on differential metabolic profiling of 12 steroid hormones and 11 adrenal toxic compounds, we could estimate which steroidogenic enzymes were affected in this mathematical model. in terms of adrenal steroidogenic inhibitors, the predicted action sites were approximately matched to reported target enzymes. thus, our computer-aided system based on systems biological approach may be useful to understand the mechanism of action of endocrine-active compounds and to assess the human adrenal toxicity of novel pharmaceutical drugs.

PMC4773560

pubmed-1325

primary plasma cell leukemia (ppcl) is a very aggressive form of plasma cell disorder. ppcl is very rare, reported in only 4-6% of patients with multiple myeloma (mm). ppcl is defined as the presence of more than 20% plasma cells in the peripheral blood and an absolute plasma cell count greater than 2 10/l, without previous evidence of mm. consistent with the high tumor burden, extramedullary involvement, such as hepatosplenomegaly, lymphadenopathy, soft-tissue plasmacytomas, or leptomeningeal infiltration, is more frequent in ppcl than in mm. despite the development of new agents, such as proteasome inhibitors or immunomodulatory drugs (imids), for plasma cell disorders, the median overall survival of ppcl although ppcl is difficult to treat, high-dose melphalan followed by autologous stem cell transplantation (auto-sct) and/or allogeneic stem cell transplantation (allo-sct) seems to improve outcomes in younger selected patients. however, patients with ppcl who had central nervous system (cns) relapse after auto- and/or allo-sct were refractory to various treatments, and had an extremely poor prognosis. we herein present a ppcl patient with cns relapse after allo-sct who underwent concurrent intrathecal therapy (it) and radiotherapy (rt) followed by maintenance therapy with pomalidomide and low-dose dexamethasone (pd). a 46-year-old man without a previous significant medical history was referred to our hospital with complaints of anorexia and generalized fatigue. laboratory findings revealed significant abnormalities, including a white blood cell (wbc) count of 19.710/l with 45% plasma cells, a hemoglobin level of 11.6 g/dl, a platelet count of 7.010/l, a creatinine level of 7.63 mg/dl, and a ldh level of 301 iu/l (table 1). on a peripheral blood smear, the plasma cells were medium-sized, with round nuclei, basophilic cytoplasm, and occasional cytoplasmic vacuoles (figure 1a). g/l, and the serum free light chain (flc) k/ ratio was 161.11 (table 1). in this case, a m-component of the igg-k type was identified by serum protein electrophoresis. moreover, flow cytometry on the peripheral blood showed that these plasma cells were positive for cd38, cd49e, cd54, cd138 and cytoplasmic kappa light chain. t(11;14)(q13;q32) in 9 metaphase cells (figure 1b) and 46,xy in 11 metaphase cells, and a fluorescent in situ hybridization analysis revealed ccnd1/igh gene fusion rearrangement. the patient was admitted to an intensive care unit (icu) for continuous hemodiafiltration (chdf). on the same day, we started induction therapy with lenalidomide (15 mg/day orally on days 1-14), bortezomib (1.3 mg/m subcutaneously on days 1, 4, 8, and 11), and low-dose dexamethasone (20 mg/day orally on days 1, 2, 4, 5, 8, 9, 11, and 12), also known as rvd induction therapy, each for a 21-day cycle. eight days after starting the induction therapy, the circulating plasma cells in the peripheral blood disappeared, and he was transferred from the icu after withdrawal of chdf. after a total of 3 cycles of chemotherapy, the laboratory abnormalities markedly subsided, consistent with a very good partial response (vgpr), because serum immunofixation was positive. he and his family consented to undergo allo-sct. at our outpatient department, because of grade 2 peripheral neuropathy, he continued induction therapy with lenalidomide (25 mg/day orally on days 1-14), bortezomib (1.3 mg/m subcutaneously on days 1 and 8), and dexamethasone (20 mg/day orally on days 1 and 8) in each 21-day cycle. after a total of 6 courses, allo-sct was performed with the bone marrow from an unrelated donor (hla-a one allele mismatch). the conditioning regimen consisted of fludarabine 120 mg/m (30mg/m on day -5, -4, -3, and -2), melphalan 180 mg/m (90mg/m on day-4 and -3), and rabbit anti-thymocyte globulin 2.5 mg/m (1.25mg/m on day -2 and -1). for the prevention of graft-versus-host-disease (gvhd), tacrolimus was started from day -1, and methotrexate was given on days 1, 3, and 6. complete donor chimerism was detected in the bone marrow on day 29. during this admission, however, serum immunofixation was positive despite normal igg and flc levels; therefore, he was still considered to have a vgpr. two months after discharge, the patient developed cervical, mediastinal, and axillary lymphadenopathy. he was therefore diagnosed with ebv-associated lymphoproliferative disease and was immediately treated with 2 cycles of rituximab monotherapy, resulting in a complete response. tacrolimus was discontinued on day 180, and there was no evidence of chronic gvhd. however, 6 months after allo-sct, he had marked cytomegalovirus (cmv) antigenemia. the protein level in the cerebrospinal fluid was 53 mg/dl, and the glucose level was 55 mg/dl; 17 monocytes per microliter were detected. cytological examination revealed that some of these cells resembled plasma cells (figure 2a). magnetic resonance imaging (mri) with gadolinium (gd) enhancement revealed a small, enhanced nodule in the lateral medulla oblongata (figure 2b, arrowhead). enhanced computed tomography (ct) showed no extramedullary tumors. moreover, we still observed complete donor chimerism, without leukemic plasma cells, in his bone marrow. the patient underwent 4 courses of weekly it, consisting of methotrexate 15 mg, cytarabine 20 mg, and prednisolone 40 mg in combination with local cranial rt (5 fractions of 2 gy for a total dose of 10 gy). after this combined treatment, his neurological symptoms resolved and the plasma cells in the cerebrospinal fluid disappeared. to prevent cns recurrence, we administered pomalidomide 4 mg/day on days 121, with 20 mg of dexamethasone weekly for each 28-day cycle. subsequently, a reduced dose of 2 mg was administered for 2 cycles, but grade 4 neutropenia occurred again. he resumed pomalidomide at 1 mg and continued the low dose without severe hematological abnormalities. twelve months after the cns relapse, both enhanced ct and brain mri with gd enhancement revealed no extramedullary tumors. in our outpatient follow-up, he maintained vgpr without major complications for more than 18 months after the diagnosis of cns relapse. the prognosis of plasma cell disorder has improved following the introduction of new agents and the development of sct, but a cure remains elusive. the long-term survival rate of ppcl is low due to the aggressive nature of the disease that comprises high tumor burden and extramedullary involvement. in the us, upfront allo-sct is still one of the treatment options for young ppcl patients without major complications. in our study, we performed upfront allo-sct with a myeloablative-conditioning regimen for our young patients without major complications. the center for international blood and marrow transplant research (cibmtr) conducted a retrospective sct study in ppcl, and demonstrated high survival rates in an autologous group compared with those in an allogeneic group. in the cibmtr study, non-relapse mortality in the allogeneic group was much higher at 3 years (42% vs. 5%), despite this group possessing a lower relapse rate (39% vs. 61%). our patient survived for more than 2 years after allo-sct, although he unfortunately suffered from cns relapse on day 350. consolidation and maintenance therapy after auto- and/or allo-sct in plasma cell disorder is still controversial. however, some clinical trials have recently shown that lenalidomide maintenance after auto-sct for mm significantly prolonged progression-free survival (pfs) and overall survival. moreover, a prospective clinical trial by the intergroupe francophone du mylome group showed that a bortezomibbased regimen followed by auto-sct and subsequent maintenance therapy with lenalidomide, bortezomib, and dexamethasone for ppcl significantly improved pfs compared with those who received a reduced intensity conditioning allograft. in the future, auto-sct and maintenance therapy combined with novel agents may be a treatment option for young ppcl patients. the prognosis of plasma cell disorders with cns involvement is extremely poor. in a retrospective study on cns myeloma conducted in 12 greek institutions, there was no significant difference in median survival of cns myeloma (called post cns-mm) between novel drug-treated patients and those given other treatments (4 months vs. 2 months). by multivariable analysis, extramedullary lesions, prior to treatment with novel drugs, and high ldh levels at myeloma diagnosis were statistically independent predictors of post cns-mm survival. these findings corroborate our observations that our patient with cns relapse of ppcl had an extremely short-term outcome. it has been reported that 19% of post cns-mm patients met the criteria for ppcl. furthermore, some of these ppcl patients developed isolated cns relapse during systemic remission after auto-sct. in a prospective study by royer et al., 2 ppcl patients experienced neuromeningeal relapse after autologous- and allo-sct, respectively, and both patients soon died despite intrathecal chemotherapy. in the present case, although vgpr was maintained by rvd induction therapy followed by allo-sct, isolated cns relapse occurred within 1 year. several drugs used in multiple myeloma, including bortezomib and lenalidomide, do not efficiently cross the blood of note, bbb penetration of lenalidomide was 11% in a rhesus monkey model, whereas that of thalidomide was 42%. in a murine model, pomalidomide displayed 39% bbb penetration and high anti-tumor activity in the cns. to the best of our knowledge, the anti-tumor effect of thalidomide is inferior to that of pomalidomide. additionally, pomalidomide is more effective in ameliorating extramedullary involvement. in our case, concurrent it and rt followed by pd was initiated after the diagnosis of isolated cns involvement after allo-sct. in addition, maintenance therapy by pd might prolong survival to prevent systemic recurrence, including cns involvement, for more than 18 months. the role of pomalidomide for ppcl patients with cns involvement should be further explored in clinical trials. our ppcl patient received rvd induction therapy followed by up-front allo-sct. however, concurrent it and rt followed by pomalidomide-based maintenance therapy well controlled his cns recurrence and prolonged his survival. to find an optimal treatment for this rare but aggressive ppcl, prospective clinical trials for this disorder should take into account the importance of myeloma drug penetration across the bbb in order to enhance the treatment of cns recurrence

primary plasma cell leukemia (ppcl) is a rare aggressive variant of plasma cell disorder and frequently presents with extramedullary disease. central nervous system (cns) involvement with ppcl has an extremely poor prognosis. we describe a 46-year-old man with ppcl treated with a combination of lenalidomide, bortezomib, and dexamethasone as induction therapy following upfront allogeneic stem cell transplantation (allo-sct). despite achieving a very good partial response, the patient suffered from an isolated cns relapse 12 months after allo-sct. he was immediately started on concurrent intrathecal chemotherapy (it) and cranial irradiation (rt). subsequently, pomalidomide and low-dose dexamethasone (pd) were given as maintenance therapy. he has been without cns recurrence for more than 18 months. our case suggests that concurrent it and rt followed by pd maintenance therapy may be an effective option to control cns relapse of ppcl after allo-sct.

PMC5337827

pubmed-1326

in this issue of the jem, two articles describe a potential role for two bacterial toxins in the modulation of the immune system by interfering with t cell signaling. using an in vitro jurkat t cell system, gerke et al. (8) report that yersinia pseudotuberculosis inhibits t cell activation and that the inhibitory activity was strictly dependent on the yersinia outer protein h (yoph), a potent tyrosine phosphatase that is delivered to host cells by a bacterially encoded type iii protein secretion system. previous studies by a number of laboratories have identified several tyrosine phosphorylated proteins as apparent targets for the tyrosine phosphatase activity of yoph in cell culture systems. these include p130cas, focal adhesion kinase, and paxillin in hela cells, and fyb/slap130, p130cas, and skap55 in macrophages (912). in general, these are proteins involved in integrin signaling, which is consistent with the proposed role of yoph as an antiphagocytic molecule. more recent studies have shown that yoph can also inhibit t cell signaling in-vitro and that this activity could be correlated with the presence of yoph and the dephosphorylation of the tyrosine kinase lck (13). (8) have now extended those studies and added the t cell signaling adaptor proteins lat and slp-76 to the list of potential yoph substrates. these adaptor proteins are essential for the transduction of signals from the t cell receptor and its associated tyrosine kinases, and their tyrosine phosphorylation is required for their activities. (8) argue that tyrosine dephosphorylation of these adaptor proteins by yoph is responsible for the observed inhibition of t cell receptor signaling after yersinia infection of cultured jurkat t cells. they also present data that argue that both of these proteins are high affinity substrates of yoph, presumably requiring small amounts of translocated toxin to be targeted for dephosphorylation. in the second article, rossi paccani et al. (14) report that anthrax toxin also disrupts t cell signaling but by a completely different mechanism. anthrax toxin, which is produced by bacillus anthracis, is composed of two enzymatic or a subunits known as edema factor (ef) and lethal factor (lf) that alternatively associate with a b subunit, known as protective antigen, which mediates their delivery into target cells (15). ef is a potent calcium/calmodulin-dependent adenylate cyclase, which causes a massive increase of cyclic amp in intoxicated cells and global disruption of cell signaling. lf is a zinc-dependent protease that specifically disrupts the mitogen-activated protein (map) kinase signaling pathways by cleaving the activating map kinase kinases mek1/2 and mkk3, mkk4, mkk6, and mkk7 (1619). (14) showed that treatment of peripheral blood lymphocytes with protective antigen in combination with either lf or ef effectively blocked t cell signaling as measured by the expression levels of the surface activation markers cd69 and cd25, the production of cytokines, and cell proliferation. given the ubiquitous presence of the anthrax toxin receptors and the essential role for map kinases and camp in t cell signaling, the results presented by rossi paccani et al. the broader issue raised by these two and other studies with other bacterial toxins that disrupt immune cell function in vitro, however, is the in vivo relevance of the findings. do these pathogens specifically interfere with t cell signaling during the course of an infection? unfortunately, in vitro studies, although useful to provide testable hypotheses, can not provide answers to these important questions (8) make a strong case for slp-76 and lat as high affinity substrates for this tyrosine phosphatase and hence of potential relevance during infection when presumably the pathogen would deliver small quantities of this toxin. indeed, it is often the case that under vitro experimental conditions, cells are exposed to toxin quantities not usually delivered during actual infections. nevertheless, it is not known how much yoph is delivered by yersinia during infection, and there is no evidence demonstrating that yersinia hampers t cell function during infection. yersinia infections are usually acute (20), arguing against the need for these pathogens to counteract the host-specific adaptive immune responses during infection. however, although rare, chronic infections are sometimes observed (21, 22), and in those cases, the ability to interfere with t cell function might be useful to the pathogen. whether this function of yoph has been specifically selected by evolution or is simply a by-product of another of its demonstrated functions, such as disruption of macrophages during acute infection, remains to be established. anthrax toxin possesses even a more difficult challenge because its biochemical activity (such as inhibition of map kinase signaling) can potentially affect a large number of cellular processes. therefore, the ability of anthrax toxin to inhibit map kinase signaling can result in the in vitro inhibition of any of the rather large number of intracellular signaling pathways that involve these kinases, regardless of their relevance during infection. for example, if added to cultured neurons, anthrax toxin would certainly prevent neurite outgrowth, a process strictly dependent on map kinase signaling (23). however, it would be hard to argue that this is a relevant function for the toxin during infection. by the same token, anthrax toxin predictably inhibited in vitro t cell signaling, a process strictly dependent on map kinase signaling. however, more experiments will be required to establish whether this activity of the toxin is important during infection. if untreated, b. anthracis infections are usually hyper acute (24); therefore, inhibiting t cell signaling may not provide a significant advantage to the pathogen. on occasions, b. anthracis can be associated with sub-acute infections (such as cutaneous anthrax; reference 24), in which case inhibition of t cell function may provide some advantages to the pathogen. in any case, because b. anthracis is most likely an accidental pathogen of mammals, it is unlikely that evolution may have played any role in directly shaping the effects of anthrax toxin on t cells. therefore, this activity might be more a byproduct, relevant or not, of other activities of this toxin in the normal ecology of b. anthracis. although every pathogen must contend with the onslaught of the innate immune responses, it is not necessarily the case that every pathogen must counteract the acquired immune response to fulfill its replication program. for most pathogens that cause acute infections, their life cycle within the host is most often over by the time the naive host mounts a meaningful acquired immune response capable of controlling the infection. in fact, most infections with pathogens that have coevolved with their hosts are indeed asymptomatic, do not lead to overt harm, and most often result in protective convalescent immunity. on the other hand, pathogens that cause persistent infections might be under strong evolutionary pressure to evolve specific mechanisms to avoid acquired immune responses. indeed, mechanisms of antigenic variation or specific inhibition of antigen presentation evolved by microbial pathogens are well documented (25). for example, many viral pathogens specifically interfere, by a variety of mechanisms, with both major histocompatibility class i and ii antigen-presenting pathways (26, 27). in addition, many viral, bacterial, and protozoan pathogens undergo rapid antigenic variation to evade the onslaught of the acquired immune response (2830). whether inhibition of t cell signaling by anthrax or yoph toxins should be added to the list of pathogenic mechanisms specifically evolved to counter the acquired immune response

microorganisms that cause persistent infection often exhibit specific adaptations that allow them to avoid the adaptive immune response. recently, several bacterial toxins have been shown in vitro to disrupt immune cell functions. however, it remains to be established whether these activities are relevant during infection and whether these toxins have specifically evolved to disrupt the adaptive immune system.

PMC2213035

pubmed-1327

membrane proteins (mps) constitute about 30% of all the proteins encoded in the currently known genomes, and play critical roles in cell signaling, ion transport, and cell-cell communications, as well as assist the folding of other mps (1). because of these biological significance, mps represent the most important class of drug targets about 50% of current molecular targets are membrane-bound (2). however, only about 2% (518 of 25,176) of the 3d structures deposited in the protein data bank (pdb; ref. 3) are for mps. and the number of high-resolution structures (from x-ray diffraction and more recently from nmr) remains even smaller, largely because of the difficulties in crystallizing mps. recently, some new ideas and experimental approaches have been introduced in the area of mp crystallization (4), all of which exploit the spontaneous self-assembling properties of lipids and detergent as vesicles (vesicle-fusion method), discoidal micelles (bicelle method), and liquid crystals or mesophases (in meso or cubic-phase method). despite these promising new methods, the current gap between need and supply of mp 3d structures makes prediction algorithms important and essential. mps come in a variety of sizes and shapes, though the available 3d structure principles are far less diverse than those of the globular proteins. from a structural point of view one is the -helix bundle protein, in which one or several -helices span the membrane; and the other is -barrel protein, in which eight or more antiparallel tm -strands form a closed barrel 5., 6 .. two recent examples 7. since jhnig and edholm in 1992 presented one of the first methods using secondary structure prediction to build suitable model structures as initial conformations for molecular dynamic studies (9), several groups have tried computational approaches to elucidate mp structures. in 1993, milik and skolnick presented a method based on the combination of a hydropathy scale for the prediction of trans-bilayer fragments with dynamic monte carlo simulation techniques (10). in 1994, taylor et al. adapted some programs originally developed for the prediction of globular protein structures to derive a method for the prediction of integral mp structures (11). each step in the method is fully automated, from the initial sequence data bank searches to the final construction of 3d models. consequently, estimates for prediction accuracy are perhaps overly optimistic. here, we summarize recent attempts within the field of computational biology and bioinformatics to predict an mp s structure. most current methods of theoretical mp structure prediction do not actually deal with predicting the 3d structure, but rather try to predict the most likely topology of the protein, that is to say, the in/out location of the n and c termini relative to the membrane, and the number and position of transmembrane (tm) segments. a high-quality model of secondary structure and topology is a prerequisite for experimental structure-function studies, and can be a starting point for attempts to model the 3d structure before molecular dynamics or simulated annealing simulations. in recent years, various accurate methods because the number of high-resolution structures of -barrel proteins is less than that of the -helix proteins, the neural network has been more frequently adopted in the -strand topology prediction. the details of some methods based on hidden markov models (hmms) are listed in table 2. many secondary structure prediction methods are based on statistical methods, physicochemical methods, sequence pattern maching, and evolutionary conservation (12). the main methods for identifing tm helices are on the basis of their hydrophobicity and known minimum length (at least 15 residues; ref. membrane propensities were defined by a statistical analysis carried out on a set of 640 tm helices, belonging to 133 mps extracted from swiss-prot (14) that have experimentally defined topologies. the five widely used prediction methods for predicting the topology of -helix bundle mps are tmhmm (15), hmmtop (16), memsat (17), phdhtm (18), and toppred (19). tmhmm, hmmtop, and memsat are all based on hmms with 5~7 types of structural states. toppred was the first topology prediction method that combined hydrophobicity analysis and the positive-inside rule. generally, these sequence-based methods for predicting the number and approximate location of tm helices within mps have about 85% accuracy. in 2003, karin meln et al. tried to construct useful reliability scores for these methods (20). 21) algorithm can provide a solution to the problem that non-transmembrane query sequences may give false positive hits (20%-30%) in the prediction process. the upgraded and modified version of the das-prediction method, das-tmfilter algorithm, has been distributed (22). the new algorithm is designed to make distinction between protein sequences with and without tm helices at a reasonably low rate of false positive prediction (~1 among 100 unrelated queries) while the high efficiency of the original algorithm locating tm segments in queries is preserved (sensitivity of ~95% among documented proteins with helical tm regions). in 2003, xia and colleagues presented a new approach, conpred_elite (23), that can predict the whole topology with accuracies of 98% for prokaryotic and 95% for eukaryotic proteins as they reported. besides the tm helix, another tm segments type is -barrel, which consists of several tm strands. unlike -helical mps, there are no simple low-resolution experiments that yield large amounts of data for -barrel mps. in fact, the overall hydrophobicity for -barrel mps is similar to that of soluble proteins (13). gromiha and colleagues combined amino acid preferences for -strands with the surrounding hydrophobicity of the respective residues to predict -strands (24). diederichs and colleagues proposed to use a neural network to predict the topology of the bacterial outer membrane -strand proteins and to locate residues along the axes of the pores (25). jacoboni and colleagues applied a method combining neural networks and dynamic programming to predict the location of membrane strands (26). the authors estimated that their system correctly predicted about 93% of all known membrane strands. more recently, martelli et al. developed a sequence-profile-based hmm model that can predict the topology of -barrel mps cyclicing with 6 types of states (27). they reported that the accuracy of per residue of the model was about 83%. lately the following protocol starting from secondary structure prediction and tm segments topology prediction are often used. secondary structure prediction followed by tm segments identification along with prediction of loops connecting the segments, and molecular dynamics or simulated annealing simulations, may be finally used to refine these primal models. during the last refinement step, the protein is often inserted into a water/lipid bilayer/water or a water/n-octane/water environment to take into account the presence of the cell membrane. charmm, gromos, amber, and cvff-insight are some widely used force fields in molecular dynamics calculation. the slow dynamics of lipid molecules in the bilayer might bring the difficulties in equilibrating the system (28). for globular proteins, the major successful methods for structure prediction include homology modeling, threading, and ab initio folding. along with lucubrating the mechanism of mp folding and increasing the number of high-resolution mp structures, these methods will been applied to the direct prediction of whole mp 3d structures. the question of how the controlled integration of an mp into the lipid bilayer takes place is still not fully worked out, and there are certainly aspects of mp structures that will probably not be fully appreciated until this step has been accomplished. some pursuers educed the viewpoint that the prediction of mp structures from amino acid sequences was, in large measure, a problem of physicochemistry (29). physical influences that shape mp structures include interactions of the polypeptide chains with water, bilayer hydrocarbon core, bilayer interfaces, and cofactors. studies on the mechanism of insertion and folding of mps into membranes are relatively rare and have been mostly performed with two model proteins: bacteriorhodopsin (br; ref. while br is a representative -helical bundle protein, ompa belongs to the class of -barrel protein. homology modeling constructs structures (targets) that are homologous to other protein(s) whose 3d structure is known (templates). because few high-resolution mp 3d structures are available to be used as templates, and the modeling can be unreliable when the sequence identity between the template and target proteins falls below 20%-30%, the applicability of homology modeling is limited. an ab initio method was presented (32), whose knowledge-based technique added a membrane potential to the energy terms (pairwise, solvation, steric, and hydrogen bonding). the method is based on the assembly of supersecondary structural fragments taken from a library of highly resolved protein structures using a standard simulated annealing algorithm. results obtained by applying the method to small mps of known 3d structures showed that the method is able to predict, at a reasonable accurate level, both the helix topology and the conformations of these proteins. the structure prediction of membrane proteins still remains an interesting scientific problem. because of the physical difference between mps and gps (globular proteins) current segment accuracy of reported algorithms are pretty high (above 90%), while the overall accuracy are still around 50%-60%, which gives birth to hand-raising methods to combine the reports from several other algorithms. the lack of both high-resolution and low-resolution experimental data of mp structures makes the algorithm development and their evaluation difficult, but the fact that most mp sequences are used as space blocks to get through the membrane bilayer that has predefined thickness makes the structure prediction of mps simple on functional aspects. new algorithms will emerge and reported algorithms will be refined to give a better answer to this problem .

there is a large gap between the number of membrane protein (mp) sequences and that of their decoded 3d structures, especially high-resolution structures, due to difficulties in crystal preparation of mps. however, detailed knowledge of the 3d structure is required for the fundamental understanding of the function of an mp and the interactions between the protein and its inhibitors or activators. in this paper, some computational approaches that have been used to predict mp structures are discussed and compared.

PMC5172438

pubmed-1328

recently, esthetic requirements in dental restorative treatment are on the rise. for this reason, recent attention-drawing material is zirconia which is a biocompatible material with stable structure. flexural strength and fracture toughness of zirconia reaches up to 900-1200 mpa and 9-10 mpam1,2 respectively, and zirconia has been proven to be much more excellent in its strength than that of glass infiltrated full porcelain which is widely used in esthetic restoration.1,2 material properties of zirconia like these show that zirconia can be substituted for metal and can be surely used even in long span fixed prosthesis.3,4 cad/cam system which was introduced to dentistry in 1980 's has brought cost and time reduction in fabricating restoration by using computer for inputting, designing and cutting the form of restoration. in order to overcome disadvantage of previous method of framework fabrication in which post-sintered zirconia block was cut, pre-sintered zirconia block is being used recently. by doing so, fabrication time and consumption of bur this method, however, has disadvantage in that approximately 15-30% shrinkage occur in the procedure of sintering and hardening the cut block using cam unit for the increased density of zirconia block.5 when using partially sintered block, therefore, it is more important to calculate shrinkage rate accurately than any other things in order to compensate shrinkage which inevitably occurs in sintering process, and also to gain precise product when using partially sintered block. it is considered that internal and marginal fit plays a key role in longevity of prosthesis. in previous studies, many authors said that 120 m or less marginal gap of prosthesis was clinically acceptable.6,7 according to a study on fit of zirconia framework which was used in all ceramic crown, marginal gap was reported to be 64-83 m,8,9 and in another study on clinical fit of 3-unit fixed prosthesis, zirconia prosthesis (lava, 3 m espe, seefeld, germany) for which cad/cam was used reportedly showed 80 m of mean marginal gap.10 the reason why marginal fit is important is because increased marginal leakage may cause secondary caries, periodontitis, pulpitis etc. and there was a report from one previous study saying the thicker the cement became due to a large internal gap, the weaker the porcelain became.11 this effect is shown also in zirconia. excessive thickness of cement increases radial crack12,13 and causes fracture of veneering porcelain.14 fracture of veneering porcelain is considered to be one of the greatest reasons for removing zirconia restoration.15-18 as opposed to this, too small internal gap may cause unstable seating of prosthesis.19 abduo et al.20 said that factors which influenced fit of prosthesis were fabrication system of zirconia, veneering, configuration, span length of zirconia etc. although the use of zirconia has increased and are being used more in multiple areas where the tooth are missing, studies on fit of zirconia fixed partial denture until the now have been limited to 3-unit fixed partial denture with nearly linear form; there has been few study on internal or marginal fit of 4 or more unit fixed partial denture with curved form.20 particularly, there was no study in which both internal and marginal fit depending on the span length were simultaneously evaluated. the purpose of this study, therefore, was to measure marginal and internal fit of single, 4-unit, 6-unit zirconia fixed partial denture core which had been fabricated using cad/cam system by using replica technique and to evaluate the effect that span length on fit while evaluating whether measured marginal gap was in clinically acceptable range or not. experimental groups were divided into single, 4-unit and 6-unit groups, and preparation of each abutment teeth were done in order to make single crown of upper right central incisor, 4-unit fixed partial denture with abutment teeth of upper right and left lateral incisor where upper right and left central incisors were missing, and 6-unit fixed partial denture with abutment teeth of upper right and left canines where upper right, left central and lateral incisors were missing (table 1). for tooth preparation, surveyor and diamond bur were used, and total taper of each abutment teeth was intended to be 6. preparation amount was 2 mm on incisal part and 1 mm on axial wall, and 1 mm width deep chamfer margin was formed. in order to prevent possible wear or fracture of master models from repetitive impression taking procedure, dentiformmodels (nissin dental prod. japan) where abutment preparation were done were duplicated and titanium master model weremade (addtech co., seoul, korea) (fig. 1). impression of titanium model were taken with conventional method by using addition silicone impression material (imprint ii, 3 m espe, st. paul, mn, usa) and readymade tray, and total 30 improved stone (fuji rock ep, gc corp, tokyo, japan) models including 10 models per each group were made (fig. 2). after scanning of the manufactured plaster model using cad/cam system, partially sintered zirconia blocks were cut and sintered, and finally zirconia cores were fabricated (fig. 3). when fabricating cores, cad/cam system of orapix (seoul, korea) and zirconia blocks were used. thickness of core and cement space were set as 0.6 mm and 40 m respectively. internal adjustment was not done so that fit of core that was fabricated by only cad/cam could be merely evaluated. after core fabrication was done, whether there were remnants inside, defects or distortions were checked and then cleaned with high pressure steaming. after positioning fabricated zirconia cores on each models, jigs for positioning the zirconia coreon the model were made using pattern resin (gc dental, japan) so that zirconia could always be positioned at a same spot on the models. after filling fit checking material (fit checker, gc dental, japan) in inner surface of core after that, pre-made resin jig was positioned above the core. while maintaining adapted position by hand pressure, it was place in universal testing machine (shimadzu corporation, kyoto, japan) immediately. universal testing machine was set to measurement mode, and its compressive force was limited to 40 n, and regular force (40 n) was maintained for 5 minutes until fit checker was completely hardened. when doing so, the silicon film of fit checker has to be fully attached to silicone core. filling inner surface of silicone film of core with regular bodied addition silicone impression material (aquasillv, densply caulk, usa) and hardening the filled material increase strength andalso enables gaining of stable film layer. each 8 measuring points were established for labial and mesio-distal side respectively (fig. 4), and cutting was done right in the center of the model labialy and mesio-distally (fig. 5). by using measuring microscope (axio, carl zeiss, rochester, ny, usa) and i-solution (imt i-solution inc., vancouver, bc, canada), thickness of fit checker was measured at 16 measuring points of each abutment teeth (fig. mean value was documented after 3 times measured by 2 experimenter for each measuring points. mean value and standard deviation value were calculated by adding measurement values of right and left abutment teeth of each group which was divided into bucco-lingual and mesio-distal. whether there was statistically significant difference between groups which were divided into bucco-lingual and mesio-distal or not was analyzed by multivariate analysis. after that, if there had been statistically significant difference, post-hoc test was done using dunnett t3 test per each measuring points. in all analyses, twenty seven specimens including nine single, nine 4-unit and nine 6-unit were used in fit measurement. there existed 1 specimen per each group of which measurement could not be done because fracture had occurred in the process of applying force after putting fit checker inside the core and placing it on abutment. statistical analysis was performed by adding each measurement values of right and left abutment teeth of each group. by doing so, number of specimen of each group became nine single, and eighteen 4-unit and eighteen 6-unit. mean value and standard deviation value of gap at bucco-lingual measuring point were calculated (table 2). as a result of multivariate analysis, statistically significant difference was shown at point 2, 4, 7 and 8. in order to investigate between which groups of point 2, 4, 7 and 8 showed the difference, post-hoc test was performed using dunnett t3 test (table 3). mean value and standard deviation value of gap at mesio-distal measuring point were calculated (table 4). the result of anova showed statistically significant differences at point 2, 4, 7 and 8 by span length factor. in order to investigate between which groups of point 2, 4, 7 and 8 showed the difference, post-hoc test was performed using dunnett t3 test (table 5). as a result of measuring point analysis which showed statistically significant difference, measurement value increased from single, 4-unit and 6-unit in order in point 2, 4, 7 when bucco-lingual analysis was done. but in point 8, the gap of 4-unit group was measured to be the smallest. in the result of mesio-distal analysis, measurement value increased from single, 6-unit and 4-unit in order in point b, zirconia is an esthetical and biocompatible material, and its physical property is so strong that it can be used for fixed prosthesis of posterior region. because of its property like this, it has become one of the most noteworthy materials in esthetic prosthetics. in order for zirconia to be used as prosthetic restoration material that can last long and function stably, not only its physical property but also the fit of prosthesis made by it should meet clinical requirements. in fabrication of zirconia prosthesis using cad/cam, completely sintered, this method has disadvantages in time and cost although fit could be more excellent than when partially sintered block which is used in most fabrication system is used. on the other hand, if partially sintered block is used, it will be better off when it comes to time and cost factor. however, if cutting is not done by accurate calculation due to shrinkage in sintering process, level of fit will decrease significantly. according to previous studies, 120 m or smaller marginal gap is clinically accepted.6,7 in the studies on fit of single or 3-unit fixed partial denture zirconia prosthesis fabricated by cad/cam system, marginal gap showed approximately 64-83 m as a result.8,9 these show that zirconia can be used for fixed partial denture restoration adequately. however, these were the studies which had been mostly proceeded on linear type fixed prosthesis which was shorter than 3-unit. study on prosthesis of which span is longer than this has not been done.10 in this study, therefore, whether fit of zirconia prosthesis is influenced by the span length was investigated using anterior teeth model. as methods for measuring marginal or inner fit, a method of direct inspection, a method of inspection after cutting, a method of evaluation by impression taking, a method of evaluation using explorer and so on although a method of inspection after cutting may be the most accurate way, there is a shortcoming in that more specimens have to be made for the increase of the number of measurement. therefore, replica technique where measurement of fit of various parts could be done easily with only small number of specimens was used in this study. this method had been considered to have low accuracy in the past,21 but it became known to be reliable compared to other methods for measuring fit by rahme et al.22 and laurent et al.23 study. specimens for measurement were made using replica technique, and thickness of fit checker was measured at 16 measuring points which were designated per each abutment teeth, and 2 experimenters recorded mean value after 3 times of measurements so that error could be minimized. as a result of measurement, overall fit was acceptable (small gap) in marginal and axial wall area, but connection part between margin and axial wall and incisal part showed relatively large gap. because it was considered that whether it was either right abutment tooth or left abutment tooth could not be a variable for analysis, mean value and standard deviation were calculated by adding measurement values of right and left abutment teeth together for statistical analysis in 4-unit and 6-unit group. in the case of this study, multivariate analysis was used because it was considered that gap of inner surface and margin could not be independent to each other although independent variable was a span length. as a result of statistical analysis of bucco-lingual (1-8) and mesio-distal (a-h) measuring points, there were statistically significant differences at point 2, 4, 7, 8 and b, d, e, f, g among the groups. from this result, the fact that span length influenced fit of zirconia core was confirmed. as a result of analysis of each measuring points which showed statistically significant difference, measurement value increased from single, 4-unit to 6-unit in order at 2, 4, 7 point, but measurement value of 4-unit group came out smallest at 8 point. as opposed to this, 4-unit group showed the largest value at b, f, g point as a result of mesio-distal analysis. this is considered to be because of small number of specimens that were used in the experiment and measurement error as well. also, there were more points which showed significant difference inmesio-distal part than inbucco-lingual part. it is guessed that the reason for this is because more error occurred in the process of shrinkage at mesio-distal part since the form of zirconia core was not bucco-lingually but mesio-distally long. although there was not enough number of specimen for more accurate multivariate analysis in statistical analysis process, kruskal-wallis test was performed to compensate it, and same result as that of multivariate analysis was gained. it can be said that the meaning of this study lays in the fact that evaluation of effect of span length on fit in fabrication of prosthesis. however, the number of specimen was not enough for securing statistical significance which is the weak point of this study. also, if fit of prosthesis had been measured after veneering, the study would have been closer to clinical setting. in some specimens, measurement was difficult because boundary of silicone film and impression material was blurred. for more accurate measurement, this method needs improvement. also, in case of fixed partial denture, fit is not something that is independent to one another. therefore, it is considered that comparative evaluation on volume will be more necessary than to just observe some part after cutting it. also, if analysis such as micro ct24 and so on is additionally used, appearance of transformation or distortion in shrinkage process will be able to be evaluated. the further studies to investigate the difference of fit among the different manufacturers with increased number of specimens will be necessary. and change of the span length influenced marginal fit and internal fit to some degree. in single or 4-unit fixed partial denture group, mean value of marginal fit was within clinically acceptable range. in 6-unit group, however, some margins showed values that were out of clinically acceptable range. therefore, it was analyzed that the increase of the span length could possibly decrease fit between zirconia core and abutment tooth when fixed partial denture was 6-unit or longer.

purposethe purpose of this study was to evaluate the effect of the span length on the fit of zirconia framework fabricated using cad/cam system. materials and methodsabutments for single, 4-unit and 6-unit fixed partial prostheses were fabricated. ten zirconia frameworks were fabricated for each group. the marginal and internal gap were presented by means of replica technique and measured by measuring microscope (axio, carl zeiss, rochester, ny) and software (i-solution, imt i-solution inc., vancouver, bc, canada). the results were statistically analyzed by multivariate analysis test and dunnett t3 test for post hoc test (=.05). resultsthere were statistically significant differences at 2, 4, 7, 8 points (mesio-distal section) and b, d, e, f, g (labio-lingual section). in some marginal reference points of 6-unit group (p<.05), the marginal gap were larger than 120 m. conclusionspan length of zirconia core may have an influence on marginal and internal fit. within the limitation of this study, the increase of span length of zirconia framework of 6 or more-unit fixed partial denture may decrease the marginal and internal fit.

PMC3675283

pubmed-1329

eccrine spiradenomas are rare adnexal tumours of the skin that originate in the sweat glands. only a few cases have been reported, being described for the first time by kersting and helming. though the majority of eccrine spiradenomas involve the head, trunk, and extremities, they can also appear all throughout the skin, mostly as painless solitary nodules. our case report is a malignant eccrine spiradenoma (mes/spiradenocarcinoma) diagnosed in an asymptomatic woman on the basis of breast cancer prevention programme. because of its rare incidence and lack of imaging workups our aim is therefore to discuss the main characteristics of it, and to review the literature available on this issue. a 48-year-old woman, asymptomatic, with no medical history of interest, presented in a mammography made as part of a breast cancer screening programme a lesion in the left upper-breast. 1) demonstrated a well-defined, isodense nodule located in the upper-outer quadrant of the left breast. longitudinal and transverse scans with colour doppler images were obtained. the images (fig. 2) showed a well-defined lobulating mass measuring 18 13 15 mm with homogeneous hypo echogenicity. in the colour doppler sonography a histologic examination of these showed a characteristic biphasic population of outer small cells with darkly staining nuclei surrounding larger cells with pale cytoplasm without epidermal connection. intraoperative radiologic control of the excised lesion showed integrity of the nodule with broad resection margins. macroscopically the sample measured 4 2.5 cm in which on serial sectioning a 2 1.5 cm white nodule was noted. microscopic examination showed cells arranged in ragged sheets, nests, cords, and solid masses along with occasional irregular glandular structures. benign areas, was seen as sharply demarcated lobules composed of 2 cell populations (peripheral basaloid cells and central pale cells) arranged in nests with focal cystic changes. adjoining to this 2-cell population focus (gradually or abruptly) proliferation cells with histopathological malignant characteristics transited to solid masses of a homomorphous large cell population with hiperchromatic and pleomorphic nuclei, eosinophilic cytoplasm (fig. inmunohistochemically, both cellular areas exhibited p63 and were positive for smooth muscle actin (sma). focal reactivity for ema and cea was found in the central, large, pale cells. luminal cells expressed muc-1, ck 7, ema, cea, ck cam5.2 and gcdfp-15. around 4050% of the cells composing the carcinomatous area showed positivity to ki-67 (fig. s-100, ck 20, vimentin, cd3, cromogranin and ck34betae12, estrogen/progesterone receptors were not expressed. these features were consistent with a low-grade mes noted to be arised from a pre-existing benign long-standing eccrine spiradenoma. the sentinel lymph node retrieved from axillary dissection was submitted for intraoperative histological examination obtaining a negative result for metastatic carcinoma. metastatic workup detection (bone scan, computed tomographic scans of the chest, abdomen, and pelvis) was also negative. the patient has been seen in follow-up every 36 months and has had yearly sonographic examinations. after 32 months of follow-up, there is no evidence of recurrent or metastatic disease. eccrine spiradenomas are usually benign, well-defined sweat-gland neoplasms that affect both men and women, showing no sex predilection (male to female ratio 1:1). age at presentation is variable, but most commonly arising at an age of 50. spiradenoma lesions tend to arise on the head; neck; trunk; and, less commonly, the arms followed by the legs. however, the development of neoplasms with eccrine sweat gland phenotype in the mammary ducts is not surprising in as much as the breast is considered a modified sweat gland. since the original report, 102 cases of mes have been documented in the literature. of these, only six are described affecting the breast. there are only three cases, including ours, diagnosed as malignant transformation in the breast (table 1). although, four of the five cases of breast spiradenomas were benign, 2 of these were reported to suffer malignant transformation, and one carcinosarcomatous conversion. reviewing the reports, the lesions usually show a typical history of a long-standing (often decades); unchanged cutaneous solitary nodule that suddenly becomes enlarged. the average size of malignant spiradenomas at presentation is 3.9 cm (range 0.515 cm) in diameter, but usually 12 cm as in our patient. it generally begets medical attention when a pre-existing undiagnosed lesion rapidly enlarges, changes colour, ulcerates, or becomes painful and tender. the imaging findings of breast eccrine spiradenomas have not been clearly demonstrated because of the rare incidence of eccrine spiradenomas and the lack of imaging workup. the findings reported were: round or oval shape, well-demarcated masses hypo, or hyper-dense in mammography. the us findings of the current case were characterized as well-circumscribed, oval-shaped, hypoechoic masses, surrounded by echogenic lines that represent the dermal layer. reported the first description of the sonographic findings of a mes in 1993 as a hypoechoic, heterogeneous lesion. mri, ct, and radiography can be used to assess the presence of metastatic foci in the case of malignant spiradenoma. although, an eccrine spiradenoma may resemble an epidermal inclusion cyst on mammography and us, these may be identified on the basis of mri findings. the mri findings of epidermal inclusion cysts and eccrine spiradenomas are different [810] and so they might be used in making the differential diagnosis. diagnosis of mes is based on histopathologic examination and requires finding a focus of benign spiradenoma within or adjacent to the malignant tumour. nowadays, high-grade tumours show multiple foci of florid squamous differentiation, including proliferation of solid masses consisting of large cells with hyperchromatic nuclei, loss of pas-positive basement membrane, sarcomatous cell metaplasia and surrounding cell reaction. the mesenchymal elements are non-specific and are often described as rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, and chondrosarcoma. on the other hand, in the absence of a benign focus, tumours can be confused with other skin malignancies. the diagnosis of these is much more difficult and requires finding benign focus on histopathology and an inmunohistochemical myoepithelial benign profile in consonance with the finding of two cell populations. in our case, histologically proliferation of cells with hyperchromatic pleomorphic vesicular nuclei, variably prominent nucleoli, increased mitoses, invasion of surrounding membrane and adipose mammary tissue characterized malignant transformation. inmmunohistochemically, the cells express immunophenotypes similar to mioepithelial cells with a variable expression of p63 and sma, and ductal differentiation cells expressing cea, ema, ck7, muc-1, ckcam5.2 y gcdfp-15. s-100 protein positivity in basal epithelial cells has been demonstrated, but was not evidenced in our case. we also identified ki-67 and d1-cyclin expression, as a result of a low-grade differentiation tumour. while the inmmunohistochemical profile of benign or malignant eccrine spiradenoma is not diagnostic of an eccrine neoplasm, it is helpful in elucidating the cellular differentiation and narrowing the differential diagnosis. radiation and hyperthermic chemotherapy can also be administered as medical treatment to prevent focal recurrence. in our case, malignant eccrine spiradenoma metastasizes (40%) to regional lymph nodes, bone, lungs, brain, and liver (in descending order of frequency). distant metastases portend an ominous prognosis, but if possible, resection should be done. we did not implement an axillary dissection, as sentinel node biopsy turned out negative. due to the high risk of developing metastases, close follow up of these patients for early detection of recurrence should be carried out. malignant eccrine spiradenoma metastasizes (40%), so a close follow up is recommended. the authors declare that there is not any source of funding for this surgery case report. written informed consent was obtained from the patient for publication of this case report and accompanying images. francisco villalba ferrer, andrs garca vilanova, carlos fuster diana, jose medrano gonzlez: surgery, follow up and surgical comment on the clinical case.

highlightseccrine spiradenomas are rare adnexal tumors of the skin. malignant transformation of benign forms is even more unfrequent.we report a case of malignant breast eccrine spiradenoma diagnosed on the basis of breast cancer prevention programme.imaging findings of these tumors have not been clearly demonstrated because of the rare incidence and lack of workup.the mri findings of epidermal inclusion cysts and eccrine spiradenomas are different and so these findings might be used in making the differential diagnosis.

PMC4601958

pubmed-1330

upper and lower limb innervation is greatly affected by brachial and lumbosacral plexus lesion, leading to loss of motor and sensory function [17]. in the neonate, such injuries are even more harmful, since surgical treatment is limited and neuronal loss is particularly enhanced by neurotrophic factors deprivation. nonexistent or limited handling of neonatal proximal lesions results in limb dysfunction and neuropathic pain [812]. experimentally, a well-accepted model to mimic axotomy injury retrograde repercussion to spinal neurons is the neonatal peripheral nerve axotomy [1318]. sciatic nerve transection, early after birth, results in significant degeneration of spinal motoneurons as well as sensory neurons present in the dorsal root ganglia. also, a number of interneurons, present in deep spinal cord laminae, degenerate due to the loss of input and/or output. importantly, exogenous treatment with neurotrophins transiently rescues a significant number of neurons, leading to the possibility of axonal regrowth and regeneration [19, 20]. to date, neonatal peripheral nerve repair following neurotmesis is largely limited due to technical drawbacks. in fact, to perform end-to-end coaptation by epineural/perineural suturing in the newborn represents a major challenge. on the contrary, reconnection of transected stumps by using biocompatible sealants may significantly facilitate the process, as well as taking the advantage of employing the adhesive as scaffold to engraft stem cells, as well as neurotrophic substances, to the injury site. fibrin sealant has been applied in neurosurgery for decades, being effective and biocompatible, with no side effects to the nervous system microenvironment. together, when activated, their interaction mimics the coagulation cascade, forming a stable and adhesive clot. regard, much effort by the center for the study of venoms and venomous animals (cevap) has been made to produce a new fibrin sealant that uses a serine protease from a brazilian snake (crotalus durissus terrificus) with thrombin-like activity. also, in order to avoid human blood, bubaline-derived blood serum cryoprecipitate has been used as the fibrinogen source. among several advantages, the nonhuman fibrin glue hampers transmission of hepatitis and hiv/aids [2327]. also, it has a customizable formulation that can be adjusted to increase or decrease fibrin clot formation and stability. although anatomical repair of spinal roots and other lesioned plexus components constitute the primary approach, additional strategies are necessary to enhance neuroprotection and to improve the regenerative response of severed neurons. this may be achieved by stem cell therapy that has shown particularly important positive effects following nervous system injury [2837]. in this regard, we have already shown evidence that mononuclear cell therapy produced neuroprotection, preservation of synapses in the spinal cord, and reduction of glial reaction following dorsal and ventral horn adult lesion [38, 39]. the present work investigated the viability and efficiency of neonatal sciatic nerve end-to-end coaptation, performed with the application of fibrin sealant. we show that both commercial and customized nonhuman fibrin adhesives perform equally well and allow motor and sensory recovery up to 12 weeks after injury. nerve stumps coaptation was performed by using either a commercially available fibrin sealant-tissucol (baxter, vienna, austria) or a patented fibrin sealant derived from snake venom, kindly supplied by the center for the study of venoms and venomous animals (cevap) of unesp (patent registration numbers br1020140114327 and br1020140114360). at the time of use, the components were previously thawed, reconstituted, mixed, and applied [2427, 31]. two-day-old (p2) neonatal lewis rats were anesthetized by hypothermia and subjected to unilateral sciatic nerve transection at mid-thigh level. animals were divided into three groups: axotomy alone (ax, n=30), in which a 2 mm segment of the proximal stump was resected in order to assure absence of contact between stumps; axotomy followed by end-to-end cevap's fibrin sealant coaptation (ax+fs, n=30); axotomy followed by end-to-end commercial fibrin sealant coaptation (ax+cfs, n=30). an overview of the surgical procedure is illustrated in supplementary figure s1 (in supplementary material available online at http://dx.doi.org/10.1155/2016/9028126). an additional nonoperated control group was specifically utilized for the motor test evaluation (n=5). also, in order to evaluate the retrograde degeneration of motoneurons following nerve transection without proximal stump resection, an additional axotomy control group was evaluated following stump reapposition (ax-sr; n=5). both fibrin sealants used herein are composed of three separate solutions: (1) fibrinogen, (2) calcium chloride, and (3) thrombin or thrombin-like (in the case of the serine protease derived from snake venom). during surgical repair, the first two components were applied and the proximal and distal stumps were approximated in an end-to-end fashion. the sciatic nerve was gently shifted to assure the stability of the coaptation and to evaluate the success of the repair. the institutional committee for ethics in animal (ceua/unicamp) and the center of experimental and ethical animal (ceea/unesp) approved all experiments (proc. number 2593-1 and 904-2011, resp.), which were performed in accordance with the guidelines of the nih and the brazilian college for animal experimentation. the animals were housed using a 12 h light/dark cycle and controlled temperature (23c), with free access to food and water. animals were anaesthetized with an overdose of kensol (xylasine, kning, argentina, 10 mg/kg) and vetaset (ketamine, fort dodge, usa, 50 mg/kg), and the vascular system was rinsed by transcardial perfusion with phosphate buffer 0.1 m (ph 7.4). for neuronal survival counting and the immunohistochemical evaluation, subjects (n=5 for each group) were fixed by vascular perfusion of 10% formaldehyde in phosphate buffer (ph 7.4). the lumbar intumescence was dissected out, postfixed overnight, washed in phosphate buffer, and stored in sucrose 10, 20, and 30% for 24 h each, before freezing. transverse cryostat sections (12 m thick) of the lesioned spinal cord segments (l4, l5, and l6) were obtained, transferred to gelatin coated slides, and stored at 22c until use. for sciatic nerve regeneration analysis, the rats were transcardially perfused with 200 ml of a fixative containing 2.5% glutaraldehyde and 1.0% paraformaldehyde in 0.1 m phosphate buffer (ph 7.4). the sciatic nerve ipsi and contralateral to the lesion were removed and stored overnight in the same fixative at 4c. the specimens were then osmicated, dehydrated, and embedded in durcupan (fluka acs, steinheim, switzerland). the resin blocks were trimmed and semithin sections (0.5 m) were obtained and stained with 0.7% sudan black in 70% of ethanol solution for morphometric analysis. counting of motoneurons survival was performed on spinal cord sections (n=5 for each group, ipsi and contralateral ventral horn, 4, 8, and 12 weeks after lesion). cryostat transverse sections were stained for 35 min in aqueous 1% cresyl fast violet solution at room temperature. the sections were then washed in distilled water, dehydrated, and mounted with entellan (merck, darmstadt, germany). the motoneurons were identified based on their morphology and location in the ventral horn (dorsolateral lamina ix). only cells with visible nucleus were counted for every twentieth section (totalizing an interval of 240 m between sections) along the lumbar enlargement, both on the ipsilateral and contralateral sides of each spinal cord. the percentage of surviving cells was analyzed by the ratio of absolute numbers of motoneurons, counted per section, on the lesioned and nonlesioned sides, respectively, and multiplying the result by 100. abercrombie's formula was used to correct the duplicate counting of neurons: n=nt/(t+d), where n is the corrected number of neurons counted, n is the number of cells counted, t is the thickness of the sections (12 m), and d is the average diameter of neuronal nuclei. since the difference in size significantly affects cell counts, the value of d was calculated specifically for each experimental group (ipsilateral and contralateral). in this sense, the nuclear diameter of 15 randomly picked neurons from each group was measured with imagej software (version 1.33, national institutes of health, usa) and the mean value was calculated. slides containing transverse spinal cord sections were incubated for 45 min in a 3% bsa solution followed by incubation with the following primary antibodies: mouse anti-synaptophysin (dako, glostrup, denmark 1: 200) rabbit anti-gfap (abcam, 1: 1500), and rabbit anti-iba1 (waco, 1: 700). the primary antibodies were diluted in a solution containing 1% bsa (bovine serum albumin) and all sections were incubated for three hours in a moist chamber at room temperature. after rinsing in pb, the sections were incubated with a cy3-conjugated secondary antiserum (1: 250, jackson immunoresearch, west grove, pa, usa) for 45 min in a moist chamber at room temperature. the sections were then rinsed in pb, mounted in a mixture of glycerol/pb (3: 1), and observed with a leica dm5500b microscope coupled with a leica dfc345 fx camera. for quantitative measurements, three representative images of the ipsilateral and contralateral ventral horn were captured from each animal for all experimental groups (n=5 for each group, ipsi and contralateral ventral horn, 4, 8, and 12 weeks after lesion). for analysis of synaptophysin immunolabeling, the integrated density of pixels was measured in eight areas surrounding each lateral motor nucleus motoneuron, in the anterior horn of the spinal cord. for the analysis of anti-glial fibrillary acidic protein (gfap) and anti-ionized calcium binding adaptor molecule 1 (iba1) antibodies, the integrated density of pixels was measured in the lateral region of the spinal cord ventral horn, as described by oliveira et al. and freria et al. the integrated density of pixels was calculated for each section and then as a mean value for each spinal cord. the data are represented as the mean standard error (se) for each group. morphometry, regenerated axon area, and counting analyses were performed by sampling at least 30% of each nerve cross-section (magnification of 1,000x) using a bright field microscope (n=5 for each group, ipsi and contralateral ventral horn, 4, 8, and 12 weeks after lesion). sampling bias was avoided by spreading the micrographs systematically over the entire cross-section, according to the procedure proposed by mayhew and sharma. these images were used for counting the total number of myelinated fibers. for each specimen, two fields were used to measure the perimeter and diameter of myelinated fibers and the myelin area, using adobe photoshop cs6 extended. these values were used to calculate the myelinated axon diameter, fiber diameter, myelin thickness (fiber diameter, axon diameter/2), and g ratio (axon diameter/fiber diameter) that indicate the relative success of the regeneration process as well as myelination process. lumbar spinal cords (n=3 for each group, ipsi and contralateral ventral horn, 4 weeks after lesion) were dissected out and stored overnight in fixative at 4c. the specimens were then trimmed, osmicated, dehydrated, and embedded in durcupan (fluka). ultrathin sections from the l4l6 segments were collected on formvar coated copper grids, counterstained with 2% uranyl acetate and lead citrate, and examined under a transmission electron microscope operating at 120 kv (tecnai biotwin g2 spirit, fei company, the netherlands). neurons with large cell bodies (~35 m in diameter), found in the sciatic motoneuron pool and cut in the nuclear plane, were identified as -motoneurons by the presence of c-type nerve terminals and chromatolysis (axotomized side). the cell surfaces were then sequentially digitalized at a magnification of 11,000x with an eagle 2k video camera (fei company, the netherlands) connected to a computer system, and the images mounted together using vector graphics software. synaptic terminals opposing the motoneuron somata were identified and their numbers per 100 m of cell membrane, as well as the membrane covering of all the terminals (calculated in percentage of membrane length), were determined using the measurement tool of image j software (version 1.33u, national institutes of health (nih), usa). the terminals were typed as f-type (flattened synaptic vesicles or flattened and spherical vesicles that contain glycine/gamma-aminobutyric acid (gaba) as neurotransmitter), s-type (spherical synaptic vesicles that contain glutamate as the neurotransmitter), or c-type (presence of a subsynaptic cistern and acetylcholine as the neurotransmitter), according to the procedure described by conradi in 1969 (supplementary figure s2). the distance between consecutive nerve terminals covering the motoneurons a total of 24 sciatic -motoneurons from three animals per group (two neurons per animal in four groups, ax, ax+fs, ax+cfs, and contralateral) were quantified. motor function was analyzed using the peroneal functional index (pfi) by the walking track test (catwalk system, noldus inc. a high speed video camera gevicam (gp-3360, usa) equipped with a wide-angle lens (8.5 mm, fujicon corp., china) is positioned underneath the walkway and the paw prints are automatically recorded and classified by the software. the paw prints from each animal were obtained twice a week from the third week of life. pfi was calculated as the distance between the third toe and hind limb pads (print length) and the distance between the first and fifth toes (print width). measurements of these parameters were obtained from the right (unlesioned) and left (lesioned) paw prints, and the values were calculated using the following formula described by bain and colleagues in 1989: (1)pfi=174.9eplnplnpl+80.3etsntsnts13.4,where n: normal or nonoperated side; e: experimental or operated; pl: print length; ts: total toe spread, or distance between first to fifth toes., ribeiro preto, sp, brazil) was utilized to evaluate sensory function recovery (n=5 for each group). for that, the rats were kept inside acrylic boxes for twenty minutes before the experiment for habituation. nociception threshold was measured at the plantar region by touching the skin with a pipette tip adapted to a force transducer, which is connected to a digital counter that shows the force, in grams, used to trigger the paw flinch. hyperalgesia was measured by stimulating the paw surface for six times, and the results were averaged and shown as a mean se. in order to avoid foot pad lesion and inflammation, the maximum stimulation weight (cut-off threshold) was set to 70 g. each data collection was carried out in duplicate and in a blinded manner. the data are presented as mean se, and the differences between groups were considered significant when the p value was<0.05 (),<0.01 (), and<0.001 (). statistical analysis was performed with graphpad prism 4.0 software. in this sense, data were subjected to anova followed by bonferroni post hoc test for parametric data or mann-whitney u test for nonparametric data. neuronal survival was investigated as the ipsi/contralateral ratio of motoneurons present at ventral horn lamina ix. after 4, 8, and 12 weeks, a severe degeneration motoneuron was observed in the ax group. coaptation groups performed equally well and presented statistically significant rescue of axotomized motoneurons (figure 1, table s1). no significant differences between the numbers of motoneurons on the contralateral side in the different experimental conditions were observed. additionally, axotomy alone with immediate reapposition of the stumps and followed by 2 mm proximal stump resection did not differ with regard to motoneuron survival (supplementary figure s3). quantitative measurements of synaptophysin immunoreactivity in the sciatic motor nuclei after axotomy (ax) and after axotomy followed by coaptation (ax+fs; ax+cfs) were carried out 4, 8, and 12 weeks after injury. axotomy group showed significant reduction of immunoreactivity ipsilateral to the lesion side. on the contrary, synaptic covering was preserved following coaptation in all survival times analyzed (figure 2, supplementary data table s2). a significant increase in astrocyte reactivity after axotomy was observed four weeks after lesion (figure 3, supplementary data table s3). similar results were observed in microglial reactivity, where groups with coaptation showed decreased microglial reaction, particularly at four weeks after lesion (figure 4, supplementary data table s4). although absence of spontaneous regeneration following axotomy alone was observed, acute coaptation (ax+fs, ax+cfs) resulted in axonal growth with sensorimotor recovery. area measurements showed no significant difference between ax+fs and ax+cfs groups, although such nerves presented reduced dimensions as compared to the contralateral (nonlesioned) samples (table 1). additionally, the estimation of total number of axons revealed no significant differences between coaptation groups (figure 5, supplementary data table s5), although significantly reduced in comparison to the normal nerve. of note, commercial fibrin adhesive displayed the worst values for all parameters analyzed at four weeks after lesion, indicating slower axonal regeneration progress (figures 6, 7, and 8). such initial difference was not present eight and twelve weeks after lesion, so that both coaptation groups performed in a similar fashion at later stages. statistical differences between groups were only observed at four weeks after sciatic nerve coaptation. in this sense, ax+fs displayed better recovery as compared to ax+cfs. such statistical evaluation is presented in detail as supplementary material: figure s4, fiber diameter; figure s5, axon diameter; and figure s6, myelin thickness. no statistical differences were depicted regarding the g ratio (figure 9), although cfs group presented mostly under myelinated fibers four weeks after injury. all analyzed motoneurons showed at least one cholinergic presynaptic terminal (type c) in apposition to the plasmatic membrane surface. lesioned neurons, affected by axotomy, showed changes compatible with chromatolysis, such as displacement of the nucleus to the periphery and decrease in cytoplasm electron density. glial projections were observed, in the ax group, intermingling the space between part of the presynaptic terminals and the postsynaptic membrane (figure 10(a)). fully apposed terminals were depicted in the other experimental groups (figures 10(b)10(d)). the total synaptic covering, which represents the motoneuron body surface in contact with presynaptic terminals, revealed reduction of excitatory terminal covering in all groups analyzed, when compared to the contralateral side (figure 10(e)). four weeks after lesion, nontreated neurotmesis (ax group), presented a 41.22% covering reduction of type s inputs when compared with contralateral side. nerve coaptation resulted in excitatory input preservation, with no statistical differences between commercial and cevap sealant (figures 10(f) and 10(g)). fibrin sealant nerve repair promoted even more prominent effects on inhibitory inputs (type f boutons), showing close to normal covering (figures 10(f) and 10(g)). axotomy alone led to greater distance between terminals that contrasted with coaptation repair, where clusters of terminals were close to each other (figures 10(h)10(k)). the recovery of motor function was studied by the walking track test, using the catwalk system (noldus inc.). statistical analysis from peroneal functional index showed a significantly greater peroneal functional index (pfi) following coaptation (ax+fs, ax+cfs), regardless the nature of the fibrin sealant (figure 11). pfi has been chosen over sciatic functional index (sfi), because measuring the intermediate toe spread (its) was not possible. we regard that to muscle atrophy since nerve lesion was performed in the neonatal period. the nociceptive recovery was analyzed by stimulation of the plantar surface of the paw with electronic von frey. contralateral paw showed withdraw reflex around 30 g. axotomy alone (ax) rats did not show withdraw behavior at the cut-off weight, indicating lack of sensory perception (70 g, figure 12(a)). both groups subjected to coaptation with fibrin sealant (ax+fs and ax+cfs) showed lower threshold withdrawal response (around 45 g, figures 12(b)-12(c)). it is well known that neonatal axotomy results in devastating neuronal loss in the spinal cord and dorsal root ganglia (drg). this is particularly because of the interruption of neurotrophic factors production and retrograde flow, mostly at the target organs. in fact, administration of neurotrophic factors, such as brain derived neurotrophic factor (bdnf) and ciliary neurotrophic factor (cntf) rescue motoneurons from degeneration. also, the use of cannabidiol (cbd), a cannabinoid with neuroprotective properties, is also able to avoid both motoneuron and drg neurons death, following neonatal peripheral nerve lesion. in this way, early neonatal nerve axotomy has been broadly accepted as a consistent model for studying neuroprotection [46, 48]. nevertheless, due to the reduced size of pups, combined with the thin and delicate structure of the peripheral nerves at perinatal stage, repair following transection is hardly accomplishable. the present work shows that end-to-end nerve coaptation can be performed in a reproducible fashion by using fibrin sealant as a connecting structure between proximal and distal stump. it is important to highlight that in humans nerve size is larger, allowing direct suturing. in this scenario, the use of fibrin glue may enhance repair stability and function as a scaffold for cell migration. in the present study, we compared two different fibrin adhesives, one commercial brand and another recently developed by cevap. the latest is based on nonhuman components, avoiding transmission of infectious blood-borne diseases. the results have shown that both sealants (commercial and cevap) are excellent for the coaptation process, although cevap's fibrin sealant has proven to be easier to handle at the moment of surgery. surgeries involving neonatal subjects ought to be particularly fast, and clotting speed is a crucial parameter. due to the fact that cevap derived adhesive uses bubaline fibrinogen, the concentration of such protein in the cryoprecipitate is specially higher than the human counterpart. in turn this in turn possibly allowed early regrowth of regenerating axons, originated from the proximal stump, resulting in improved morphometric parameters, four weeks after repair. in line with the positive results described in the present work, the fibrin sealant from cevap has been tested in humans, for autologous skin grafting of the nasogenian sulcus showing promising results. moreover, it has been tested for the immobilization of free periodontal gingival grafts in lower premolars and for the treatment of chronic venous ulcers [5054]. besides, recent studies also show the efficacy of this new sealant on peripheral nerve regeneration [55, 56], treatment of skull defects, and following ventral root avulsion. it has been shown that this scenario can be partially reversed by local or systemic administration of trophic substances, such as neurotrophins, antioxidants, cannabinoids, and gangliosides [47, 59, 60]. nevertheless, the regrowth of axons from the spinal motoneurons and drg neurons towards the periphery is dependent on anatomical restauration of pns structures. we show that coaptation at the neonatal stage is neuroprotective by itself and generates a permissive scaffold through which regeneration can be successfully accomplished. the results shown herein indicate significant preservation of motoneurons in the spinal cord, assessed by direct counting at 4, 8, and 12 weeks after lesion, reinforcing the long lasting positive effects of the repair procedure. nevertheless, since nerve repair was carried out at p2, motor and sensory evaluation could only be initiated from the third (catwalk) or fourth (von frey) week of age due to animal size and sensory-motor coordination. in turn, our data reflect already established recovery that happened before behavior tests could be started, what explains the rather constant peroneal and von frey results. importantly, restauration of neonatal lesion, which mimics obstetric brachial plexus injury, led to preservation of synapses, as seen by synaptophysin immunostaining. reduction of astrogliosis and microglial reaction also contribute to synapse stability and functionality, correlating with substantial motor and sensory recovery in ax+fs and ax+cfs groups up to twelve weeks after surgery. in this regard, improved peroneal index following coaptation was observed with either fibrin sealant treatment, when compared with axotomy alone. such improvement indicates reestablishment of muscle innervation as well as suprasegmental control [61, 62]. after an injury, retraction of presynaptic boutons that appose spinal motoneurons takes place during the first week after lesion. this event reduces synaptic transmission [6469] and allows the axotomized neurons to focus metabolism on survival and regrowth of a new axon and dendrites. herein, synaptic covering was significantly preserved in coaptation groups, in line with the immunohistochemical observations (synaptophysin). a quantitative analysis of the synaptic covering for each type of presynaptic terminal showed significant preservation of s and f type of synapses. however, the proportion of preserved inhibitory synapses was greater than excitatory. such scenario, according to lind et al., facilitates neuroprotection by reducing glutamatergic excitotoxicity [70, 71]. besides the prevention of excitotoxicity, the number of preserved presynaptic terminals/100 m in apposition to the alpha-motoneuron membrane was also increased in both coaptation groups. synapse dynamics also depend on astrocytes and microglia that act in favor of maintenance of homeostasis that is necessary to transmission and synaptic plasticity. one well-known retrograde effect of peripheral axotomy is the development of central astrogliosis and microglial reaction, directly interfering on synapse stability [7275]. gfap and iba1 are highly expressed proteins in astrocytes and microglia, respectively, and are upregulated after lesion [73, 76]. the fact that coaptation downregulates glial reaction at lumbar spinal cord level is in line with the positive anatomical and behavioral findings previously discussed. overall, the present data suggest that acute repair of neonatal peripheral nerve with both fibrin sealant analyzed, namely, a commercial brand and a nonhuman derived adhesive produced by cevap/brazil, promotes neuroprotection and regeneration of motor and sensory axons. also, the present study demonstrates that neonatal end-to-end nerve coaptation is feasible and may in turn be of use for repairing obstetric brachial plexus injuries.

brachial plexus lesion results in loss of motor and sensory function, being more harmful in the neonate. therefore, this study evaluated neuroprotection and regeneration after neonatal peripheral nerve coaptation with fibrin sealant. thus, p2 neonatal lewis rats were divided into three groups: ax: sciatic nerve axotomy (sna) without treatment; ax+fs: sna followed by end-to-end coaptation with fibrin sealant derived from snake venom; ax+cfs: sna followed by end-to-end coaptation with commercial fibrin sealant. results were analyzed 4, 8, and 12 weeks after lesion. astrogliosis, microglial reaction, and synapse preservation were evaluated by immunohistochemistry. neuronal survival, axonal regeneration, and ultrastructural changes at ventral spinal cord were also investigated. sensory-motor recovery was behaviorally studied. coaptation preserved synaptic covering on lesioned motoneurons and led to neuronal survival. reactive gliosis and microglial reaction decreased in the same groups (ax+fs, ax+cfs) at 4 weeks. regarding axonal regeneration, coaptation allowed recovery of greater number of myelinated fibers, with improved morphometric parameters. preservation of inhibitory synaptic terminals was accompanied by significant improvement in the motor as well as in the nociceptive recovery. overall, the present data suggest that acute repair of neonatal peripheral nerves with fibrin sealant results in neuroprotection and regeneration of motor and sensory axons.

PMC4942656

pubmed-1331

desktop computer and laptop use is becoming common, and computing-related musculoskeletal symptoms are considered important health problems in university students and office workers1,2,3,4. previous studies have indicated that improper sitting posture during visual display terminal (vdt) work (e.g., desktop computer or laptop use) can induce musculoskeletal disorders, especially low-back pain3, 4). carter and banister3 suggested that increased tension in ligaments and discs during a slumped sitting posture may lead to low-back pain. cross-legged sitting, a commonly adopted posture during daily living and vdt work, has been reported to induce a slumped sitting posture5, 6. lee et al.5 showed that the cross-legged sitting position leads to a greater slumped posture compared with an upright sitting posture during vdt work. to prevent the unwanted slumped posture, it is crucial to identify factors that contribute to slumped posture during cross-legged sitting. limited hip flexion range of motion (rom) could be one possible risk factor for excessive lumbar flexion. because the lumbar spine and hip joint are connected via the pelvis, limited hip flexion can cause greater lumbar flexion through pelvic posterior tilt during the trunk flexion-related posture7. although it seems reasonable that limited hip flexion rom leads to greater lumbar flexion during the cross-legged sitting posture, no study has investigated how hip flexion rom influences the kinematics of the lumbar spine in the cross-legged posture during vdt work. furthermore, previous studies have not determined whether greater lumbar flexion during vdt work with a cross-legged sitting posture changes trunk muscle activities as measured by electromyography (emg). only one emg study revealed decreased emg activity in the internal oblique (io) muscles during the static cross-legged sitting posture compared with the static normal sitting posture8. thus, the aim of the present study was to compare the lumbar flexion angle and trunk muscle activity in individuals with and without limited hip flexion rom during vdt work with a cross-legged sitting position. all subjects showed right-leg dominance and performed computer work more than 20 hours per week. measurements of the right hip flexion rom were used to classify subjects into the experimental or control groups. according to previous findings9, sufficient hip flexion rom was defined as more than 120 of hip flexion, and limited hip flexion rom was defined as 110 in this study. subjects with more than 120 of right hip flexion rom (one female, six males) were classified into the control group, and subjects with no more than 110 of right hip flexion rom (eight males) were classified into the experimental group. subjects were excluded if they had acute low-back pain, orthopedic damage, or lower extremity injury during the last 6 months. in addition, individuals with hip flexion rom between 110 and 120 were also excluded in this study. the inje university faculty of health science human ethics committee approved this study, and each subject signed an informed consent form before participation. to measure hip flexion rom, subjects were placed in the supine position on a table, and an examiner passively flexed the right hip of subjects until further hip flexion was limited by firm end feel9. the fulcrum of a goniometer was placed on the greater trochanter, and the proximal and distal arms were aligned with the lateral midlines of the pelvis and femur, respectively. the lumbar flexion angle was measured using eight vicon mx-t10 motion capture systems (vicon motion systems ltd., three reflective markers were placed on the bilateral anterior superior iliac spines and on the midpoint between the bilateral posterior superior iliac spines for the pelvic segment. additionally, four reflective markers were attached to the first and second lumbar spinous processes and 3 cm bilaterally from the second lumbar spinous process for the lumbar segment. the lumbar flexion angle was calculated by assessing the anterior rotation of the lumbar segment with respect to the pelvic segment using the cardan angle10. the trunk muscle activity of the bilateral rectus abdominis (ra), external oblique (eo), and io muscles was recorded using a synchronized surface emg system (delsys inc., prior to attachment of the electrodes, skin preparation was performed by shaving the hair and cleansing with an alcohol swab. each electrode was attached along the direction of the muscle fiber based on placements suggested by criswell11. emg signals were acquired at a sampling rate of 1,000 hz with a bandwidth of 20450 hz and converted into root-mean-square (rms) data. to normalize emg data of the trunk muscles, reference voluntary contraction (rvc) data of trunk muscles were collected when subjects were seated on a chair in a comfortable sitting posture with 90 of hip and knee flexion for 40 s. the rvc maneuver was repeated three times, and the mean value of the average muscle activity for the middle 30 s of the three trials was used to normalize trunk muscle activity. prior to vdt work, a laptop (xnote r400, lg, seoul, korea) was placed on a 73-cm-high desk, and subjects sat on a height-adjustable chair without a backrest with 90 of hip and knee flexion. the examiner confirmed the 90 hip and knee flexion position using a goniometer. for vdt work with a cross-legged sitting posture, subjects were instructed to cross the right leg over the left by putting the right knee on the left knee5, 8. following cross-legged sitting, subjected performed typing work in which they copied some text provided on the monitor by the korean version of hansoft. the subjects typed for 1 min for each trial, and three test trials were conducted with 30-s rest periods between trials. the average values of the lumbar flexion angle and emg activity of the trunk muscles for the middle 40 s of each trial were collected, and the mean values of three test trials were used for data analysis. the subjects characteristics (age, height, weight, hip flexion rom), lumbar flexion angle and trunk muscle activity during vdt work with cross-legged sitting in the experimental and control groups were compared using independent t-tests., chicago, il, usa) was used, and the statistical significance level was set at p=0.05. the general characteristics of the subjects in the control group (mean age 25.1 1.5 years; mean weight 62.6 5.7 kg; mean height 171.9 6.9 cm) and the experimental group (mean age 23.6 1.5 years; mean weight 67.4 4.1 kg; mean height 175.1 4.5 cm) were not significantly different (p>0.05), except for the hip flexion rom (123.6 2.4 versus 102.5 3.8; p<0.001). the lumbar flexion angle was significantly greater in the experimental group compared with the control group during vdt work with cross-legged sitting (p=0.017) (table 1table 1. comparison of the lumbar flexion angle and trunk muscle activity between the two groups during visual display terminal work with cross-legged sittingvariablemean sdcontrol groupexperimental grouplumbar flexion ()15.2 7.023.3 4.3*right ra activity (% rvc)101.2 5.9100.5 8.2right eo activity (% rvc)98.7 6.2113.3 30.4right io activity (% rvc)91.4 7.9109.4 22.3left ra activity (% rvc)101.3 7.1101.7 8.2left eo activity (% rvc)98.3 5.0115.3 35.0left io activity (% rvc)99.8 10.897.2 11.3rvc, reference voluntary contraction; ra, rectus abdominis; eo, external oblique; io, internal oblique.*however, there were no significant differences in emg measures of trunk muscle activity between the control and experimental groups (p>0.05). rvc, reference voluntary contraction; ra, rectus abdominis; eo, external oblique; io, internal oblique.*in the present study, the subjects with limited hip flexion rom showed greater lumbar flexion compared with subjects with sufficient hip flexion rom during vdt work with cross-legged sitting; however, these differences in hip flexion rom did not influence emg activity in trunk muscles. the lumbo-pelvic-hip complex is connected via a kinematic link termed the lumbo-pelvic rhythm7, 12. during forward bending, the lumbar spine is flexed anteriorly with respect to the pelvis, while the pelvis is flexed anteriorly on the femur12. when lumbar flexion is limited, greater hip flexion is required with pelvic anterior tilt throughout the lumbo-pelvic rhythm7. in other words, greater lumbar flexion is caused throughout the pelvic posterior tilt when hip flexion is limited. the results of our study imply that greater lumbar flexion compensated for insufficient hip flexion in subjects in the experimental group during vdt work with cross-legged sitting. despite significant differences in lumbar flexion between the experimental and control groups, emg activity in the trunk muscles was not significantly different between the two groups. snijders et al.8 reported that the cross-legged sitting position decreased emg activity in io muscles due to increased stability in the sacroiliac joint. the cross-legged sitting posture is performed by the combined motion of hip flexion and adduction. increased tension in the biceps femoris, gluteus maximus and piriformis by hip flexion and abduction influences sacroiliac joint compression and tension in the sacrotuberous ligament, which can contribute to increased stability in the sacroiliac joint13,14,15. although the hip flexion angle may have been influenced by differences in hip flexion rom in subjects in this study, sufficient hip adduction may have been possible for all subjects during vdt work with cross-legged sitting. we suggest that the sacroiliac joint compression by hip adduction during cross-legged sitting may not differ between subjects with and without limited hip flexion rom, resulting in the absence of significant differences in emg activity in trunk muscles between the two groups. first, our study included a small sample size; therefore, it is difficult to generalize our results. however, a previous study by lee et al.5 showed that trunk kinematics were immediately changed after assuming a cross-legged sitting posture during vdt work, and the trunk flexion angle then did not significantly change for 30 min. based on previous findings, we consider that the kinematic data of the lumbar spine and emg data of the trunk muscles during vdt work with cross-legged sitting for short periods may provide meaningful information for individuals who prefer cross-legged sitting.

[purpose] the purpose of this study was to compare the lumbar flexion angle and electromyography (emg) measurements of trunk muscle activity in individuals with and without limited hip flexion range of motion (rom) during visual display terminal (vdt) work with cross-legged sitting. [subjects] the 15 participants included a control group with sufficient hip flexion rom (n=7) and an experimental group with limited hip flexion rom (n=8). [methods] all subjects performed vdt work with cross-legged sitting. the lumbar flexion angle was measured using a three-dimensional motion capture system, and the trunk muscle activity was recorded using a surface emg system during vdt work with cross-legged sitting. the differences in trunk flexion angle and trunk muscle activity between the two groups were analyzed using independent t-tests. [results] the lumbar flexion angle was significantly greater in the experimental group than the control group, although trunk muscle activity did not differ between the two groups. [conclusion] these findings suggest that limited hip flexion leads to greater lumbar flexion during cross-legged sitting.

PMC3885834

pubmed-1332

a 76-year-old woman with hypertension was admitted to the hospital with complaints of chest pain and dyspnea. arterial blood gas analysis revealed the following findings of hypoxemia and hypocapnia: po2, 36.7 mmhg; and pco2, 30.4 mmhg. the patient's cardiac enzyme values were: creatine kinase-mb, 105 ng/ml; and troponin i, 0.38 ng/ml. the b-type nitriuretic peptide test result was 64. a diagnosis of recent myocardial infarction was made based on the following electrocardiographic findings: t-wave inversions and q waves in leads ii and iii and a vf, and t-wave inversions in leads v1 to v4. transthoracic echocardiography revealed akinesia in the apex free wall of the right ventricle, moderate pulmonary hypertension (right ventricular systolic pressure [rvsp], 55 mmhg), and a 2.72.8 cm mobile mass in the free wall of the right atrium. pulmonary computed tomography (ct) angiography revealed pulmonary emboli occluding the pulmonary arteries in the upper and lower lobes on both sides, and a suspicious intra-atrial cardiac tumor with pulmonary thromboembolism was revealed according to the radiologic findings (fig. to detect other possible causes of the pulmonary embolism, abdominal ct and tumor marker tests were performed, the results of which were all normal. the patient was diagnosed with pulmonary embolism due to the mass in the right atrium based on the result that the patient's d-dimer was<0.35. however, the patient refused surgical treatment. after 26 months, she was intubated due to severe dyspnea and was then referred to ajou university hospital. transthoracic echocardiography revealed: 1) hypokinesia in the right ventricle, 2) a dilated right ventricle, 3) mild pulmonary hypertension, 4) a 3.52.8 cm mass in the free wall of the right atrium, which had grown compared to previous findings, and 5) tricuspid regurgitation. pulmonary ct angiography revealed emboli occluding the right pulmonary artery and segmental arteries in the upper, middle, and lower lobes with a right atrial mass. the radiological characteristics were similar to that of a previous study (an intra-atrial cardiac tumor with pulmonary thromboembolism) (fig. pulmonary perfusion imaging showed decreased perfusion in the majority of the upper and lower right lobes, in part of the anterior segment of the left upper lobe, and in part of the superior and posterior basal segments of the left lower lobe (fig. transesophageal echocardiography revealed a 4.82.6 cm hypermobile mass with multilobular heterogeneous echogenicity in the upper lateral aspect of the free wall of the right atrium, which was not affecting the blood flow in the superior vena cava, inferior vena cava, or tricuspid valves (fig. 2c, coronary angiography showed findings of moderate coronary artery disease, which was treated with drug therapy alone. we decided to perform removal of the right pulmonary arterial emboli and the mass in the right atrium. the emboli in the left pulmonary artery decreased in size compared to the previous ct findings, and it was not considered a critical lesion because it was confined to the inferior lingular segmental artery. a standard sternotomy incision was made. the patient's body temperature was lowered to 23 (moderate hypothermia) through cardiopulmonary bypass, and then the myxoma with a 2 to 3 mm pedicle on the free wall of the right atrium was excised along with adjacent normal tissue. at 23, we opened the right pulmonary artery, which revealed that the myxoma segment was not adhered to the intima of the artery (fig. an embolus within the right pulmonary artery was removed en bloc in a bloodless surgical field (fig. 3b). after we confirmed that there were no further masses in the arteries supplying each segment, the right pulmonary artery and atrium were closed. histopathological examination revealed that both the mass resected from the right atrium (4.33.53 cm) (fig. 3c, white arrow) and the embolus removed from the right pulmonary artery (721.5 cm) (fig. in addition, the size of the right atrium had decreased and the pulmonary artery pressure had slightly decreased to 35 mmhg. however, tricuspid regurgitation remained stationary (grade 2/4). imaging studies, the right lung had improved, but the left lung showed no changes. the patient is currently on out-patient follow-up without any significant symptoms. according to the echocardiography performed in december 2010, there was no evidence of pulmonary embolism, and the rvsp was 35 mmhg. pulmonary emboli are mainly caused by deep vein thromboses and embolus in the right heart or at the tip of a catheter. chitwood were the first to perform a successful resection of a myxoma through cardiopulmonary bypass. the recurrence rate of pulmonary embolism after resection has been reported to be 0.4% to 5%. pulmonary emboli frequently recur when 1) surgical resection is incomplete, 2) emboli detached during surgery adhere to the intima of the heart, 3) emboli are of multicentric origin, 4) there is a family history of myxoma, or 5) a new myxoma develops from myxoma precursor cells. because myxoma tissue is extremely friable, it is frequently detached and it adheres to a new site [2-5]. approximately 75% of all the myxoma cases occur in the left atrium, 23% in the right atrium, and 2% in the ventricle. myxoma can manifest obstruction symptoms due to blood flow blockade, symptoms due to embolism, arrhythmia, and other generalized symptoms. syncope or sudden death can develop when pulmonary or systemic circulation is disturbed or when blood flow to the atrioventricular valve is blocked. thrombosis can be induced by myxoma segments, thrombi that has formed within the tumor, or infected lesions within the tumor. embolism occurs in 30% to 45% of the patients with myxoma of the left atrium. emboli originating from myxoma in left atrium are mainly found in the brain, kidney, and branches of the aorta and lower extremities. embolism occurs in approximately 10% of the patients with myxoma in the right atrium, and even pulmonary embolism can occur as in our case. in our case, the pulmonary embolism that had been diagnosed in may 2008 was different from the one detected by pulmonary ct angiography in march 2006. the former is thought to have been induced by thrombi formed by the myxoma in the right atrium, but not by myxoma segments, because the ct density (region of interest [roi] property) was significantly higher in the myxoma detected in may 2008 (roi, -20 for the myxoma in the right atrium as well as for the myxoma in the right pulmonary artery) than in the myxoma detected in march 2006 (roi, 80). in march 2006, heparin administration at admission and maintenance anticoagulant therapy with coumadin resolved thrombi in the pulmonary artery on both sides were performed; however, in may 2008, the myxoma mass in the right atrium was separated and occluded the right pulmonary artery, causing severe dyspnea that required endotracheal intubation. removal of myxoma in the right side is important for preventing pulmonary embolism, maintaining contractibility, and restoring dilation functions of bilateral atria. special precautions should be taken during tumor resection in order to prevent thrombus formation and pulmonary embolism. jones et al. proposed that complete resection of myxoma should be performed under adequate exposure and minimal manipulation. furthermore, pulmonary embolectomy should be considered in cases of myxoma associated with pulmonary embolism in order to improve patient symptoms. we reported a case of a 76-year-old woman with myxoma in the free wall of the right atrium. the patient's initial bilateral pulmonary embolism was developed due to thrombi formed by the myxoma; however, the embolism that occurred again two years later was due to myxoma segments.

a 76-year-old woman with hypertension was admitted to the hospital with complaints of chest pain and dyspnea. an echocardiogram and pulmonary computed tomography angiography showed right atrial myxoma complicated with pulmonary thromboembolism. an operation to resect the right atrial myxoma and pulmonary embolism was recommended; however, the patient refused and was discharged with anticoagulation therapy. two years later, she developed dyspnea. radiological studies and echocardiography showed similar results with the previous findings. the patient underwent mediastinotomy with resection of the right atrial myxoma and pulmonary embolectomy. as there are few reports on right atrial myxoma complicated with pulmonary embolism, we report a successful case of surgical removal of right atrial myxoma and pulmonary embolism.

PMC3573168

pubmed-1333

cancer remains one of the major causes of death in children between the ages of 1 15 years.1 pediatric cancers differ markedly from adult cancers in their nature, distribution and prognosis. pediatric oncologists face unique challenges because treatment with irradiation, surgery and chemotherapy can adversely affect the children's growth and development. though lower compared with the incidence of some adult cancers, it comes next to accidents as the leading cause of death among children in the developed world.1 the patterns of childhood cancer in america and europe are almost the same, with leukemia and tumors of the central nervous system accounting for over one-half of the new cases. however, there is a dearth of data on the incidence and patterns of childhood cancer in africa. although many papers have been published on this in some african countries,29 reports on the patterns and incidence of childhood cancer in sudan are very few. the objective of this study is to determine the patterns of childhood cancer in gezira state, central sudan. the patterns of cancer were studied focusing on the prevalence of tumors according to age, sex, geographic and ethnic distribution and relating the cancer to environmental and genetic causative factors. all children with cancer, aged one to 15 years diagnosed by means of histological or cytological examination and admitted to the institute of nuclear medicine and molecular biology and oncology from may 1999 december 2004, were included in the study. gezira is the second largest state in sudan, with an estimated population of 3,962,000 with a 50:50 male to female ratio (49.3:50.7) according to the last population census (1993). gezira state was considered the richest state in the country before the discovery and extraction of oil. the institute of nuclear medicine, molecular biology and oncology (inmo) which was founded in 1993, has a new department established in 1997 to manage and care for cancer patients in a modern multidisciplinary approach. this is the second oncology centre in sudan and it caters for patients with cancer from gezira state and the surrounding states in the central region of sudan. the results of this study showed a pattern of childhood cancer in patients admitted to inmo during the period (may 1999 dec. 2004). lymphoma was the most prevalent (42.8%) followed by acute lymphoblastic leukemia (19.8%) and kidney tumors (12.8%). prevalence of childhood cancer over the period may 1999 december 2004 at inmo distribution of childhood cancer according to place of residence and gender distribution of children according to their tribes types of cancer by age of children admitted to inmo over the period may 1999-december 2004 association between common types of cancers and child tribes the prevalence of tumor in children in our study was higher among boys (64.7%) than girls (35.3%). most of the children admitted with cancer came from the rural areas (66.1%) compared to 33.9% from urban areas. the distribution of children according to their tribes showed that the majority of them belonged to central sudan tribes (41.8%), followed by kordofan and northern sudan tribes, (both of them recorded 20.3%). children from the darfur tribes constituted 14.8%, while those from eastern sudan tribes were only 2.7%. the results showed that lymphoma, acute lymphoblastic leukemia and bone tumor commonly occurred in children above five years of age in contradistinction to kidney tumor and retinoblastoma which commonly occurred in children younger than 5 years. the prevalence of non-hodgkin lymphoma in males were found to be 65% while it was 35% in females. there was also a significant association between the children's tribes and the types of cancers at a level of 5%. although the causes of childhood cancers are largely unknown, a few conditions can be explained with specific chromosomal and genetic abnormalities, and ionizing radiation exposure. environmental causes have long been suspected by many scientists but have been difficult to determine because it is difficult to identify past exposure levels in children particularly during potentially important periods such as pregnancy or even the time prior to conception. in addition, each of the distinctive types of childhood cancers develops unique clinical course in terms of age, race, gender and many other factors.1 it has been shown that in many developing countries, the reported prevalence of childhood cancer in boys is substantially higher than in girls. the ratio of boys to girls registered with childhood cancer, increased with decreasing gross domestic product and with increasing infant mortality, suggesting that boys are increasingly more likely to be affected than girls with increasing economic disadvantages.10 the ratio of boys to girls in our study is 1.8:1 which agrees with the african trend but differs from the trend in western countries where the female to female ratio is 1:1.1012 the pattern of the cancer in gezira-sudan is like other african countries, with lymphoma as the commonest followed by acute lymphoblastic leukemia and kidney tumor. this was also the pattern found in a study conducted in khartoum in the early nineties.1316 the distribution of the most common three cancers according to age and gender in our study, is in consonance with international trends for lymphoma as a common cancer in children aged more than five years, with non-hodgkin lymphoma being twice more common in males than in females, while hodgkin is five times more common in males than in females. in this study, acute lymphoblastic leukemia differed from the international trend in that it was more common among the children aged more than five years. however, the gender distribution was similar to the international pattern which has females and males equally affected. the prevalence of kidney tumors as evident in our study is in accord with the international pattern which shows that the tumor is prevalent in children less than 5 years of age with slight difference in male to female ratio 1.7: 1. there was a significant relationship of the prevalence of different cancers in relation to the different sudan tribes. in this study it was evident that the: patterns of cancer in gezira state is like other african patterns but different from those of western countries.lymphoma is the most common cancer in gezira state, with males three times more affected than females. children above five years of age were shown to be the most commonly affected.in contrast to western countries leukemia is more prevalent in children above five years of age. patterns of cancer in gezira state is like other african patterns but different from those of western countries. lymphoma is the most common cancer in gezira state, with males three times more affected than females. in contrast to western countries leukemia is more prevalent in children above five years of age. there is a need for research to determine the annual incidence of different cancers in children in gezira and determine mortality and five year survival rates.a detailed study of environmental risk factors is necessary since gezira state has the largest agricultural scheme in africa and middle east. there is a need for research to determine the annual incidence of different cancers in children in gezira and determine mortality and five year survival rates. a detailed study of environmental risk factors is necessary since gezira state has the largest agricultural scheme in africa and middle east.

introduction: cancers form one of the major causes of death in children between the ages of one and 15 years. they differ markedly from adult cancers in their nature, distribution and prognosis. the patterns of childhood cancers in america and europe are almost the same, with leukemia and central nervous system tumors accounting for over one-half of the new cases. in contrast, lymphoma is the most common prevailing cancer of this age group in africa. objective:the objective of this study is to determine the patterns of childhood cancers in gezira state, central sudan. it is a retrospective study using hospital records. all children with cancer, aged 1 15 years diagnosed by means of histological or cytological examination admitted to the institute of nuclear medicine, molecular biology and oncology from may 1999 december 2004 were included in the study. results:the results showed a pattern of childhood lymphoma as the most common cancer (42.8%) followed by acute lymphoblastic leukemia (19.8%) and kidney tumor (12.8%). the prevalence of cancer was found to be higher among boys (64.7%) than girls (35.3%) with a rate of 1.8:1. most of the children admitted with cancer were from rural areas (66.1%) compared to (33.9%) from urban areas. conclusion:lymphoma, acute lymphoblastic leukemia and bone tumor commonly occurred in children above 5 years in contradistinction to kidney tumor and retinoblastoma which was prevalent in children less than 5 years of age.

PMC3410067

pubmed-1334

it was observed in 1910 that a sufficiently high concentration of guanosine could form a gel, unlike the other nucleobases, and in 1962 it was discovered that four guanosine can self-assemble to form a hydrogen-bonded square, with bonds between the n1o6 and n2n7 positions. this structure is known as a g-tetrad or g-quartet. like any nucleobase, there is also a strong propensity for these structures to stack on each other via-interactions, forming four-stranded helices called g-quadruplexes, with the phosphate backbone perpendicular to the plane of the g-quartets. the four strands may be from separate molecules, or they may be from only 2 or 1, with loops joining them together [37]. they form with great thermal stability, and have been found experimentally to form from genomic sequences in critical regions such as telomeres, gene promoters and utrs, [9, 10] and to have physiological effects in each of these regions. in telomeres, their formation reduces the activity of telomerase, the upregulation of which has been associated with 85% of cancers, and has led to much pharmaceutical interest. g-quadruplexes in gene promoters, such as the oncogenes c-myc and c-kit, [12, 13] have been shown to control transcriptional activity in vitro, although interestingly their formation can lead to the increase or decrease of activity in different systems. it has been shown that g-quadruplex formation in the 5 utr can decrease translational activity, and there have been suggestions of other physiological effects. a wide variety of proteins have been found to interact specifically with them, and they have been shown experimentally to form in vivo [1618]. g-quadruplexes have also been employed as biosensors (e.g., for thrombin) and in other nanotechnological applications (e.g., [20, 21]). some of these uses are reviewed in. in parallel with the experimental work being developed, computational techniques have also been developed to predict which sequences will form g-quadruplexes [2224]. there are a variety of different algorithmic rules that can be used to predict which sequences can form g-quadruplexes, [25, 26] although some are more widely used and accepted. there is not sufficient evidence for any of them to be held as absolutely true, and it is only recently that any work has been done to try to predict relative stabilities of possible g-quadruplex structures, rather than just whether they could form or not. despite this limitation, computational methods have led to a number of discoveries, including the observations that g-quadruplexes are relatively rare in the human genome, but more prevalent than expected in gene promoters. some of the computational discoveries have been recently reviewed [25, 26]. the field as a whole has grown very significantly in recent times, with a roughly exponential rise in publications (see figure 1), including over 350 in 2009. a dedicated book has been produced, together with special issues of some journals focused on this topic, and some databases on particular aspects of g-quadruplexes. a series of international conferences has been initiated, the first two hosted in louisville, ky [29, 30]. at the first of these, it was suggested that a central and coherent website to store and provide data related to g-quadruplexes should be produced, and we volunteered to provide such a repository, hosted at the url http://www.quadruplex.org/. here, we describe the features available at that website, and in particular the core databases to describe predicted g-quadruplexes, and a new tool to estimate the thermal stability of these structures computationally. we also describe the other online sources of predictive data for g-quadruplexes, so that researchers may chose the most appropriate tool for their work. the core quadruplex database (quaddb, http://www.quadruplex.org/?view=quadbase) provides both static and searchable data for researchers on computationally predicted g-quadruplexes (putative quadruplex sequences, pqs). these have been generated as previously described, using our favoured predictive algorithm, which identifies sequences on either strand of the form (g3+n17g3+n17g3+n17g3 +). this has been shown experimentally to be a good predictor of in vitro g-quadruplex formation. it aims to identify specific g-quadruplexes that may form, providing a testable in vitro hypothesis that can be tested using simple biophysical methods. for any researcher interested in identifying pqs in specific sequences, we provide the quadparser program pre-compiled for ms windows and mac os x with detailed instructions. the program is customisable, so that different patterns can be searched for. different loop length constraints, g-tract lengths and so forth may all be set, so that the algorithm can be adjusted to fit with the particular context desired. quadparser has a variety of output styles for different uses, and reads sequence data in fasta format. the data search section allows a researcher to identify any pqs in gene promoters (defined as the 1 kb upstream of the tss) or utrs for their gene of interest. the output provides full details of the gene, including genomic parameters, and the location and sequence of pqs in the appropriate regions of every transcript of the gene. figure 2 displays the output when searching the human genome for pqs in the promoter or utrs of c-kit (hgnc nomenclature kit). currently, searches may be performed against the human, chimpanzee and mouse genomes. as a convenient alternative to gene-by-gene searches or using the quadparser program we currently offer this data for human (builds 34, 35 and 36 for back compatibility), chimpanzee (2.1), mouse (37), rat (3.4), dog (2), chicken (2), zebrafish (7), fruitfly (5.4), roundworm (180) and yeast (1.01) genomes. in each case the data provides a genomic coordinates for each pqs, together with the strand, sequence and a unique identifier. the thermal stability of g-quadruplexes varies with the concentration of monovalent cations, specifically na and k. however, even for fixed concentrations, the exact details of the sequence, and hence the structure formed, make a very large difference. g-quadruplexes can vary from those which are too unstable to form at 5c to those which will resist temperatures above 95c. it is therefore necessary not just to predict which sequences can form g-quadruplexes at all, but also the stability with which such sequences can form. such experiments are relatively easy to perform, and have led to a series of studies of different aspects of the relationship between sequence and stability [3134]. however, this does not enable prediction of unmeasured sequences, forcing researchers to make informed guesses as to the stability of novel sequences. we recently developed a bayesian learning algorithm that is capable of making accurate predictions of thermal stability for new sequences, having been trained on a collection of measured sequences. we provide an interface to this system at http://www.quadruplex.org/?view=quadpredict, enabling researchers to make easy predictions of melting temperatures under various conditions for any desired sequence. one feature of the bayesian inference we use is that in addition to predictions of the melting temperature, we also provide uncertainties in the values for each sequence. in general, the uncertainty increases for sequences that are highly unlike those in our training set. this therefore enables researchers to decide rationally how much faith to place in a particular prediction. we intend to develop the training data further, and have already employed a rational active learning protocol to collect more data and reduce the uncertainties below that originally presented. we will continue to do this, and also provide an opportunity for researchers to contribute their own data, so that the bayesian inference can be increasingly accurate. we hope that depositing data publicly may become a standard requirement for publication of g-quadruplex thermal data. we allow researchers to discover whether particular sequences they are interested in are already in our database of measurements, with information about exactly how such an experiment was performed. we hope that these facilities will prove useful to all those working in this field. as well as those interested in biological aspects of g-quadruplexes, we feel this facility may be particularly helpful for those working in nanotechnology or materials science, providing them with a method of rationally selecting g-quadruplex-forming oligonucleotides. there are a number of other tools that may be used to predict the existence of g-quadruplexes in dna, and links to these are provided from http://www.quadruplex.org/. bagga and coworkers use a similar algorithm to quadparser called qgrs mapper. it has different default parameters, in particular looking at sequences with fewer consecutive guanines and longer loops, but essentially looks for much the same sequences. interestingly, it includes a scoring parameter for different possible g-quadruplexes that can be formed. although this is loosely based on empiric evidence, it is not clear how the g-score produced, which ranges up to a maximum of 105, relates to stability. to the best of our knowledge, no empiric tests have been performed testing the validity of the g-score even as a ranking list, but it is still a useful formulation of established rules of thumb. as well as the qgrs mapper, which also provides the facility to search by genes, they also provide specialised databases, grsdb2 and grs_utrdb, for searching pre-mrnas and utr sequences. maiti and coworkers offer a site called quadfinder, which implements essentially the same algorithm as quadparser. (at the time of writing it does not appear to be functioning.) at the same institute, chowdhury and coworkers have a site called quadbase, again using essentially the same algorithm. they focus on cross-species analysis, offering an ortholog analysis for finding conserved g-quadruplexes, across either prokaryotes (proquad) (and see) or eukaryotes (euquad). it should be noted that the conservation required is by presence, and no sequence comparison is performed. lastly in this category is the greglist database of potential g-quadruplex regulated genes, which lists all human genes that have a g-quadruplex in the 1 kb region upstream of the transcription start site. a completely different approach to g-quadruplex prediction is taken by the maizels lab [42, 43]. whereas other methods aim to predict specific g-quadruplex sequences, largely driven by the desire of structural biologists to have structures to study, and by the desire of medicinal chemists to have a defined form to target, the g4 calculator from eddy and maizels accepts that many of these structures are highly polymorphic in vivo. as a result, they do not aim to predict individual structures but look at the density of sequences likely to lead to g-quadruplex structures. given that this is an entirely orthogonal approach, it is striking that in many cases, particularly working on the gene functions that are likely to be regulated by g-quadruplexes, very similar conclusions arise from using this approach as the quadparser model. we strongly recommend that for any large-scale genomic studies, both approaches are used to corroborate the results found. computational methods have been of great use in understanding the role that g-quadruplexes may play in biology, unveiling their function in gene promoters [27, 42] and in regulating translation. they have also revealed that stable g-quadruplexes are generally located in nucleosome-free regions. stability predictions have been used to develop experimental methods to directly visualise g-quadruplexes using afm. we anticipate that greater availability of ever more reliable tools will both improve the quality of informatic research in this area and make it increasingly easy for experimentalists to access computational results.

g-quadruplexes are four stranded nucleic acid structures formed around a core of guanines, arranged in squares with mutual hydrogen bonding. many of these structures are highly thermally stable, especially in the presence of monovalent cations, such as those found under physiological conditions. understanding of their physiological roles is expanding rapidly, and they have been implicated in regulating gene transcription and translation among other functions. we have built a community-focused website to act as a repository for the information that is now being developed. at its core, this site has a detailed database (quaddb) of predicted g-quadruplexes in the human and other genomes, together with the predictive algorithm used to identify them. we also provide a quadpredict server, which predicts thermal stability and acts as a repository for experimental data from all researchers. there are also a number of other data sources with computational predictions. we anticipate that the wide availability of this information will be of use both to researchers already active in this exciting field and to those who wish to investigate a particular gene hypothesis.

PMC2915886

pubmed-1335

while over 90% of vulvar malignancies are squamous cell carcinomas, other histologic types include melanomas, basal cell carcinomas, sarcomas, extramammary paget's disease, and bartholin gland adenocarcinomas. ectopic breast tissue in the vulva was first described by hartung in 1872, and since that time, several cases of both benign and malignant lesions arising in vulvar mammary tissue have been reported (van der putte, 1994). the histology of the mammary-like tissue has multiple forms, ranging from simple, wide, slightly coiled tubular glands with a smooth outline, to more complicated forms in which the coiled structure has numerous branches and acini forming lobules. similar to normal breast tissue, both epithelial and myoepithelial cells are present and the glands are subject to both the dysplastic and malignant changes that are seen in normal breast tissue (van der putte, 1994). we report a case of a mammary-like invasive carcinoma presenting as an asymptomatic vulvar nodule. a 65 year old, multiparous, postmenopausal female status-post total hysterectomy with bilateral salpingo-oophorectomy 39 years prior for symptomatic myomatous uterus, presented for her annual well woman exam. she was without complaints and review of systems was negative; specifically, she denied any recent history of unintended weight loss or gain, fever, chills, nausea, melena or hematochezia, vaginal bleeding or discharge, abdominal pain or fullness. her last mammogram was 12 years prior, after which she was diagnosed with right breast ductal papilloma which was excised. in addition to her prior hysterectomy, gynecologic history was significant for estrogen replacement therapy for 20 years, which she stopped after developing what she reported as two years prior to presentation, she had such a lesion removed from her right labia that had been present for one year and pathology was benign, per patient. on further discussion, she reported that the lesion had returned, was not painful, and was not associated with any further growth for the last six months. clinical breast examination did not demonstrate evidence of palpable masses, retraction, skin changes, or axillary adenopathy. genital examination revealed a mobile, soft, non-tender, 2.5 0.75 cm nodule at 11 o'clock on her right labia majora. excisional biopsy was performed in clinic, which revealed ductal carcinoma in situ with positive margins. the patient then underwent a wide local excision of the area, which pathologically revealed invasive carcinoma with negative margins. the specimen from the wide local excision revealed a surface which was predominantly dark brown and wrinkled with a smooth, white, flat lesion measuring 0.8 0.3 cm; the lesion was 1.2 cm from the 12 o'clock margin and 0.4 cm from the nearest (3 o'clock) margin. on serial sectioning, the lesion was firm and white, and extended to a depth of 0.3 cm. microscopic examination revealed invasive carcinoma, at least 1.6 mm in greatest dimension (fig. 1), arising from within a background of mammary-like glands of the vulva demonstrating residual focal dcis. the invasive lesion was noted to come within 1.3 mm of the margin, but all margins were negative for invasive disease. immunohistochemical (ihc) stains including smooth-muscle heavy chain (smm-hc), cd 10, and p63 were performed and while there appeared to be some myoepithelial cells present along the smooth contoured edge by smm-hc only, there were other areas without any definitive basal cell/myoepithelial cell staining (fig. 2). the area of concern demonstrated an irregular growth pattern (no longer lobulocentric) and was not associated with the prior biopsy site changes. the tissue was then tested for hormone receptor status, and was found to be estrogen receptor (er) positive, but progesterone receptor (pr) and her2/cep17 negative. secondary to narrow margins, re-excision was performed as well as an ipsilateral groin sentinel lymph node dissection, both of which were negative for dcis or malignancy. after referral to a medical oncologist, she was also administered letrozole, an aromatase inhibitor, as adjuvant therapy for the er positive cancer. a relative paucity of reports has documented complications of ectopic breast tissue in the vulva, beginning with hartung in 1872. mammary ridges were theorized to have developed in a line from the axilla to the groin, and while the large majority of these ridges would normally regress, it was possible for rudimentary tissue to persist and develop into supernumerary mammary tissue (van der putte, 1994). these mammary-like glands have an unknown function, but were distinguished from eccrine and apocrine glands. the glands are simple tubular structures and are sparsely concentrated; however, in some women they are more numerous and assume more complicated forms that resemble mammary tissue (van der putte, 1994). the more complicated forms contain the basic coiled structure, but are obscured by numerous branches and acini which form lobules. they are distinctly different from eccrine and apocrine glands by multiple factors including: 1) the type of epithelium, 2) the formation of diverticula, branches, and lobuli, 3) the shedding of clusters of epithelium, and 4) the expression of receptors for estrogen and progesterone. additionally, mammary-like glands are also noted to be different than normal mammary tissue derived from the mammary ridges for multiple reasons as well: 1) their simpler configuration, 2) the higher number of glands than would be expected in rudimentary elements from mammary ridges, and 3) the direct relationship to eccrine glands. furthermore, based on human embryologic studies, the mammary ridges could not involve the anogenital area, as at the height of the formation of the mammary ridges in 9 mm and 10 mm embryos, the labia majora are still far from their first appearance (van der putte, 1994). however, ectopic breast tissue has been described in the vulva, as well as in any other area of the body. both ectopic breast tissue and mammary-like glands are susceptible to the physiologic, dysplastic, and malignant changes seen in normal breast parenchyma (castro and deavers, 2001), as there have been reported cases of invasive ductal carcinoma, ductal carcinoma in situ (dcis), lobular carcinoma, mucinous adenocarcinoma, phyllodes tumors, and fibroadenomas in accessory breast tissue in the vulva (lopes et al., 2006). although ectopic mammary tissue in the vulva is believed to be present in two to six percent of women, few cases of invasive ductal carcinoma have been reported in the literature (kazakov et al., 2011). the most common clinical presentation is a painless, solitary nodule, arising most often in the labia majora. patient age at presentation has been between 45 and 84 years, with most being 60 or older (kazakov et al., most carcinoma cases are histologically consistent with invasive ductal carcinoma, which is also the most common histology in orthotopic breast tissue (kazakov et al., 2011). this still leaves a differential diagnosis of primary mammary carcinoma, carcinoma of a skin appendage, or metastatic mammary carcinoma. recognition of an in situ component is the best clue to establishing the primary origin in the skin, as was seen in our case, rather than a metastatic process (kazakov et al., 2011). secondary to the rarity of these lesions, there is no widely accepted recommended management strategy. a recent review only identified approximately 25 cases reported in the literature (benito et al., 2013). management strategies have ranged from wide local excision for dcis to radical vulvectomy with bilateral inguinofemoral nodal dissection for invasive carcinomas (irvin et al., 1999). adjuvant therapies have included radiation, anthracycline-based chemotherapy, and hormonal therapy, in an attempt to decrease recurrence and improve survival. patients that did not receive adjuvant therapy had a median survival of 4 months (lopes et al., 2006). only 1 patient out of 8 who received adjuvant therapy had died at the time of the previous publications, and the median survival was greater than 22 month (lopes et al., 2006, irvin et al., 1999). not surprisingly, there have been no clinical trials to evaluate the effectiveness of chemotherapy and radiation in such a small number of patients, so information on treatment has been, and should continue to be, extrapolated from treatment of cancers in the breast i.e. excisional procedure with or without lymph node dissection, followed by other adjuvant therapies as needed. there is little to no data reporting on the utility of sentinel lymph node biopsy in this clinical scenario. breast carcinoma is almost exclusively managed using sentinel lymph node mapping (krag et al., 2010), while promising data support the use of this technique for vulvar cancer (levenback et al., 2012). vulvar cancer is ideally suited for sentinel lymph node mapping secondary to the predictable lymphatic drainage of the vulva, the low false negative rate, and the substantially reduced morbidity (e.g. lymphedema) compared to a full groin lymph node dissection (levenback et al., 2012). we demonstrate this to be a potentially feasible technique for primary breast adenocarcinomas arising within the vulva in mammary-like glands, and in the context of such a rare histology, may be included in the management options of this disease.

highlightsprimary invasive breast carcinoma can be found arising from within mammary-like glands in the vulva.there is no standard management strategy for this rare disease; treatment recommendations should be similar to that for primary breast carcinoma.the use of sentinel lymph node biopsy may offer another management option for this disease.

PMC3895283

pubmed-1336

the 25 years millennium development goals target number 4 (mdg4) of the united nations required the reduction of the mortality rate of children below 5 years of age (u5mr) by a factor of 67% by the end of 2015. this translated to the saving of extra 520 babies per day out of the estimated 781 that died every day in nigeria. this was a huge and difficult task that required proactive thinking and development and implementation of revolutionary ideas given the antecedents of such a struggling low-resource country as nigeria. all kinds of money-guzzling programmes that were based on foreign but internationally standardised methods have been executed in nigeria from the inception of the mdg4. various international donor agencies identified with a number of paediatrics healthcare needs donated money and collaborated with nigerian ministries of health at federal (fmoh) and state levels for the eradication of diseases and epidemics and general improvement of child health. large sums of money have been spent in a wide spread recruitment and engagement and reengagement of almost the same sets of expertise in a variety of changing patterns by fmoh. unfortunately the u5mr has remained high as all these efforts have made little progress towards this ultimate goal. the literature has revealed that a large number of babies die within the first 28 days of life (neonatal period) in many tropical countries. this includes nigeria where over 660 newborn babies were estimated to die every day according to a more recently published report. it has also been demonstrated that neonatal mortality rate (nnmr) accounted for up to 40% of the total u5 mortality. this was quite significant than to be ignored as was realised by some latin american countries. by simple mathematics, it could be explained that any measures taken to reduce nnmr would contribute significantly towards the realisation of mdg4. however, most of the international and national collaborative programmes repeatedly targeted older children without significant impact on the neonates. unfortunately, even if the rest of older u5 children were all saved without a significant reduction in nnmr, the mdg4 target of 67% would still not be realised. many years on from inception, nigeria could not demonstrate significant mdg4 progress going by the ten-year timeline between ibe's increasing admission delivery and ogunlesi et al. kangaroo-mother-care (kmc) techniques of keeping babies warm may scarcely work for big neonates. however extreme preterm babies (< 29 weeks ga) or extreme low-birth-weight babies (< 1000 g) might likely die without proper incubator intervention. a typical nigerian special-care-baby-unit (scbu) could have a daily census of up to 45 neonates on admission at the same time of which 15 or more would be incubator-dependent very-preterm or extreme-preterm babies [7, 10]. such common scenario implied a basic requirement of minimum 20 functional incubators at any given time for a standard nigerian scbu. sadly no referral centre could demonstrate the availability of up to 4 functional incubators for a consecutive time period of two years. it was rather common to see a large number of dysfunctional and obsolete incubators littering the hospital walkways, workshops, dump sites, and scrap yards whilst the scbus remained empty. the unaffordability of reliable incubator systems in low-income settings was a well-known fact as a modern incubator sold in excess of 25,000. poor spare parts supply chain and unstable and erratic power supply constituted some of the harsh operating conditions for the nondurability of these systems whenever any few could be procured. since such large number of functional incubators must be available on a continuous long term bases to guarantee improved and sustainable neonatal survivability, nigeria should have reconsidered other options in order to save its babies. individual referral centres in nigeria could have adopted nonconventional measures to create new standards that might save more babies and hence enable the realisation of mdg4 target. the aim of this work was to investigate how remedies to some identified issues of concern might have changed practice and overall neonatal survival rate in few hospitals in nigeria. a collaborative medical outreach was launched in 2003 as a private research initiative to apply some willing nigerian tertiary/referral hospital neonatal centres to develop affordable incubators and techniques that could tackle lack of functional systems at the centres. the application of recycled incubator technology (rit) developed gradually and became popular among a number of leading teaching hospitals scattered across the landscape of nigeria. in a resolution, the committee of chief executives of federal tertiary health institutions (ccefthi) of nigeria endorsed the new application and moved into collaboration in using this to save more babies in a few prospective special care baby centres in nigeria. the new application, though in insufficient number in these centres, provided continuous services that soon revealed the other practice deficiencies that contributed to poor outcome other than lack of incubators. these were individually experimented upon by the provision of prospective solutions before adoption as standard remedies. all participating centres readily adopted the proposed concern remedies, albeit with varying degrees of implementation with which the participating hospitals distinguished themselves based on their various outcomes. in order to investigate how these remedies might have corroborated with the level of practice outcomes from each centre a centre could score a maximum of 5 points on each of the concerns remedies if fully implemented or nothing if completely ignored. the recruitment of hospital centres into this scholarly project followed a natural desire of the hospital management of each prospective centre to reduce their prevailing high neonatal mortality rate (nnmr). earlier publications demonstrated the trial, introduction, and standardisation of an incubator recycling technique that has safely been applied with high reliability and safety records for up to ten years [1012]. the systems were often fully restored at costs that were smaller than 25% of the costs of modern incubators. the recycled-incubator-technology (rit) systems were constructed using internet-sourced generic components arranged by design and applied to the old casings. the new low-cost systems so-produced were capable of 10 years of life expectancy and easily maintainable by locals [10, 12]. finished products have been applied in the main referral hospitals that presently serve up to 20 states in nigeria (figure 1). the rit was applied to extensively improve the capacity of outreach centres in terms of the number of functional incubators available to handle the high volume of admission delivery that was on the increase. a good example was the lagos university teaching hospital (luth) that grew from no available functional incubator in january 2007 to become nigeria's largest centre with 38 units of functional incubators and 8 units of neonatal resuscitaires (total 46 units) by december 2013. these systems were distributed within 3 large hospital wards that looked after babies born within the hospital (inborn), babies referred from outside (outborn), and paediatrics surgical ward, respectively. the concern of incubator availability in each centre was scored as follows: 04 functional incubators received zero (0 pt), 59 (1 pt), 1014 (2 pts), 1519 (3 pts), 2024 (4 pts), and 25 or more (5 pts). the introduction of rit systems and the associated outreach amongst nigerian hospitals particularly popularised attainment of sustainable and consistent presence of functional incubators in any nigerian centre. prior to this, the use of the very old and crude technique of hot-water-bottles was the most popular method of providing warmth to the neonate at many premier teaching hospitals in nigeria. the sustained presence of functional incubators at the centres soon exposed the other deficiencies of nursing and clinical skills associated with incubator care. it became necessary to develop two levels of elective courses for clinicians and nurses in order to instruct on the various unacceptable practices being exposed. most of these awful practices ultimately led to infections and cross-infections, as primarily witnessed and evidenced from patient's case-notes, thereby impoverishing overall outcome (figure 2). at the time of preparing this paper, over 1600 candidates had passed through level-1 course and this equipped candidates with a more advanced skill of patient-specific thermoneutral control in cases of very prematurity and very low birth-weight. a perceived improvement on overall outcome through these courses in some centres made the training become an important component for career progress for the staff of the centres. courses were executed as frequently as the hospitals called for this and up to two streams in a year. centres were given 1 pt for each course organised between april 2011 and march 2013 up to a maximum of 5 pts. many centres accepted rit as affordable option of restoring proper neonatal incubation in the scbus. this led to the desire to increase their incubator capacities beyond what the available old/abandoned systems could number to adequately accommodate the increasing admission of incubator-dependent neonates. hospitals were assisted to obtain casings of used or obsolete models at give-away prices through some foreign agencies. the obtained systems were then restored to functionality using rit components and methods, hence making more incubators available for the babies (figure 3). no assessment score was assigned to this as this application reflected in the scoring of concern-1. academics within individual centres were invited to form local research groups and supported to carry out investigative studies of observable phenomena relating to temperature control in the neonate (figure 4). a minimum of one possible research project was proposed to each participating centre accompanied with the provision of scholarly encouragements and affordable material support. this was a difficult concern as the culture of this kind of investigative research was unpopular amongst nigerian clinicians and nurses. only two individual centres were able to complete and publish any studies [9, 16]. some of the successful efforts resulted in multicentre publications because the national outlook of this outreach made it possible to extract data on the same phenomenon across centres from different regions of nigeria for comparison [11, 14]. each centre was awarded 1 pt for every journal publication up to a maximum of 5 pts. sustainability of the neonatal incubator practice amongst nigerian scbus would be a difficult one if the question of maintenance of spare parts was not addressed. local independent artisans were assessed and recruited for training wherever available within the cities where the participating hospitals were located. these included welders, painter, electricians, perspex-craftsmen (figure 5(a)), and tailors. these were trained on how to use their own skills and tools at the comfort of their own workshops to reproduce samples of various items of parts they were given. this approach made these parts affordable and readily available whilst creating jobs for the other members of the public. this method had initially been used to assemble a full locally made incubator that has been in use for over 5 years at the federal medical centres (fmc) owerri (figure 5(b)). this was not assigned any centre scoring point because it was not based on how well a hospital performed. neonatal apnoea episodes were highly frequent conditions in every nigerian scbus corroborating with the country's well-known high neonatal mortality rate. apnoea might happen as a result of various physiological and clinically diagnosed or undiagnosed factors wilting the neonate. there was no presence of apnoea monitors in any of the centres at the inception of the present outreach. the neonates were so many but attending nurses so few per rota shift, so eye balling was inherently inefficient. nursing staff strength of the centres was so poor that none of the centres could demonstrate any better nurse-to-neonate ratio of 1: 10. a typical nigerian busy centre could witness persistent apnoeic attacks on up to 5 babies at the same time. this real life scenario suggested a possibility of losing up to 4 or all of the 5. this was because at the average of 10-neonate-workload, a nurse attending to the first apnoeic baby might not be aware of the other 4 early enough to save them. therefore the hospitals were encouraged to (1) work towards 1: 4 ratio as a minimum operational standard by employing more nurses and (2) install individualised cot/incubator fixable apnoea monitors. market research for an appropriate design and affordable baby breathing/motion monitor was carried out. this yielded a recommendation to apply the bm02 apnoea monitoring system (jablotron, czech republic) on all cots and incubators as minimum standard. nurse-to-neonate ratio of up to 1: 4 was given the full 5 pts, up to 1: 6 (4 pts), 1: 7 (3 pts), 1: 8 (2 pts), and 1: 9 (1 pt). apnoea monitors installation up to 3 nos received 1 pt, up to 5 nos (2 pts), up to 10 nos (3 pts), up to 15 nos (4 pts), and up to 20 nos (5 pts). the introduction of rit-incubator and its subsequent application in expanding the capacity of nigerian scbus was intended to ensure availability of adequate number of functional incubators to save more babies. this aim soon began to be frustrated by impoverished mains power supply to operate the incubators when needed. neonatal incubation being a life support intervention ought not to be switched off to preserve the neonate's life. unannounced power cuts in nigeria were very frequent and rampant and could last longer than 10 hours for each episode. hence, neonatal hypothermia defined as body temperature below the physiological range of 36.5c37.4c continued to be a serious concern even when baby managed to secure accommodation in available incubator. the idle period in-between power availability was hence narrowed by the introduction of power-banking technique as a practice standard. 3.5 kva/48 amps inverter-battery system (fussion series, su-kam industries india) for every group of up to 8 incubators and resuscitaires. this was capable of sustaining these systems for up to 10 hours after power failure. if power-bank capacity was increased to power up to 16 or more incubators then the full 5 pts were awarded. incubators were operated nonstop, day-in day-out, so long as there was an occupant neonate. hence, this needed to be regularly serviced to guarantee the prevention of avoidable breakdowns. nigeria had a well-known culture of running systems and appliances to destruction due to neglecting professional maintenance. this often led to long periods of system out-of-use whilst management scrambled all over the place in search of repair or replacement. fac was introduced as a service of professional advice when needed accompanied with on-site routine system assessment/maintenance. this was a collaboration of professional care that managements were encouraged to enter into via the signing of agreements or memorandum-of-understanding. this automatically empowered a highly reputable third party dedication that ensured the sustainability of the centre's incubator capacity. professional advice was executed whenever this was called for via electronic communication throughout the year. a score of 5 pts was assigned to centres with unbroken fac service within the last 3 years leading up to march 2013. annual performance competition was introduced in 2010 to award prizes to (1) the nation's overall best scbu manager (normally the chief nursing officer of the winning scbu) and (2) the hospital with the best managed collective group of scbus. each centre was assessed twice a year at 6-month interval for an average annual score but without prior notification of when this would happen in order to capture the actual practice standards of the centre. the 1st and 2nd best hospitals and the best overall manager each received a trophy and various amounts in cash reward. the announcement of the worst scbu of the year was introduced in 2013. the quest to be announced as best scbu or best manager soon triggered competitiveness that seemed to have positively affected overall practice outcome across the participating centres. heads of hospital managements were also taking pride in showcasing their strengths in different aspects of the concerns pursuit. the full 5 pts down to 1 pt were given to any centre that achieved 1st up to 5th position in each of the inclusive 3 years in this analysis (i.e., 20112013); average of scores for the three years was awarded. it was expected that positive impact of these concern remedies over time could alter prevailing status of indices such as neonatal mortality rate (nnmr), length of hospitalisation of surviving neonates, and patient influx. the centres were assessed through retrospective data collected from their patient admission/discharge registers. by standards this was supposed to be a summary note-book that captured information such as dates of admission and discharge, birth-weight, gestation age, and overall outcome (dead or alive). assessment did not require patient's identity to be revealed; however, all participating hospitals were invited to follow their institutional ethical rules to submit their data. three other big referral hospital centres that were not participating in the outreach were also invited to submit assessment data as the invited control centres represented the common practice status of a typical nigerian scbu and very similar to the states of the outreach centres before intervention began. submitted sets of data were used to measure three basic indices of clinical success, that is, overall average neonatal mortality rate (nnmr), average length of hospitalisation for the surviving neonates, and the associated patient-traffic (influx). the class of babies that might require incubator intervention for survival, incubator-dependent-neonates (idn), was defined based on the nigerian standards of neonatal classification. this included babies born earlier than 37 weeks of gestation (preterm) or those of birth-weight less than 2500 g (low birth weight). intrauterine growth restricted (iugr) babies within this boundary were also included provided birth-weight was less than 2500 g. other quantified parameters were the fractional size of idn babies in the presented population and the mortality rate of the idns (idnmr). the total number of idns that died within 48 hours of presentation (d48 babies) was calculated and used to assess how overall nnmr was affected by these. one set of data was to be submitted by each participating centre to measure these performance indicators for the last 24 months leading up to march 2013, tagged post-rit period. outcomes were compared amongst outreach centres and also against outcomes from control centres. the idn class was further examined to identify the very low birth parameter cases; that is, very-lbw (1500 g) and very-preterm (32 weeks ga) cases. this was applied to investigate how very-low birth parameter cases might be currently affecting overall idn survival statistics at the hospitals. the managements of a very few tertiary hospitals in nigeria accepted to try out the new ideas of recycled incubator technology (rit) initially. all the neonatal care centres responded positively with enthusiasm as the various procedures were being initiated in each centre. the present coverage of the use of rit systems or procedures across hospital centres in the states of nigeria is shown in figure 6. many doctors and nurses enrolled for the training courses at various times; some repeated the courses and the associated examinations until they passed them. a good number of the hospitals signed the agreements and operated the failure-preventive audit culture (fac) component of the outreach (figure 7). the managements of few hospitals ensured unbroken fac services, some lasting up to 8 years to the time of this report. fac was broken a number of times in other hospitals due to administrative successions that never considered this very important but restarted after it became obvious that stoppage had resulted in many scbu disasters. we found that centres with less unbroken fac operated with more consistent or increasing incubator capacities (figures 7 and 8). we observed that there was better structural transformation in hospitals where the management showed higher level of commitment to the entire outreach projects by strictly following their 6-month pac reports and implementing them. periodic interdepartmental reshovelling of nursing staff as practiced in nigeria no longer affected scbu trained nurses in hospitals where management mandated the proactive participation of the nursing department in the outreach project. there was better improvement of nurse-to-neonate ratio in these hospitals too. we observed that the best performing matrons/nurses were the most regular on courses and seminar attendance. the poorly performing centres in the national competition were mainly those that paid little attention to training courses irrespective of the growth of their incubator capacity. only two individual centre groups were able to fully complete an investigative research and publish the original journal articles [9, 16]. only 5 of 15 invited centres submitted their data for inclusion in the present analysis. these were from tertiary hospitals located in the southern nigeria (c1 and c2), middle-belt (c3), and north (c4). centre (cc), a tertiary hospital in southern nigeria (figure 9). average neonatal mortality (nnmr) across the outreach centres was computed at 114 deaths per 1000 neonatal admissions (114/1000). when d48 mortality was excluded, death rates were twice higher than these at the control centre (figure 9). typically, we computed that up to 64% of overall deaths of incubator-dependent-neonates (idn) at the respective centres occurred within 48 hours of presentation. there were very few survivals amongst babies born before 30 weeks gestation or below a birth weight of 1000 g. data also revealed that nearly all of the babies dying within the 48 hour window were either average length of surviving idn hospitalisation at the outreach centres was 20.5 days (average centre range: 832 days). this was not quantified at the control as supplied data lacked such details. the recycled incubator technology (rit) and associated outreach concerns in this study have demonstrated the significant capability of nigerian special care baby units (scbus) in achieving improved newborn survival. an earlier publication to assess the impact of this outreach in 2009 studied performances of the same group of hospitals that were involved in the present analyses. this revealed that facility-based average nnmr had dropped from 254/1000 to 198/1000 babies. this figure was only a reflection of what the country could have achieved if all the nigerian hospital scbus adopted the same techniques as those analysed in the study. the nnmr of our control centre in the present study (250/1000) was very similar to the 254/1000 that was quantified as national average at the inception of the present outreach project as reported by amadi et al.. this is a validation that seemed to suggest that facility-based nnmr within a typical tertiary centre in nigeria might remain within this high figure until the statuses of the concerns that have been identified in this study were positively altered. at the inception of invitation to submit data for a retrospective assessment of the present outreach, all the hospitals invited to submit raw-data accepted with enthusiasm promising to keep up with the provided deadline. however, many of these either failed to comply or out rightly excused themselves from the study for various weak reasons, some of which might have stemmed from the fear of exposing suspected relative poor performance. the few submissions however had the advantage of coming from hospitals in the southern, northern, and middle-belt of nigeria, giving a good national coverage. our present study has shown that there has been further improvement on the overall outcomes of facility-based performance indices of the outreach centres. notably, the average nnmr within the facilities has dropped from the figure of 198/1000 as reported by amadi et al. to 114/1000 in the present study. this significant leap must have been a result of the unprecedented dedication to capacity expansion, training, and implementation of fac audit reports by the centres that have been assessed. analysis was initially designed to use a scoring technique to comparatively measure the performances of entire centres in each of the concerns and to relate these to their respective outcomes. unfortunately, the analysed data came from just a few of the participating hospitals for which very strong conclusions might not be drawn. however these were the top performing hospitals in the use of the concern remedies (figure 11), and clinical and nursing practices in the centres had not been altered in any other direction since the 2009 assessments of amadi et al.. hence result could only demonstrate that these concerns were the very vital components that brought about a significant reduction in nnmr as compared to the control. we are not able to accurately quantify the relative contribution of each concern remedy towards the overall lowering of nnmr as these top centres applied varying levels of priority to different concerns. for example, the fmc owerri was enabled to execute up to 7 concerns but particularly favoured continuous retraining of their scbu staff while luth lagos maximised 5 concerns and particularly favoured incubator capacity expansion up to 38 units of functional incubators and 8 units of resuscitaires. this boosted luth's overall baby influx, a turnover of over 150 neonates in some months between patients being admitted in the out-born, in-born. and paediatric-surgery the admission registers of these high performing centres revealed higher influx of neonates as compared to situations prior to the present outreach. overall baby admission at our control was comparable to other centres and had the third largest neonatal admissions (figure 12). this suggested that the population within the catchment zone of this hospital had significant willingness to seek specialist intervention. however our control recorded the least relative number of idn presentations amongst the admitted babies. this indicates a higher hesitation in the presentation of idn babies to the control centre despite the people's good level of willingness to seek the hospital, perhaps as a result of very low success rate (figure 13). the control centre (cc) saved the least number of idns (74 neonates) amongst the 5 individual centres over the 2-year period that was analysed. this was far smaller than the worst record from any of our outreach centres despite the control's locational and academic advantages as a teaching hospital (figure 14). the clinical team at our control centre argued that the reasons for the poor outcomes might be due to depletion of both infrastructure and skilled manpower as most of the incubators in our scbu were broken down and the few functioning are in serious need of maintenance; also skilled nurses who have training in neonatal care were seldom given the opportunity to remain in the neonatal ward due to reshovelling of nurses around departments irrespective of the special training a nurse might have. our outreach centre (c4) was probably the most remotely located federal referral hospital in nigeria being situated at extreme northeastern region that was well known for higher infant mortality rates and people's unwillingness to seek hospital care. this centre was highly disadvantaged in terms of its very poor relative number of qualified doctors and nurses, without any fulltime consultant paediatrician. illiteracy was relatively higher amongst mothers; hence these were very unlikely to seek early medical intervention, orchestrating the common situations of very poor neonatal vital-signs at the point-of-admission. despite all these c4 's index in terms of idnmr was below average rate and was only outperformed by one centre (figure 13). it was our opinion that the overall reduction of average nnmr from 254/1000 to the present 114/1000 amongst our outreach centres was a demonstration of the effectiveness of the provided concern remedies. the present study has also demonstrated the information regarding the neonatal deaths occurring within 48 hours (d48) of presentation. we would suggest that our analysed outreach centres had almost pushed the boundaries of neonatal mortality to the limits except for the d48 babies which constituted up to half of the total mortality in some centres. most (83%) of the d48 babies were either very lbw or very preterm cases. a further investigation is hence recommended to urgently study the manner of care given to this class of patients, especially in the first 7 days of life, in order to synthesise any possible techniques that might guarantee better survival rate. the corporate nigeria might not have achieved the mdg4 target; however our study has shown that there were pockets of individual hospital centres that have already surpassed this target. for example, our outreach centre c3 has achieved overall nnmr of 89/1000, a reduction of 65% in terms of the national facility-based average as computed in 2009. it would have been a possibility for nigeria to corporately attain the mdg4 goal had the country paid more attention to the very simple but high impact approaches that were implemented at these few centres.

millennium development goal target on infant mortality (mdg4) by 2015 would not be realised in some low-resource countries. this was in part due to unsustainable high-tech ideas that have been poorly executed. prudent but high impact techniques could have been synthesised in these countries. a collaborative outreach was initiated to devise frugal measures that could reduce neonatal deaths in nigeria. prevailing issues of concern that could militate against neonatal survival within care centres were identified and remedies were proffered. these included application of (i) recycled incubator technology (rit) as a measure of providing affordable incubator sufficiency, (ii) facility-based research groups, (iii) elective training courses for clinicians/nurses, (iv) independent local artisans on spare parts production, (v) power-banking and apnoea-monitoring schemes, and (v) 1/2 yearly failure-preventive maintenance and auditing system. through a retrospective data analyses 4 outreach centres and one control were assessed. average neonatal mortality of centres reduced from 254/1000 to 114/1000 whilst control remained at 250/1000. there was higher relative influx of incubator-dependent-neonates at outreach centres. it was found that 43% of mortality occurred within 48 hours of presentation (d48) and up to 92% of d48 were of very-low birth parameters. the rit and associated concerns remedies have demonstrated the vital signs of efficiency that would have guaranteed mdg4 neonatal component in nigeria.

PMC4129921

pubmed-1337

thyroid lesions containing gross fat include heterotopic fat nests, thyrolipoma, teratoma, liposarcoma, and thyroid cancers of the papillary or follicular type (234567). most of these cases are only described upon obtaining pathological data after surgery; radiologic methods are of limited use (89). with the approval of the institutional review board of our hospital, we reported the case of an intrathyroid nodule composed mainly of gross fat detected by computed tomography (ct) and confirmed by lobectomy as a follicular variant of papillary thyroid cancer (fvptc) with mature fat. a 58-year-old woman presented with an incidentally detected mass in the thyroid on routine ultrasonography (us) at a primary clinic. the patient had a 10-year history of diabetes mellitus and hypertension, and was currently taking medication. physical examination revealed a firm mass of approximately 3 cm in size in the right anterior neck. laboratory tests revealed that serum anti-microsomal antibody, anti-thyroglobulin antibody, thyroglobulin, calcitonin, t3, free t4, and thyroid-stimulating hormone levels were within normal ranges. ultrasonography revealed an ovoid lesion with a smooth margin of approximately 2.9 2 1.3 cm in size. the nodule was generally hyperechoic, with the peripheral portion more hyperechoic than the central area. the perithyroidal fat, muscle, and vertebral bodies were therefore not clearly visualized (fig. doppler us did not show any vascular signals in the nodule. based on these findings a board-certified radiologist performed us-guided fine needle aspiration using a freehand technique with a 23-gauge needle and a 5-ml disposable plastic syringe. cytologic smears were made on glass slides and immediately fixed in 95% alcohol for both papanicolaou staining and may-grunwald-giemsa staining. cytology revealed atypia of undetermined significance or a follicular lesion of undetermined significance with negative for braf mutations and positive for an nras 61 mutation. the thyroid ct protocol at our hospital includes pre-contrast, early, and delayed phases. early phase images (40 seconds) were scanned from the hyoid bone to the thoracic inlet to cover the entire thyroid and level iii-vi lymph nodes. delayed scans (90 seconds) covered the area from the skull base to the upper mediastinum. all scans were acquired with a slice thickness of 2.5 mm and reconstruction increment of 2.5 mm. pre-contrast ct showed markedly low attenuation (mean ct number, -80 hounsfield units [hu]), in a well-defined mass with fine reticular and thick septa-like structures in the parenchyma of the right mid-to-upper portion of the thyroid (fig. 1b). to measure the enhancement pattern of the tumor, 25 mm of the region of interest was placed in the area of the fatty mass, including the reticular and thick septa-like soft tissue lesions. the tumor was enhanced in the early phase (mean ct number, 16 hu) and was washed out in the delayed phase (mean ct number, -13 hu) (fig. the tumor was ovoid in the longitudinal direction, had a lobular appearance in the axial plane, and was completely encapsulated in the thyroid parenchyma, with no evidence of extrathyroidal extension. based on these findings, we suspected a fat-containing, neoplastic lesion of the thyroid gland, such as thyrolipoma, teratoma, lipoma, low-grade liposarcoma, or a follicular tumor with stromal fat. considering the results from cytopathology and the gene mutation analysis it showed a yellowish, homogeneous, and well-demarcated nodular lesion measuring 2.5 2.2 1.5 cm at the upper-to-mid pole. microscopic examination revealed a well-circumscribed, thin, fibrous, and capsulated nodule with mature fat. there were atypical follicular cells with nuclear enlargement, nuclear overlapping, chromatin clearing, and nuclear grooves without papillary structures in the nodule (fig. on immunohistochemistry, the tumor cells stained positive for hbme-1 (dako, glostrup, denmark, 1:100) and galectin-3 (novocastra, newcastle upon tyne, uk, 1:200). finally, we tested for the v600e and k601e mutations in exon 15 of the braf gene and codons 12, 13, and 61 of the kras and nras genes. in our case, the tumor was a yellowish, homogeneous, and well-demarcated nodular lesion on gross examination, with negative value of attenuation on ct, suggesting fat. however, it was difficult to recognize the tumor as a fat containing mass on us examination. the hyperechoic nature of the mass and the curtain-like hyperechoic shadowing posterior to the mass may suggest rich fatty component. we suspected that the hyperechoic shadowing might have been due to a reverberating artifact caused by innumerable fat globules and septa of the tumor, similar to the artifact frequently observed along the thick abdominal walls that consist of several layers of fat globules surrounded by connective tissues, skin, and fascia. computed tomography and gross specimen findings of our case were very similar to those of thyrolipoma, a benign fat-containing tumor (810). thyrolipoma is considered a variant of follicular adenoma, which is characterized by a well-circumscribed and encapsulated nodule resulting from the proliferation of thyroid follicles admixed with mature fat (1011). this case could have been misdiagnosed as a follicular neoplasm or thyrolipoma, as the tumor cells did not show a papillary growth pattern upon microscopic examination and the gross features were very similar to thyrolipoma. two reports provided the ct and magnetic resonance imaging findings of thyrolipoma (812). the mass predominantly had fat attenuation and distinct margins in the thyroid gland. however, the authors only performed pre-contrast ct on the lesion. therefore, it is uncertain whether thyrolipoma and thyroid cancer with massive stromal fat can be differentiated by ct with contrast enhancement. in our case, the tumor was enhanced in the early phase and slightly washed out in the delayed phase. fat-containing thyroid cancer of the papillary or follicular type, have only been reported from pathological studies (2456). fat tissue was observed only by microscopy, so it was assumed that small amounts of fat tissue could not be detected by ct. we also assumed that the detectability of the fat component is variable, as it depends on the total number of fat cells in the mass. mature teratoma can be recognized if the fat-containing mass contains calcification, bone or cartilage, and cystic components. however, this is not possible, as radiological images do not always exhibit all these attributes (7). ct images of liposarcoma (3) in the thyroid gland are very similar to those in our case, and differentiating between teratoma, thyrolipoma, liposarcoma, and papillary thyroid cancer with massive fat may be impossible using radiology alone. some investigators have insisted that fat cells are derived from displaced remnants of embryonic structures in the thyroid. in amyloid goiter, schrder and bcker (1) postulated that adipose tissue may be derived from metaplasia of stromal fibroblasts due to either impaired circulation and diffusion or tissue hypoxia caused by amyloid deposition. derienzo and truong (5) suggested that the fat cells in thyrolipoma and follicular carcinoma might be neoplastic tissue because 1) fat did not exist in the normal thyroid, 2) fat was a major component of the tumor, 3) there was no evidence of degenerative changes such as dystrophic calcification, cystic or hemorrhagic degeneration, or amyloid deposition, 4) fat was widely distributed in the tumor, and 5) there were similar fat-containing tumors in other organs such as thymolipoma, parathyroid adenoma, and fat-containing fibroadenoma of the breast. our case showed similar microscopic features to theirs in agreement with the suggestion that the fat cells might be neoplastic cells themselves. in summary these findings included a hyperechoic mass with a smooth margin with a reverberating artifact and no detectable doppler signal on us. ct revealed a well-encapsulated fat-attenuation mass with enhancing septa-like structures. fvptc should be included in the differential diagnosis of fat-containing focal lesions in the thyroid, in addition to thyrolipoma, teratoma, liposarcoma, and small focal fat nests near the capsule. moreover, fine needle aspiration cytology with gene analysis may be helpful in determining the nature of fatty tumors before surgery.

thyroid cancer may have small adipose structures detected by microscopy. however, there are no reports of thyroid cancer with gross fat evaluated by radiological methods. we reported a case of a 58-year-old woman with a fat containing thyroid mass. the mass was hyperechoic and ovoid in shape with a smooth margin on ultrasonography. on computed tomography, the mass had markedly low attenuation suggestive of fat, and fine reticular and thick septa-like structures. the patient underwent a right lobectomy. the mass was finally diagnosed as a follicular variant of papillary thyroid cancer with massive stromal fat.

PMC4644758

pubmed-1338

a sound, agreeable or disagreeable, is a stimulus discerned by the sense of hearing. disagreeable or undesired sounds were described as noises, which may cause undesirable masking of sounds, may interfere with speech and communication, may produce pain, injury and brief or perpetual loss of hearing. the acoustic environment of learning-teaching activities at a dental college is characterized by high noise levels in relation to other teaching areas, due to the exaggerated noise produced by the use of dental equipment's by many users at the same time. the sources of dental sounds that can be treated as potentially damaging to hearing are high-speed turbine handpieces, low-speed handpieces, high-velocity suction, ultrasonic instruments and cleaners, vibrators and other mixing devices, model trimmers and also worth mentioning are air conditioners. in dental learning areas, teachers and students exposure to noise constitutes a health risk and has both auditory and non-auditory effects. the non-auditory effects include hypertension, sleep disturbance, decreased learning performance, stress reactions, interference with communication and concentration, annoyance, mental fatigue and a reduction in efficiency. while auditory effects may include permanent hearing loss, and short term exposure to loud noise can cause a temporary change in hearing or a tinnitus. hearing loss caused by noise is referred to as noise induced hearing loss. according to the national institute for occupational safety and health (niosh), exposure to noise levels above 80 db is associated with these consequences, which depends on the intensity of the noise, distance to the source, total duration of noise and the individual's age, physical condition and sensitivity. furthermore, noise has an adverse impact on patients as a factor that may cause fear. exposure to noise is measured in units of sound pressure levels called decibels, named after alexander graham bell, using a-weighted sound levels [db(a)]. the a-weighted sound levels closely match the perception of loudness by the human ear. niosh has recommended that all worker exposures to noise should be controlled below a level equivalent to 85 db(a) for 8 h/day to minimize occupational noise induced hearing loss. noise pollution is one of the most important situations requiring a solution by the contemporary world. niosh has recognized noise as 1 of the 10 leading causes of work related diseases and injuries. in dental learning areas, teachers and students are vulnerable to different noise levels while working and teaching in dental clinics and laboratories. the aims of this study were to measure noise levels produced during the different dental learning clinics, by equipment's used in dental learning areas under different working conditions and by used and brand new handpieces under different working conditions. in pre-clinical and clinical areas, the microphone was placed at ear level at a distance of 15 cm from a main noise source to simulate the auditory position of the operator. the noise levels of the equipment's were measured at the corner of the learning area, which may consider the less noisy part of the learning area to eliminate as much as possible the interference with the external noise. the noise levels were measured over about 20 s interval and the maximum and minimum intensities in decibel was recorded. this was repeated three times sequentially in the same day, so we recorded 6 measurements for each equipment, one maximum and one minimum for each time interval. the noise levels of equipment's used in laboratories, pre-clinical and clinical areas at the dental college of damascus university were measured under different working conditions. the laboratories, pre-clinical and clinical areas will be henceforth referred to as dental learning areas in this study. the equipment's of which the noise levels were measured in clinical areas were: ultrasonic scaler, turbine, contra angle handpiece, micro motor handpiece, low volume suction pump, high volume suction pump and amalgamator (capsule). the measurements were taken with the equipment only turned on (without cutting) and during cutting operations. ultrasonic scalers with or without suction pump and suction pumps running free and when they touch mucosa were measured for noise levels. the noise level of micro motor handpiece was measured by setting it at 35.000 rpm. the noise levels of different dental learning clinics was measured by placing the noise level meter at the center of the clinic during the middle-third of working time (between 11 a.m. and 1 p.m.), which nearly represents the highest noise hours. the number of equipments, which were used at the same time, was 20 equipments. noise levels were measured in seven clinics: operative, fixed prosthodontics, removable prosthodontics, endodontics, pedodontics, oral surgery and periodontics. the sound levels were measured with a precision sound level meter (beha unitest 93517, germany) with a microphone in dental learning areas. the sound level is measured on the a scale, which was designed to mimic the response of the human ear. at the dental laboratories, the microphone was placed near the technician's ear at a distance of 15 cm from a main noise source to simulate the noise intensity reaching the eardrum and another reading was taken 2 m away. this was to simulate the person within a 2 m radius of the operator who is also exposed to the same noise. the equipments of which the noise levels were measured in the dental laboratories were: stone trimmer, automatic molding machine, manual molding machine and sandblaster. the data were collected, tabulated and statistically analyzed using t-tests with significance level set at 5% using the statistical package for the social sciences program version 13(spss inc., chicago, il, usa). the results of the noise levels of equipments measured in dental laboratories at two distances of 15 cm and 2 m are shown in table 1. noise levels [db(a)] of dental laboratory engines the noisiest laboratory equipment recorded in this study was by the sandblaster with an la(eq) of 93.32 1.99 at 15 cm distance. the results of the noise levels of the equipments measured in pre-clinical and clinical areas are shown in table 2. noise levels [db(a)] of the equipments measured in pre-clinical and clinical areas the results indicated that the maximum sound levels of equipments in dental clinics and laboratories were 92.2 db and 96 db, respectively. in dental clinics, the highest noise was produced by the micro motor handpiece while cutting on acrylic (92.2 db) and the lowest noise (51.7 db) was created by the ultrasonic scaler without suction pump. the highest noise in laboratories was caused by the sandblaster (96 db at a distance of 15 cm) and the lowest noise by the stone trimmer while only turned on (61.8 db at a distance of 2 m). there was significant differences in noise levels of the equipments used in dental laboratories and dental learning clinics (p=0.007). the mean noise levels of the equipments used in dental laboratories were much higher than those used in dental clinics. the mean noise level for dental laboratories engines at a distance of 15 cm from a main noise source was 81.62 db, compared with the mean value of 73.75 db for dental clinic equipments (p=0.009). the laboratory engines had the highest noise levels (at a distance of 15 cm from a main noise source-81.62 db), whereas the noise levels in high-speed turbine handpieces (75.44 db) (p=0.003) and the low-speed contra angle handpieces (68.31 db) (p<0.01) were decreased. the noise level of a contra angle handpiece at the clinical areas was significantly lower than at the pre-clinical areas (p=0.019). noise levels of turbine in cutting activities compared to non-cutting showed that the noise levels measured during the cutting activities were significantly higher to those found when only turned on (p<0.01). the average value of the difference was equal to 5.38 db(a) and 19.45 db(a) for brand new and used respectively. there was no significant difference in noise levels of contra angle handpiece in cutting activities compared to non-cutting activities in both pre-clinical and clinical areas for brand new and used ones. the average value of difference for turbine and contra angle handpieces (only turned on) was equal to 6.09 db(a). furthermore, there was no significant difference in noise levels of micro motor handpiece in cutting activities compared to non-cutting activities. there was no significant difference in noise levels between low and high volume suction pump. furthermore, there was no significant difference in noise levels of ultrasonic scaler without suction pump and with suction pump. noise levels of used equipments compared with brand new equipments in the clinics showed that the noise levels produced by used turbine were significantly higher than those produced by the brand new turbine (p=0.045) [figure 1]. there was no significant difference in noise levels between brand new and used contra angle handpieces in both pre-clinical and clinical areas. average noise levels of used and brand new dental turbine compared in this study, the high-speed turbine was significantly noisier than low-speed contra angle (p=0.001). the average value of difference for the high-speed turbine against low-speed contra angle handpiece was equal to 7.14 db(a) [figure 2]. mean noise levels of high-speed turbine and contra angle handpieces the results of the noise levels at the center of the dental learning clinics are shown in table 3. the highest noise level for all dental clinics was at the pedodontic clinic (67.37 5.56 db(a)) (p=0.019). in this study, noise levels of the equipments used in dental learning areas under different working conditions were measured. the noise levels measured in this study were similar to that measured in other international studies of noise in dentistry. noise levels for suction pump were 69.41-81.51 db(a) in this study, whereas in the united kingdom they were 68-70 db(a), in portugal they were 70-74 db(a) and in india they were 79-81 db(a). noise levels for the turbine were 66.72-84.16 db(a) in this study, whereas in the united kingdom, portugal, india and saudi arabia were 70-75 db(a), 68-76 db(a), 75-81 db(a) and 69-76d b(a), respectively; for contra angle handpiece they were 66.12-70.5 db(a) in this study, whereas in united kingdom, portugal, india and saudi arabia were 72-75 db(a), 69-75 db(a), 70-76 db(a) and 65-71 db(a), respectively. the noise levels of turbine measured during the cutting activities were significantly higher to those found when only turned on. presented average values of+10 db(a), in similar conditions of measurement in portugal and saudi arabia, respectively. the significant level of difference in noise levels of used turbines compared to brand new turbines could be an indication of bearing failure. the bearing resistance is affected by wear, not only of the metal surfaces, but also of the ball-cages, when roughness contributes to friction. in general, the used turbine was noisier at an average of about 9.11 db(a) difference more than the brand new one [figure 1]; therefore, the hearing damage risk may be lesser among dentists who use brand new turbine. in this study, the high-speed turbine was the noisiest equipment compared to low-speed contra angle. this is concordant with antecedent studies mentioning that the high-speed turbine handpiece generates a higher noise level than the low-speed handpiece. maximum sound pressure levels of the noise created by the dental drill was 91.9 db by the brand used dental turbine while cutting on a tooth, which has a risk of damage to the dentists hearing. the noise level of a contra angle handpiece at the clinical areas was lower than at the pre-clinical areas, which may have been because students rarely used the maximum speed of the air contra angle handpiece during dental treatment, while in the pre-clinical area it was always used at the higher speeds.. dental laboratories in dental teaching institutions were the areas of highest noise levels when compared to other dental learning areas. the effect of noise on learner comfort affecting the work performance and mental efficiency has been researched. noise can induce learned helplessness, increase arousal, alter the choice of task strategy and decrease attention to the task. suggest the classification given by cavanaugh to set a limit value in places of learning in dental teaching institutions. accordingly, 56 db(a) could be adequate as the upper limit value for a relaxed communication at a normal tone at 3 m. all the evaluated areas presented a value higher than this maximum. the highest noise level recorded for all dental clinics was at the pedodontic clinic. this may be due to the children who are normally crying during the oral health treatment. comparison of noise levels in this study with some european limits indicated that they did not comply with these laws. some international legal limits for equipment noise levels in la(eq) (db[a ]) are: italy 40, france 38, sweden 35, portugal 46 and india 50. in this study, there were some high recorded measurements of noise levels, which have a risk of damage to the dentists hearing (exceeding the limit of risk of hearing loss of 85 db[a ]) such as stone trimmer, manual molding machine, sandblaster (at distance 15 cm and 2 m), low volume suction pump, turbine (brand used) and micro motor handpiece. therefore, a necessary reduction of exposure in sound levels is required for acoustic comfort. based on the above study it can be concluded that the noise levels detected in this study were considered to be close to the limit of risk of hearing loss (85 db(a)); a necessary reduction of exposure in sound levels is required for acoustic comfort.

background: in dental practical classes, the acoustic environment is characterized by high noise levels in relation to other teaching areas. the aims of this study were to measure noise levels produced during the different dental learning clinics, by equipments used in dental learning areas under different working conditions and by used and brand new handpieces under different working conditions. materials and methods: the noise levels were measured by using a noise level meter with a microphone, which was placed at a distance of 15 cm from a main noise source in pre-clinical and clinical areas. in laboratories, the microphone was placed at a distance of 15 cm and another reading was taken 2 m away. noise levels of dental learning clinics were measured by placing noise level meter at clinic center. the data were collected, tabulated and statistically analyzed using t-tests. significance level was set at 5%. results: in dental clinics, the highest noise was produced by micro motor handpiece while cutting on acrylic (92.2 db) and lowest noise (51.7 db) was created by ultrasonic scaler without suction pump. the highest noise in laboratories was caused by sandblaster (96 db at a distance of 15 cm) and lowest noise by stone trimmer when only turned on (61.8 db at a distance of 2 m). there was significant differences in noise levels of the equipment's used in dental laboratories and dental learning clinics (p=0.007). the highest noise level recorded in clinics was at pedodontic clinic (67.37 db). conclusions: noise levels detected in this study were considered to be close to the limit of risk of hearing loss 85 db.

PMC4275629

pubmed-1339

the growing resistance of pathogens is one of the biggest public problems worldwide.1 multidrug-resistant bacterial strains can cause severe infections as they are no longer responsive to most conventional antibiotics.2,3 to combat these pathogens, efforts have been extended to develop a new generation of antibiotics. host defense peptides for their immunomodulatory properties, are cationic amphiphilic peptides, which are the first line of defense to protect organisms from microbial infection.48 it has been demonstrated that naturally occurring or synthetic amps can be a new functional class of antibiotics.9,10 amps are antimicrobial agents based on their activity against the prokaryotic membrane. these agents adopt globally amphipathic conformations upon initial contact with bacterial membranes rich in anionic phospholipids. the conformations, which resemble detergent-induced micelle formation, result in total membrane disintegration in which their cationic and hydrophobic side groups segregate into distinct regions. this finding indicates that amps are potential antibiotic agents with a different antimicrobial mechanism, and that this activity mainly depends on their physical mechanism.11 the structural and sequence diversity of amps include amphipathic -helices (eg, cathelicidins), -sheets with 24 disulfide bridges (defensins and protegrins), extended conformation (indolicidin), and beta-loop peptides (brevinin).1215 among the amps, human defensins and cathelicidins play an important role, linking innate and acquired immunities (figure 1).8 importantly, amps are therapeutic agents with a lower tendency to elicit antibiotic resistance than conventional antibiotics. currently, the main reasons for the limited practical application of amps include their very high susceptibility to proteolytic degradation by microbial enzymes, toxicity due to high amounts of drug needed for therapy, relatively short half-life, and their high production cost.16 the design and synthesis of peptide mimics (peptidomimetics) have been developed to mimic the structure, function, and mode of action of host-defense amps, which act on bacterial cell walls or membranes and can potentially circumvent those obstacles. antimicrobial peptidomimetics display antibacterial activity against a broad-spectrum of bacteria, including drug-resistant strains, and are less susceptible to resistance development in bacteria. a number of antimicrobial peptidomimetics have been developed in the last decade, such as -peptides,1719 peptoids,2025 arylamide oligomers,26,27 and -turn mimetics.28,29 recently, a new class of antimicrobial peptidomimetics termed aapeptides because they contain n-acylated-n-aminoethyl amino acid units derived from chiral peptide nucleic acid backbones have been developed. they are highly resistant to proteolytic degradation and their amphipathic structures can mimic the bactericidal mechanism of amps.3032 currently, different antimicrobial aapeptides have been developed, such as -aapeptides and -aapeptides.33,34 this review aims to describe recent progress in the discovery of peptidomimetics as new generation antimicrobial agents and discusses future directions for antimicrobial peptidomimetics in the emergence of multidrug-resistant bacteria. to improve the antimicrobial activity of peptidomimetics, the relationship between the structure and function of these peptides must be considered. interestingly, antimicrobial peptidomimetics may be designed by joining amphiphilic peptide building blocks. in this regard, a potent and broad-spectrum antimicrobial activity can be fine-tuned by changing the ratio of cationic/hydrophobic groups via the introduction of hydrophobic building blocks, suggesting that the structure activity relationships in antimicrobial peptidomimetics indicate the balance of forces required for bactericidal activity.3538 to date, peptidomimetics have been designed by cyclization of linear peptides or coupling of stable unnatural amino acids. in addition, hu et al,30 and niu et al31,32 reported the development of a new class of peptidomimetics termed aapeptides, and depending on the position of the side chain (connected to either the -c or -c in relation to the carbonyl group), two subclasses of these peptides (-aapeptides and -aapeptides, respectively) have been designed (figure 2). previous studies have indicated that focused libraries of linear aapeptide (including both -aapeptides and -aapeptides) sequences have been developed so that these sequences might mimic natural linear amps and adopt globally amphipathic conformations upon initial contact with bacterial membranes.39,40 moreover, it has been reported that the antimicrobial activity of some aapeptides is still generally comparable, or even superior, to the amp magainin as well as a previously reported linear -aapeptide 1 against several bacterial strains.31 interestingly, padhee et al39 reported that a focused library of different linear -aapeptide sequences such as 1 and 2 has been prepared to minimize the hemolytic activity, but with a potent and broad-spectrum antimicrobial activity to arrest the growth of both gram-positive and gram-negative bacterial pathogens. in addition, these authors showed that 1 and 2 are the most potent antimicrobial -aapeptide peptidomimetics with broad-spectrum activity, especially toward clinically relevant strains including the multidrug-resistant strains vancomycin-resistant enterococcus faecalis and methicillin-resistant staphylococcus aureus (mrsa).39 furthermore, a focused library of linear -aapeptide sequences containing -1, -2, -3, and -4 has been prepared.40 in this context, it has also been demonstrated that compared with -1, -2 and -3, -4 contains enhanced bactericidal activity against gram-positive strains, indicating that some -aapeptides are very potent and supporting their potential development as antimicrobial agents to treat gram-positive bacterial infections.40 however, they are quite toxic to blood cells as well as other mammalian cells. in fact, the antimicrobial activity of -aapeptides is likely to be enhanced if the overall hydrophobicity increases, which at the same time also leads to increased hemolytic activity and cytotoxicity. interestingly, the hemolytic activity and cytotoxicity can be minimized by introducing more cationic residues. on the other hand, focused libraries of lipo aapeptides (including both -aapeptides and -aapeptides) sequences have been developed, and the lipid tails of these lipopeptides are important for biological activity to facilitate bacterial membrane interaction, giving them broad- spectrum activity against both gram-positive and gram-negative bacteria.30,31 a focused library of lipo antimicrobial -aapeptides sequences has been prepared, including 3 and 4.32 interestingly, the development of cyclic -aapeptides that mimic function of amps has been reported.41 these cyclic peptides have enhanced antimicrobial activity compared with their linear antimicrobial aapeptides, as their structures adopt a semirigid backbone conformation, resulting in a more stable amphipathic structure. structure activity relationships of cyclic antimicrobial -aapeptides incorporating a global distribution of cationic and hydrophobic residues are in development. in this context, group amphiphilic building blocks can be joined, and the resulting oligomers are cyclized.42 since peptidomimetics interact nonspecifically with their target membranes, the addition of a positive charge by adding arginine, lysine, or histidine residues to the peptide sequence is required for initial electrostatic attraction with negatively charged bacterial membranes, whereas hydrophobic bulk guides insertion into the bacterial membrane. recent developments in peptidomimetics that are formed through insertion into the amino acid backbone or heteroatom replacement indicate that several peptidomimetics form structural designs such as helices, sheets, turns, and loops via noncovalent interactions. to prepare aapeptides, the current literature indicates that different approaches have been developed.32,43 originally, the synthesis of these peptides was achieved using the building block strategy (figure 3).3032 in this approach, 9-fluorenylmethyloxycarbonyl-protected peptide building blocks were prepared and then assembled on a solid support to provide the desired peptide sequences.4447 another approach termed this approach is a solid-phase synthesis of the peptides, which eliminates the need of building block preparation; thus, chemically diverse functional groups can be conveniently introduced into the desired peptide sequences.43 at present, it has also been reported that preorganized secondary structures including helical or sheet-like conformations within peptidomimetics are unnecessary in the antibacterial activities of these peptides.39,40,4850 in contrast, the presence of backbones with certain flexibility can lead to a potent and broad-spectrum antimicrobial activity, indicating the importance of the conformational rigidity in the molecular design of antimicrobial peptidomimetics.5153 in this context, peptidomimetics have more dihedral angles compared to canonical peptides, and this molecular design induces high flexibility. previous studies have indicated that in the molecular design of the amphipathic helical 21-mer peptide ([ kaakkaa]3; amino acid sequence: k a a k k a a k a a k k a a k a a k k a a), the peptide s cytotoxic activity is highly dependent upon the spatial positions of tryptophan and cationic residues within the hydrophobic sector of an -helix.54 more recently, the synthesis of enzymatically resistant versions of amps by partial substitution of l-residues with nonnatural d- or b-residues has been developed. in this regard, mcgrath et al55 synthesized a lysine-leucine or klotho peptide known as (klaklak)2, which had low toxicity toward mammalian cells, with high antimicrobial activity. furthermore, these authors demonstrated that d(klaklak)2, a variant of this molecule, is bactericidal against several gram-negative species, including escherichia coli, klebsiella pneumoniae, and acinetobacter baumannii. interestingly, a strain of k. pneumoniae, which was resistant to conventional antibiotics, was susceptible to this peptide with the minimal inhibitory concentration of 75 g/ml. in addition, this peptide has stronger fungicidal activity.55 in the remainder of this review, the discussion will highlight the discoveries that have led to our current understanding of the development of peptidomimetics in the context of their use as therapeutic agents. peptidomimetics represent an important field in pharmacology as they circumvent the limitations of amps used in therapy. therapeutic applications of antimicrobial peptidomimetics have also been considered in regard to their high resistance against enzymatic degradation.5659 regarding antimicrobial peptidomimetics which are currently in phase ii clinical trials, choi et al60 have designed small arylamide foldamers that mimic amps. importantly, these authors also demonstrated that hydrogen-bonded restraints in the structure of arylamide increase activity toward s. aureus and e. coli. on the other hand, the pharmaceutical company lytix biopharma as (troms, norway) has recently commenced phase i/iia clinical trials with another antimicrobial peptidomimetic known as lytixar tm (also known as ltx-109) for nasal decolonization of mrsa (http://www.lytixbiopharma.com). this peptidomimetic, containing a modified tryptophan derivate as lipophilic bulk, displayed a combination of high antibacterial activity against methicillin-resistant staphylococci and staphylococcal biofilms.57 another antimicrobial peptidomimetic, which is currently in phase ii clinical trials for the broad spectrum treatment of mrsa infections, is brilacidin (also known as pmx-30063).61 this peptidomimetic was synthesized by polymedix (radnor, pa, usa) and was then acquired by cellceutix (beverly, ma, usa) in september 2013. it is important to consider that this peptide is a potent bactericidal with broad-spectrum activity, not only against gram-positive bacteria such as s. aureus and enterococcus faecium but also against gram-negative species such as e. coli. interestingly, in a phase ii study involving patients with acute skin infections, 215 patients were treated with three different doses of brilacidin, and brilacidin s favorable profile was noted in 65%87% of patients.61 another antimicrobial peptidomimetic known as pol7080 has also been developed. researchers at the university of zurich and polyphor ltd (allschwill, switzerland) have developed this peptidomimetic by means of a novel approach to peptide preparation named protein epitope mimetic technology. this peptidomimetic specifically targets pseudomonas aeruginosa through a mode of action that is different from the membrane-disrupting activity of the parent compound.29 currently, pol7080 is being prepared by the company polyphor, and in preclinical studies, this antimicrobial peptidomimetic was highly active on a broad panel of clinical isolates, including multidrug resistant pseudomonas bacteria, with a potent bactericidal activity in a mouse septicemia infection model. p. aeruginosa is an opportunistic bacteria which causes serious infections in patients with reduced immune systems (eg, those having acquired immunodeficiency syndrome or cancer). at present, phase i clinical studies29 of the peptidomimetic pol7080 have been completed in healthy individuals in europe, demonstrating the clinical safety and tolerability of this peptide (http://www.polyphor.com/products/pol7080). regarding an important role of antimicrobial peptidomimetics in the prevention of virus infections and the treatment of cancer metastasis, antimicrobial peptidomimetics that inhibit virus replication as well as possess antitumor activity against different cancer cell lines have been designed. importantly, the structure of these peptidomimetics has been explored by using a peptidomimetic library in order to obtain higher plasma and metabolic stabilities. furthermore, the use of nanotechnology as delivery tool for both classes of peptides will be presented later in the review. as shown in previous reports, some -aapeptides are effective in arresting the growth of both gram-positive and gram-negative drug-resistant bacterial pathogens.50 in addition, it has been demonstrated that -aapeptides can mimic the human immunodeficiency virus (hiv) tat peptide by binding to hiv-1 rna with a high affinity, comparable to their peptide counterparts.44,46 moreover, it is well known that the cxc chemokine receptor 4 (cxcr4) is a coreceptor of hiv-1 infection in human cells.62,63 in this context, development of peptidomimetic ligands for cxcr4 as therapeutic agents for hiv-1 infection and cancer has been reported.6466 in fact, the peptidomimetic arg(*)-arg-nal(2)-cys(1x)-tyr-gln-lys-(d-pro)-pro-tyr-arg-cit-cys(1x)-arg-gly-(d-pro) (*) (pol3026) is a novel specific beta-hairpin mimetic cxcr4 antagonist, with potent anti-hiv activity.67 this peptide has a ten-fold increase in potency, with a good bioavailability profile following subcutaneous administration.67 interestingly, ligand binding site mapping using a panel of cxcr4 mutants demonstrated that the new analog d(1-10)-vmip-ii-(9-68)-sdf-1 (rcp222) share the interactive amino acids on cxcr4 with hiv-1 glycoprotein 120.68,69 to date, peptidomimetic ligands have served as inhibitors of stromal cell-derived factor-1 since these peptidomimetics are involved in the interactions of hiv-1 envelope glycoprotein and stromal cell-derived factor-1 with membrane ligands of cd4+human cells.7074 another peptide with anti-hiv activity is alx40-4c. the structure of this peptide was designed from the basic hiv-1 transactivation domain for the inhibition of tat tar interaction.75 the anti-hiv effects of this peptide are elicited through selective binding to cxcr4.76 in addition, zhou et al77 reported that the human apj, a g protein-coupled seven-transmembrane receptor, is essential for its coreceptor activity for hiv-1; thus, it is an alternative target of alx40-4c to block hiv glycoprotein 120 from binding to the cellular membrane. on the other hand, regarding the development and improvement of nanoparticles for stabilization and delivery of antiviral peptides, a nanoformulation of the amphiphatic -helical peptide p41 (a positively-charged analog of c5a peptide, derived from the hepatitis c virus protein) has been designed to treat hiv/hepatitis c virus coinfection, indicating the potential of this nanoformulation for stabilization and delivery of antiviral peptides, while maintaining their functional activity.78 currently, cancer is a major concern in relation to human mortality, and all types of cancer are characterized by irregular cell growth. antitumor drugs are subject to differences in target tissue and absorption, which can be particular to each patient. in addition, acquired drug resistance is considered the widespread cause for tumor recurrence.79 at present, some radiolabeled -aapeptides have been used as tracers for positron emission tomography, indicating a therapeutic application as anticancer agents.45 in addition, peptidomimetics have been the basis for a number of studies performed to discover new novel anticancer agents.79,80 in this regard, the in vivo inhibitory effects on the growth of tumor cell xenografts in nude mice by the cyclic pentapeptide fc092 ([ d-arg2]-fc131), a cxcr4 antagonist, have been reported.81,82 the intrinsic relationship between its structure and its high specificity to tumor cells is likely playing the key role in the cytotoxicity of peptidomimetics. these characteristics allow the peptidomimetics to bind to cancer cells and disrupt the negatively-charged tumor cell membrane, which is derived from a greater than normal expression of anionic molecules such as sialic acid-rich glycoproteins or phosphatidylserine.83 importantly, these chemical differences aid the electrostatic interaction of the positively-charged peptide and the negatively-charged tumor cell membranes.80 studies have reported of amps that are effective against bacteria and cancer cells but not against normal mammalian cells such as cecropins from insects and magainins from amphibians.84,85 on the other hand, signal transducer and activator of transcription (stat) proteins are a family of cytoplasmic transcription factors. phosphorylation induces their homo- or heterodimerization, and an important function of these dimers is to control gene expression. stat3 is frequently activated in many human cancer cell lines and is involved in cancer development and progression. importantly, dysregulation of stat3 can lead to increase in its activity and contribute to tumorigenesis., it has been reported that an oxazole-based small-molecule stat3 inhibitor, which modulates stat3 stability, induces significant antitumor cellular effects.86 one primary goal of drug delivery for cancer therapy is to increase the amount of drug delivered to the tumor site and decrease its exposure to healthy tissues.87 recent advances in microencapsulation technologies have been used to enhance drug protectivity, availability, and distribution by employing different biodegradable delivery platforms like liposomes, dendrimers, nanoemulsions, polymeric nanocarriers, and nanoparticles. these nanoformulations can be used to control drug/molecule release and enhance targeted delivery and effectiveness.88 in this regard, wang and zhang89 encapsulated a polypeptide isolated from the unicellular green algae chlorella pyrenoidosa, which exhibited the highest inhibitory activity on human liver hepg2 cancer cells (49%), and they named the polypeptide chlorella pyrenoidosa antitumor polypeptide. the main mechanism of action of this peptidomimetic is condensation/fragmentation of nuclear chromatin.89 the in vitro release of this peptide against gastric cancer cells provided a basis for the development of encapsulated antitumor peptides. the peptidomimetics klaklakklaklak and the isoasp-gly-arg (or isodgr) peptides serve as potent tools for developing new antitumor peptides. they can selectively kill cd13/v3+breast cancer cells in both in vitro and in vivo experiments by inhibiting angiogenesis by binding to v3 +, which is increased on tumor cells.90 currently, the antitumor role of the analgesic-antitumor peptide (agap) isolated from the scorpion buthus martensii has been reported. this protein, consisting of a small ubiquitin-related modifier linked with a hexahistidine tag from e. coli, was used as an antitumor peptide, and the main mechanism of action of this peptidomimetic is through cell cycle arrest.91 the recombinant system agap showed considerable inhibition of lymphoma and glioma propagation.91 interestingly, using sw480 human colon cancer cells, it was proposed that recombinant agap induces cell cycle arrest in the g0/g1 phase, attended by the decrease in the s phase without significant change in the g2/m phase.91 together, these studies strongly suggest that the use of peptidomimetics is a potent tool for developing new antitumor peptides. the main limitations in the use of these peptides are their poor bioavailability due to insolubility related to their intrinsic physicochemical properties, potential toxicity to host cells, tissue distribution, and poor pharmacokinetic issues. despite these disadvantages, it is important to consider that further studies are needed to investigate the cost of large scale production of peptidomimetics and the transition of these peptides from the laboratory to the clinic to confirm that they provide an effective new class of therapeutic agents. however, the combination of the therapeutic use of peptidomimetics and conventional therapy against cancer (eg, chemotherapy, radiotherapy, or surgical procedures) can help in overcoming drug resistance in cancer cells. increased funding and innovative research approaches to prepare peptidomimetics are required for practical use of these peptides as therapeutic agents. substantial progress has been achieved in the past decade with respect to the development of antimicrobial peptidomimetics that mimic the bactericidal activity and mode of action of amps. since several classes of peptidomimetics have great potential as a new generation of antimicrobial agents due to their low immunogenicity and enhanced stability compared with amps, in the near future, it will be important to resolve issues of hemolytic activity and cytotoxicity of some antimicrobial peptidomimetics. it will also be possible to gain further insight into the development of molecular design in peptidomimetics and to explore its medical and pharmacological applications. in fact, fine-tuning the biological activity of peptidomimetics may be readily achieved with the introduction of a variety of additional hydrophobic building blocks. in addition, mechanistic studies are needed to evaluate the possibility for antimicrobial peptidomimetics to induce drug resistance in bacteria, and research will be focused on the development of antimicrobial peptidomimetics against gram-negative bacteria, as they are generally more difficult to kill than gram-positive bacteria. finally, an important challenge over the next decade will be to develop new potential drug delivery systems for peptidomimetics. in this context, nanoformulation approaches have emerged as an important tool to improve the delivery and stability of antimicrobial peptidomimetics. in conclusion, peptidomimetics possess significant properties that support their inclusion in the generation of new antimicrobial agents. moreover, antimicrobial peptidomimetics have several advantages over amps, including enhanced stability, cell specificity, and better tolerability. furthermore, the synthetic flexibility of these molecules allows fast structure modifications to create novel antimicrobial peptidomimetics, having particular pharmacological properties. finally, structure activity relationships clear the way to establish peptidomimetic libraries, which can lead to the development of novel antimicrobial agents. a better understanding of the structural properties of peptidomimetics will potentially facilitate the practical use of these peptides as important therapeutic agents.

antibiotic resistance is an increasing public health concern around the world. rapid increase in the emergence of multidrug-resistant bacteria has been the target of extensive research efforts to develop a novel class of antibiotics. antimicrobial peptides (amps) are small cationic amphiphilic peptides, which play an important role in the defense against bacterial infections through disruption of their membranes. they have been regarded as a potential source of future antibiotics, owing to a remarkable set of advantageous properties such as broad-spectrum activity, and they do not readily induce drug-resistance. however, amps have some intrinsic drawbacks, such as susceptibility to enzymatic degradation, toxicity, and high production cost. currently, a new class of amps termed peptidomimetics have been developed, which can mimic the bactericidal mechanism of amps, while being stable to enzymatic degradation and displaying potent activity against multidrug-resistant bacteria. this review will focus on current findings of antimicrobial peptidomimetics. the potential future directions in the development of more potent analogs of peptidomimetics as a new generation of antimicrobial agents are also presented.

PMC4155802

pubmed-1340

the first pandemic reportedly occurred in 412 bc,, and the first attributed to influenza in 1580., since then, 31 influenza pandemics have been described; the five most recent in 1889, 1900, 1918, 1957 and 1968,, ,,, were separated respectively by 11, 18, 39 and 11 years. the present threat of a new influenza pandemic is at the origin of renewed interest in the 1918 spanish flu, as it was undoubtedly the most deadly influenza pandemic in modern history. recently, murray et al. extrapolated, in their paper published in 2006, the potential global pandemic influenza mortality occurring in 2004, based on data from the 19181920 influenza pandemic. a first american report in 1927 suggested that the main 19181919 wave was responsible for 21 millions of deaths worldwide. a revised estimation of influenza pandemic mortality given in 1991 was 247393 million, and another published in 2002 even set the death toll up to 100 million to take into account the lack of data in a large part of the world. the lack of reliable mortality data in a large part of the world leads to reliance on anecdotal evidence and extrapolation of mortality rates for entire countries from punctual information obtained in highly specific settings., concerning europe, we are aware of only three published papers reporting estimates of the mortality burden.,, herein, we analyzed the mortality data obtained during the pandemic, in 14 european countries, which accounted for 75% of the european population in 1918. monthly allcause deaths of civilians 19061936 were obtained from a book published, in 1954, by the french national institute of demographic study, which compiled official demographic and vital statistics from several countries, including 14 european countries. for each country, allcause death counts were available on a monthly basis, except for england and wales, for which only quarterly data are available. the observed data were standardized to mortality rates per 10 000 inhabitants using the corresponding yearly populationcount estimates. we used a periodic equation to model the monthly baseline mortality rate (b t) of years 19061922: with, respectively, n=12 or n=4 when monthly or quarterly data were analyzed. in this model, the baseline mortality rate depends on time t through a constant term 0, a secular trend 1, annual predictors 1 and 1, semiannual predictors 2 and 2 and an error term t. the unknown parameters were estimated at maximum likelihood from the fit of a linear regression to data from a training period, extending from 1906 to 1917. the estimation was based on the time series truncated from the values observed during the epidemic periods, defined as the 3 consecutive month period with the highest incidence. more precisely, from each annual august 1july 31 window, we removed the trimester with the highest mortality to take into account seasonal influenza, and the associated annual excess mortality. july period was used because seasonal influenza epidemics occur from fall to spring in europe. the baseline model described above we defined excessmortality periods as those starting when observed mortality exceeded the upper limit of the prediction interval [threshold=e (b t)+196 sd (e t)], for at least 2 consecutive months (one trimester for england and wales) and ending when the observed mortality fell below the upper limit of the prediction interval. excess mortality was calculated as the difference between observed and expected baseline deaths during the excessmortality period (figure 1). the crosshatched area represents excess mortality, calculated as the difference between observed (black line) and predicted (continuous gray line) mortality, from the beginning of the pandemic period (light gray shading; first month during which observed deaths exceeded the pandemic threshold, dashed gray line) until the end (first month during which observed deaths fell below the pandemic threshold). for finland, we subtracted from excess deaths, the 30 000 deaths attributed to the finnish civil war (january may 1918). data for the civilian mortality rates in germany presented a step pattern for the period 19141918, probably reflecting mismatched definitions of population counts and deaths as a result of changing geographical borders. therefore, we first replaced the linear trend of mortality rates during 19141918 by those of the surrounding years (19061913 and 19191936), and obtained excess mortality as described above. we also compared the excess mortality in each of the 14 countries during the 19181919 pandemic with the excess mortality during the rest of the years (19061917; 19201922) to determine how worse the 1918 period was compared with those seasons. cumulative excess deaths during the 19181919 period were 198 million in 14 countries in europe accounting for 75% of the population, an increase of 86% more deaths from baseline. extrapolating that figure to the rest of europe, the estimated total mortality in europe during the 1918 pandemic would be 264 (198/075) million deaths, i.e. 11% of the entire population estimated at 250 million in 1918. the highest cumulative excess/predicted mortality ratio was observed in italy (+ 172%) during the pandemic period (peaked in october 1918), following by bulgaria and portugal (+ 102% each), spain (+ 87%), the netherlands (+ 84%), sweden (+ 74%), germany (+ 73%), switzerland (+ 69%), france (+ 66%), norway (+ 65%), denmark (+ 58%), scotland (+ 57%) and england and wales (+ 55%), with the lowest ratio being seen in finland (+ 33%)(table 1). the highest excessmortality rate (per 10 000 inhabitants) cumulated throughout the entire excessmortality period was observed in portugal (233/10 000 inhabitants), followed by italy, spain, bulgaria, switzerland, finland, france, germany, sweden, netherlands, norway, england and wales, scotland and denmark (figure 2). excess mortality in 14 european countries represents the excess mortality calculated, based on expected baseline mortality. existence of a later peak in april 1920 (not taken into account to calculate excess deaths throughout the pandemic period). south gradient in the progressively darker shades of gray,*(/10 000 inhabitants). overall, the excess mortality found during the pandemic was 35 times higher than the excess mortality found in the rest of the 19061922 period. this ratio ranged between 16 (finland) and 61 (portugal) (table 2). excess mortality (%) comparison between the 19181919 pandemic periods and the rest of the years (19061917; 19201922) in each of the 14 countries we found a statistically significant negative correlation (spearman s rank correlation =066; p=0013) between excess mortality and latitude (figure 2), meaning that northern countries experienced significantly less mortality, while southern europe suffered significantly more excess deaths. countries were clustered according to their mortality patterns (figure 3): countries with one sharp mortality peak (e.g. portugal, spain, italy, bulgaria), countries with two major peaks (e.g. switzerland, scotland, netherlands and france), country with several successive peaks (only finland). monthly (or trimesterly) mortality rate*(ordinate) in 14 european countries from 1917 to 1921 (abscise). lines and shading are as defined in the legend to figure 1,*(/10 000 inhabitants). excess mortality was time dependent: the start and stop months of the excessmortality period varied across the continent and occurred in four waves (figure 4, table 1). moreover, an early first wave of deaths, occurring between march 1918 and july 1918, was observed in six countries (bulgaria, portugal, germany, finland, switzerland and spain) (figure 4). importantly, the excessdeath curves could be temporally superposed, albeit not of the same intensity, for 19181919, with mortality peaking in all countries within a 2month window (october november 1918, figure 3), defined as the second wave (figure 4). in five countries (spain, denmark, finland, germany and switzerland), a late peak between january 1920 and april 1920 was recorded. that fourth wave in 1920 was not taken into account in estimating the cumulative excessdeath rates for the entire pandemic period the limitation of any study focused on mortality burden at the epoch of the spanish pandemic depends on the exhaustiveness of the data. during this period, data were often scattered in various places or sources, making their collection for analyses difficult. this is the reason why earlier publications were based on various documents dating from 1918 to 1919 (medical journals, daily newspapers and archives). in this study we took advantage of the existence of vital statistics compiled in european countries by demographers, using a homogeneous method. we used the information available on the monthly variations of death figures to estimate the pandemic mortality burden, based on the values of a 12 years, were for example computed by simply making the difference between the 1918 and 1920 yearly mortality data and the values observed in the preceding and succeeding 3year periods. the comparison of the datasets used in the different mortality assessments, and the results obtained, are shown in figure 5. the changes in borders and the mass population movements related to world war 1 may be one reason for these discrepancies. for example, in germany, we checked that using the raw data (not corrected for the mismatches of populations and deaths) would have lowered our estimation of 426 574 to 340 000, changing the excess mortality estimate from 73 to 67%. in total, however, the agreement between estimates are more remarkable than the discrepancies. for example, the total european burden was estimated at 2 300 000 in the two first reports, published in 1991 and 2002, based respectively on 18 countries and 21 countries, at 2 005 569 deaths in the paper from murray et al. (total of their 13 european values) versus 1 980 950 deaths in this report. comparison of published mortality estimates in europe (number of deaths) during the 19181919 influenza pandemic. circle: johnson and mueller (j); dark circle: ansart et al. (a); square: patterson and pyle (p); triangle: murray et al. as the 14 european countries we have studied in this paper represented 75% of the european population at that time, one can deduce that 26 million excess deaths (11% of the total population) occurred in europe during the period when spanish flu was circulating. american excess deaths during the same period were estimated at 550 000 (corresponding to 065% of the american population) by glezen wp. however, johnsonmueller put the us figure at 675 000 deaths and more recently murray et al. at only 400 000 deaths (047% of the total population). whatever is taken as estimation of the american pandemic death burden, it is well below the european estimations we provide and others have provided. a possible explanation is that, at the end of ww1, europe was characterised by massive civilian and army movements, a health care system put at its minimum and frail populations. this has very likely heavily impacted the european death toll. finally, we measure the indirect impact of the influenza virus on the mortality. herein, we analyzed allcause mortality and not only influenza or pneumonia deaths. indeed, we were obliged to base our estimation on the available monthly data on concerned total mortality, not influenza and pneumonia (p&i) mortality however, there is a striking linear relationship between the overall annual influenza and pneumonia mortality records, for each of the 11 countries where such data were available, and the total number of excess deaths (figure 6). this is consistent with the hypothesis that p&i mortality is a relatively constant proportion of the total number of deaths, and supports our use of the total number of excess deaths to quantify the death toll of the pandemic. p&ideaths plotted versus allcause excess mortality in 11 european countries*. spearman s rank correlation =094; p<10.*germany, spain, france, italy, norway, netherlands, portugal, sweden, switzerland, england and wales and scotland. there was a high level of variability between the excess of mortality experienced in the 14 countries we studied, with a minimal value of 33% in finland and a maximal value of 172% in italy. all factors that can be related to the mortality burden such as the existence of coinfectious diseases, especially bacterial pneumonia and tuberculosis, or socioeconomic status were unlikely to have a north south gradient. possible geographic determinants of the influenza mortality are cold temperature and vitamin d deficiency relative to low sunlight exposure, but they would have implied a higher mortality in northern countries, not the opposite as we observed. we observed a high degree of synchronism in europe, with the highest mortality peak occurring in all countries within a 2month window (oct nov 1918) (3, 4). all but two countries (denmark and italy) experienced at least a twowave pattern. in addition to this common feature, six countries had an earlier first wave of excess mortality before october 1918 (bulgaria, portugal, germany, finland, switzerland and spain) and five countries (spain, denmark, finland, germany and switzerland) had one late excessmortality waves in february and march 1920. the very peculiar profile of mortality in finland could be explained by their civil war that caused 30 000 deaths in early 1918 (that could not be excluded directly from our analysis because of their unknown spatiotemporal distribution). a fourth wave having occurred in the early spring 1920 similar to the one we found was reported before in specific settings. including 6 additional months in the calculation of excess mortality (until july 1920), would have increased the burden estimates as follows: 405 800 (+ 153 744) deaths in spain, 60 500 (+ 5768) in finland, 13 600 (+ 3044) in denmark, 510 200 (+ 83 663) in germany and 39 000 (+ 9080) in switzerland. where the 1918 pandemic first emerged is still being debated. a recent analysis of the 1918 h1n1 genome failed to single out a particular location., the origin has been successively proposed in asia, in a british army post in france in 1916,, in usa,, ,, or in spain. the european origin was not supported by taubenberger et al. in their 1997 report on the sequence of the 1918virus genome extracted from autopsy samples (obtained from the armed forces institute of pathology in washington, dc) of 28 us servicemen who died of the flu during their military service. our findings can provide clues as to the origin of the pandemic and do not make plausible an european origin: indeed, if the pandemic would have started somewhere in europe, we would have expected to see a spatiotemporal spread outwards from this hypothetical origin, while the data show that all the european countries reached simultaneously their epidemic peaks. all authors contributed to the study and agree with the contents of the manuscript. conceived and designed the study: sa, ajv, pyb, cp. collected the data: sa. contributed materials, analysis tools: fc, af. wrote the paper: sa, ajv.

background the origin and estimated death toll of the 19181919 epidemic are still debated. europe, one of the candidate sites for pandemic emergence, has detailed pandemic mortality information. objective to determine the mortality impact of the 1918 pandemic in 14 european countries, accounting for approximately threequarters of the european population (250 million in 1918). methods we analyzed monthly allcause civilian mortality rates in the 14 countries, accounting for approximately threequarters of the european population (250 million in 1918). a periodic regression model was applied to estimate excess mortality from 1906 to 1922. using the 19061917 data as a training set, the method provided a nonepidemic baseline for 19181922. excess mortality was the mortality observed above this baseline. it represents the upper bound of the mortality attributable to the flu pandemic. results our analysis suggests that 264 million excess deaths occurred in europe during the period when spanish flu was circulating. the method provided space variation of the excess mortality: the highest and lowest cumulative excess/predicted mortality ratios were observed in italy (+ 172%) and finland (+ 33%). excessdeath curves showed high synchrony in 19181919 with peak mortality occurring in all countries during a 2month window (oct nov 1918). conclusions during the spanish flu, the excess mortality was 11% of the european population. our study highlights the synchrony of the mortality waves in the different countries, which pleads against a european origin of the pandemic, as was sometimes hypothesized.

PMC4634693

pubmed-1341

dexamethasone, see scheme 1 for molecular structure, is a synthetic member of the glucocorticoid class of steroid drugs that has anti-inflammatory and immunosuppressant effects. it is more potent than cortisol in its glucocorticoid effect, while having minimal mineralocorticoid effect. based on the who's list of essential medicines, dexamethasone is of the most important medications needed in a basic health system. it is used to treat many inflammatory and autoimmune conditions, such as rheumatoid arthritis and bronchospasm [2, 3]. in addition, it is given in small amounts before and/or after some forms of dental surgery and useful to counteract allergic anaphylactic shock, if given in high doses. it is present in certain eye drops and as a nasal spray and certain ear drops [4, 5]. with respect to the widespread use of the drug, developing the rapid, low cost, and reliable procedure for quantitation of it in real samples with different matrices is necessary. various reports have been found for quantitative determination of dexamethasone in real samples with different matrices. they are including spectrophotometry, liquid chromatography [7, 8], liquid chromatography-mass spectrometry [911], and electrochemical methods. the methods have limitations such as high cost and hard operation [711] and low repeatability. kinetic spectrophotometric method that has advantages such as high sensitivity, sufficient accuracy, simplicity, speed, and the necessity of less expensive apparatus makes it as an attractive method for the determination of trace elements in samples with different matrices such as foods and biological and pharmaceutical [14, 15] samples. in continuing of our research interest for the determination of drugs, a simple the method is based on the catalytic effect of dexamethasone on the orange g-bromate reaction system. the developed method has been successfully applied for the determination of dexamethasone in pharmaceutical and biological samples. to the best of our knowledge, we do not have any report for the determination of dexamethasone using kinetic spectrophotometric method. a single beam agilent uv-vis spectrophotometer (8453, usa) with 1 cm glass cell was used to measure the absorbance. a thermostated water bath (heidolph, germany) was used to keep the temperature of all solutions at the working temperature (25.0 0.1c). dexamethasone as dexamethasone phosphate (sigma) stock solution 100.0 mg l was prepared just before use by dissolving appropriate amount of dexamethasone phosphate in water and diluted to the mark in a 100 ml calibrated flask. a solution of orange g (6.6 10 mol l) was prepared by dissolving 0.2985 g of orange g (merck) in water and diluting to 1.0 l with water. sulfuric acid solution (4.0 mol l) was prepared by appropriate dilution of conc. sulfuric acid (merck). a 0.05 mol l of potassium bromate solution was prepared by dissolving 8.3504 g of kbro3 (merck) in water and diluting to 1.0 ml in a calibrated flask. the catalyzed reaction was studied spectrophotometrically by monitoring the change in absorbance of the reaction mixture at 478.5 nm (max). for this purpose, to a series of 10 ml volumetric flasks, 0.8 ml of 6.6 10 mol l of orange g solution, 1.5 ml of 4.0 mol l sulfuric acid solution, and the standard solutions containing 0.020.54 mg of dexamethasone were added. then, 1.0 ml of 0.05 mol l of bromate solution was added and diluted to the mark. a time measurement was started just after adding the last drop of the bromate solution. after thorough mixing, a portion of the solution was transferred to a glass cell. the absorbance of catalysed reaction (a s) was measured against water at max and 20c for time interval 30420 s. the measurement in the absence of dexamethasone was repeated to obtain the values for the uncatalysed reaction (a b). finally, the difference in the absorbance change was considered as the response (a=a s a b). ear-eye drop (0.1%) and injection solution (8.0 mg) were used as pharmaceutical samples. each sample was diluted properly and appropriate amount of the solution was used in each analysis. they were spiked with dexamethasone and solid phase extraction technique with c18 cartridge (supelco inc., 10 ml) was used for purification and preconcentration of dexamethasone from the samples. the purification and preconcentration procedure was performed as discussed in. according to the procedure, orange g, a yellowish synthetic azo-based dye, widely used as color marker for following the electrophoresis process, ph indicator, dyeing of textiles, paper, and leather, and preparing coloring inks. the capability of the reaction system (orange g, sulfuric acid, and bromate) for the determination of dexamethasone was evaluated by following the change in absorbance in absence (figure 1) and presence (inset of figure 1) of it. comparison of the two spectra indicated that trace amounts of dexamethasone can increase the reaction rate seriously. therefore, the proposed reaction system can be used for the determination of dexamethasone. the suggested reaction mechanism for proposed reaction system the uncatalysed reactions that resulted in blank signal (a b) was carry out in a cyclic way by these reactions: (1)orange gred+bro3+6h+orange gox +br+3h2o (2)6bro3+10h++12br6br2+6h2o (3)orange g(red)+br2+h+orange gox+2br in the presence of dexamethasone (4)dexamethasonered+br2+h+2br+dexamethasone(ox) where red and ox are the reduced and oxidized forms of the reactant, respectively. since dexamethasone along with the orange g participates on br generation, the possibility of br2 generation was increased (reaction (2)) and resulted in increasing the possibility of decolorization of orange g (reaction (3)). therefore, the change in absorbance in presence of dexamethasone was increased dramatically. in order to obtain the maximum sensitivity as employing the proposed procedure, the effect of orange g concentration on the rate of reaction was studied over the range 52.879.2 mol l. as it can be seen in figure 2, the sensitivity was increased up to 66.0 mol l of orange g. at higher concentrations, the reaction rate was decreased which may be attributed to the dye aggregation. thus, 66.0 mol l of orange g as optimum concentration was selected for further study. the effect of sulfuric acid concentration on the catalyzed and uncatalyzed reactions was studied over the range of 0.6 to 0.8 mmol l (figure 3). the maximum sensitivity was obtained at 0.76 mmol l, whereas at higher concentrations the sensitivity was decreased. protonation of orange g at higher acid concentrations that makes its oxidaion to be quite difficult resulted to the disorder. therefore, 0.76 mol l of sulfuric acid was used for further study. the effect of bromate concentration on the reaction rate was studied in concentration range 4.55.5 mmol l. as shown in figure 4, the net reaction rate was increased up to 5.0 mmol l which was selected as being the optimum concentration of oxidant. under optimum experimental conditions, the effect of the temperature on the reaction rate was studied in the range of 15 to 45c. increasing the temperature up to 20c caused an increase in the sensitivity, whereas at higher temperatures it decreased. the optimum time was found by measuring the change in the absorbance during 30540 s. the reaction rate increased up to 420 s, and in longer times the reaction rate was almost constant. calibration graph was constructed by plotting the response against dexamethasone concentration. using the recommended procedure and under optimized conditions that are outlined above, calibration graph was linear over the range 0.254.0 mg l of dexamethasone including two segments of 0.212.0 and 12.054.0 mg l. the regression equation of the two segments gives (5) and (6), respectively. (5)a=0.0154dexamethasone+0.0071 r2=0.9987, (6)a=0.0032dexamethasone+0.1574 r2=0.9984, where a is the difference in the absorbance between the blank and the sample and [dexamethasone] is the dexamethasone concentration in mg l and r is the correlation coefficient. the limit of detection (3s b/m; s b is the standard deviation of the blank signal and m is the slope of calibration curve) was 0.14 mg the tolerance limit was defined as the concentration of the added species causing an error more than 5% on analytical signal. the obtained results show that chloride and nitrite have seriously interfering effect, whereas they do not exist in real sample matrix. also, the interfering effect of cortisol that has the same effect on dexamethasone and can be coexisting with the analyte in real samples matrices was investigated. the tolerance limit of 8.3 fold than 2.0 mg l of dexamethasone which was not more than the upper limit of cortisol in blood confirms the proposed method is free from the cortisol interfering effect. evaluating the reliability and analytical applicability of the developed method makes it potentially useful for the quantitative determination of dexamethasone in real samples with different matrices. the experimental t values for eye-ear drop (1.72 and 1.73) and injection solution (1.74 and 0.88) are different from the critical value (4.30, 95% confidence level, and two degrees of freedom). regarding the difference between the experimental and critical t values, the systematic error for the determination of dexamethasone in pharmaceutical samples using the developed method is negligible. also, the procedure was used for the determination of dexamethasone in urine and serum samples. after sample preparation (section 2.4.2) they were spiked with different amounts (2.0, 8.0, and 20.0 mg the recoveries of the spiked urine and serum samples vary over the range 99.4102.0% and 99.0100.7%, respectively. the recovery values are near 100% and confirm that the systematic error during the quantitative determination of dexamethasone in biological samples is slight. successive applications of developed method for drug determination in pharmaceutical preparations and urine samples were performed. therefore, the developed method is free from interfering effect of matrix effect and suitable for analysis of dexamethasone in different samples. this study reports a sensitive and relatively selective spectrophotometric method for the determination of dexamethasone using a new reaction system. the developed method possesses distinct advantages over other existing methods in cost, simplicity, ease of operation, and applicability to real samples analysis. also, the reliability of the method permits the analysis of pharmaceutical and biological samples satisfactorily.

dexamethasone is a type of steroidal medications that is prescribed in many cases. in this study, a new reaction system using kinetic spectrophotometric method for quantitative determination of dexamethasone is proposed. the method is based on the catalytic effect of dexamethasone on the oxidation of orange g by bromate in acidic media. the change in absorbance as a criterion of the oxidation reaction progress was followed spectrophotometrically. to obtain the maximum sensitivity, the effective reaction variables were optimized. under optimized experimental conditions, calibration graph was linear over the range 0.254.0 mg l1. the calculated detection limit (3s b/m) was 0.14 mg l1 for six replicate determinations of blank signal. the interfering effect of various species was also investigated. the present method was successfully applied for the determination of dexamethasone in pharmaceutical and biological samples satisfactorily.

PMC4337262

pubmed-1342

diabetes mellitus type 2 is strictly related to cad (coronary artery disease); in fact 7080% of diabetic patients die of cardiovascular complications; moreover, these patients have a risk of myocardial infarction about four times higher than that found in the general population [13]. several noninvasive techniques are available for this purpose, including stress ecg and spect (single photon emission computed tomography) [4, 5] and most recently coronary ct. in fact the latest ct generation with 64 slices has emerged as a truthful alternative to conventional ca (coronary angiography), with excellent diagnostic accuracy allowing us to identify and quantify the degree and extent of coronary artery disease, including the study of wall arteries [69]. previously published results have proved the high values of sensitivity, specificity, and negative predictive value (almost 100%) of ct for the assessment of coronary disease, even in patients treated with stent and bypass [10, 11]. however, in case of important coronary calcifications, the ct examination presents several limitations in residual vessel lumen evaluation. otherwise, mdct (multidetector computed tomography) sometimes shows some limitations in the grading of coronary stenosis due to motion artefacts or severe vessel calcifications. it is well known that diabetes causes a large amount of vessel calcification, resulting in lower diagnostic accuracy of ct in the detection and evaluation of coronary stenosis in diabetic patients. in this case, it could be useful to work with hybrid imaging, merging the anatomical images of ct to the functional images of spect, overcoming the limits of the two techniques. our purpose was to combine the results of the mdct morphological data and the spect data using hybrid imaging to overcome the limits of the mdct in the evaluation of coronary stenosis in diabetic patients with large amount of calcium in the coronary arteries. between january 2009 and december 2011, we enrolled 120 consecutive diabetic patients (84 males and 36 females), mean age 67 years old (range 5078), and performed a coronary ct. all the patients had one or more cardiac symptoms such as stable angina, atypical chest pain, and dyspnea. we evaluated the cardiovascular risk factors for each patient, looking for hyperglycaemia (glycated haemoglobin>7.5%), hypertension (bp>130/85 mmhg), hypercholesterolemia (cholesterol>190 mg/dl), obesity (bmi 30 kg/cm), smoking, and family history of cad (table 1). twenty patients with a history of known cad (1 with previous bypass graft surgery, 19 with previous successful angioplasty) were included, while patients with a serious arrhythmia, known allergy to iodinated contrast agents and kidney failure, were excluded. invasive coronary angiography was performed in patients in which the ct examination results were doubtful. afterwards the imaging fusion results were compared to the coronary angiography evaluation. all patients, provided informed consent to the examinations and the study was approved by the local ethics committee. we used a 64-slice ct scanner (lightspeed vct, general electric medical systems, milwaukee, wi, usa) and a retrospective synchronization technique. patients with a heart rate higher than 65 bpm were previously (almost 5 days before) treated with oral beta-blocker therapy. a preliminary unenhanced scan was done to determine the scan extent and to calculate the calcium score (smartscore protocol). the acquisition stack extended from the ascending aorta superiorly (approximately 1 cm above the tracheal bifurcation) and the heart apex inferiorly (lower portion of the lowest hemidiaphragm), therefore enabling the evaluation of the entire cardiac volume. scan parameters for the unenhanced baseline scan were beam collimation 8 2.5 mm, slice thickness 2.5 mm, table feed 1 cm/4 slices, tube rotation speed 0.35 s, tube voltage 120 kv, intensity 300 ma, fov 25 cm, craniocaudal scan direction. a second image stack was then acquired after intravenous administration of iodinated contrast material using a dual-head automated injector (stellant, medrad, pittsburgh, pa, usa). a dose of 80 ml of nonionic iodinated contrast material (iomeron 400, bracco, milan, italy) was administered through an 18-gauge needle cannula placed in an antecubital vein, followed by 40 ml of saline solution, both at a rate of 5 ml/s. to synchronize the beginning of the scan with the arrival of the contrast agent in the coronary arteries, the bolus-tracking technique was used. parameters for the contrast-enhanced scan were beam collimation 64 0.625 mm, slice thickness 0.625 mm, reconstruction increment 0.625 mm, table feed 2.9 mm/rotation, tube rotation 0.35 s, tube voltage 120 kv, intensity 400800 ma, fov 25 cm, craniocaudal scan direction. image reconstruction was carried out using three temporal windows at 70%, 75%, and 80% of the cardiac cycle, corresponding to the r-r interval or mid to end diastole. in the event of motion artefacts due to sudden changes in heart rate, other reconstruction windows were used (from 40% to 65% of the r-r cycle). the ct datasets were analysed by the agreement of two independent, experienced readers, who used axial source images, multiplanar reformations, volume rendering, and thin-slab maximum-intensity projections on a remote workstation (advantage workstation 4.4; ge healthcare). each coronary was judged as negative for coronary disease in the presence of one stenosis<50% and positive in the presence of one stenosis 50% [1416]. gated-spect was performed on a double-headed camera system (spect-tc vg millennium, ge healthcare, usa). we adopted the single day protocol: stress-rest protocol with 300 mbq tc-mibi or tetrofosmin at peak exercise during bicycle ergometry or dipyridamole infusion and 900 mbq at rest (n 203, 77.2%) after 3 hours. spect images were acquired 1 hour after the radiopharmaceutical injection both for the rest and the stress phases. thirty-two images of 25 sec per frame (matrix 64 64, zoom 1.33) were acquired using the step and shoot technique (90 g/head). energy discrimination was provided by a 20% window centred over the 140 kev photon peak of tc. transverse images were reconstructed by the filtered back projection method, with a butterworth filter (order 10; cutoff 4.0) for processing and a ramp filter for back projection using a xeleris console. the left ventricular myocardium was divided up into 17 segments; each of the 17 segments was scored according to the guideline for semiquantitative analysis (semiquantitative scoring system: the five point model: 0=normal; 1=mildly reduced-not definitely abnormal; 2=moderate reduced-definitely abnormal; 3=severe reduced; 4=absent radiotracer distribution). the fusion between stress-spect images and ct axial images was done using the cardiac iq fusion protocol on a remote workstation (advantage workstation 4.4; ge healthcare). this software allows a perfect matching between the images using as reference points the ct myocardium outlines and the spect epicardial surface of the left ventricle; furthermore this software cuts the aorta and the veins. several 3d protocols permit a better visualization of the coronaries, the wall septum, and the right ventricle. the result of the fusion is an image with a high spatial resolution and a better definition of the coronary stenosis. these hybrid images have been evaluated separately by a radiologist and a nuclear medicine doctor with eventual agreement. cca was performed using a philips flat-panel system with multiple projections (medical philips system, the netherlands). stenoses were evaluated as a percentage of the reference diameter determined in two orthogonal projections, taking the mean of the two samples as the final value. data were expressed as means plus one standard deviation (sd) or as percentages. diagnostic accuracy for evaluating significant stenoses was calculated by comparing spect/tc with cca images. the comparison between groups was obtained by analysis of variance or the test, as appropriate. mean heart rate of the patients during the examination was 65 bpm (range 5677 bpm). after the ct scans, 12 (10%) patients were excluded because of motion artefacts due to an increase in heart rate during the acquisition. the quality of the images of the other 108 (90%) patients was excellent in 76 patients (70.2%), good in 16 patients (15%), sufficient in 8 patients (7.4%), and low in 8 patients (7.4%). considering the three main coronary branches in the overall count, left anterior descending (lad) artery, right coronary artery (rca), and left circumflex artery (lcx), 324 coronaries were evaluated. a total of 52 vessels (32.8%) had stenoses>50%, in particular 28 (8.6%) were in the lad, 16 (4.9%) in the rca, and 8 (2.4%) in lcx; 228 coronaries (70.4%) were found to have nonsignificant stenoses (< 50%) (table 2). we observed that 44 coronaries (13.6%) were not evaluable or doubtful due to the presence of a large amount of vessel calcification (table 2). the mean calcium score value in not evaluable or doubtful coronaries was 823 36. considering the 324 areas corresponding to the ct coronary arteries in particular, 44 (13.6%) perfusion defects were located in the anterior and septal wall, 28 (8.6%) in the inferior wall, and 8 (2.5%) in the lateral wall. furthermore, gated-spect showed 232 (71.6%) areas without perfusion defects and 12 (3.7%) doubtful cases because of artefacts due to mammary gland attenuation in the female patients (table 2). the 52 (16%) coronary arteries with significant stenosis at ct were confirmed as perfusion defects at spect. of 228 coronaries (70.4%) found to have nonsignificant stenosis at ct, 212 (65.4%) were confirmed as normal perfusion areas while 16 (4.9%) were described as perfusion defects at spect. of 44 (13.6%) nonevaluable coronaries at ct, 12 (3.7%) were described as perfusion defects at spect, 20 (6.2%) as normal perfusion areas, and 12 (3.7%) as doubtful cases because of artefacts due to mammary gland attenuation in the female patients (table 3). of 324 coronary arteries and corresponding areas, the hybrid spect/ct evaluation showed 92 (28.4%) of areas with hypoperfusion and 232 (71.6%) with normal perfusion (table 2). in the first group of 52 (16%) coronaries with significant stenosis at ct and perfusion defect at spect, hybrid images demonstrated a hypoperfusion (figure 1). in the second group of 228 (70.4%) with nonsignificant stenosis at ct, hybrid images demonstrated a normal perfusion in 212 (65.4%) cases and a low hypoperfusion in 16 (4.9%) cases (figure 2); of these 16 cases, 12 (3.7%) cases were described at ct as stenosis of 4050%, while the other 4 cases were seen at ct as nontransmural myocardial necrosis (with known history of non-st infarction). in the third group of 44 (13.6%) doubtful coronaries at ct, hybrid images demonstrated 20 (6.2%) normal perfusion areas and 24 (7.4%) hypoperfusion areas (seen at spect as 12 perfusion defects and 12 doubtful cases), described as nonquantifiable calcified stenosis at ct and were finally evaluated as significant stenosis (table 3). the third group of 44 coronaries was also studied with a conventional ca, confirming the results of hybrid images (24 significant stenoses and 20 nonsignificant stenoses). the patients with 24 significant stenoses also underwent a ptca (percutaneous transluminal coronary angioplasty) with stenting. therefore the evaluation of hybrid images permitted the identification of all the stenoses and solved the doubts related to the 44 coronaries not well evaluated at ct and spect separately, with a diagnostic accuracy of 100% in comparison versus ca (table 3). myocardial perfusion scintigraphy represents the most widely used technique validated in the prognostic stratification of diabetic patients with known or suspected ischemic heart disease in order to predict the short and medium terms (generally 1-2 years) for cardiac events such death and myocardial infarction [18, 19]. however, the perfusion images obtained with spect show a good sensitivity and a low specificity in the detection of patients with cad. the diagnostic accuracy of myocardial scintigraphy is affected by different variables. unlike positron emission tomography (pet), single-photon emission computed tomography (spect) provides no correction for attenuation. this causes the presence of false positives in some areas (false hypoperfusion obtained from attenuation of photons through the tissues of the body), along the passage from the heart to the gamma camera. mostly the artefacts are related to the hepatic uptake, due to the interposition of the diaphragm and, in female patients, in the anterior-lateral wall due to the mammary gland; because of this reason, in our study, spect was unable to assess the myocardial perfusion in 12 female patients. furthermore spect reported some false positives when a patient can not reach 85% of the theoretical maximum heart rate during the exercise testing; this event occurs in obese patients, in older patients, in patients with arterial disease of the lower limbs, and in subjects treated with beta-blockers. in these cases because of these reasons, there is a high variability of values of sensitivity and specificity reported in previously published meta-analysis [1821]. specificity and sensitivity have been improved by the introduction of gated technique: especially in women, the number of false positives can be reduced, observing at the same time the perfusion and the functionality of the myocardial region. however, sometimes the myocardial region that seems to have a normal perfusion after a stress test could hide a perfusion defect and would not be able to determine a myocardial stunning [21, 22]. coronary ct, when used in selected populations of patients with low-intermediate cardiovascular risk, has been shown to provide essential morphological information about the coronary tree. the diagnostic accuracy of cardiac ct is widely demonstrated by high values of sensitivity, specificity, and negative predictive value [23, 24]. actually, the most important limit in the evaluation of the vessel lumen is the presence of large coronary artery calcification, causing a blooming type artefact, due to the hardening of the x-ray beam during the passage through the calcified plaques. this artefact could cause a significant invalidation of lumen visualization determining false positive in coronary ct and consequently sending a certain number of patients with stenosis<50% to undergo a ca. in our study the patients, being diabetic, had large calcifications of the coronary arteries, creating doubts in the interpretation of quantitative coronary stenosis in 44 coronary arteries. during the postprocessing phase, the use of some convolution filters (kernel) can decrease the blooming artefact; in particular applying the high-resolution filters (bone and detail), the relative density of calcium is reduced, the margins of calcification plaque are better delineated, and the width of the vascular lumen increases. however, in a large number of cases, the quantification of stenosis caused by calcified plaque remains still difficult and it is often necessary to send the patient to ca examination or a spect. part of medical literature has tried to define the agatston values above which performing ct would be inappropriate. however, although sensitivity and specificity are reduced, the overall diagnostic accuracy is not reduced and, in our study, it has been implemented with the fusion imaging ct/spect. the skillful use of hybrid images, obtained by merging ct and spect images, is to have both morphological and functional data with a high spatial resolution, allowing a better stratification of diabetic patients, identifying the patient with a significant stenosis and hypoperfusion who needs revascularization and avoiding a ca in patients with unquantifiable calcification plaques at ct and spect [26, 27]. the hybrid images can overcome the limits of coronary ct, permitting the evaluation of large vessel calcification, metal stents, and small size vessels. however, this problem usually does not occur in the evaluation of noncalcified native coronary arteries; in fact ong et al. (2006) demonstrated that in patients with calcium score<142 agatston score the interpretability of the coronary tree was 93.6% against 86.9% of those patients with values>142 agatston score. in our experience, hybrid spect/ct images provide help in the evaluation and grading of calcified stenoses in ct and overcome the problem of the abnormal uptake of the radiopharmaceutical by the breast tissue in spect. in particular in the group of 44 (13.6%) doubtful coronaries at ct, hybrid spect/ct images demonstrated 20 (6.2%) normal perfusion areas and 24 (7.4%) hypoperfusion areas (seen at spect as 12 perfusion defects and 12 doubtful cases because of breast tissue), described as unquantifiable calcified stenosis at ct and finally evaluated as significant stenosis. therefore the evaluation of hybrid images permitted the identification of all the stenoses and solved the doubts related to the 44 coronaries not well evaluated at ct and spect separately, with a diagnostic accuracy of 100% in comparison to ca. these results are extremely interesting when applied to a group of patients such as diabetics, in which, until now, the limit of the coronary ct examination in the evaluation of calcified coronary was high. on the other hand, the correct identification of an area of myocardial hypoperfusion at spect examination is enhanced by the morphological information of the coronary arteries with a significant decrease in the number of false positive and an increase of the prognostic value of scintigraphy. in fact, spect provides a high prognostic accuracy in the prediction of cardiac event (especially in the short-term followup), although it gives only functional information. the perfusion defects can be localized and assigned to a specific coronary artery only if the spect exam is complemented by a morphological assessment. according to medical literature, hybrid imaging adds diagnostic clinical value, improving patient risk stratification and showing a higher sensitivity and specificity in the detection of the haemodynamically relevant coronary artery stenoses compared to the side-by-side analysis [2931]. furthermore the improvement of diagnostic accuracy was obtained with the latest ct scanners, as high-pitch dual-source ct coronary angiography and 320-row cardiac ct, which allow to improve the imaging of coronary plaque and obtain the evaluation of myocardial viability with a high diagnostic accuracy, a reduced radiation exposure and amount of contrast agent, even in patients with heart rates of>80 bpm or atrial fibrillation [3235]. our study has the limit of a high exposure of radiation because the patients underwent two different examinations (ct and spect) and because we used a retrospective gating during the ct scan; nevertheless, the actual use of the prospective gating and the application of new advanced reconstruction techniques that reduce image noise and improve low contrast detectability and image quality can give higher diagnostic performance of ct scan at very significant lower dose [36, 37]. another limitation of the present study was the only anatomical and not functional evaluation of coronary stenoses. therefore, the usefulness of the fusion between ct and spect images is to overcome the limits of ct and the limits of spect with a high diagnostic accuracy, especially in patients with high cardiovascular risk and high presence of physical limitations, like the diabetic patients.

purpose. our purpose was to combine the results of the mdct (multidetector computed tomography) morphological data and the spect (single-photon emission computed tomography) data using hybrid imaging to overcome the limits of the mdct in the evaluation of coronary stenosis in diabetic patients with large amount of calcium in the coronary arteries. method and materials. 120 diabetic patients underwent mdct examination and spect examination. we evaluated 324 coronary arteries. after the examinations, we merged ct and spect images. results. ct evaluation: 52 (32.8%) coronaries with stenosis 50%, 228 (70.4%) with stenosis<50%, and 44 (13.6%) with a doubtful evaluation. spect evaluation: 80 (24.7%) areas with hypoperfusion, 232 (71.6%) with normal perfusion, and 12 (3.7%) with a doubtful evaluation. of 324 coronary arteries and corresponding areas, the hybrid spect/ct evaluation showed 92 (28.4%) areas with hypoperfusion, and 232 (71.6%) with normal perfusion. conclusion. hybrid ct/spect imaging could be useful in the detection of significant coronary stenosis in patients with large amount of coronary calcifications.

PMC4045525

pubmed-1343

african oil bean (pentaclethra macrophylla benth) is a member of the family leguminosae. it is popular in nigeria where it is known by several names such as apara in yoruba, ukana in efik, and, the most prominent, ugba/ukpaka in igbo. ugba is a popular igbo condiment and delicacy made from traditional household solid state fermentation of african oil bean seeds. the fermentation is a mixed culture alkaline process involving a variety of microorganisms [1, 2]. although a variety of microorganisms are involved in the process, only the bacillus spp. the methods of production and also the length of fermentation vary from one producer to another and with the final intended use resulting in nonuniform products. however, the basic method involves boiling the seeds for up to 12 hours, removing and slicing the cotyledons, soaking/washing the sliced cotyledons in several changes of water, and wrapping the sliced cotyledons for the fermentation to take place. beans that have been fermented for 2-3 days are taken as a snack delicacy directly or following seasoning and some further processing. well fermented beans (up to 5 or more days) are added to soup as flavouring. prepared in different ways, ugba is an essential food item for various traditional ceremonies, and in instances it may be used as meat substitute in certain soups/gravies particularly for the rural poor. flavour is considered the quality index of ugba and plays an important role in consumer acceptability. although fermented african oil bean seeds have typical flavour and appealing organoleptic quality, differences in flavour range and intensities exist. these vary perhaps as attributes of producer organisms and are due to the various volatile compounds produced by the fermenting population. a great deal of work has been implemented in the microbiological characterisation of the fermentation process. however, unlike asian and pacific fermented seasoning, no work has yet been reported, to our knowledge, on the flavour components of ugba and their evolution during the fermentation process. in fact, this knowledge gap is not restricted to ugba but appears to cut across the entire gamut of african fermented foods, particularly of the genre of seasoning agents. knowledge of the volatile and flavour characteristics of the fermented oil bean seeds and their evolution will aid in the quality control of the process, modification of the flavour, and eventual synthesis of the ugba flavour products for possible use in the seasoning of various factory processed and even home foods. this is important in view of declining production of seeds, due to deforestation, and the need to extend use of the appealing aroma of ugba to other food types. the aim of this study was to identify the volatile flavour components of african oil bean seeds during natural and pure culture fermentation and to relate these to the fermentation progress. african oil bean seeds were purchased from dealers in a local market in nsukka, enugu state. the seeds were first boiled for 68 hrs to soften the coat and enhance peeling. the cotyledons were then sliced longitudinally into 4.5 cm 0.1-0.2 cm slices, washed and boiled in water for 1-2 hrs, and drained and soaked in water for 1012 hrs. after soaking, the slices were washed in several changes of water to reduce bitterness and allowed to drain in a basket. the slices were then wrapped in clean dry banana leaves (musa sapientum linn.) in 4050 g wraps. the wraps were allowed to ferment for 3 days at room temperature (28 2c) to produce ugba. one kilogram (1 kg) of african oil bean seeds was boiled dehulled, peeled, washed, and sliced for the traditional process. the slices were further washed and 20 g portions were placed into conical flasks and capped tightly with cotton wool and aluminium foil. the flasks containing the slices were sterilized by autoclaving at 110c for 10 mins. pure cultures of b. subtilis and b. megaterium isolated from the traditional process and identified by standard microbiological procedures were inoculated independently in 20 ml sterile 0.1% peptone water and incubated for 24 hrs. after incubation, the cultures were centrifuged and the culture pellets were washed repeatedly with sterile saline and distilled water. 0.1 ml of the cells were emulsified in 9.9 ml distilled water and pour plate method was used to ascertain the population of the cells and to check for culture purity. each flask was separately, aseptically inoculated with 1 ml suspension (5.0 10 ml) of b. subtilis or b. megaterium individually using a sterile pipette and mixed with the sample by simple manual mixing. fermentation was allowed to proceed for 72 hrs at room temperature with occasional manual mixing by shaking the flask. to check for purity of the process during the period of fermentation, 1 g of the fermenting slices was removed at 24-hour interval and at the end of fermentation and agitated in 9 ml 0.1% sterile peptone water. smear of the sample was streaked on nutrient agar plate for isolation of the particular organism acting as a single pure culture. triplicate pure culture flasks were homogenised and sampled for analysis of volatile compounds. only pure culture processes were analysed for volatile compounds. samples for analysis were collected from naturally fermented ugba and also from ugba fermented with pure cultures of b. subtilis and b. megaterium. one gram (1 g) of the fermenting sample was ground and homogenized in 9 ml diethyl ether to extract volatile flavour compounds. 1 filter paper was used to separate the mixture; then millipore filtration using membrane filter of 0.45 m pore size was used to remove particulate matter from the resulting filtrate. the resulting filtrate was placed in small ampoules, sealed, and stored at 30c until being required for analysis. analyses were done on gc-ms (qp 2010 plus; shimadzu corp, japan) operated in electron-impact (ei) mode with an ionization voltage of 70 ev. sample aliquot of 1 l was injected and analyses were done with split injection mode. the gc column oven temperature was 60c and injector temperature was 250c with 53.2 ml/min flow and interface temperature of 250c. oven temperature was held at 60c for 2 mins, ramped to 180c and held for 3 mins, and finally programmed at 280c and held for 5 mins. the ion source temperature was 200c and the solvent cut time was 2.50 mins. gc chromatograms of the fermenting african oil bean seeds were examined and each peak identified was confirmed by direct comparison of their chromatographic retention times, retention indices, and mass spectra with those of the authentic compounds and/or data in the nist05 mass spectral library. at the end of the traditional fermentation, the produced ugba had a very light brown colour and exhibited classical aroma of the product, with a light, but sharp, ammoniacal smell. there was no visible sign of any slime on the slices which remained firm but slightly softer than the control/starting material. a total of 36 aroma compounds made up of 12 hydrocarbons, 10 esters, 5 alcohols, 2 phenols, 2 ketones, and one of each of furan, amine, acid, thiophene, and lactone were identified at different fermentation periods during natural fermentation (table 1). at the end of the pure culture process, the products of fermentation with two organisms were generally similar in colour, both being very light brown with no visible slime on the slices. the product of the b. subtilis fermentation, however, had only a slightly deeper ammoniacal smell than that of b. megaterium. both products tasted similar to the product of natural process but were slightly bitterer than the natural process. similarly, the product of b. megaterium was bitterer than that made with b. subtilis. the extract of oil seed fermented with b. subtilis yielded 30 aroma compounds comprising 10 hydrocarbons, 8 esters, 3 alcohols, 2 sulfur compounds, 2 amines, and one of each of acid, aldehyde, phenol, ketone, and furan (table 2). on the other hand, sample fermented with b. megaterium produced 29 aroma compounds consisting of 9 hydrocarbons, 10 esters, 2 nitrogenous compounds, 2 ketones, 3 alcohols, and one of each of lactone, aldehyde, furan, and amine (table 3). volatile compounds have been shown to be partly responsible for the aroma of several fermented foods [711]. they are probably important players in the flavour of ugba, as they have been shown to be important in a variety of bacillus spp. fermented foods. ugba flavours are formed during fermentation as a result of breakdown of plant compounds as well as formation of microbial metabolites by the active populations responsible for the fermentation process. the ammoniacal flavour observed in ugba is believed to result from the alkaline degradation of protein component of the seed by the fermenting bacillus spp.. many of the aroma compounds identified in this study derived from the breakdown of lipids (fatty acid), proteins (amino acid), and other bioavailable compounds in ugba through the activities of the microbial enzymes. hydrocarbons, esters, alcohols, phenols, ketones, furan, and amines were detected in all the samples examined and the consistency of these various volatiles would suggest roles for them in the final flavour of the product, although minor. however, neither the flavour thresholds of these compounds in this product nor their precise individual contributions are yet known. this may explain their abundance as volatile compounds in the fermented african oil bean seeds, since the seeds are rich in oil. nakamura et al. suggested that hydrocarbons do not play a significant role as flavour compounds in roasted sesame since they possess a relatively weak aroma. if this be the case in ugba, then the principal flavour compounds may be other compounds coproduced in the fermentation process. it is to be expected, as with all complex products, that the final flavour of ugba will derive from the interaction of a variety of compounds. methyl esters of fatty acids are one of the largest group of volatile aroma compounds produced during the fermentation in both mixed culture natural fermentation and pure culture processes. these were largely produced during 3648 hr of fermentation and persisted in various forms to the end of the process. they are produced enzymatically during fermentation by the methanolysis of acyl-coa that is formed during fatty acid synthesis or degradation. these compounds have been reported to be responsible for quality sensory properties of various fermented foods. 9-octadecenoic acid, methyl ester; pentanoic acid, methyl ester; 11,14-eicosadienoic acid, methyl ester; and methyl 12-methyl tetradecanoate are methyl esters of fatty acids which occurred commonly and in significant amounts (from the peak areas) in all the samples. histamine was identified at 0 hr fermentation and cyclopentanamine was identified in the sample fermented with b. subtilis at 36 hr. the presence of these compounds may be attributed to the enzymatic decarboxylation of amino acid in the seeds during fermentation. the presence of nitrogenous compounds, butyl isocyanide and acetonitrile, in the sample fermented with b. megaterium at 12 hr and 48 hr, is indicative of deaminase activity during the fermentation. unfermented ugba has been reported to contain cyanide and other toxic compounds/antimetabolites as has been reported in a variety of oil seeds. some volatile sulfur compounds in food are obtained by a variety of enzymatic reactions and/or as products of secondary metabolism of leucine and cysteine. methane sulfonic anhydride and t-butyl sulfinate, o-methyl, were present at 12 hr and 60 hr, respectively, in the sample fermented with b. subtilis. considerable amounts of various alcohols were identified during the fermentation in all samples. of these, many alcohols have been described as possessing undesirable odor which partially contributes to raw or beany a small number of aldehydes, ketones, and acids were identified in this study. it is conceivable that these compounds contribute to the final flavour of fermented african oil bean seeds. aldehyde can also be obtained as metabolic by-products of the enzymatic transamination or oxidative deamination of amino acid. 2,4-hexadienal and 14-heptadecenal are aldehydes identified in the samples fermented with b. subtilis and b. megaterium at 60 hr of fermentation. although b. subtilis and b. megaterium are present in natural fermentation process the profile of volatiles differed between pure culture and traditional one. this is understandable, as the mixture of other microorganisms present in the natural process may modulate the variety, quantity, and persistence of different volatile compounds. phenol, 2,4-bis(dimethylbenzyl) phenol, and 2,4-bis(dimethylbenzyl)-6-t-butylphenol identified during natural fermentation were not detected in pure culture fermentation and may be attributed to the smoked banana leaves used in wrapping the slices prior to fermentation. although relatively small amounts of aldehyde, ketone, furans, amines, lactones acids, and thiophenes were identified in this study, they are expected to play important roles in the aroma of ugba because of their low detection/aroma thresholds as has been reported in roasted chicory, in the dry scented thai flowers used in tea production and in roasted malaysian almond nuts. a vast array of volatile compounds has been identified in fermented pentaclethra macrophylla seeds (ugba). the variety and quantity of these volatiles change with the time of fermentation and the participating populations. the dynamic nature of evolution of volatile compounds is interesting for modulating the flavour of the final product. it remains to be demonstrated, the relative amounts, flavour thresholds, and specific contribution of the various compounds to the final flavour of the ready to eat fermented seeds. clearly, a proper understanding of the roles of the various flavour compounds will assist in the modulation of ugba flavour for the various possible uses of this important fermented seasoning and for a possible use in food processing industries and homes as a seasoning.

fermented african oil bean (pentaclethra macrophylla benth) seed is a successful and well studied seasoning and snack in parts of western africa. gc-ms analysis of fermenting seeds revealed a mixture of several volatile aroma compounds which changed with time and starter organism. during natural mixed culture process 36 volatile compounds including 12 hydrocarbons, 10 esters, 5 alcohols, 2 phenols, 2 ketones, and one each of furan, amine, acid, thiophene, and lactone were identified. when bacillus subtilis was used in pure culture, 30 compounds comprising 10 hydrocarbons, 8 esters, 3 alcohols, 2 amines, 2 sulfur compounds, and one of each of acid, aldehyde, phenol, ketone, and furan were identified. sample fermented with b. megaterium produced 29 aroma compounds comprising 9 hydrocarbons, 10 esters, 2 nitrogenous compounds, 2 ketones, 3 alcohols, and one of each of lactone, aldehyde, furan, and amine. methyl esters of various long chain fatty acids may be key aroma compounds, based on consistency and persistence. qualitative or quantitative contribution of individual compounds may only be determined following flavour threshold analysis.

PMC4745521

pubmed-1344

recent results from the national institutes of health (nih) frequent hemodialysis network (fhn) daily trial and the following rehabilitation, economics and everyday-dialysis outcome measurements (freedom) study have provided confirmation of the clinical and quality of life (qol) benefits of short daily hemodialysis (sdhd) when compared to conventional thrice weekly in-center hemodialysis (chd). the fhn study was conducted in-center; however, sdhd in the usa is more commonly performed as a home therapy and is not offered as a standard treatment modality in most dialysis clinics. although it has been nearly 50 years since the early pioneers such as belding scribner and stanley shaldon began dialyzing patients in their homes, peaking at 40% of all us dialysis patients in 1973, the popularity of home hemodialysis (hd) has been limited in the usa for a number of reasons, including lack of suitable equipment and increased cost. the last few years, however, have seen a revival in home hd, resulting in renewed interest in the various forms of more frequent therapy, including sdhd. the revival of home dialysis was led in the mid 1990s by robert uldall, andreas pierratos and others in canada, gradually spreading throughout the usa. this re-emergence has been made possible by recent advances in technology, including portable and more user-friendly machines designed specifically for use in the home environment. in 2005, the fda cleared the first hd machine for home use in the usa, the nxstage system one. fda clearance was based on an open-label, prospective, two-treatment two-period crossover study on 32 patients to compare sdhd performed in-center (prescribed six times/week) versus sdhd performed at home. the study was conducted at six centers in the usa under an investigational device exemption. this study showed sdhd was delivered as efficiently in the home environment as in-center, with 98.5% of treatments performed successfully in-center versus 97.3% at home. notably, the composite rate of intra-dialytic and inter-dialytic adverse events was significantly higher during the in-center phase when compared with the home phase (5.3 versus 2.1 adverse events/100 treatments; p=0.007), suggesting hd therapy is at least as safe at home as in-center. when comparing clinical parameters from the period immediately preceding the study when patients were treated with conventional thrice weekly center hd, home sdhd was associated with reductions in blood pressure, antihypertensive medications and interdialytic weight gain. since the clearance of the nxstage system one in the usa, the number of patients now performing home dialysis is estimated to be 5000, with the majority of these patients performing sdhd. although this represents a considerable increase over the past 5 years, it is still only 1% of the total end-stage renal disease (esrd) population. sdhd offers the promise of improved clinical outcomes in patients with kidney failure, and given the positive results recently released from both the fhn and freedom studies outlined below, sdhd has the potential to become a more viable and popular choice amongst many esrd patients. results recently published from the fhn daily trial, funded by the nih, showed that in-center sdhd (prescribed six times/week), provided significant benefits in both composite co-primary outcomes of death or 12-month change in left ventricular mass (lvm) and death or 12-month change in the rand-36 physical health composite (phc) score. specifically, the fhn daily trial showed patients randomized to sdhd reduced their lvm by 16.3 35.3 g (p<0.001) and improved their phc score by 3.3 8.9 points (p=0.004), after 12 months. however, patients on sdhd were more likely to undergo interventions related to vascular access, a result which will require further elucidation with the ongoing analysis of the trial. the fhn trial will be a tremendous source of data to further dissect the advantages seen with daily therapy in the coming years. the freedom study is an ongoing prospective cohort study investigating the clinical and economic benefits of sdhd. the objectives of the freedom study are to compare a cohort of patients starting sdhd using the nxstage system one to a matched cohort of patients receiving chd for all-cause hospitalizations and non-treatment-related medical expenditures. using a 10-to-1 ratio, totaling 5000 patients, the matched thrice weekly in-center hd cohort will be obtained from the us renal data system (usrds) database. in addition, changes in qol measures, urea kinetics, management of anemia, bone and mineral metabolism, nutrition, vascular access interventions and use of medications will be examined. the freedom study will involve up to 70 clinical sites and 500 participants, with a minimum 1-year follow-up. study participants complete qol surveys at the time of study enrollment, at 4 and 12 months and every 6 months thereafter. interim data recently released from the freedom study has shown that sdhd, performed in the home environment, is associated with significant improvements in several important clinical and qol measures when compared to conventional thrice weekly in-center hd. interim results from the freedom study investigating the effect of sdhd on depressive symptoms were recently published. depressive symptoms were examined in 248 participants at enrollment and at 4 and 12 months by administering the validated beck depression inventory (bdi)-ii survey. the study protocol requires that the site investigator be notified immediately of a bdi score>10, with mild and moderate to severe depressive symptoms defined as bdi scores of 1115 and>15, respectively. in summary, sdhd was associated with a significant improvement in mean bdi score over 12 months [11.2, 95% confidence interval (ci) 9.612.9, versus 7.8 (95% ci 6.59.1); p<0.001], in the per-protocol (pp) cohort. similar results were found in the intent-to-treat (itt) cohort. for participants with moderate to severe depressive symptoms, the bdi scores almost halved over 12 months [24.4 (95% ci 19.429.4) versus 12.6 (95% ci 8.017.2); p<0.001]. it should be noted that a bdi score>15 previously has been shown to be highly predictive of a diagnosis of clinical depression. the percentage of participants with depressive symptoms (bdi score>10) significantly decreased during 12 months (41 versus 27%; p<0.03). similarly in the fhn study, patients participating in the daily arm did show a trend toward improvement after 12 months when compared to baseline (12.6 8.7 versus 10.4 8.5, p=0.1). although this trend was not statistically significant, one might speculate that undergoing daily dialysis therapy at home may contribute to this improvement. considering the practical feasibility of daily dialysis both from a logistical and a patient comfort perspective, one might hypothesize that the home setting may further enhance the benefits of sdhd experienced in-center. the long-term effect of sdhd on post-dialysis recovery time was also assessed in the freedom study by administering the following previously validated question: how long does it take you to recover from a dialysis session and resume your normal, usual activities? interim results were recently published and confirmed that sdhd is associated with a significant decrease in post-dialysis recovery time [476 min (95% ci 359594) versus 63 min (95% ci 3295); p<0.001]. the percentage of participants experiencing prolonged post-dialysis recovery time (> 60 min) also significantly decreased over the 12-month period (81 versus 35%; p<0.001). the fhn daily trial showed significant improvements in control of hyperphosphatemia for patients on sdhd. a significant reduction in mean serum phosphate was also reported in an interim report from the freedom study, along with significant reductions in the calcium phosphate product, serum creatinine, serum potassium and a trend toward a reduction in blood urea nitrogen. both the fhn daily trial and an interim analysis from the freedom study showed no change in serum albumin levels. well-known uremic symptoms, including restless legs symptoms (rls) and poor sleep quality are common in the hd population. poor sleep quality and rls have both been linked to increased risk of death for these patients [9, 10]. a recent interim report from the freedom study demonstrated initiation of sdhd at home is associated with significant improvement in rls and sleep disturbances. results from 235 patients found 40% suffered from rls at baseline, which was associated with poorer sleep quality and respiratory disturbances. among patients with rls, the mean irls (international restless legs severity scale) score improved significantly at month 12, after adjustment for use of rls-related medications (18 versus 11, p<0.0001). among patients with moderate-to-severe rls (irls score 15), there was an even greater improvement in the irls score (23 versus 13, p<0.001). over the 12-month period, there was decline in the percentage of patients reporting rls (35 versus 26%, p=0.05) and those reporting moderate-to-severe restless legs symptoms (59 versus 43%, p=0.06). there was a similar and sustained improvement in several scales of the medical outcomes study sleep survey over 12 months, after adjustment for presence of rls and use of anxiolytics and hypnotics. recent results from both the fhn daily trial and freedom study have shown that sdhd, performed either in-center or at home, is associated with several important clinical and qol benefits when compared to conventional thrice weekly hd. the improvements in left ventricular mass, control of hypertension and improvement in hyperphosphatemia demonstrated in the fhn trial, in conjunction with the positive interim findings from the freedom study, including improvements in depressive symptoms, post-dialysis fatigue, various laboratory parameters, restless leg syndrome and sleep disorders, confirm the anecdotal benefits of sdhd seen and reported by many practicing nephrologists and their patients. until now, a complex variety of reasons has hindered the more widespread use of sdhd in the usa and globally. however, the results of these studies may provide a new impetus for esrd patients and nephrologists to reconsider the paradigm of thrice weekly center hd as the default therapy. such a paradigm shift will likely increase the possibility for improved clinical outcomes and overall patient care for those affected by esrd.

thrice weekly in-center hemodialysis is the standard of care for dialysis patients with end-stage renal disease (esrd). however, there is ongoing debate as to whether more frequent hemodialysis, with its readier management of both toxin and fluid removal, benefits patients. new evidence from recent studies, both in center dialysis and in home haemodialysis patients, adds further confirmation of improved cardiovascular outcome and quality of life in patients undergoing short daily hemodialysis. a paradigm shift in esrd care delivery may be facilitated due to new technology enabling daily therapy at home.

PMC4421457

pubmed-1345

along with the introduction of effective therapeutic agents such as somatostatin analogue (ssa), multityrosine kinase inhibitor and mammalian target of rapamycin inhibitor, much improvement has been made in the treatment of neuroendocrine tumors (net). especially, the promid and radiant studies proved the effectiveness of these new therapeutic agents in the treatment of mid gut and pancreatic net, respectively. however, in the case of metastatic gastric net, there is neither an established treatment strategy nor a prospective study because gastric nets are not only rare but also mostly diagnosed in the early stage, which can be controlled by surgical resection. therefore, gastric net has been considered a part of the gastroenteropancreatic net category and there have been no studies exclusively aimed at gastric net. in the treatment of metastatic net, a treatment approach according to the presence of hormonal symptom, embryological origin of primary site and pathologic grade is usually accepted as a principle. however, in the case of gastric net, a separate treatment approach depending on a specific classification, which can be recognized based on clinical and histologic characteristics, is considered. in the classification of gastric net, type i (74% of the gastric nets) is associated with chronic atrophic gastritis and hypergastrinemia, and type ii (6% of the gastric nets) is associated with multiple endocrine neoplasia type i, zollinger-ellison syndrome and hypergastrinemia. lastly, sporadic type iii gastric neuroendocrine carcinoma (nec; 13% of the gastric nets) is gastrin-independent and carries the worst prognosis. in this paper, we describe the case of a female gastric net patient with multiple liver metastases who was unresponsive to biologic therapy and cytotoxic chemotherapy. in addition, we look at the therapeutic strategy for metastatic gastric net together with a literature review. upper endoscopic examination revealed a localized, fixed and firm subepithelial tumor (diameter, 2.2 cm) in the peripyloric area, but there was no evidence of gastric ulcer in the entire stomach (fig. a stomach computed tomography (ct) was performed and revealed two non-enhancing, low-attenuated liver masses in s4 (2.2 cm) and s5 (1.6 cm) suggesting metastasis (fig. laparoscopic antrectomy with gastrojejunostomy and liver biopsy were performed, but hepatic metastatectomy was not tried due to multiplicity. the biopsy result of both gastric mass and metastatic hepatic nodule revealed a well-differentiated nec (net g2 by 2010 who classification). pathologically, the tumor was 37 21 mm in size with a negative margin (distance from resected margin: 0.5 cm) and infiltrated the muscular propria layer. microscopically, the tumor cells were medium-sized and uniform in shape and showed moderate cellularity with mild mitosis (< 1/50 per high-power field) (fig. on immunohistochemical staining, the tumor cells were positive for chromogranin a and cd56a (fig. 3d), and the serum gastrin level was increased to 1,832 pg/ml (normal range 0108). the vitamin b 12 level was 350 pg/ml (normal range 211911), chromogranin a level was 356.29 ng/ml (normal range 2794), 5-hydroxy indole acetic acid level was 3.04 mg/day (normal range<10) and serotonin (5-hydroxytryptamin) was 48 ng/ml (normal range<200). however, ct and mri did not localize any tumor assumed of gastrinoma. considering the gastrin level and histologic grade moreover, she complained of abdominal pain with diarrhea and was therefore treated with long-acting repeatable (lar) sandostatin at a dose of 20 mg intramuscularly every 28 days. however, abdominal ct, which was performed 3 months later, revealed progression of the hepatic metastases without tumor recurrence in the operated bed (fig. the patient received an etoposide plus cisplatin (ep) combination chemotherapy (etoposide 100 mg/m on days 13, cisplatin 75 mg/m on day 1). despite 3 cycles of ep chemotherapy, response evaluation identified a stable disease status, and eventually, her disease progressed after 6 cycles of ep chemotherapy (fig. 2c). because of the patient's economic situation and lack of grounds, targeted agents such as sunitinib and everolimus were infeasible. therefore, we chose a combination therapy with interferon- (ifn) and sandostatin lar. on response evaluation after 3 months of treatment, she showed favorable response with regression of liver metastasis (fig. since oberndorfer first denominated the carcinoid tumor in 1907, there has been much improvement in the classification and treatment of net. especially the introduction of the new who pathologic classification system, which is based on the ki-67 labeling index and a few landmark studies such as promid and radiant, provided a useful guide to the oncologists dealing with gastroenteropancreatic net. based on these studies, ssa is known to be effective in the treatment of midgut nets, and cytotoxic chemotherapy such as the ep regimen is known to be effective in the treatment of foregut nets, including pancreatic nets. however, net can arise in most organs of the body and can show a variety of clinical features according to its origin and the degree of differentiation. moreover, in the case of nets that originate from rare primary sites, standard treatment strategies have not been defined yet. although a division according to the embryological origin, which divides nets into foregut, midgut and hindgut nets, has been used in practice, it can not guarantee the homogeneity between the different organs inside the same embryological category. that is to say, although gastric net embryologically originates from the foregut, we do not know exactly whether it shows an aspect similar to net of the pancreas. moreover, gastric net has a few distinctive features in its pathogenesis and natural course. gastric net is known to be derived from the enterochromaffin-like (ecl) cells, which produce histamine, express the gastrin receptor and regulate gastric acid secretion. gastrin, which is released by g cells in the antrum of the stomach, duodenum and the pancreas, stimulate the release of histamine in ecl cells. therefore, neoplastic changes in ecl cells are often associated with elevated serum gastrin levels. based on this unique pathogenetic background, a separate classification system for gastric net was first defined by rindi et al. in 1993. according to this classification, gastric net can be subdivided into types i, ii and iii, depending on the gastrin level and source of hypergastrinemia. a treatment strategy according to that system because of the relatively benign clinical course, low metastatic potential and low histologic grade of type i and ii gastric nets, the treatment strategy for these types of nets was focused on local control, and the principal treatment was conservative, including endoscopic follow-up or endoscopic mucosal resection. even in the case of multicentric tumor with liver metastasis, antrectomy and the consequent normalization of gastrin levels was considered sufficient for the control of the remnant lesion. as for medical treatment, biologic therapy such as ssa and ifn a few studies also demonstrated a therapeutic value of octreotide in nets of gastroduodenal origin [6, 7]. besides that, khuroo et al. showed that long-acting ssa injections reduced serum gastrin and serum chromogranin a levels with a 50% reduction in the visible number of tumors in 3 patients with type i metastatic gastric carcinoids. ifn- and-have also been used in the treatment of gastrointestinal nets, regardless of functionality. immune-mediated cytotoxicity, inhibition of progression of the cell cycle from the s to the g2 phase and inhibition of tumor angiogenesis were proposed as main mechanisms of ifn. however, systemic side effects such as flu-like symptoms and hypothyroidism hindered widespread usage as a first-line treatment. according to the european neuroendocrine tumor society (enets) guidelines, ssa is known to be valuable for the subgroups of patients with slowly progressive and low proliferative nets (g1) of gastroduodenal origin, and in cases of metastatic g3 necs or well-differentiated but rapidly progressive metastatic disease, combinations of etoposide and cisplatin are recommended. however, it is not certain whether the histologic grade depending on the rate of cellular proliferation correlates with the classification based on gastrin levels and how to integrate the gastric net and who classifications into the same treatment strategy for gastric nets, especially in cases of metastatic disease. moreover, there is the opinion that dysplastic cells are less dependent on gastrin stimulation and do not regress in response to reduced circulating gastrin levels. indeed, a few metastatic cases with a bad prognosis have been reported in type i or ii gastric nets [11, 12]. in our case, the biopsy results showed a well-differentiated, g2 nec with 4% of ki-67 mitosis and an elevated gastrin level of 1,832 pg/ml. however, there was no evidence of endocrine neoplasia at the other site. based on the histologic grade and gastrin level, type therefore, we expected good response to ssa, but somatostatin did not achieve response and disease progression was faster than expected. however, after 6 cycles of ep chemotherapy, the tumor eventually progressed within only 2 months. because the general condition of our patient was infeasible for another cytotoxic chemotherapy, we again used ssa for the relief of functional symptoms and stabilization of the tumor. in addition, we added ifn- and obtained favorable response. according to previous studies comparing ssa or ifn alone with a combination of the two drugs, response was not superior in any of these groups, whereas the side effects leading to an interruption of the therapy were more frequent in the combination group. however, according to kolby et al., the combination of ifn- and ssa reduced tumor progression better and for a longer time than either agent alone and was well tolerated because ssa can reduce side effects of ifn-, although the study was aimed at midgut net. one review article about the combination of ifn- and ssa presented data showing an advantage of additional ifn- after progression following ssa alone. however, there is not enough statistical evidence for an upfront use of the combination of ifn- and ssa. in summary, for the treatment of metastatic gastric net, both the clinical classification according to the gastrin level and the pathologic grade should be considered. ssa can be considered a first-line medical treatment for type i and ii metastatic gastric net. however, the combination of ssa and inf- can be an effective option in cases showing aggressive tumor behavior or after failure of ssa alone.

despite remarkable progression in the treatment and classification system of neuroendocrine tumor (net), some questions have remained unanswered. the lack of an established treatment strategy for gastric net is one of the problems. because of its paucity, gastric net is not discussed in independent, large-scaled prospective studies and tends to be excluded from clinical trials. moreover, a separate classification system and some distinguished clinical features render the treatment of gastric net more complicated. here, we present a case of a female gastric net patient with g2 proliferation index and multiple liver metastases. based on the histologic grade and a high serum gastrin level, we initially treated her with somatostatin analogue. however, the patient did not respond. after that, cytotoxic chemotherapy with the etoposide plus cisplatin regimen only showed response in the short-term period. however, combination therapy with octreotide and interferon brought about significant regression of the tumor. herein, we present our case together with a literature review of the treatment of metastatic gastric net.

PMC4025115

pubmed-1346

oral leukoplakia (olep) is defined as a white plaque of questionable risk that is diagnosed after excluding other known diseases or disorders that carry no additional risk for cancer. olep is a clinical term that requires the application of strict diagnostic criteria for its classification. olep may present a wide spectrum of histopathological patterns, including hyperkeratosis, epithelial dysplasia, carcinoma in situ, and squamous cell carcinoma (scc). the ki67 protein is localized to the nucleus in all actively dividing cells, and its expression level is proportional to the severity of dysplasia in olep. the upregulation of ki67 in both dysplastic and malignant tissues qualify ki67 as a reliable marker of cell proliferation with prognostic significance. cyclooxygenase (cox)-2 is an enzyme that catalyzes the synthesis of prostaglandins (pg) whose expression is associated with carcinogenesis owing to its roles in apoptosis, angiogenesis, inflammation and immunosuppression, invasion, and metastasis. cox-2 expression was investigated for its relation with specific markers of these multiple mechanisms and correlations between high levels of cox-2 expression and carcinogenic progression were reported. additionally, cox-2 was reported to activate several classes of chemical carcinogens and its peroxidase function was contributed to the activation of procarcinogens. the mode of action of cox-2 in carcinogenesis was linked to pgs, especially of the e series. due to their effects on proliferation, angiogenesis, immune surveillance and apoptosis, increased synthesis of pgs are believed to be important in the pathogenesis of cancer [8-10]. cox-2 was found to be overexpressed in various carcinomas as well and high levels have been detected in scc compared to normal epithelium [11-14]. the aim of this clinicopathological study was to assess the prognostic value of cox-2 and ki67 expression for oral leukoplakia by determining their association with histomorphological data. to model the process of malignant transformation, samples of normal oral mucosa and squamous cell carcinoma were also investigated. tissue samples were collected from olep patients who were referred to the department of oral diagnosis and radiology in the faculty of dentistry at marmara university, istanbul, turkey. informed consent was obtained from the patients and the study was approved by the ethics committee of marmara university (mar-yc-2006-0123). patients using non-steroidal anti-inflammatory drugs at the time of diagnosis and in the month prior to their clinical examination were excluded from the study because of the potential effects of these agents on cox-2 levels. smoking and alcohol habits were recorded. with respect to the former, patients were divided into two groups. smokers were individuals who were smoking regularly at the time of diagnosis and for at least 1 year prior. non-smokers comprised patients who reported never having smoked as well as former smokers who had previously smoked but were not doing so at time of diagnosis. individuals who had one or more drinks three or more times per week were defined as habitual drinkers and placed in the alcohol consumption group, with one unit of an alcoholic drink corresponding to 12 g of ethanol. clinically, leukoplakia was subdivided into homogeneous (c1) and non-homogeneous (c2) types. c1 was defined as flat, thin, and uniformly white; c2 referred to predominantly white, or white and red lesions that were irregular in shape, flat, nodular, or exophytic. a conservative excisional biopsy was performed for any lesion smaller than 2 cm in diameter; larger lesions were treated according to histopathological results by applying previously described management protocols. biopsied specimens were evaluated in the department of tumour pathology at the institute of oncology of istanbul university. the final diagnosis was confirmed by histopathological examinations and any other definable lesions were excluded. olep samples were divided into four subgroups according to histopathological features: hyperkeratosis (hk) and-when epithelial dysplasia was present-three oral intraepithelial neoplasia (oin) subgroups classified according to severity as mild dysplasia (oin1), moderate dysplasia (oin2), and severe dysplasia and carcinoma in situ (oin3). a total of 20 samples, presenting 10 samples of intact normal oral mucosa (n) and 10 samples of scc were retrieved from the archives of the department of tumour pathology and used as negative and positive control groups, respectively. the control tissue samples were used only in the immunohistochemical evaluation in order to form a gradually increasing model for the process of malignant transformation (figure 1) and not evaluated in terms of habits of smoking and alcohol consumption. olep=oral leukoplakia; nsaid=non-steroidal anti-inflammatory drugs; hk=hyperkeratosis; oin=oral intraepithelial neoplasia; n=normal mucosa; scc=squamous cell carcinoma. the 50 tissue samples were embedded in paraffin and serial sections were cut at a thickness of 5 m that were collected on charged slides, then permeabilized and dried overnight in an autoclave at 56 c. after deparaffinization, sections were subjected to antigen retrieval by heating in a microwave four times for 5 min in citrate buffer (ph 6.0), then cooling to room temperature and washing in phosphate buffered saline (pbs) for 5 min. endogenous peroxidase activity was quenched by incubating the sections in 3% h2o2 followed by washes in distilled water. slides were incubated for 2 h with rabbit anti-cox-2 (thermo scientific, lab vision corporation, fremont, ca 94538-6406, usa) and rabbit monoclonal anti-ki-67 (thermo scientific, thermo fisher scientific, anatomical pathology, fremont, ca 94538, usa) antibodies, while negative control sections were treated with pbs. all slides were incubated with biotinylated goat anti-rabbit secondary antibody (tr-015-bn; lab vision corp.) for 25 min, followed by a streptavidin peroxidase reagent for 25 min; the chromogenic substrate 3-amino-9-ethylcarbazole was used to visualize immunoreactivity. sections were counterstained with mayer s hematoxylin, covered with a coverslip, and evaluated under a light microscope (olympus, tokyo, japan). quantification of cox-2 and ki67 staining cox-2 and ki67 staining was evaluated by a pathologist who was blinded to the experimental protocol. slides were scanned at low magnification under a light microscope and five areas with the highest degree of staining were selected for cell counts. only nuclear staining was accepted as positive for ki67. for cox-2, cytoplasmic and membranous staining the number of cells in the epithelium and in the tumour island of scc specimens was counted. the immunoreactivity score was calculated as the mean percentage of positively stained cells to the total number of cells. note the strong reaction in the cytoplasms of oral intraepithelial neoplasia cells marked by arrows. positive staining was observed in the nucleus of oral intraepithelial neoplasia cells marked by arrows. kruskal-wallis test was used to compare variables among multiple groups with mann-whitney u test (bonferroni corrected) as a post-hoc test. fisher s exact and fisher-freeman-halton exact tests were used for qualitative comparisons of data. the means (m) and standard deviations (sd) of ages, expressions of cox-2 and ki67 were expressed as (m [sd]). clinicopathological findings of olep patients clinicopathological characteristics of the study group are shown in table 1. patients ranged in age from 30 to 75 years, (54.33 [11.03]), with 19 presenting hk (13 (68.4%) males, 6 (31.6%) females) and 11 presenting oin (7 (63.6%) males, 4 (36.4%) females). the mean ages of the hk and oin patients were similar (55.05 years vs. 53.09 [12.93] years) (p=0.966). the occurrence of oin was associated with a non-homogeneous clinical aspect (p=0.004), but was also observed in lesions with a homogeneous appearance (three oin1 and two oin2 patients). the homogeneous-type olep was more frequently observed in the hk group (p=0.004). clinicopathological characteristics of the study group statistically no significant, mann whitney u test. sd=standard deviation; hk=hyperkeratosis; oin=oral intraepithelial neoplasia; n=number of samples; m=male; f=female; s=smoker; ns=non-smoker; u=using; nu=non-user; c1=homogenous type; c2=non-homogenous type. there was no difference between groups in terms of smoking and alcohol consumption (p=0.156; p=0.327; respectively). lesions were more often localized on the tongue for oin while buccal mucosa localization was higher for hk (p=0.002) (table 2). localization of oral leukoplakia lesions by histopathological subgroups n=number of samples; hk=hyperkeratosis; oin=oral intraepithelial neoplasia. immunohistochemical findings cox-2 and ki-67 expression increased relative to the degree of lesion severity in the histopathological subgroups, with the scc group having the highest numbers of cox-2-and ki67-positive cells (table 3). the mean number of cox-2 expressing cells was lowest in n tissue samples, higher in the scc and oin than in the hk group, and higher in the scc than in the oin group (p=0.0001). cox-2 and ki67 expressions in olep samples and controls statistically significant at the level p<0.05, kruskal wallis test. sd=standard deviation; olep=oral leukoplakia; n=normal mucosa; hk=hyperkeratosis; oin=oral intraepithelial neoplasia; scc=squamous cell carcinoma. among oin subgroups ki-67 expression was detected in the nuclei of cells and in ascending order; these were n (5.33%), hk (36.01%), oin (39.49%), and scc (74.66%). there was no significant difference between the hk and oin groups, but a difference was found between scc and oin groups (p=0.0001) (table 3). the prevalence of malignant transformations of olep is between 0.13% to 17.5% for observation periods ranging from 1 to 30 years. several factors are associated with an increased risk of malignant transformation, and the clinical appearance of olep is considered a major benchmark. the occurrence of oin is relatively low for the c1 clinical type, and higher rates of malignant transformation have been reported for the c2 type. in the present study, 5/23 (21.7%) c1 and 6/7 (85.7%) c2 olep cases presented oin. however, given that oin was detected in c1 cases, a biopsy is recommended as a mandatory procedure for all olep types. tobacco usage in different forms is the most common antecedent for olep lesions, with suggestions that some lesions may regress upon cessation of tobacco use. according to one report, the risk for malignant transformation among individuals with scc that had ceased using tobacco for more than 10 years was similar to that of non-smokers. the patients in the present study were predominantly smokers (80%), and two patients in the non-smokers group had ceased smoking for more than 10 years and both presented with hk. nonetheless, due to dynamic nature of olep, regression is not expected in all olep cases. paradoxically, some olep lesions in non-smokers were reported to have worse prognosis than in smokers, and women without smoking habits had a higher risk of malignant transformation among the groups examined in one study. consistent with these findings, all the oin 3 patients in the present study were non-smokers. it is suggested that the high-risk nature of lesions in non-smokers is associated with intrinsic factors such as inherited or acquired predisposition, for which a more aggressive treatment strategy is recommended. alcohol consumption is another risk factor for olep, but its most potent effects are exerted through synergy with tobacco usage, with geographical variations reportedly playing a role. in this study, a slightly higher rate of alcohol consumption was detected in oin than in hk (27.3% vs. 10.5%), although this difference was not statistically significant. the increased proliferative capacity in olep has been confirmed by a higher expression level of ki67, which was proposed as a marker for the occurrence and severity of oin in oral mucosa. its association with lesion severity underscores ki67 expression as a prognostic factor for olep, which was suggested in the present study; the highest ki67 expression was observed in scc, followed by oin and hk, indicating that this marker of proliferation may be a predictor of progressive malignancy. on the other hand, although there was a gradual increase in ki67 expression, no significant difference was detected between the hk and oin groups. cox-2 is an inducible enzyme whose expression is low or negative in most tissues, but a few hours after a single stimulation the enzymatic activity of cox-2 was reported to increase more than 10-fold and then return promptly back to the basal level. this expression is influenced by growth- and tumour-promoting factors, inflammatory stimuli, and oncogenes under a variety of pathophysiological conditions. cox-2 expression has been implicated in the malignancy of scc of the tongue as well as oin and cox-2 overexpression was found to be predictive of patient survival in scc. elevated levels of cox-2 were also observed in the majority of dysplastic squamous epithelia and scc, in contrast to the weak expression detected in normal esophageal tissue. another study reported progressively higher cox-2 levels in normal mucosa, oin, and scc. consistent with these results, here it was found that cox-2 expression was lowest in normal mucosa, higher in hk followed by oin, and highest in scc. nevertheless, as a limitation of the present study, the number of the study group is low (30 samples of olep) and the subgroups are not equal (2 samples of oin3). therefore, further studies are required with larger populations in order to indicate cox-2 and ki67 as prognostic markers of olep. our results suggest that cyclooxygenase-2 and ki67 expression may have a prognostic value for evaluating the malignant transformation of oral leukoplakia, and that the etiopathogenesis of oral leukoplakia should be investigated in terms of risk factors other than smoking and alcohol consumption. semih ozbayrak has worked in the field of oral medicine and dentomaxillofacial radiology in department of oral diagnosis and radiology, faculty of dentistry, marmara university, istanbul, turkey (1986-2013) and provided indispensable advice and guidance throughout this investigation.

abstractobjectivesoral leukoplakia is a precancerous lesion of the oral mucosa. the upregulation of ki67 and cyclooxygenase-2 has been reported in both dysplastic and non-dysplastic tissues. the aim of this clinicopathological study was to investigate the prognostic value of ki67 and cyclooxygenase-2 expression for oral leukoplakia. material and methodsa total of 50 samples were investigated and the study group consisted of 30 oral leukoplakia samples. samples of 10 intact oral mucosa and 10 squamous cell carcinoma were included as negative and positive control groups, respectively. epithelial dysplasia was defined as oral intraepithelial neoplasia (oin) and classified into subgroups 1-3. tissue samples were assessed immunohistochemically for ki67 and cyclooxygenase-2 expression. clinicopathological correlations of oral leukoplakia patients were also investigated. resultsall oin 3 patients were non-smokers (p<0.05), and homogeneous oral leukoplakia lesions also presented oin. both cyclooxygenase-2 and ki67 expression increased with the severity of lesions, which defined different subgroups (p<0.05), except there was no significant difference between the hyperkeratosis and oin groups for ki67 expression. conclusionscyclooxygenase-2 and ki67 expression may have a prognostic value for the malignant transformation of oral leukoplakia.

PMC4516855

pubmed-1347

osteosarcoma is a malignancy of mesenchymal cells that have the ability to produce osteoid or immature bone. osteosarcoma of the head and neck is relatively rare and constitutes only around 8.4% of all osteosarcomas, while in the jaw there is an estimated incidence of 0.7 per million population. gnathic osteosarcomas are broadly categorized into central (intramedullary) and peripheral (surface) subtypes. surface tumors are further divided into parosteal well-differentiated (low-grade), periosteallow- to intermediate-grade and high-grade surface osteosarcomas. in 1949, geschickter and copeland were the first to describe parosteal osteosarcomas under the heading of parosteal osteoma a group of parosteal bone tumors of which the seemingly benign clinical and histologic aspect was belied by frequent relapse and metastasis. unni et al., defined the juxtacortical osteosarcoma as a distinctive type of malignant bone tumor that originates on external surface of the bone specifically in relation to periosteum and/or immediate connective tissue. the first report of parosteal osteosarcoma in craniofacial site was reported in 1961 by som and peimer. it has predilection to occur in the 2and 4decade mainly affecting distal femur. in the long bones females have a greater risk than men (3:2), whereas in jaws men are more affected as (1.75:1) compared to women. in the jaws it can be mistaken for benign lesions as the clinical behavior and radiographic appearances are usually benign. histologically, parosteal osteosarcoma is well-differentiated and is characterized by spindle cell stroma with minimal atypia and rare mitotic figures separating irregular trabeculae of woven bone. parosteal osteosarcoma of craniofacial area behave similar to long bone counterparts with slow growth and low grade malignancy that do not tend to metastasize. this paper describes a case of parosteal osteosarcoma and emphasizes the need for differential diagnosis of this rare entity with benign reactive and neoplastic osseous lesions. a 22-year-old female patient presented with the complaint of swelling in the right side of the face since 9 months, which was not associated with pain, discharge or neurological symptoms. on extraoral examination, diffuse swelling of about 4 4 cm in midface region was noted on the right side of the face. intraorally, swelling involved right maxillary buccal aspect of the ridge from second premolar to third molar measuring around 3.5 2.5 cm in dimensions, extending anterioposteriorly from the mesial aspect of maxillary second premolar to distal aspect of third molar and superioinferiorly from the marginal gingival level to the vestibule causing obliteration, without any palatal expansion. exophytic bony swelling on right maxillary alveolar ridge occlusal radiograph revealed dome-shaped swelling with radiopaque shadow and patchy radiopacity on buccal aspect of maxilla [figure 2]. axial and coronal computed tomography (ct) scans revealed well-defined, hyperdense bony mass with patchy radiopacity emanating from the buccal cortical plate with broad base. three-dimensional ct (3d-ct) showed lobulated exophytic bony mass attached with broad base to the buccal cortical plate formed by the network of multiple inter coalescing striae of trabeculae [figure 3c]. the h and e stained sections showed parallel array of bony trabeculae in a background of spindle cells and angular cells which showed minimal atpia. areas of osteoid admist the angular and spindle cells were also detected [figures 4-6]. the patient was treated with partial maxillectomy and is under follow-up since the past 1.5 years. occlusal view showing well-defined radiopaque lesion with patchy radiopacity (a) axial ct showing hyperdense radiopaque lesion, slightly dense at base than at periphery. (c) 3d-ct showing exophytic bony mass showing network of multiple intercoalescing radiating striae. 3d-ct=three-dimensional computed tomography irregular osteoid matrix separated by spindle and angular cells. (h&e stain, 100) photomicrograph showing thick often parallel (streamers) array of bony trabeculae in a spindle stroma. parosteal osteogenic sarcoma is an uncommon malignant tumor. in a large case series of 998 osteosarcomas of entire skeleton, dahlin et al. bras et al., reviewed seven cases of gnathic parosteal osteosarcomas of which five were reported in mandible and two in maxilla with an average age of 35.4 years. simon et al., in there view of 12 parosteal osteosarcomas, reported five in maxillary and seven in mandibular region with an average age of 34 years. a peak incidence between the ages of 20 and 30 years with 2:3 male to female ratio however, we report parosteal osteosarcoma in a 22-year-old female patient, in the maxillary region. to date only 27 cases of juxtacortical osteosarcomas have been reported of which 12 were of parosteal variant, nine were of periosteal variant and six were of periosteal/parosteal type. the present case brings the total number of parosteal osteosarcomas of jaws to 13, a rarity in itself in the literature. concurrent with the literature presenting features in our case were a painless, slowly growing, exophytic osseous swelling with nonlobular outer surface. pain was mentioned as dull aching in half of the reported cases and it appeared as exophytic hard nodule on the attached gingiva appearing as soft tissue epulides mimicking benign osseous proliferative growth. occasionally surface ulceration was noted. radiographically, parosteal osteosarcomas are radiodense, lobulated, cauliflower-like or oval masses with a broad stalk attached to the external cortex of the underlying bone. parosteal variant is considered to arise from the outer layer of cortex, therefore does not elevate the periosteum like periosteal variant. between underlying bone and the tumor, a thin radiolucent cleavage plane called string sign, a characteristic but not a constant finding, is seen in only 30% of the cases. string sign is a radiolucent line histopathologically corresponding to unmineralized thickened periosteum interposed between the cortex and the tumor mass. it is more difficult to demonstrate in the jaws because their anatomical form does not allow the necessary radiographic projections to be obtained. various other radiographic patterns like cotton wool image with distinct margin, patchy radiopacity and tumor mass with diffuse hazy opacification with few fine bony spicules radiating from the lower border of the lesion also have been reported. in contrast, periosteal osteosarcoma presents with a radiographically intact cortex with no involvement of the underlying marrow cavity. the tumor matrix is not as radiographically dense or homogenous as parosteal osteosarcoma and has a poorly defined periphery. since treatment and prognosis varies with both the variants of surface osteosarcomas, radiologic differentiation is important. computerized tomography scan helps to demonstrate the tumor confinement, attachment to the cortex and involvement of marrow. ct and magnetic resonance imaging (mri) images are extremely useful for staging, detecting the presence of cortical erosion, regions of dedifferentiation, intramedullary extension or satellite lesions in the surrounding soft tissues. scintigraphy and chest ct are useful in confirming the solitary nature of the tumor and absence of metastatic disease. in the present case, many interlacing coalescing trabeculae of varying thickness forming a meshwork from the lower border of the intact cortex as seen in 3dct was an interesting finding not reported in previous cases. radiographic differential diagnosis includes tori, exostosis and peripheral osteomas more commonly; and other rare entities like nora's lesion (bizarre periosteal ossifying periostitis), osteochondroma, myositis ossificans and hyperostosis. tori and exostosis occurring at the same site are painless, slow growing masses with limited growth potential and can attach to cortex of jaw via pedicle or wide base. often the correct diagnosis is established on histologic basis. peripheral osteomas tend to be more radiodense because of absence of cartilagenous areas and the rare trabeular straie tend to be fine and closer together, like the edge of the feather. however, the aggressive behavior with rapid expansion and medullary involvement should help in ruling out their presence. osteochondroma, a rare entity in jaws, shows continuity of cortex and underlying medullary bone with the base of the lesion. hyperostosis, an uncommon lesion encountered under the pontics in the posterior region can be confused with parosteal variant. histologically, it is well-differentiated and characterized by spindle cell stroma with minimal atypia and rare mitotic figures separating irregular trabeculae of woven bone. with time, the trabeculae often coalesce and form a large mass of solid bone. about 4050% of parosteal osteosarcomas exhibit foci of cartilage. in the present case fine lace-like osteoid tissues were present among hyperchromatic tumor cells with foci of cartilaginous tissue. microscopic evaluation of the specimen helps in screening the dedifferentiation areas and areas that represent high grade behavior. approximately 10% of parosteal osteosarcomas dedifferentiate to high-grade osteosarcoma with a corresponding worsening of prognosis. the histologic differential diagnosis of parosteal osteogenic sarcoma of the jaws includes nora's lesion, benign fibro-osseous lesions like fibrous dysplasia and ossifying fibroma, low-grade intraosseous osteosarcomas and osteomas. nora's lesion histopathologically shows characteristic cartilage capping and distinct zones which distinguishes it from parosteal osteosarcoma. fibrous dysplasia and ossifying fibroma apart from their histopathology, the intramedullary location negates the possibility of them in differential diagnosis. osteoma in conjunction with mature bone lacks the spindle cell proliferation seen in parosteal osteogenic sarcoma and do not show cellular polymorphism, anaplasia and mitotic activity. low-grade intraosseous osteosarcoma shows overlapping histologic features with parosteal osteosarcoma. in most of the cases, minimal local reccurence occurs with wide excision of adjacent bone and periosteum by removing satellite lesions and preventing local spread along the bone surface. now, chemotherapy following wide excision is the recommended treatment protocol for recurrent cases and those exhibiting highly malignant features on histological examination. low-grade parosteal osteosarcoma has an overall good prognosis and has low metastatic potential. local control is more limited in the maxilla than the mandible and thus mandibular parosteal osteosarcomas overall have better prognosis than those of the maxilla. therefore maxilla has high recurrent rate. with the number of recurrences, the incidence of intramedullary involvement and dedifferentiation to high-grade sarcoma increases. to conclude, this case illustrates the importance of correlation of clinical, radiological and histopathological features in confirming the diagnosis of parosteal osteosarcoma. because of the less aggressive biological behavior

parosteal osteosarcomas are rare, low-grade juxtacortical variant of osteosarcoma, especially in the jaws, representing 1.6% of all bony malignant tumours and upto 5% of all osteosarcomas. only 12 cases of intraoral parosteal osteosarcomas have been reported in the english literature. in the jaws males are more commonly affected with peak occurrence at 39 years and nearly equal site predilection for maxilla and mandible. radiographically, parosteal osteosarcomas are radiodense, lobulated masses with a broad stalk to the cortex of the bone with no periosteal reaction and medullary invasion. microscopically, shows well-differentiated tumor with minimum atypia and rare mitotic figures separating trabeculae of woven bone. unlike classical and periosteal osteosarcoma, it is considered to have a good prognosis. a case report of this rare entity in 22-year-old female patient with bony hard, painless swelling of 9 months duration in maxillary premolar-molar region is presented. the need for differential diagnostic approach is emphasized from other seemingly benign clinical entities.

PMC4409192

pubmed-1348

the online version of this article (doi:10.1007/s40121-014-0043-9) contains supplementary material, which is available to authorized users. a member of the togaviridae family, infection with the chikungunya virus (chikv) has emerged as a major public health concern, resulting in outbreaks of fever and debilitating arthralgia in the caribbean and globally. the first autochthonous (native) transmission of chikv in the caribbean was confirmed in december 2013 on the french island of saint martin. rapid spread and local transmission of chikv occurred in the caribbean and the americas within 9 months (fig. 1). this has resulted in 651,344 suspected and 9,182 laboratory-confirmed chikungunya cases in the caribbean and the americas as reported by the centers for disease control and prevention. the effective management of the rapid spread of chikungunya is integrally related to the geographic distribution and re-emergence of competent mosquito vectors, evolving chikv genotypes, susceptible human populations, environmental influences, and social mobilization. understanding the contribution of these factors is important to improving the control of the global transmission of this disease. fig. 1introduction of chikungunya to the caribbean by epidemiological week first reported december 2013 to august 2014. data from paho/who statistics introduction of chikungunya to the caribbean by epidemiological week first reported december 2013 to august 2014. there are three distinct chikv genotypes: west african, east central south african [ecsa; or indian ocean lineage (iol)], and asian [1, 5, 6]. these genotypes represent the evolution of the virus in keeping with the geographic distribution over the years. in africa, this virus is transmitted through enzootic (sylvatic) and urban (human mosquito cycle) cycles among non-human and human primates by aedes mosquitoes species. previous authors report the probable existence of the chikv in africa for hundreds of years, with the first suspected epidemic documented in 1779. subsequent reports included outbreaks in nigeria (1964), the ivory coast (1981), and senegal (1983) associated with the west african strain. the resurgence of chikungunya in the twentieth century was first documented in 1953, in the newala district of tangaiynika (tanzania). the chikv continued to spread through the suspected vectors aedesfurcifer/aedescordellieri, to other south african countries, inclusive of rhodesia (zimbabwe; 1961, 1962, and 1971). epidemics associated with the ecsa lineage subsequently occurred in cameroon (2006) and gabon (2007) with aedesafricanus and aedesalbopictus reported as the principal vectors, respectively. in gabon in 2007, concurrent chikv and dengue fever epidemics were also experienced, with chikv transmission linked to the a.albopictus mosquito. the iol strain originated in coastal kenya in 2004 and 2005 (affecting 75% of the lamu island s population, via the vector aedesaegypti) and then spread to the reunion island (affecting one-third of the population) and was subsequently introduced to other islands of the indian ocean and continental india, via the vector a.albopictus [1, 57, 12]. it is believed that the 2007 outbreak in italy was initiated through the importation of the chikv by a traveler from reunion island. other countries in which international travel facilitated the spread of chikv included france (from travelers from reunion island; 77%), comoros (14%), and mauritius (9%). autochthonous transmission in france, however, was not detected until the year 2010, in association with a. albopictus. studies have reported a high efficiency of transmission of chikv in the temperate climates by the a. albopictus species. there are several aedes species which are competent in the transmission of the chikv, with a.aegypti and emerging populations of a.albopictus identified as responsible vectors in asia. the asian strain identified in thailand in 1958 was transmitted by the suspected vector, a. aegypti. this asian strain was also reported in outbreaks in india (19621965) and southeast asia (19581960 and 19621964), also in association with the a. aegypti mosquito. epidemics of the 1980s and 1990s in southeast asian countries (philippines, thailand, myanmar, and indonesia) were associated with the asian endemic/epidemic chikv lineage. since then, the asian endemic/epidemic chikv lineage has continued to circulate sporadically, transmitted primarily among humans by a.aegypti and was again documented in the 2006 outbreak in malaysia [1, 7]. emergence of an iol strain with resulting replacement of the endemic asian strain occurred during the southeast asia epidemics of 20062009 [1, 7]. it is thought that the e1 envelope glycoprotein mutation of the endemic asian genotype resulted in the decreased adaptability of the chikv to a.albopictus, with decreased transmission, and the facilitation of chikv evolution and strain replacement. the caribbean region continues to encounter challenges with the control of mosquito populations including a.aegypti and other aedes species. the aedes mosquito was introduced to the caribbean through the african slave trade. following its introduction, a significant increase in the a. aegypti population density was associated with the intercontinental exchange activities of the second world war. public health interventions of national health ministries and the pan american health organization in the 1950s and 1960s successfully reduced a.aegypti vector indexes by 1972. this success, however, was short lived and the re-infestations of the latin american and caribbean regions by a.aegypti in the late 1970s was accompanied by the introduction of a.albopictus in the early 1980s to some caribbean countries. although both a.aegypti and a.albopictus may be found in the caribbean, a.aegypti remains the principal vector responsible for the transmission of chikv and dengue in this region. to date, the chikv lineage identified in the 2014 caribbean outbreak belongs to the old asian lineage. the presence of the asian lineage chikv, compounded by a nave caribbean population and high prevalence of a.aegypti, makes the probability of establishment of endemicity of chikungunya in the caribbean high. this risk of establishment of endemicity extends to most tropical and subtropical regions of latin america areas and the southern usa, which is presently experiencing challenges with a.aegypti re-infestation. in the months preceding the caribbean s first case, china and the philippines accounted for 27.5% of travelers into the caribbean. there were ten cases reported in spain from travelers from haiti and dominica republic during april to june 2014. the expansion of the chikv to north and south america from the caribbean is of great concern. in their review of air travel from the caribbean, noted that the final destination of 52.1% of international travelers from the chikungunya-infected caribbean territories was the usa. the most likely cities to be travelled to from the caribbean were new york (13.8%), paris (11.7%), miami (7.8%) and san juan, puerto rico (3.9%). it is expected that the change of climatic conditions, compounded by the increase of the warmer weather in temperate zones, will facilitate the further spread of this disease. the months june through to november are designated as the hurricane season in the caribbean, or the wet season. the wet season has been shown to be associated with the increased breeding of the a.aegypti mosquitos. florida reported its first two cases of autochthonous chikv transmission in july 2014 and, as of september 9, 2014, remains the only us state with autochthonous transmission. florida now has eight cases of local transmission and 184 travel-associated laboratory-confirmed chikungunya cases as reported to arbornet. the impact of the introduction of chikv into the caribbean region, a known tourist destination exchange of an estimated 20 million persons annually of which 9 million are us residents, remains to be seen. the acquisition of chikv by travelers to the caribbean enhances the spread to neighboring caribbean countries and globally. as of epidemiological week 35, among the caribbean countries affected, those with the highest incidence rates (incidence/100,000 population) include: guadeloupe (16,465), martinique (15,087), saint martin (french part 13,401), saint barthelemy (11,707), dominica (5,068), dominican republic (4,128), and haiti (627) (fig. 1). the emergence of chikungunya in the caribbean unfolds a story of the dynamic evolution and interaction of microbes and risk factors underlying the determinants of health. the interaction of economic trade and its role in the transmission of vectors and infectious agents needs to be reviewed and policies reinforced and implemented.. continued education to increase the awareness of this disease is needed to curtail the population growth of mosquitoes and to mitigate the spread of chikungunya by travelers to the usa and other regions. improved awareness of the epidemiology and clinical manifestations preventative measures and epidemiological monitoring of the evolving chikv epidemic in the caribbean must be improved and sustained. this article does not contain any new studies with human or animal subjects performed by any of the authors. lizette mowatt and sandra t. jackson have no conflict of interest to declare with respect to this article. this article does not contain any new studies with human or animal subjects performed by any of the authors. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

chikungunya is a mosquito-borne virus that has shown increased prevalence in the caribbean since october 2013. there have been several outbreaks throughout asian and african countries over the past few decades with global travel and tourism having a major impact on the further spread of this disease. improved policies and practices for preventative measures and epidemiological surveillance must be implemented to prevent the continued transmission of chikungunya.electronic supplementary materialthe online version of this article (doi:10.1007/s40121-014-0043-9) contains supplementary material, which is available to authorized users.

PMC4269617

pubmed-1349

there has been a striking upsurge in tuberculosis (tb) vaccine research in china in recent years. more than 80 papers reporting such research have been published since 2004 (figure 1), many in english language journals. this has been driven and funded largely by chinese government agencies, since the scale of the tb problem and the difficulties in implementing fully effective control measures were recognized., we can note that there are at least two additional restricting factors limiting the progress of the field in china: few research groups have access to facilities for safely working with animals infected with a dangerous pathogenic organism such as the tubercle bacillus; infrastructure for undertaking vaccine clinical trials to international standards is sparse. in consequence, the universal problem of how to select among the promising approaches and candidate vaccines is particularly acute in china. nevertheless, the basic research problems are being tackled with some enthusiasm and creativity, and the new 5-year program to improve tb control that was announced by the chinese ministry of health on 1 april 2009 will have an impact. the antigens investigated to date and the locations of research groups are listed in tables 1 and 2, respectively. a substantial body of work has gone into characterizing the immune responses to be found in tb infection and following vaccination. the twin purposes of identifying candidate antigens for use in vaccines and diagnostic tests and of defining immunological markers for latent infection and disease states drive these studies. these studies have not always been followed up with tests of protective or therapeutic efficacy in animal infection models. the increased immunogenicity of mouse dendritic cells transfected with antigen heat shock protein 65 (hsp65) has been tested in wuhan and in hong kong, and differences have been found in the polarizing effect of intracellular mycobacterium bovis bacille calmette guerin (bcg) vaccine bacteria on antigen presentation by human adult and cord blood dendritic cells. various forms of recombinant bcg expressing early secreted antigen 6 of mycobacterium tuberculosis (esat6), or antigen 85b (ag85b) and antigen rv3425, or ag85b and esat6 with tumor-necrosis factor-, or esat6 and human granulocyte macrophage-colony stimulating factor (gm-csf) have shown enhanced immunogenicity. recombinant m. smegmatis expressing m. tuberculosis culture filtrate protein 10 (cfp10)/esat6 fusion protein was found stimulatory for macrophage inducible nitric oxide synthetase. recombinant m. smegmatis and bcg strains have been made that can express cloned antigens at a range of different levels under the control of modified fura gene promoters. other bcg recombinants expressing esat6, esat6/interleukin 2 (il-2) or ag85b/esat6 fusions have been made. a range of known antigens has been tested for immunogenicity as mixtures, fusion proteins and peptides. these include chimeric ag85b/esat6 with adjuvants monophosphoryl lipid a (mpl) and trehalose 6,6'-dimycolate, hsp16.3 with dimethyl-dioctadecyl-ammonium bromide/mpl (dda/mpl) adjuvant, fusion protein m. tuberculosis protein 64 (mpt64)esat6, resuscitation-promoting factor b (rv1009), or cfp10, esat6 or rpfe (rv2450) with nitrocellulose, or rv3772 and rv3425 with incomplete freund's adjuvant; or ag85b/mpt64190198/mtb8.4 plus a novel adjuvant of dda with bcg extract. in-silico analysis of putative mhc class 1-restricted epitopes present in antigens encoded within the region of difference 1 (rd-1) to rd-16 regions of m. bovis genome has revealed potential high-affinity hla binders and profiles of human humoral responses to 38-kda, mtb48, cfp10/esat6 antigens have been defined. screening of human immune sera against an expression library of m. tuberculosis open reading frames revealed three novel antigens among the top 20 most strongly recognized: rv1987, rv3807c and rv3887c. more than a dozen studies have used dna vaccination as a means of delivering antigens in immunogenicity tests. many have shown th1-biased immunogenicity without additional adjuvanting, for example with ag85b, ag85b/esat6 fusion, esat6/cfp10 fusion, mtb8.4/38-kda/ag85b fusion and epitopes from esat6, ag85a, cfp10 and ag85b inserted within hsp65. targeting the expressed product for degradation via the ubiquitin pathway has been used to enhance mhc class 1 presentation of epitopes from mpt64 and 38-kda, mpt64, and esat6. enhanced responses to encoded antigens have been obtained by additionally encoding cytokines, such as interleukin 21 (il-21), with ag85a or ag85a/esat6, and gm-csf with ag85a. inclusion of dna expressing il-12 enhanced prime/boost responses to bcg and to plasmids expressing ag85a and esat6. esat6 dna priming and protein boosting has also been shown to give enhanced th1 responses. many antigen preparations have been tested for their capacity to protect against challenge infection with virulent m. tuberculosis. subcutaneous esat6/cfp10, or esat6/mpt64 fusion proteins on nitrocellulose protected mice against h37rv challenge, but not as effectively as bcg. hsp65/il-2 fusion protein was found to elicit better protection than hsp65 given with dda/mpl adjuvant and protection was equivalent to that produced by bcg. similarly, either hspx, a dormancy associated antigen, or synthetic epitope 91104 gave protection equivalent to bcg when given with dda/mpl./mpt64190198/mtb8.4 fusion protein with dda adjuvant boosted protection against challenge in bcg primed mice. a similar fusion protein in which mtb8.4 was replaced by hspx gave a similar boost, but boosting with a mixture of the two fusion proteins was even better. a fusion protein of esat6 and ag85a also significantly boosted protection against h37rv in mice. recombinant bcg expressing the ag85b/mpt64190198/mtb8.4 fusion protein gave slightly better protection to mice against h37rv challenge than parent bcg or bcg expressing rag85b alone. recombinant bcg expressing a fusion protein of human interleukin (hil)-12p70 and esat6 showed increased immunogenicity but less protective effect. recombinant salmonella typhimurium that both expressed esat6/ag85b fusion protein and delivered it as a dna vaccine when given orogastrically gave protection similar to subcutaneous bcg and the combination was superior to either vaccine alone. the earliest reports indicated protection in mice superior to bcg when a divalent construct expressing both ag85b and mpt64, or a mixture of plasmids expressing ag85b, mpt64, mpt63 and esat6 was used. the protection given by a mixture of three plasmids expressing mpt83, ag85b and esat6 was enhanced by including dda adjuvant and an encoded fusion protein of ag85b and mpt64 was superior to the separately encoded antigens. encapsulation in poly(lactide-co-glycolide) microspheres with dda enhanced the protective efficacy in mice of dna-encoding ag85b/mpt64/mpt83 fusion antigen, and strikingly the dna mixed with dda was superior to bcg in protecting cattle against challenge. inclusion of a plasmid expressing il-2 improved protection by this plasmid or by plasmid expressing mtb8.4. a mixture of plasmids encoding ag85b, mpt64, mpt70 and tb10.4 boosted protection by bcg, as did a plasmid expressing a cfp21/mpt64 fusion protein. ag85b or ag85a were superior to esat6 when compared separately for protection as dna vaccines. dna expressing a fusion of mpb64/ag85b/esat6 was superior to a mixture of plasmids expressing the separate antigens and gave protection equivalent to bcg. the protective effect of dna expressing hsp65 against bcg challenge was enhanced by incorporating epitopes of esat6, ag85a/b and cfp10 within the hsp65 backbone. the protective effect of hsp65 dna against h37rv challenge was increased by expression as a fusion with hil-2, but did not surpass that of bcg. expression of ag85b fused to bovine herpes virus 1 vp22 protein, which facilitates dissemination of antigen to adjacent cells, resulted in protection against h37rv challenge in mice that was better than protection by bcg. few studies have been conducted with animals other than mice: a mixture of ag85b, hspx and cfp10/esat6 fusion together with cpg and aloh as adjuvant was immunogenic in mice but gave little protection against challenge with h37rv in guinea pigs; in contrast, combined dna vaccines encoding antigens ag85b, mpt64 and mpt83 given with dda appeared to be better than bcg in protecting cattle. interest in therapeutic vaccination has been sustained by clinical studies of a commercial chinese product, m. vaccae extract. recent meta-analyses of published data concluded that this product gave significant benefit in preventing tb in people at high risk (13 studies), but there was only a minor benefit from treating new tb cases (54 studies). in a unique and contrasting approach, a recombinant m. smegmatis delivering dna expressing human granulysin and murine il-12 was recently found to be therapeutic against h37rv infection. most research into therapeutic vaccines for tb has focused on the use of naked dna vaccination. dna had a significant therapeutic effect against h37rv in mice and the fusion hsp65/il-2 was significantly better. treatment of infected mice with a dna vaccine expressing a fusion protein of mycobacterial hsp70 and leukocyte cluster of differentiation antigen 80 substantially reduced acid-fast bacteria and pathology in liver and spleen, whereas bcg had no effect. although treatment with dna expressing ag85b was therapeutic, treatment with dna expressing mpt64 was not, and the mixture was less effective than the ag85b vaccine on its own, and inclusion of dna expressing il-12 gave a slight additional benefit. mice infected with a clinical isolate resistant to isoniazid and rifampicin responded well to treatment with the drugs plus dna expressing chimeric ag85a/esat6, and appeared to respond better to treatment with the drugs plus dna expressing ag85a than to the dna vaccine alone; ag85a dna alone was at least as effective as ag85a plus rifampicin in treating mice infected with a strain resistant to rifampicin and isoniazid, ag85a an immunogenic mixture of dna vaccines expressing ag85b, mpt64 and mpt83 has been found to work as a potent adjunct to isoniazid plus pyrazinamide therapy in mice and to be therapeutic in cattle infected with m. bovis, reducing both pathology and bacterial numbers; a mixture expressing ag85b, mpt64 and hsp65 was similarly effective. inclusion of immunostimulatory nucleotide motifs in the gene transcript enhanced the therapeutic efficacy of a plasmid expressing hsp65 when tested against h37rv in mice. it is evident in considering this body of recent tb vaccine research in china that much of it has been tactical in nature, establishing credentials both nationally and internationally, and building new research bases. additionally, there are creative and insightful studies of particular relevance to the needs of china. researchers are now able to exploit cutting edge technologies in designing new tb vaccines and are increasingly able to test the vaccines in relevant animal models of infection. the evidence that dna vaccines can provide effective therapy for tb in cattle may be a significant pointer to the future. the potential benefits of adding immunotherapies/therapeutic vaccines to tb chemotherapy have been recognized in china, but both preventive and therapeutic approaches against human tb await development of clinical trial capacity for their proper assessment.

it is now privately acknowledged that there may be little if any perceptible impact of the national bacille calmette guerin (bcg) vaccination program on disease prevalence, despite the extensive coverage of the newborn infant population and likely benefit in the early years of life. a better preventive vaccine than bcg is now being sought by chinese researchers. urgency has been added to the control problem by the emergence of multidrug-resistant tuberculosis (tb). furthermore, expensive second-line drugs seem unlikely to be made available by the government to treat drug-resistant cases, so attention in addition has turned to the potential of immunotherapy as an adjunct to chemotherapy. research trends are summarized here.

PMC3636423

pubmed-1350

the so-called feminization of medicine is likely to have important implications in patient-physician relationship, local and societal delivery of care, and the medical profession itself. when specialty choices are examined, women are proportionately over represented in the primary care fields. they are underrepresented in most surgical fields, with the exception of obstetrics and gynecology, where women now comprise the majority of practicing physicians. why anesthesiology is not a more popular specialty choice among women is difficult to determine. lifestyle issues are often cited as an important consideration in the selection of a specialty for residency training, and anesthesiology is often included in lists of specialties said to be associated with favorable lifestyles. however, data indicate that anesthesiologists rank among the busiest specialists, with an average of 61 h/week committed to professional activities and an average of 59 h/week dedicated to patient care. income expectations and personality factors gender discrimination and sexual harassment may be important factors. some factors that may influence women on the choice of anesthesiology as a career is sparse or even no contact with anesthesiologists during the preclerkship medical school curriculum. careers in medicine historically demanded a selfless emphasis on caring for one's patients, sometimes at the expense of one's marriage, children, and personal life. as women entering the medical field is increasing in numbers, the conflicts between career and family became more prominent. thus, many challenges must confront woman physicians in balancing their multiple roles as physician, mother, and spouse. women are underrepresented in anesthesia and with an effort to equalize the numbers of men and women within the medical school, one might expect the number of women entering anesthesiology to increase concomitantly mansoura is the capital city of dakahlia which is one of the most important egyptian provinces and is famous with many great medical centers. the total number of anesthesiologists in mansoura city at the time of our study was 224 and the proportion of women among them was 25.9%, and the proportion of women among the doctors in general was nearly 51%. the aim of our work was to evaluate the implications of anesthesiology as a profession on personal and family life of women anesthesiologists. we also wished to elicit the differences between academic and nonacademic women anesthesiologists with respect to these effects and the effect of women anesthesiologists on the profession. a descriptive comparative study was conducted by surveying women anesthesiologists in mansoura, egypt during the period between january and may 2013. the survey involved woman anesthesiologists affiliated with mansoura university hospital (academic anesthesiologists) and others working in hospitals related to ministry of health at mansoura (nonacademic anesthesiologists). during the study period, a convenience sample of 51 women were recruited, and 46 agreed to participate in the study with a nearly 90% response rate. nonparticipation was due to lack of interest in the study, absence during the study period and incomplete questionnaires. approval to conduct our study was granted by our institution's review board and from the management of the involved hospitals. the researchers introduced themselves to participants and informed them about the aims of the study. the content validity was determined by consulting a panel of experts. to ensure reliability, the questionnaire was pretested through a pilot study on a group of doctors who were not included in the final analysis and cronbach alpha coefficient of internal consistency was 0.87. the constructed survey instrument was finally edited in english version and consisted of four sections: the first section elicited some personal information of the respondents (sociodemographic characteristics). the second part consists of 6 items exploring the potential reflection of anesthesiology on family life of women (implications on family life), the third contains 12 items asking about the possible reflection of anesthesiology on woman personality (implications on personality and personal life) and the final section elicits 11 items reflecting the perceived impact of women on anesthesiology as a profession (job implications). the responses were coded using the variables in the responses to determine the coding guide. to facilitate quantification and analysis of data, respondents had options of strongly agree scored as five; agree scored as four; undecided scored as three; disagree scored as two and strongly disagree scored as one on a five point likert scale. data were analyzed using spss program (statistical package for social sciences) version 13 windows (ibm, inc., chicago, illinois, usa). descriptive statistics (i.e., mean, frequency, percentage, and standard deviation) and inferential statistics (i.e., chi-square test, fisher's exact, and t-test) were used. for comparison in order to evaluate the agreement among academic and nonacademic, items with multiple response levels were collapsed into binomial variables of agree and disagree. a score of three (undecided) on likert scale was considered as disagree. the age of the anesthesiologists with an academic career was not significantly different from those without academic career (33.1 vs. 31.8 years). nearly, one-fifth of our woman anesthesiologists were single (21.7%). the number of children in the two groups was not significantly different [table 1]. sociodemographic characteristics of the studied woman anesthesiologists the implications on family life were significantly much more afflicted by an academic career compared to nonacademic career in the form of: delayed marriage (87% vs. 44%), delayed first baby (78% vs. 30%), child rearing (96% vs. 30%), maternity rights (91% vs. 26%) and poor fulfillment of family demand (83% vs. 44%) respectively [table 2]. implications on family life among the studied woman anesthesiologists the majority of studied women reported personality changes. the positive implications on personality and personal life of anesthesiology as independence, empowerment and positive social interaction were not significantly different between the two groups. furthermore, most of anesthesiology drawbacks on personality (aggression, nervousness, depression, stressful attitude, and drug experimentation) were significantly more cited by women with academic careers. however, the financial limitation was significantly more complained by the noncareer anesthesiologists (p=0.032). the other negative effects observed in a large number of women in both groups were affection of feminine attitude, sense of discrimination, and poor social acceptance [table 3]. implications on personality and personal life of among the studied woman anesthesiologists anesthesiology was perceived privileged due to women's employment in the field by soothing the work environment, more emotional reaction to patient complaints and increased perfectionism (perceived by about 91%, 89% and 76% respectively). on the other hand, findings revealed that increased conflict with the surgeons (98%), poor surgeon acceptance (87%), poor patient acceptance and recurrent change of work schedule (80% each) were the most common items perceived by women as negative implications on the field of anesthesiology as a result of women's employment. difficult to manage night shifts was more significantly mentioned by academic career anesthesiologists compared to the others (p<0.001) [table 4]. we can not deny that rearing and caring at home are the primary role for any woman whatever her level of education, especially in arab countries. biologically, the most appropriate time for the woman to have children coincides with the period when career demands are most intense, making the balancing of career and family demands particularly difficult during this period. this critical period usually overlaps with the medical study and training, particularly with financial limitations and high work demands. the implications on family life were significantly much more expressed by women anesthesiologists with academic career, and more than 50% of both women groups perceived that their family life was exposed to negative implications. although only 9% of women with delayed marriage blamed anesthesiology work as a predisposing factor for their pregnancy problems, however this needs consideration. these findings were concomitant with a previous study which reported that most doctors recommended postponing pregnancy until after the completion of training. in contrast, another survey study conducted on pediatricians found that they gave birth to their first child during residency. our results could be explained by the fact that most of the academic career anesthesiologists have an intense schedule with teaching, research and regular or duties. the present findings were also coinciding with the study that found woman resident physicians experienced more pre-eclampsia and preterm labor than other women. unfortunately, our results emphasized that the interest of female doctors in academic medicine is reduced. this could be explained by many concerns that may range from balancing their multiple roles to the worry of physician-mother on the child's safety while she is away from home. furthermore, the view of some colleagues to a woman doctor's pregnancy and family commitments as evidence of a diminished dedication to medicine and career can be a contributory factor., support some of the previous concerns by concluding that decreased academic progress was related to childbearing. the present work showed that the maternity rights were affected for more than 58% of our women anesthesiologists, and this was significantly higher among career group. in egypt, even with the presence of a law that allows 3 months of full paid maternity leave, some women-doctors due to fear of demotion in their career, are pushed to sacrifice their maternity rights. it was surprising to know that many american physicians do not take the entire 6 weeks of maternity leave to which they are entitled. this action was partly explained by the concern of their colleagues attitudes toward their staying home with their infant or by their feeling guilty about being away from their patients and work. regarding implications on personality, the present study established that our ladies were privileged by their choosing anesthesia as a career, where most of them felt independent and empowered besides developing positive social interaction. previous studies support our finding by demonstrating that woman had greater adaptability to clinical situations, better vigilance, perceptual speed and associative memory that might give them an edge over males to recall more details. university staff in general has a better income compared to nonacademic which is a privilege granted by higher education committee in egypt to encourage the graduated physician to be a university staff. this can partly explain the significant financial problems encountered by nonacademic anesthesiologists in comparison to those in academic careers. the sense of discrimination due to gender is considered as a major factor that has a great negative impact on our woman anesthesiologists and represents 78% in this study. gardner et al., have reported that although woman anesthesiologists in south africa have higher career satisfaction; gender discrimination and harassment were found to be more likely among women. in contrast, a canadian study revealed an absence of gender discrimination. regarding the implications on profession the current study showed that around 98% of woman anesthesiologists complained from increased conflict with surgeons and this may give an explanation, not only for the high stressful attitude of two-third of our sample but also for the women perversion from anesthesia specialty. regardless of gender bias, conflicts with surgeons could be explained by the facts that surgeons have often been perceived by other physicians with negative features such as aggressive, dominating, cold, impatient, and selfish. these conflicts were commonly attributed to poor communication, and limited contact between males and females in our culture, especially in a society like mansoura. in this study, alternative change of work schedule, delayed response to on call and difficult to manage night shifts were reported by 80%, 54% and 52% of the studied groups, respectively. one study concluded that 85% of women were more prone to modify their job tasks or alter their career for the benefit of their families and children compared to only 35% of male physicians. job stress and its implications were so clear in our study, and this was supported by a previous egyptian study which observed more fatigue and psychological distress among anesthesiologists. kinzl et al., have reported that woman anesthesiologists showed higher concentration and limited possibilities to control work environments as compared to their male colleagues. it is not surprising, that our findings revealed high significant domination of an academic career over noncareer woman anesthesiologists regarding most of the anesthesiology drawbacks reflected on women, which were discussed in this study. hence, we can use our findings to explain the results of another study, which reported that the percentage of women faculty members in all specialties who reach the designation of full professor insulates well-behind that of men, and this is definitely true for anesthesiology, the findings of our study are discouraging as literature has revealed that the healthy development of anesthesiology requires a vital increase in the number of women affiliate to academic anesthesiology. gender disparities in academic promotion leading to less likelihood of external grant funding and fewer publications than men could be a contributory factor. furthermore, the junior partners are assigned to more work pressures, less desirable pay and they are frequently scheduled for later hours, on weekends and during holidays. one of the major limitation is the cross-sectional design of the study that was based on self-reported questionnaires provided by women. furthermore, the small sample size may be another limitation to detect the accurate gender associations. also the exclusion of male's opinions from the survey; and the sociocultural factors of this study that make the number of woman anesthesiologists limited may interfere with the generalizability of our results to anesthesiologists in other societies. however, as we try to recruit most women anesthesiologists working in mansoura hospitals, the results have external validity of women working as anesthesiologists in mansoura, egypt. the study delineated the implications on the personality and family life of women who selected academic anesthesiology as a specialty and career. furthermore, anesthesiology as a profession is significantly benefited by women joining in the academic ranks.

background and aims: we hypothesized that feminization of medicine has an impact on the choice of anesthesiology as a specialty. however anesthesiology is still not a more popular choice among women in medicine. we aimed to evaluate the implications of anesthesiology as a profession on personal and family life of women anesthesiologists; the differences between academic and nonacademic women anesthesiologists with respect to these effects and the effect of women anesthesiologists on the profession. materials and methods: this study surveyed a total of 46 woman anesthesiologists; both academic and nonacademic institutions in our country between january and may 2013. a convenient sampling method was used. a constructed self-reported questionnaire was developed to explore the 29 items of woman-anesthesiology-related implications (family, personal, and job), in the form of close-ended questions. results:negative implications of anesthesiology on personal and family life of woman were common. with the exception of financial limitation, academic group was significantly more affected. increased surgeons conflict (98%), poor surgeon acceptance (87%), poor patient acceptance and recurrent change of work schedule (80% each) were the most common perceived negative impacts of women in the field of anesthesiology. some positive implications were perceived as independent, positive social interaction, empowerment, soothing work environment, emotional reaction to patient complaint, and increased perfectionism (nearly 94%, 98%, 87%, 91%, 89%, and 76%, respectively). conclusion: serious implications exist for the personality and family life of women who chose anesthesiology as a specialty and career, and most of these implications were significantly more prevalent among women with an academic career. furthermore, anesthesiology as a profession was significantly affected by women joining the department.

PMC4353160

pubmed-1351

wiihabilitation is a new idea that came about after introduction of nintendo wii fit (wii) for rehabilitation1. the nintendo wii is a low-cost commercial gaming system that provides an attractive means of facilitating exercise2, 3. nintendo wii fit games encourage lower extremity movement, challenge balance, and require the player to remain in a standing position during play. the wii has been used across a variety of clinical specialties and may potentially be a useful in the rehabilitation of a wide variety of conditions. virtual reality-based training is a feasible and suitable therapeutic intervention for dynamic balance in older adults5, 6. the nintendo wii has been used as a tool that provides a novel way to interact with games in order to promote physical activities. it directs the user s motor response to be either specific or global to train motor abilities such as the range of motion of different limbs and whole body balance training7. addition of the wii balance training program to conventional exercise programs has been recommended to improve ankle muscle strength in patients with functional ankle instability at a low cost8. it has also been reported that virtual reality programs improve the static balance and dynamic balance of subjects with functional ankle instability and can be used safely and efficiently by patients9. the foot and ankle make up complex anatomical structures that contribute significantly to the function of the whole lower limb10. ankle joint stability involves co-contractions of surrounding muscles, recognized as agonist/antagonist activity, to facilitate reciprocal planar motion and protects and ensures efficient function of the joint11. the strength ratios between antagonistic muscle groups have received considerable attention from clinicians during examinations and in monitoring of rehabilitation progress13. also, they are thought to be clinically relevant because co-activation of opposing muscles across a joint may be important in maintaining dynamic joint stability14. the agonist/antagonist ratio was advocated to answer the dilemma regarding a means of more objectively evaluating and comparing the muscle balance (or imbalance) around a joint. ratios for various muscle groups in both the upper and lower extremities have been developed and have become an improved gold standard for evaluation15. impaired foot function can give rise to many problems, such as strains on ligaments, poor co-ordination, and impairment of the function of the muscles12. injuries to the ankle account for a large portion of the injuries to the lower extremity. it has been reported that approximately 80% of ankle sprains lead to injury recurrence and instability16. ankle injury programs for prevention and treatment have disadvantages relating to time and cost. repeated episodes of ankle sprain often occur despite introduction of many rehabilitation programs. further research clearly is needed in this area in terms of both treatment and suggestions to reduce the rate of ankle sprains17. to the knowledge of the authors, there are no previous studies investigating the effectiveness of nintendo wii fit training on muscle strength ratios of ankle muscles. additionally, many studies have investigated the inversion/eversion ratio, but patients still suffer from ankle instability. furthermore, it has been reported that the concentric dorsiflexion/plantar flexion strength ratio was affected in chronic ankle instability (cai) patients. therefore, this study was conducted to investigate the effect of nintendo wii fit training on the dorsiflexion/plantar flexion strength ratio as a new concept in the field of prevention and rehabilitation of ankle injuries. thirty- two healthy male college students from the faculty of physical therapy, cairo university, participated in the study. they were randomly assigned, using simple blind randomization by using procedures such as coin tossing into two equal groups: an experimental group (group i) and control group (group ii) (table 1table 1.general characteristics of the participants in the experimental and control groupsexperimental group (n=16)control group (n=16)age (years)19.6 1.319.6 1.6weight (kg)76.9 5.672.9 6.8height (cm)173.2 5.5171.8 5.7). participants in group i performed the nintendo wii fit training program. participants in group ii were instructed to avoid participation in any strength training programs until after the post-training testing. participants were included in the study if they had good ankle muscle strength (grade four), normal flexibility of the ankle muscles, and a normal foot posture index. participants were excluded if they had a history of previous ankle surgery or ankle injury, had a history of vestibular disorder, had previously participated in a strengthening or athletic training program, or had pronated or supinated foot postures. the foot posture index total score reference values were as follows: normal=0 to+5, pronated=+ 6 to+9, highly pronated 10 +, supinated=1 to 4, and highly supinated 5 to 12. the ethical committee of the faculty of physical therapy, cairo university, approved this study. all participants signed an informed consent form assuring that their participation was voluntary. a brief orientation session about the protocol of the study, its aims, and the tests to be performed was provided to each participant. nintendo wii fit includes over 40 games covering strength training, yoga, aerobics, and balance. the wii training program in this study included an initial balance game (table tilt game) for 2/3 of session s total duration followed by a muscle strength game (standing rowing squat game) for 1/3 of the session s duration. the sessions lasted up to 30 minutes and were performed 3 times per week for 6 weeks. the selected games target agonist-antagonist muscle contraction specifically dorsiflexion and plantar flexion. the participant tries to move a ball into a hole in a tilting platform. the platform tilted according to weight redistribution on pressure sensors in a wii balance board (wbb). this game allowed motion in antroposterior and mediolateral directions around the ankle joint. furthermore, it trained the participant to control the position of the center of gravity (cog) during rapid changes of body movement. the standing rowing squat game is a muscle conditioning game that requires the player to perform squats by flexing the knees from a standing position until the flexed knee reach the required level. the required level was determined by cog movement displayed on the screen as a red dot that must be kept in a blue area in the final position of each squats. they increase joint compressive forces leading to increased joint stability, increased muscle co-activation, and decreased joint shear forces18. they also dynamically recruit most of the lower-body musculature, including the quadriceps femoris, hip extensors, hip adductors, hip abductors, and triceps surae19. a biodex system 3 isokinetic dynamometer (biodex medical systems, shirley, ny, usa) was used for measuring the peak torques/body weight (nm/kg) of the ankle dorsiflexors and plantar flexors to calculate the dorsiflexion/plantar flexion strength ratio. each participant was instructed to perform 5 sets of ankle plantar flexion and dorsiflexion through a 60 range of motion (rom) (from 40 plantar flexion to 20 dorsiflexion) an angular velocity of 60/sec. this is the best angular velocity for measuring muscle strength because it is easy, safe, and a large number of motor units are recruited at low angular velocities20. the test procedures were performed twice, once before and once after the wii training program in the experimental group and at the beginning of the study and then after 6 weeks in the control group. statistical analysis was performed with ibm spss statistics for windows version 20 (ibm corp., armonk, ny, usa). 2 2 mixed design analysis of variance (anova) was conducted to compare the isokinetic ankle dorsiflexion/plantar flexion strength ratio between the experimental and control groups before and after the training. initially and prior to final analysis, data were screened for normality assumption using shapiro-wilks normality test. the mean values of the ankle dorsiflexion/plantar flexion strength ratio decreased significantly (f=7.41, p=0.01) between before and after the training. the mean values of the ankle dorsiflexion/plantar flexion strength ratio were significantly decreased in the experimental group after the training when compared with before the training. however, there was no significant difference between before and after the training period in the control group (table 2table 2.comparison of the mean changes in the ankle dorsiflexion/plantar flexion strength ratio between the experimental and control groupsexperimental groupcontrol groupbeforeafterbeforeafterankle dorsiflexion/plantarflexion strength ratio (%) 78.8 31.757 18.2*66.1 31.758.5 28.6*significant difference within group (p<0.05)).*significant difference within group (p<0.05) nintendo wii fit was released just over six years ago as a mean of improving basic fitness and overall well-being. it has been adopted by a number of practitioners to assist in the treatment of various impairments. however, much work remained to be done to identify which type of participants would benefit from it, which system features are critical, and what types of training routine levels would work best. in addition, there have been few studies with randomized controlled designs that have investigated its effect on agonist/antagonistic muscle ratios and how this kind of training should be carried out. the results of the current study revealed a significant decrease in the ankle dorsiflexion/plantar flexion strength ratio after the training compared with before the training in the experimental group. meanwhile, there was no significant difference between before and after the training period in the control group. this might indicate an effect caused by the wii training program as the control group did not receive training. in the present study, the decreased strength ratio can be attributed more to the increased peak torque of the plantar flexors than the dorsiflexors, as the mean sd values for the peak torque of the plantar flexors increased from 54.1 22.85 nm/kg before the training to 96.1 30.83 nm/kg after the training, an improvement of 42%. on the other hand, the mean nm/kg before the training to 55.73 15.37 nm/kg after the training, an improvement of 17.27%. this may be explained by wii training acting as a form of neuromuscular training with feedback. neuromuscular training is defined as a multi-intervention program with a combination of balance and strength training21, 22. it improves mechanical leg muscle strength due to training-induced gains in neuromuscular function including strength gains23, 24. previous literature reported that lower extremity functional retraining depends not only on lower-limb mobility and bilateral coordination, but also requires other motor skills, such as balance control21. therefore, better controlled movement of joint results from increased muscle strength4. for this reason, combined strength and balance training should be incorporated into lower limb rehabilitation to augment the potential outcomes of training and rapid recovery. moreover, the squat is considered one of the best exercises for improving quality of life because of its ability to recruit multiple muscle groups in a single maneuver26. the squat has biomechanical and neuromuscular similarities to a wide range of athletic movements and thus is included as a core exercise in many sports routines which designed to enhance athletic performance28. the feedback provided in wii training is very important for user motivation and positive reinforcement2. this feedback in the form of either knowledge concerning performance or knowledge concerning results, has been found to enhance motor skill learning29. motion about the ankle joint is controlled by combined actions of at least one pair of opposing muscles (the tibialis anterior and gastrocnemius). therefore, the calf muscles are important in maintaining an erect posture and have a major role in controlling the ankle joint, which is often one of the first joints to help the body restore its equilibrium after perturbation31. pronator weakness, evertor weakness, and calf dysfunction are all terms used to describe the cause of chronic ankle instability cai15. cai patients have higher ankle dorsiflexion/plantar flexion strength ratio) resulting from lower plantar flexors strength32. in the same context, findings of the current study showed a decreased ankle dorsiflexion/plantar flexion strength ratio. for this reason, cai patients may benefit from the wii training principles, as the training program combines the benefits of balance training, closed kinetic chain exercise, and feedback effects. neuromuscular training programs are implemented with the aim of optimizing performance, preventing injury, or providing rehabilitation in functional ankle instability patients33. mcguine and keene34 showed the effectiveness of neuromuscular training in reducing sport-related injury risk. any neuromuscular training program should incorporates a variety of physical activities that supports increasing strength and balance36. this study was limited to healthy male adults who had no ankle deficits, and it only lasted for six weeks. the selected games for the training protocol were (rowing squats and table tilt) activities from nintendo wii fit. based on the findings of the current study, wiihabilitation is recommended as a preventive measure and treatment modality for ankle instability in sedentary individuals and athletes. thus, the field of physical therapy may improve the rehabilitation protocol for ankle injuries by introducing wiihabilitation as a novel method for rehabilitation. further studies should be carried with wider age categories, as this would be valuable for determining the responses of different age categories to wii training. it is recommended to explore the correlation between the effect of wii training and the results of functional performance tests. additionally, extending the training period beyond two months may be advisable to elicit more significant effects.

[ purpose] this study was conducted to investigate the effect of wiihabilitation on the ankle dorsiflexion/plantar flexion strength ratio in adults. [subjects and methods] thirty-two healthy male volunteers were randomly assigned to two equal groups (experimental and control). participants in the experimental group received a wiihabilitation training program for six weeks. data were collected using a biodex system 3 isokinetic dynamometer. peak torques of the dorsiflexors and plantar flexors were measured at an angular velocity of 60/sec which in turn were used to derive the ankle dorsiflexion/plantar flexion strength ratio. [results] the mean values of the ankle dorsiflexion/plantar flexion strength ratio decreased significantly between before and after the training in the experimental group, meanwhile there was no significant difference between before and after the training period in the control group. [conclusion] wiihabilitation has an impact on the ankle dorsiflexion/plantar flexion strength ratio, so it can be considered an effective training tool in terms of the ankle strength ratio. thus, it could be recommended for both prevention and rehabilitation of ankle instability patients.

PMC5088142

pubmed-1352

endoscopic surgery, either by laparoscopy or thoracoscopy, was achieved through a trocar that inserted into the cavity where the target organ was located. all surgeons were obliged to use trocars as long as they adopted the co2 gas insufflation method. because the first trocar was always inserted through the abdominal or chest wall, a risk has always been present of inadvertently injuring an abdominal or thoracic organ, as well as the possibility of bleeding. reusable trocars have the potential advantage of costing less than disposable trocars, if they can be used many times without losing the sharpness of the knife. dull reusable trocars might tear the abdominal wall much more than a sharp disposable trocar on insertion. injury on insertion of the trocar has been among the possible major complications of laparoscopic surgery. the safety shield system in which the knife popped in and out across the tip of the trocar was later developed, and this improvement has decreased injuries. however, a problem that surgeons can not ignore has remained in regard to the procedure for inserting the trocar: bleeding from abdominal wall muscles torn by the trocar knife. drops of blood along the trocar conceal the target organ from laparoscopic view and create problems for surgeons. in a french survey, we developed an ultrasonic (us) vibrating trocar, the sharpness of which was not diminished even with more than 900 repetitive insertions, overcoming the disadvantages of commercially available trocars, and also preventing bleeding from the tract at insertion. however, the friction due to movement of the obturator generated heat in the adjacent tissue along the insertion tract. therefore, we examined the degree of histological damage along the insertion tract and the recovery of abdominal wall tension by comparing port-site skin tensile strength with each trocar, in a porcine model. a generator and ultrasonic vibrating trocar (olympus optical co., tokyo, japan) were used in this study. the frequency of the generator was set at 23.5 khz, the vibration amplitude at 150 m. the ultrasonic trocar is composed of an obturator, which conducts ultrasonic vibratory movements, and a trocar (figure 1). the diameter of the obturator is 12.6 mm, and the diameter of the trocar is 14.8 mm. swine were purchased from sumichiku limited, and six 9- to 11-week-old female swine were raised on specific-pathogen-free food for young swine purchased from shimizukou feed company. general anesthesia was achieved with midazolam (yamanouchi, japan) 0.2 mg/kg and medetomidine hydrochloride (meiji seika, japan) 40 g/kg for presedation, ketamine hydrochloride (sankyo, tokyo, japan) 10 to 15 mg/kg for induction, and isoflurane (dainihon pharm. sodium sulbactam/sodium ampicillin (1.5 g) was administered intravenously during the operation. veterinary ampicillin was administered orally (2 g/day) for 5 days after the operation. the phantom was a block of swine muscle 200 mm x 300 mm x 35 mm. the ultrasonic trocar attached to a digital push-pull gauge (aiko engineering co., tokyo, japan, model-9500) (figure 2) was pushed into the muscle block, and the strength of the force was measured and recorded by a computer. four trocars were compared: a new ultrasonic trocar (usn), a conventional trocar (uso) that had been inserted 900 times, a 12-mm dilating tip (dt) trocar (ethicon endosurgery, usa) and a 12-mm versaport (ve) trocar (us surgical co., tokyo, japan). the force required for insertion into the abdominal wall could be varied according to the means of insertion. therefore, we kept in mind the necessity of arranging the times for insertion so as to be the same length, and compared the levels of force among the trocars. it took an average of 6.5 seconds to completely insert the new and conventional us trocars; the average insertion time was 6.6 seconds for the dt trocar and 6.4 seconds for the ve trocar. the average force was calculated and statistical significance was evaluated with the student t test. an 18-mm incision was made in the skin of a pig, and the us trocar, dilating tip trocar, and versaport trocar were inserted into the abdominal cavity through the incision. trocars were then removed, and the wound was closed with 2 skin staples. bleeding from under the skin was equally controlled by monopolar electrocautery with all 3 types of trocars. in the actual procedure, 1 vertical full-thickness skin stitch up to the muscle fascia layer was placed 1 cm from the wound line, and the suture was retracted with a digital push-pull gauge. the force at which the wound opened 5 mm was recorded as the break strength. the port site not used in the tensile strength evaluation was saved for pathological examination. the full thickness of the abdominal wall around the port was removed, fixed with formalin, and stained with hematoxylin-eosin (he). the temperature around the port site was measured and recorded with a thermal video system tvs 8000 (nihon avionics, tokyo, japan) to detect temperature elevations. statistical analysis was performed with the student t test, and p<0.05 was considered significant. we compared the force necessary to insert the trocar through swine muscle (table 1). the new and conventional us trocar insertion forces were significantly less than the forces of the other 2 trocars, but the insertion procedure was performed in almost the same time. the dt and ve trocars required the same level of force. required force at insertion. usn=new us trocar; uso=conventional us trocar; dt=dilating tip trocar; ve=versaport trocar. the respective break forces at 1 and 2 weeks were 2.4 0.7 kg (n=4) and 4.8 0.3 kg (n=4) for the us trocar, 1.3 0.2 kg (n=4) and 5.7 0.6 kg (n=3), for the dt trocar; and 2.9 0.9 kg (n=4) and 3.9 0.4 kg (n=4) for the ve trocar (figure 3). the break forces for the us and ve trocars were higher than that of the dt trocar at 1 week, while that of the dt trocar was highest at 2 weeks.. tensile strength at the us trocar site was greater than at the sites where the other 2 tro-cars were used. degenerative changes caused by burning were found in the muscle tissue when the us trocar was used. no difference was found between the us and dt trocars in the epidermal layer (figure 4). the width of the scar was greater at the us port site than that at the dt port site. we measured temperature changes produced by the us trocar because the other 2 trocars did not produce friction that generated heat. skin temperature around the port site started to increase after the trocar was inserted, and rose to 78.8c just before the peritoneum was broken. we developed an ultrasonic trocar that does not lose blade edge sharpness, and prevents the problem of bleeding. the insertion force of the new us trocar through the abdominal wall was 34% of the force required by commercially available dt trocars. we examined the force with the conventional us trocar, which had been used 900 times. the conventional us trocar insertion force decreased to only 22% less than that of the new us trocar, maintaining ease of insertion as compared with other trocars. the value of the insertion force for the conventional us trocar was only 42.3% of that of the dt trocar, and 45.8% of that of the ve trocar. the us trocar maintained its capacity even with 900 insertions, and this novel instrument had the added advantage that the heat generated by friction between the ultrasonic vibrating trocar and adjacent tissue induced protein denaturation and thereby decreased bleeding. the force of the us trocar insertion was less than that required by the dt and ve trocars. this meant that the us trocar was inserted through the abdominal wall more easily than either the dt or the ve trocar. when we did not insert the trocar smoothly through the abdominal wall, we pushed the trocar more forcefully than usual. the insertion force through the abdominal wall by the us trocar was less than that required by either the dt or the ve trocar. the us trocar had the advantage of a lower probability of abdominal organ injury when not vibrated, even when an organ was touched, because the edge of the us trocar tip was made nontraumatic in order to decrease injuries. therefore, we speculated that the us trocar might decrease the probability of injuring an abdominal organ, or vessels, or both, and thereby decrease potentially fatal complications. this observation indicated that we had developed a reusable trocar with the advantage of cost-effectiveness. bleeding from the port requiring conversion to open surgery was reported in 10 out of 537 cholecystectomies in the japan society of endoscopic surgery 1998 survey. we have used us tro-cars in clinical practice since 1998 and have performed 35 cholecystectomies, 5 gastrectomies, and 10 colonic resections. we have never encountered bleeding when inserting the us trocar. this advantage may be attributable to the nontraumatic tip of the us trocar, which decreased the likelihood of injuring vessels and may also be ascribed to the ultrasonic vibrating tip dividing tissues and thereby preventing vessels from being torn. the us trocar produced friction that generated heat in adjacent tissues. despite the wider scar generated by the us trocar, as compared to the dt trocar, in he-stained specimens, the tensile strength of port site at 1 week was 2.4 kg, which was no less strong than those of the others. the strength at 2 weeks was 4.8 kg and was almost the same as the 5.7 kg achieved with the dt trocar and was stronger than the 3.9 kg achieved with the ve trocar. these findings confirmed the absence of clinical problems in regard to the tensile strength of the port site with the us trocar. however, this thermal scar might allow surgeons to add a stitch between the fascia split made by the us trocar. the skin burn injury associated with temperature elevation has the potential to delay the healing process. shortening the time of contact between the obturator and tissue could decrease the degree of the burn injury. this was accomplished by making the trocar surface smoother and thinner, which decreased resistance during insertion and allowed the trocar to be inserted quickly. the issue of the burning effect on the peritoneum remains to be resolved, but it is anticipated that resolution will be achieved with a smoother and thinner trocar. we developed a reusable ultrasonic vibrating trocar that could be bloodlessly and smoothly inserted more than 900 times through the abdominal wall.

reusable trocars have the advantage of being more cost-effective than disposable trocars. however, the reusable trocar does lose its sharpness on insertion with repetitive insertion. nonreusable trocars are expensive, but the sharpness of the knife facilitates insertion. nonreusable trocars have a safety shield system designed to decrease abdominal organ injury, though the potential problem of bleeding from the abdominal wall port site has yet to be resolved. we therefore developed a novel ultrasonic vibrating trocar that does not lose its sharpness even with repetitive insertion. this trocar prevents bleeding by means of an ultrasonic cavitation effect. the ultrasonic vibrating trocar has the advantage of ease of insertion, and the force required for new reusable trocar insertion was only 34% of the force required for insertion of commercially available nonreusable trocars. the force required for multiply used conventional reusable ultrasonic vibrating trocar insertion, ie, 900 insertions, was maintained at less than 46% of the force required by the corresponding nonreusable trocars. bleeding from the abdominal wall was prevented by an ultrasonic cavitation effect.

PMC3043436

pubmed-1353

hepatitis b virus (hbv) infection is an important global health problem [1, 2] especially in asia, africa, southern europe, and latin america. about 2 billion people are infected with hbv worldwide [47], among them, 400 million suffer chronic hbv infection. in brazil, the hbsag positivity ranges from 1.6% to 8.5%, corresponding to approximately 3 to 16 million infected individuals [813]. the residual hbv risk in screened blood transfusion ranges from 1: 10,700 to 1: 62,734 and is markedly higher compared to europe, north america [17, 18], australia, and japan. further, the identification of blood donors with occult hbv infection (obi) individuals negative for hepatitis b antigen (hbsag) with detectable circulating hbv dna has created concern related to the safety of the blood supply [21, 22]. hepatitis b virus continues to offer the greatest risk of transfusion-transmission infection despite hbsag screening of blood donations. residual risk of hbv transfusion-transmission results from occult hbv infection, window period donations, and possible escape variants. there is a need to develop cost-effective algorithms using available serological and nat methods to increase blood safety without compromising the availability of blood. false-positive results in anti-hbc testing and false-negative results in nat methods can hamper the identification of occult hbv infection. in anti-hbc testing, false-positive result to anti-hbc are attributed to nonspecific reactions associated with competitive anti-hbc enzyme immunoassay, possiblly due to iga or igm-related molecules produced from nonspecifically activated hbv-specific b lymphocytes or cross-reactivity with other serum components. in donor testing using nat pooling methods, the low viral load found in most obis may affect blood safety and screening algorithms in which confirmation depends upon dna detection. the reasons for low hbv dna levels in absence of detectable hbsag in obi remain undefined, but it is conjectured that both host and viral factors are important in suppressing viral replication and maintaining control of the infection [5, 6, 23]. t-cell mediated responses contribute to the control of viral replication, protect against reinfection, and may lead to spontaneous resolution of infection in obi. patients who successfully clear the virus present a t-cell response to hbsag, core and polymerase proteins [25, 26]. additionally, increased hbcag- and hbeag-specific t-helper cell responses are also seen in patients with viral clearance. the t-cell response in individuals with an anti-hbc only profile showed a typical protective memory when compared to anti-hbc-negative blood donors that have not been exposed to hbv. cellular and humoral immune response pressure against hbv envelope proteins are the principal mechanisms originating obi. in this context, the aim of this study was to develop algorithms/methods to confirm anti-hbc test results. a new method is proposed: detection of hbcag-specific t-cell responses. the blood donor population from colsan-associao beneficente de coleta de sangue (so paulo, brazil) was tested between january 2010 and october 2011. we selected 2,126 donors found positive only for anti-hbc from a total of 125,068 donations. during the process, obi donors were identified, and their hbcag-specific t-cell response was analyzed and compared to donors with chronic (hbsag positive) and recovered (anti-hbc only) infection. as control subjects, we enrolled 100 healthy blood donors that had no reactivity for hbv including hbv pcr, and who were nonreactive for hepatitis c virus (hcv), human immunodeficiency virus (hiv), human t-lymphotropic virus (htlv), syphilis, and chagas diseases. all subjects gave their informed consent, and the study was approved by the unifesp ethical committee. blood units collected at colsan-associao beneficente de coleta de sangue (so paulo, brazil) were screened for the presence of serum anti-hbc and hbsag using a commercial enzyme linked immune-assay (elisa) (biomrieux, france). hbe, anti-hbe, anti-hbs, and core igm testing was performed using the elfa system (biomreiux, france). all the samples were also screened by elisa for possible hcv, htlv, hiv infections, syphilis, and chagas diseases (elisa) (biomrieux, france). serum samples were stored at 20c and thawed immediately before use. for detection of hbv dna, dna was extracted and subsequently subjected to a commercial test for hbv-dna (cobas ampliprep/cobas taqman hbv test, v2.0) with a range from as low as 20 iu/ml to as high as 1.7 e+08 iu/ml. peripheral blood mononuclear cells (pbmcs) were freshly isolated from heparinized blood by ficoll-hypaque density gradient centrifugation with lymphoprep (axis-shield, oslo, norway) as previously described. subsequently, cells were resuspended in complete rpmi-1640 medium (gibco, invitrogen, beijing, china), which contained 10% heat-inactivated fetal bovine serum (gibco, invitrogen, australia), 2 mm l-glutamine, 100 u/ml penicillin, and 100 g/ml streptomycin. t-cells were cultured at 10 cells per ml in rpmi-1640 (gibco, invitrogen, beijing, china) supplemented with 10% heat-inactivated human ab serum, 2 mmol/l l-glutamine, 100 u/ml penicillin, and 100 g/ml streptomycin and incubated overnight at 37c and 5 percent co2. recombinant hbcag (rhbcag) expressed in escherichia coli are based on cloned hbv dna of hbv genotypes a, b, c, d, and f was purchased from diasorin, saluggia, italy. seventeen overlapping 20-mer peptides covering the entire hbv core sequence (amino acids 1 to 183) were synthesized with a multiple-peptide synthesizer using standard 9-fluorenylmethoxy carbonyl chemistry (syro, multisyntech, bochum, germany). recombinant-hbcag (10 g/ml) was added, and cells were further incubated for 5 days. control cultures included 2 g/ml phytohemagglutinin (pha), medium alone, and recombinant hcv ns5 protein, (american research products, belmont, ma). results were expressed as counts per minute (cpm) for pha-stimulated cultures and as stimulation index (si), for example, the ratio between median cpm in the hbcag-containing cultures and median cpm of control hcv ns5-containing cultures. the 99th percentile of hbcag-specific si in 10 healthy blood donors was 2.01. for practical purposes, the antigen used for the human ifn- elispot assays was the same used in hbcag t-cell response (rhbcag). multiscreen-ip 96-well plates (millipore, billerica, ma) were coated overnight at 4c with 10 g/ml anti-human ifn- monoclonal antibody (1-dik; mabtech, sweden). plates were washed seven times with dpbs and blocked with rpmi 1640 supplemented with 10% fetal bovine serum for 2 hours at room temperature. freshly isolated or frozen-thawed after washing, 50 l of 1 g/ml biotinylated monoclonal antibody to inf- (7-b6-1; mabtech, sweden) was added to each well and incubated for 2 hours at room temperature; plates were subsequently washed seven times and 50 l per well of 1 g/ml streptavidin-alkaline phosphatase (mabtech, sweden) was added, and plates were further incubated for 1 hour at room temperature. plates were washed seven times, and 100 l per well of bcip/nbt (diluted by distilled water; zymed bcip/nbt substrate kit, invitrogen, camarillo, ca) was added. after 10 minutes, the colorimetric reaction was stopped by distilled water and washed three times with distilled water. plates were air dried, and the immunospot s4 macro analyzer (cellular technology ltd, usa) was used for spot counting. results were expressed as numbers of spot-forming cells (sfc) per 10 pbmcs. the number of specific ifn--secreting cells was calculated by subtracting the value of the unstimulated control from the value of the stimulated sample. the positive control consisted of pbmcs stimulated with 10 g/ml of pha, and the criteria for a positive response for the ex vivo elispot assays were more than 5 sfc per well and more than twice the number of sfc than the unstipulated control well. therefore, in this study, more than 20 sfc per 10 pbmcs would be a positive response. comparison of paired data from patients at different times of followup was performed by wilcoxon's signed-rank test. correlation between hbv dna and hbc-specific si was assessed by spearman's test. a total of 2,126 of 125,068 donations, (1.7%) were positive for anti-hbc, and none of them had detectable hbsag in the serum. reactivity only hbsag was found in 125 (0.1%) donations, and hbsag plus anti-hbc was observed in 563 (0.45%) donations. both hbsag and anti-hbc were absent in 122,254 (97.75%). among 2,126 (hbsag/anti-hbc+) only 21 samples had detectable hbv dna. among the 125 samples with primary infection (hbsag+/anti-hbc), 27 samples had detectable hbv dna. among 563 samples with chronic infection (hbsag+/anti-hbc+), 438 had detectable hbv dna (figure 1). between january 2010 and july 2010, when hbv dna screening was performed using minipools of 6 samples, we did not observe positive samples in a total of 12.000 screened samples from blood donors with hbsag and anti-hbc negative. in the 21-month study period, 21 blood donors with anti-hbc alone profile were identified as obi (1 in each 5955 donors). in the same period, the number of blood donors with reactivity for hbsag and anti-hbc was 113 (1 in each 1106 blood donors). among the group of 2,126 samples with anti-hbc only, test reactivity (represented as co/s) ranged between 0.008 and 0,975. the samples were divided in two groups based on a threshold of co/s=0.1 and further tested for hbv dna. 21 samples had hbv dna detectable and co/s 0.1 and then was classified as obi, and 2,105 anti-hbc only segregating into 1,293 anti-hbc co/s 0.1 and 812 co/s<0.1 (figure 1). the specificity of the anti-hbc response in these two groups of anti-hbc only samples was investigated further by analyzing the hbcag t-cell response through t lymphocyte proliferation and inf- response to specific and nonspecific stimuli. the proliferative response of peripheral blood lymphocytes to nonspecific (pha) and specific stimuli (hbv core antigen; hcv ns5 antigen) was analyzed in hbv carriers, obi, healthy donors, and two groups based on a threshold of co/s 0.1 and co/s 0.1. the relevant finding was the observation that anti-hbc only subjects with co/s 0.1 (n=1.293) did not have either hbcag-specific t-cells or detectable hbv dna in any analyzed samples. false-positives for anti-hbc, corroborated by the absence of reactivity to anti-hbc in the subsequent donations. the median signal value obtained in the false-positive group was equal to healthy blood donors (si<3,0). all anti-hbc only with co/s 0.1 (n=833) presented hbcag-specific t-cell responses; however, 21 out of 833 presented detectable hbv dna (obi). the median si obtained in hbv carriers and the obi group was 13,6 and 12,2, respectively. the subjects (n=812) with co/s 0.1 and hbcag t-cell response and undetectable hbv dna are classified as spontaneous hbv resolvers. spontaneous hbv resolvers showed a large peripheral blood mononuclear cell (pbmc) response (si=36,2) to hbcag when compared to hbv carriers and obi (si=12,2) (p<0.001) (figure 2). in the subset of 21 obi subjects, 9 collected samples viral load was quantified in all donations and fluctuated between<10 and 118 iu/ml, whereas in three samples hbv dna was not detected, but hbcag t-cell response remained positive in all the study period (figure 3). elispot-inf- assays were positive in hbc only with co/s 0.1, obi, and positive control. the magnitude of t-cell responses to hbcag in hbc only with co/s 0.1 ranged from 1,020 to 1,628 iscs/10 pbmcs, whereas among obi ranged from 132 to 548 iscs/10 pbmcs, among positive controls from 160 to 582 iscs/10 pbmcs, and among hbc only co/s>0.1 and healthy blood donors from 8 to 15 iscs/10 pbmcs. the frequency of positive ifn- elispot responses in spontaneous hbv resolvers indicated a higher chance to have demonstrable hbv-specific t-cell responses than among positive controls (p<0.001) and among obi (p<0.001) (figure 4). serological markers of hbv exposure or vaccination (anti-hbs) were observed in 9.1% (3/40) from primary infection (hbv carriers); 21% (21/100) of healthy donors; 23,5% (303/1293) of hbc only with co/s 0.1; 32.4% (263/812) of hbc only with co/s 0.1; and 10,2% in obi. reactivity to igm anti-core was observed in 2.5% (1/40) and 5.0% (2/21) of hbv carriers and obi, respectively. hbe antigen was observed in all blood donors with obi and in hbv carriers but not in healthy donors and hbc only. however, 10.5% (2/21) of obi and 12.5% of (5/40) hbv carriers showed anti-hbe-positive. in healthy donors and hbc only, no reactivity to anti-hbe and core igm was observed (figure 5). hepatitis b virus presents a higher residual risk of transmission by transfusion than hepatitis c virus (hcv) or human immunodeficiency virus (hiv), especially in countries of south america, like brazil, where the prevalence of hbsag in the general population ranges from 2 to 20%. in this study we found hbv dna detectable in 21.6% of blood donors with primary infection (hbsag (+) anti-hbc () and 77.8% of chronic blood donors (hbsag (+) anti-hbc (+)). these data are consistent with the observation by several authors about hbv persistence in chronic infection [29, 30]. the persistence of hbv dna was observed like obi in 0.98% of the 1.7% of blood donors which were anti-hbc positive, corroborating results of previous studies that observed 3.3% obi subjects into of 5.6% of blood donors with anti-hbc alone profile. the same assays for detection of hbsag and anti-hbc were used in both studies, and the detection limit in hbv dna pcr was similar. in the general blood donor population, the prevalence of obi in this study was 1 in 5,955 (0.017%), and, in the same period, hbv carrier rate was 1 in 1,106 blood donors. the obi among blood donors varies from country to country in different parts of the world. for example, in the european countries such as poland, italy, spain, and germany, the obi prevalence rates of 0.006%, 0.22%, 0.05%, and 0.0006%, respectively, have been reported by candotti et al.. in addition, it seems that the prevalence of obi in blood donors has a regional variance, not only internationally but also nationally. this national discrepancy can be observed in brazil, where hbv prevalence differs regionally, varying from 27% in the north east to 275.6% in north west. in this context, testing for anti-hbc is important, principally in the detection of cases where hbsag is negative and hbv dna detectable (obi). however, a high frequency of reactivity to anti-hbc is observed in brazil ranging from 1.0% to 5.0% in northeast, midwest, southeast, and south of brazil but can reach alarming levels of 75.6% in the amazonia region. in this study, we observed a 1.7% of frequency of reactivity to anti-hbc in the absence of hbsag, according with frequency found in southeast of brazil. however, the presence of hbv dna observed was low representing only 0.98% of the 1.7% of anti-hbc only blood donors. these data raise the question what is the meaning of anti-hbc reactivity in absence of detectable hbv dna in blood donors? in the serological routine in blood bank, it is not uncommon to find isolated reactivity for anti-hbc and hbv dna undetectable in first donation followed by nonreactive anti-hbc results in the next donation, suggesting that the initial result was false-positive. the determination of the frequency of true anti-hbc only in blood donors is very important to blood safety strategy and to indentify recovered infection, escape mutants, and obi. algorithms including multiple successive assays are necessary but not sufficient to identify true reactivity to anti-hbc. to overcome this difficulty, japanese blood banks have set up an anti-hbc cutoff agglutination title of 32 and, below this level, donations are considered noninfectious (presumably false-positive or with undetectable anti-hbs). actually japanese blood bank defers all donors with any reactive result for anti-hbc. in this study we had relevant findings about the correlation between threshold (co/s) values in anti-hbc competition assay and hbv dna detection. among anti-hbc only subjects with detectable hbv dna, all had co/s 0,1. however, among blood donors with co/s>0.1, none had detectable hbv dna. distinguishing in this group between hbv exposure and false-positive to anti-hbc appears to be defined by the hbcag t-cell response. we found that, among the anti-hbc only group with co/s<0.1, and in the presence of hbv dna, all donors had a response for hbcag t-cell in all subjects that we classified as occult hbv infection. in anti-hbc only co/s>0.1 and in the absence of hbv dna when there was an hbcag t-cell response, we classified these individuals as having spontaneous hbv clearance, and in absence of hbcag t-cell response we classified them as false-positive to anti-hbc. these findings are corroborated by the difference in magnitude of hbcag t-cell response between the groups (hbv carriers and healthy groups), obi subjects with serial collected samples and elispot-inf- data. we found a large hbcag t-cell response for spontaneous hbv clearance, positive controls, and obi, all subjects with previous hbv exposure when compared healthy group. we observed hbv dna fluctuation in 9 cases of obi with serial collected samples; however, the presence of hbcag t-cell response was observed in all serially collected samples. in the follow-up of cases of obi, we observed fluctuation of viral load but the hbcag t-cell response was always positive. this is very important to the identification of obi because it eliminates several problems that are due to false results in hbv dna detection, like viral load, assay sensitivity, fluctuation of viral load, repeat of the hbv dna detection, and other factors (mutant, variants, mosaic, and quasispecies). in the other 11 remaining obis, we observed linear hbv dna detection in two subsequent donations. these blood donors will be followed for more two donations, and the results will be analyzed. in transfusion medicine, assays to detect anti-hbc hbcag specific t-cell response showed high sensibility in distinguishing true positive for false-positive to anti-hbc. the results observed in hbcag- t-cell response corroborate with elispot-inf- data. we observed that spontaneous hbv resolvers presented higher frequency of positivity in this assay when compared to hbv carrier, healthy donors, anti-hbc false-positive results, and obi (p>0.001). in addition, the magnitude of t-cell responses to hbcag in hbv spontaneous resolvers was clearly higher when compared to hbv carriers, healthy donors, anti-hbc false-positive results, and obi (p>0.001). these data suggest that antibody titers indeed correlated with hbcag- t-cell response and elispot-inf-. the presence of immune response was found in elispot-inf- in spontaneous hbv resolvers, and obi and hbv carriers are according to findings of zerbini et al. who reported that the presence of memory t-cell response in anti-hbc positive individuals with obi could be important for maintaining viremia under control but persistent. despite all the evidence pointing to efficiency of hbcag t-cell response to confirm anti-hbc result in the competition assay, the discrepancy of hbcag t-cell response in the obi and positive groups when compared to findings of zerbini and cols in 2008 and bes and cols in 2012 was an important point to elucidate. we suggest that the discrepancy between our results and these researchers could be principally due to process of selection of study groups, because if we introduce the group classified for us as false-positive to anti-hbc, the magnitude of hbcag t-cell response is equal between obi and positive control, if it is considered to be the median of stimulation index found in true positive and false-positive to anti-hbc, agreeing with zerbini and bes studies. hbcag t-cell response proved to be an important test to eliminate false-positive results for anti-hbc and that could be used in the research and in blood bank routine. in brazil, the cost of discarding anti-hbc false-positive donation is high, and the cost to use hbcag t-cell response in the laboratory that had previous laminar flow cabinet and co2 incubator is low, five times smaller than the cost of false-positive donation. we can not forget the stigma in the donor to receive a result with positive serology and indirect cost for the public health service to conduct additional tests in order to confirm the positive anti-hbc result. the presence of hbcag t-cell response in the absence of hbv dna in cases of obi suggests that hbcag t-cell response could be a complementary or in specific cases an alternative to nat. even with detecting other serological markers of hepatitis b like core igm, hbeag, anti-hbe and anti-hbs, the identification of obi in anti-hbc only is difficult. firstly, we investigated the concentration of anti-hbs in obi, spontaneous hbv resolvers, and hbv carrier, and we observed that frequency was 10.2%, 32,4%, and 9.1%, respectively. several studies showed that some individuals with obi or hbv, who have recovered from hbv infection, produce neutralizing anti-hbs. in these same individuals we may observe continuous hbv dna replication at low levels that are detectable for years in the liver, peripheral blood mononuclear cells or serum [32, 33, 3639]. the frequency of anti-hbs found in spontaneous hbv resolvers was higher than hbv carriers and obi probably due to important immunity role of anti-hbs in protection against hbv infection. the presence of anti-hbs in obi has been reported by many authors with frequency varying from 0.5% to 15% still tested positive for serum hbv dna, through at a very low titer which is in accordance according with our findings in this study. another serological marker studied was anti-hbe, an important marker of hbv chronic carriage. we observed in obi (10.5%) and hbv carrier (12.9%) the presence of anti-hbe in lower concentration when compared to levels of anti-hbc. this data could be explained by the variable proportion of samples with hbc alone profile, in which anti-hbe is detected. although, with lower titers of anti-hbe compared to anti-hbc, there comes a point when the former is no longer detectable, leaving the latter as the only serological marker of infection. hbeag was observed in all blood donors with viremia, including obi and hbv carrier. igm anti-hbc is the first antibody detectable in acute hbv infection, which is usually detected within 1 month after appearance of hbsag. the presence of igm anti-hbc with high index value usually indicates a recent hbv infection, and this antibody usually disappears within 6 months. however, the index value of igm anti-hbc may increase to levels usually detectable in acute infection in 1020% of chronic hepatitis b patients with acute exacerbation or hepatitis flare that often leads to a misdiagnosis of acute hepatitis b. in our study we observed that the frequency of high index value of igm anti-hbc was 5.0% in obi and 2.5% in hbv carrier, suggesting a recent hbv infection. our data suggest that hbcag-specific t-cell response could be used to confirm anti-hbc serological status distinguish previous exposure to hepatitis b virus and anti-hbc false-positive.

background. during routine donor screening in the blood bank, it is not uncommon to find isolated reactivity for anti-hbc in the absence of detectable hbv dna in a first donation but absence of reactivity to anti-hbc in subsequent donations, suggesting a false-positive result for anti-hbc. study design and methods. the blood donor population was screened between january 2010 and october 2011. we selected 2,126 donations positive only for anti-hbc from a total of 125,068 donations. during the process, obi donors were identified, and their hbcag-specific t-cell response was analyzed and compared to donors with chronic (hbsag positive) and recovered (anti-hbc only) infection. we analyzed correlations between signal levels (co/s) in the competitive assay for anti-hbc and hbv dna detection. results. in the 21-month study period, 21 blood donors with anti-hbc alone were identified as obi (1 in each 5955 donors). the relevant finding was the observation that anti-hbc only subjects with co/s 0.1 did not have either hbcag-specific t-cells or detectable hbv dna and obi subjects presented with co/s 0.1 and hbcag t-cell response. in the subset of 21 obi subjects, 9 donors remained positive for hbcag t-cell response after four collections. in all 9 samples, we observed hbv dna fluctuation. conclusion. our data suggest that hbcag-specific t-cell response could be used to confirm anti-hbc serological status, distinguishing previous exposure to hepatitis b virus from anti-hbc false-positive results.

PMC4437383

pubmed-1354

autoimmune hepatitis (aih) is an immune-mediated chronic liver disease characterized by hepatocellular inflammation, necrosis, and fibrosis, which can progress to cirrhosis and fulminant hepatic failure if left untreated. aih has a female predominance occurring in all ages and races, and both the median and mean age of initial disease presentation are in the forties, similar to many other autoimmune diseases. diagnostic criteria are based on demonstration of characteristic autoantibodies, elevated immunoglobulins, and histological features of hepatitis in the absence of viral liver disease. standard treatment of aih includes corticosteroids alone or in combination with azathioprine (aza) that aims at the normalization of transaminase and immunoglobulin g (igg) levels in serum which correlate with histological disease activity. it is reported that although majority of the patients respond well to the standard therapies, about 20% of patients do not due to either intolerance or refractory disease. in treatment failure, alternative immunosuppressive and biological agents including mycophenolate mofetil (mmf), tacrolimus, cyclosporine and anti tnf antibodies should be considered. the patient we report here is a 48-year old woman who was diagnosed with autoimmune hepatitis and was treated with standard therapy corticosteroids and aza. however, she could not tolerate the side effect of aza induced pancytopenia and skin rash. mmf in combination with corticosteroid was opted as second-line treatment and the patient was able to achieve remission with these treatments. a 48 year-old woman was admitted to department of hepatology due to fatigue and jaundice which lasted for approximately 10 days. she had history of heavy drinking (60 g of alcohol every day) and herbal tea intake (solomons seal tea twice a week) for recent 6 months. on physical examination, icteric sclera was observed but there was neither hepatomegaly nor splenomegaly. upon admission, the laboratory findings were as follows: alanine aminotransferase (alt) level 472 iu/l, aspartate aminotransferase (ast) level 556 iu/l, alkaline phosphatase (alp) 123 iu/l, total bilirubin 3.44 mg/dl, direct bilirubin 1.91 mg/dl and prothrombin time internal normalisation ratio (inr) 1.05. viral markers including hepatitis a antibody immunoglobulin m (hav ab igm), hepatitis b surface antigen (hbsag) and hepatitis c antibody (hcv ab) were negative. since the patient was a middle aged woman, further assessments to rule out autoimmune hepatitis were also performed. anti-smooth muscle antibody (sma) and antinuclear antibody (ana) were positive with the ratio of 1:640 for ana and igg was increased to 1,940 mg/dl. liver biopsy was performed and the histologic findings revealed mild portal inflammation with lymphoplasmacytic, neutrophilic and eosinophilic infiltration along with minimal interface activity (fig. 1). as the histologic findings were not typical for autoimmune hepatitis along with spontaneous slow decrease in ast (48 iu/l) and total bilirubin (0.64 mg/dl) levels the patient was followed up as out-patient without treatment. follow up ana was positive with the ratio of 1:1,280 and igg level was further elevated to 2,420 mg/dl (fig. 2a, b). based on the lab findings and fluctuation of ast and alt levels, diagnosis of autoimmune hepatitis type she was initially prescribed with prednisolone 30 mg which was tapered to 5 mg in in 4 months and was maintained in combination with aza 25 mg daily. the patient showed treatment response with decrease in serum ast and alt levels along with decrease in total igg levels after 3 months of treatment (fig. after 3 months of maintenance treatment, aza-related side effect, pancytopenia and alopecia developed. hemoglobin level decreased to 9.6 g/dl, white blood cell count decreased to 2,150 mm/l (absolute neutrophil count 377 mm/l) and platelet count decreased to 82,000 mm/l (fig. 2c). furthermore, hair loss occurred 2 months after treatment with aza which aggravated until the patient had to wear a wig. therefore, aza was discontinued after 5 months of treatment and was maintained on 5 mg of prednisolone daily. after discontinuation of aza, complete blood count returned to normal range within 1 month while hair growth returned to normal 2 months later. although the symptoms subsided after quitting aza, there were wax and wane in ast and alt levels after 27 months of steroid monotherapy, exacerbation of aih occurred. the steroid dose was escalated to 30 mg again in combination with 50 mg of azathioprine. unfortunately, after 20 days of combination treatment, aza-related side effect occurred again, especially severe alopecia was noted, unabling the patient to maintain on standard treatment. as remission was not achieved with first-line treatment, steroid monotherapy was maintained for another 2 months which was decreased to 15 mg during that duration and mmf 1 g per day was added thereafter. after administration of mmf, her symptoms had subsided and normalization of liver enzymes and igg level was achieved. the patient is still on 5 mg of steroid and 500 mg of mmf and is stable for 12 months after the acute exacerbation of aih. autoimmune hepatitis (aih) was first reported in 1951 as a fluctuating persistent hepatitis of young woman with marked elevations of serum immunoglobulins. the median and mean age of initial disease presentation are in the forties and the etiology of aih is unknown, but it carries all features of an autoimmune disease: genetic predisposition, spontaneous disease fluctuations, circulating autoantibodies, auto-reactive t cells, inflammatory infiltrations in liver, and a good response to immunosuppressive agents [1,5,7-9]. aih can be diagnosed when compatible clinical signs and symptoms, laboratory abnormalities (increased serum ast or alt, and total igg), serological (autoantibodies), and histological (interface hepatitis, lymphocytic and lymphoplasmacytic infiltrates in portal tracts and extending in the lobule, hepatic rosette formation, or chronic hepatitis with lymphocytic infiltration) findings are present; and other conditions that can cause chronic hepatitis, including viral, metabolic, cholestatic, hereditary, and drug-induced diseases, have been excluded. studies reported that when aih is left untreated, as many as 40% of the patients with severe disease die within 6 months and of the remaining 40% develop cirrhosis. therefore, when clinical, laboratory or histologic features of active liver inflammation is present, immunosuppressive treatment should be initiated. standard treatment includes corticosteroids alone or in combination with aza that aims at the normalization of transaminase and igg levels in serum which correlate with histological disease activity. in adults with aih, standard treatment can be started with prednisolone (starting with 30 mg daily and tapering down to 10 mg daily within 4 weeks) in combination with azathioprine (50 mg daily or 1-2 mg/kg body weight) or prednisolone alone (starting with 40-60 mg daily and tapering down to 20 mg daily within 4 weeks). although most of the patients respond very well to immunosuppressive treatment, 20% of patients do not respond to or are intolerant to standard therapy. the overall frequency of aza-related side effect is 10%, which can be improved after dose reduction or discontinuation. aza is a non-selective immunosuppressant that acts by inhibition of several enzymes involved in purine synthesis and this contributes to side effects including bone marrow suppression, nausea, rash, alopecia, arthralgias, neoplastic and malabsorption. the frequency of cytopenia in azathioprine treated patients with autoimmune hepatitis is 46%, and the occurrence of severe hematologic abnormalities is 6%. thiopurine s-methyltransferase (tpmt) is part of a cascade of enzymes responsible for the metabolism of thioprine drugs including aza, 6-mercaptopurine (6-mp) and 6 thioguanine (6-tg). some studies reported that activity of tpmt could be used to predict those who would respond to treatment and those who would show hematologic side effects as the patients with deficient tpmt activity are at severe risk of developing bone marrow suppression [15-17]. however, tpmt testing is not routinely performed as it is time consuming and not widely available in the past 15 years alternative immunosuppressive and biological agents for those who can not tolerate standard treatment were evaluated. unfortunately, the treatment options for patients who failed to respond to standard therapy is still limited. mmf, an ester pro-drug of mycophenolic acid which acts as a noncompetitive inhibitor of inosine monophosphate dehydrogenase, the rate-limiting enzyme involved in the de nevo synthesis of purines were studied as an alternative to standard treatment. according to the aasld practice guidelines, mmf or cyclosporine have had the most empiric use as alternative medications and mmf (2 g daily orally) is the most promising current agent where improvement of hepatitis can be expected in 39-84% of patients. several studies suggested mmf as an alternative treatment for those who are either refractory or intolerant to steroid and azathioprine combination therapy. study by zachou et al. prospectively studied the efficacy and safety of mmf in treatment nave aih patients where 88% of 59 patients responded to the treatment and even allowed rapid steroid tapering with eventually withdrawal of steroid. another study by sharzehi et al. based on retrospective cohort including 90 patients showed that those who were unable to continue corticosteroid and azathioprine well tolerated mmf with 88% of patients maintaining complete remission. these studies initiated mmf at a dose of 1.5-2 g/day in the former while 1g/day in the latter study. the reported side effect was gastrointestinal symptoms including nausea, vomiting and diarrhea, rash and hair loss but no evidence of bone marrow suppression was observed. our patient did not experience any of the mentioned side effects after 10 months of mmf treatment. although the second-line therapy with mmf seems promising, the response rates varied according to reason for stopping aza treatment where patients with aza intolerance had higher response rates to mmf than those who had shown insufficient response to aza (43% vs. 25%). in our case, pancytopenia and alopecia occurred after 3 months of standard treatment (corticosteroid and aza). unfortunately after 27 months of corticosteroid monotherapy, hepatitis flare recurred and was stabilized with addition of aza but again the same side effect, pancytopenia and alopecia developed. like other studies, alternative treatment with mmf was considered as a good option and we expect better treatment response for our patient as she had shown sufficient response to aza.

autoimmune hepatitis (aih) is an immune-mediated chronic liver disease characterized by hepatocellular inflammation, necrosis, and fibrosis, which can progress to cirrhosis and fulminant hepatic failure. the standard treatment for aih includes corticosteroids alone or in combination with azathioprine. although most patients achieve remission using the standard regimen, some patients do not respond due to either drug intolerance or refractory disease; in such cases alternative immunosuppressive agents should be explored. the second-line therapies are cyclophilin inhibitors such as cyclosporine a or tacrolimus, and nowadays mycophenolate mofetil (mmf) is widely used if azathioprine-based therapies are not tolerated. although these are recommended as an alternative to the first-line regimen, there is insufficient evidence for the efficacy of second-line therapies, with the evidence based mainly on expert opinion. therefore, we report an aih patient receiving the standard regimen in whom remission did not occur due to side effects to azathioprine, but was successfully treated with mmf in combination with corticosteroids as an alternative to the standard regimen.

PMC4946399

pubmed-1355

hepatitis b virus (hbv) can cause chronic and acute infection and remains a global health problem with a considerable morbidity and mortality. it was estimated that there are currently more than 350 million carriers all over the world. chronic hepatitis b virus (hbv) infection is a major cause of hepatocellular carcinoma (hcc). hbv chronic carriers, including those carrying vaccine-escape mutants, have a greater than 100-fold increased relative risk of developing the tumor. despite characterization of integrated hbv genome in chromosomes of hcc, a comprehensive understanding of the underlying mechanism needs further elucidation. tumor progression is a complex multistage process, making the cells from normal to malignant by a series of alterations including cell adhesion and movements. one of the important characteristics of the tumor cells is that they have the ability to escape from the normal controls during proliferation, differentiation, apoptosis, and migration. in metastasis, tumor cells detached from their place of formation, move, and form new tumors in distant healthy tissues. cell migration is a key activity of many important normal and abnormal biological processes, including tumor cell metastasis. adhesion of tumor cells to host cell layers and subsequent transcellular migration are important processes in cancer invasion and metastasis, which involves the extracellular matrix (ecm) degradation [810]. high motility and cell migration might be the premonition of malignancy of chronic viral infection. the rho small gtpases, which contains the rho, rac, cdc42 subfamilies, regulate cell migration through the reorganization of actin cytoskeleton, which is considered as a driving force of cell motility [11, 12]. the rho gtpases act as molecular switches cycling between active gtp-bound forms and inactive gdp-bound forms. such cycling is regulated by guanosines nucleotide exchange factors (gefs) and gtpase-activating proteins (gaps). in addition, rho gtpases activation has been linked to virus replication such as hiv-1, hsv (herpes simplex virus) and hbv [1517]. rho a, rho c, and rac1 have been reported significantly elevated in a variety of tumors, especially in the more metastatic forms [18, 19]. during the tumor progression, the loss of the cell characteristics results in morphological changes, such as cell-cell adhesion, gene expression, and motility rate. for example, the adhesion velocity has been shown to be affected by hbv replication. our laboratory has recently reported that hbv-replicating hepg2 cells display similar morphology with those expressing the constitutively activated rac1, mediated by hbx. hbx can directly interact with a rac1 nucleotide exchange factor pix, by an sh3 binding motif and affect the cell morphology. our data are in line with earlier reports on the potential invasion ability of hbx through indirect activation of matrix metalloproteinase (mmps) [21, 22]. we now report that the rac1 gtpase was involved synergistically with hbx in the migration of hbv-replicating cells. the replicative hbv genome comprising a 3.5 kb linear fragment was constructed as described in mammalian expression vector pcdna3.1 (invitrogen, usa), using a two step cloning strategy. wild-type replicative hbv genome was used as template to generate proline to alanine mutation by site-directed mutagenesis as described in. four prolines in hbx on the replicative genome were mutated into alanine: esrgr29plpg33plgalppas42p43pivptdh to esrgr29alpg33algalppas42a43aivptdh. the plasmids of rac1 wide type, constitutively activated rac1, and dominant negative rac1 were constructed into a mammalian vector pxj40 with a ha tag at n-terminal as described in [24, 25]. hepg2 cells were maintained and passaged in gibco dulbecco's minimal essential medium, supplemented with 10% fetal bovine serum, 1% antimycotic, and controlled 5% co2. rna of the hepg2 cells transfected with various plasmids was extracted using rneasy mini kit (qiagen, usa) as described in. the concentration and quality of rna were obtained by the absorbance at od260 and the absorbance ratio of od260 and od280. rt-pcr was performed to confirm gene expression from the replicative hbv genome as described in. the primers used for rt-pcr were as follows: -actin forward: 5-cttagttgcgttacaccctttc-3, -actin reverse: 5-accttcaccgttccagtttt-3, hbsag forward: 5-tcaccatattcttgggaacaa-3, hbsag reverse: 5-gttttgttagggtttaaatg-3, hbcag forward: 5-atctcctagacaccgcctca-3, hbcag reverse: 5-ttccaaattattacccaccc-3. hepg2 cells transfected with individual constructs were collected 48 hours after transfection. western blot analysis was carried out using specific anti-ha or anti-actin antibodies as described. cell motility was assessed by transwell migration assay kit (bd falcon, usa). the inserts were put into the 24-well plate and both sides of the membrane of the inserts were coated with collagen (bioscience, usa) for more than 2 hours. 36 to 48 hours after transfection, the hepg2 cells were trypsinized and cell number was adjusted to 20,00030,000 cells per 200 l volume by cell numbering. the cell suspension was deposited onto the collagen-coated membrane in each insert in the 24-well plate. the plate was put into 37c, 5% co2 incubator and cultured for 8 hours to overnight. this was needed to allow cell migration to take place, from the upper side to the lower side of the collagen-coated membrane. the medium was then removed and the upside cells of the inserts were gently mopped with cotton bars. the cells at the lower side of the membrane were fixed with 100% methanol for 15 minutes. the cells were stained with 0.04% giemsa solution (sigma aldrich, usa) for 1015 minutes. four fields were randomly selected in each insert for counting and microscopic images were taken. hepg2 cells were transfected using nucleofector to achieve high efficiency of transfection, as indicated by the high percentage of cells expressing egfp (figure 1). the expression of hbsag from the replicative genome in transfected hepg2 cells was assessed by rt-pcr, whereas the expression of wild-type rac1, rac1, rac1 was determined by western blot analysis using anti-ha antibody.how ever -actin was used as an internal control. results were shown in figure 2 and table 1 showed the normalized densities of the bands shown in figure 2(a). the density levels of rac1 wt were used as standards, other bands were normalized based on it. it indicated that all three constructs of rac1 gtpase were successfully expressed in hepg2 cells at comparable level. in addition, both hbsag and hbv core antigen (hbcag) could be detected in hepg2 cells transfected by replicative hbv genome (lane 2, figure 2(b)) and p-a mutant hbv (lane 3, figure 2(b)), with the empty vector pcdna3.1 used as control (lane 1, figure 2(b)). the motility of hepg2 cells expressing rac1 alone, and those expressing both rac1 and replicative hbv genome was then analysed by transwell assay, as indicated by the number of cells migrated to the lower side of the collagen-coated membrane (see section 2). seven sets of constructs were used in this analysis: wild-type rac1 alone; constitutively activated rac1 alone; dominant negative rac1 alone; wild-type rac1 and wild-type hbv genome; wild-type rac1 and p-a mutant hbv genome; wild-type hbv genome alone; and p-a mutant hbv genome alone. results shown in figure 3 indicated that hepg2 cells displayed differential motility depending on the transfected constructs, with the highest number of cells (indicated by arrow heads) in cells transfected either with rac1 alone (figure 3(b)) or with wild-type rac1/wild-type hbv genome (figure 3(d)). a graphical representation of the motility assay was summarized in figure 4 after cell counting and analysis. hepg2 cells transfected with the constitutively activated rac1 showed a 2.5-fold increase in cell motility (lane 2, figure 4) as compared with those expressing wild-type rac1 alone (lane 1, figure 4) or those expressing the dominant negative rac1 (lane 3, figure 4). interestingly, cells co-transfected with wild-type rac1 and replicative hbv genome showed similar motility as those transfected with constitutively activated rac1. this suggested that the cell motility was enhanced by hbv replication, likely via activation of wild-type rac1 to its gtp-bound state as recently reported. to validate our findings, the p-a mutant hbv genome (with four proline residues mutated to alanine residues in the proline rich domain) was co-transfected with the wild-type rac1. results shown in figure 4 (lane 5) indicated that the p-a mutant hbv genome abrogated the cell motility significantly, to a level comparable with expressing cells. meanwhile, the expression of replicative hbv genome itself enhanced the cell migration also (lane 6), which may imply the endogenous rac1 activation by the hbv replication. not surprisingly, the p-a mutant hbv genome abrogated the cell motility (lane 7). how ever the cell migration level caused by endogenous rac1 was less than that caused by the overexpressed rac1 v12, which suggested that endogenous wild-type rac1 was not sufficient for the activation caused by hbv replication. and such activation was confirmed by the augmentation of cell movements induced by overexpression of rac1 wt. in other words, the endogenous rac1 was enough to be activated by hbv replication and passed away cellular signal inducing high cell motility, but the over-expressed rac1 can even increase such activation in a certain degree. taken together, our results suggested that the cell migration can be caused by hbv replication through the activation of rac1 gtpase. rho gtpases comprise a family of proteins which are highly conserved from lower eukaryotes to plants and animals. rho gtpases were implicated in various cellular processes including cytoskeletal reorganization, cell cycle and division, motility, angiogenesis, and phagocytosis. for cytoskeletal reorganization, processes such as cell adhesion, cell migration, cell polarity, spindle formation and locomotion were covered. it has been reported that the hbx activates erks and pi-3k/akt pathways25, and further activates matrix metalloproteinase-919 with a potential function of invasion and metastasis. we have recently demonstrated the same activation of erk1/2 and akt by hbx using sh3 binding domain located in hbx. our recent investigation has also indicated that rac1 gtpase can be activated by hbv replication, which in turn sustains hbv replication. interestingly, the activation of rac1 and hbv replication has been found to be significantly decreased in cells transfected with p-a mutant hbv genome. our findings can be further validated by careful design of hbx knockdown in the context of the whole hbv genome, without affecting the proper viral replication. furthermore, the involvement of rac1 can be supported by titration experiments in which the same amount of hbv genome is introduced into the cells with various amount of rac1 (activated v or inactivated n). findings in this report have provided an extension on the consequences of the rac1 activation by hbv replication, in terms of cell motility. to our knowledge, this is the first report to show the potential relationship between proline rich domain in hbx (in the context of hbv replication) and cell motility, and should shed new lights on the understanding of the mechanism of hcc development.

hepatitus b virus (hbv) is a major cause of the development of hepatpcellular carcinoma (hcc). one of the significant characteristics of tumor progression is cell migration which is reflective of cytoskeletal dynamics. the rho gtpases contribute to a multiple cellular processes, including the cellular cytoskeletal reorganization and motility. it has been found that some rho gtpases have oncogenic activity and can promote cancer cell invasion. here we discuss one of the rho gtpases, rac1 can be activated by hbv replication and such activation results in the high motility of hbv-replicating cells. the enhanced cell motility can be interestingly alleviated by the mutation at the sites of proline rich domain located in hbx. our findings may provide new insights on the mechanism of hcc development associated with chronic hbv infection.

PMC2744885

pubmed-1356

the scope of newborn screening has expanded greatly in the 52 years since robert guthrie published his microbial assay for phenylalanine and the use of dried blood spots in screening for phenylketonuria. though guthrie and others went on to develop blood-spot screening methods for further disorders none were taken up very widely until the development of immunoassays and the ability to screen for congenital hypothyroidism, now the most widely practiced screen worldwide. immunoassay-based screens for other disorders followed, cystic fibrosis and congenital adrenal hyperplasia in particular, but none have achieved general acceptance. more recently, tandem-mass-spectrometry (ms-ms), with its ability to measure a wide range of metabolites simultaneously, has opened up a further range of disorders, many of which are very rare but can be incorporated with screening for phenylketonuria at little additional cost. thus, in large parts of the usa screening covers over 50 conditions (1) though this total is inflated counting sickle-associated haemoglobin variants as separate diseases. european countries with equally well-developed medical systems have very different screening policies: in 2009, the maximum number of conditions covered was 30 and some countries were screening for four or less (2). there was little consensus regarding information provision, informed consent, storage and disposal of residual specimens, screening technologies and disease definition. the reasons for these differences are not always obvious though clearly funding issues and the availability of an adequate clinical infrastructure must play a part. all formal policy reviews have acknowledged the ten principles enunciated by wilson and jungner in 1968 (3). these are couched largely in qualitative terms, there should be a suitable test which should be acceptable to the population and there should be an accepted treatment. nevertheless, the uk national screening committee has until very recently emphasized the importance of having firm numerical data and evidence from a high-quality randomized controlled trial before accepting a new condition (4). however, it is recognized that for clinically heterogeneous and relatively rare disorders, such as those now readily accessible through ms-ms screening, a degree of compromise is required. medium-chain acyl-coa dehydrogenase deficiency was accepted on the basis of a large-scale pilot study (5) and preliminary studies on five additional disorders detectable by ms-ms are underway. the approach in the usa has been quite different, with federal policy based largely on the results of a survey carried out by the american college of medical genetics (6). this canvassed opinions (rather than data) from a wide range of professional and lay personnel in the usa and abroad and commissioned literature reviews on specific diseases from practicing academic clinicians. this resulted in the identification of 29 conditions for which screening should be mandated and an additional 25 conditions that would be identified as part of differential diagnosis. the work was subsequently taken forward and amplified by the secretary s advisory committee on hereditary disorders in infants and children (7), forming the basis for both federal and state legislation. screening policy is ultimately decided at state level and based on explicit legislation, with significant regional variations in both the number of disorders covered (1) and practices such as how long the blood collecting cards are retained after screening has finished (8). this overt political dimension leaves policy-makers exposed to pressure from groups such as the save babies through screening foundation and there is almost a degree of competition as to which state screens for the most disorders. in countries like the uk where screening is offered as part of the national health service with no direct legislative basis policy tends to attract less public attention though nevertheless the uk screening programmes for cystic fibrosis and sickle cell diseases were introduced following direct pressure at the political level rather than through the formal advisory channels. the wilson and jungner analysis was based mainly on adult disease and largely neglected the family dimension of newborn screening: the value of genetic information and the way that prompt diagnosis, even of an incurable condition, can ease the family journey. family impact is the main rationale behind the welsh screening programme for duchenne muscular dystrophy which has been well-accepted locally but requires considerable professional input and is not supported in the rest of the uk. cystic fibrosis is an obvious example as in the absence of screening diagnosis is often considerably delayed, leading to significantly worse outcomes. diagnostic odyssey and has no warning of the possibility of future children also being affected. attitudes of at-risk couples towards antenatal diagnosis of cystic fibrosis are ambivalent (9) but overall the introduction of newborn screening has resulted in a significant decrease in the number of babies born with the condition (10). even before antenatal diagnosis became possible families with a phenylketonuric child diagnosed by screening tended to limit family size subsequently (11). however, in general policy-makers focus their attention on the value of newborn screening to the baby concerned and have mixed views about wider genetic implications. genetic exceptionalism is the view, strongly held by some civil liberties groups, that genetic information is special and must be treated differently from other types of medical information. dna analysis may reveal carrier status in individuals with no family history of the disorder concerned and in some countries is prohibited without a specific request from a registered medical practitioner. such sensitivities are reflected in some of the screening protocols, particularly those for cystic fibrosis. several screening programmes have retained the original two-step irt approach and require a second blood sample despite the convenience and greater specificity of analysing the cftr gene on the initial sample. programmes that do use dna analysis vary greatly in the mutation panel used: some restricting it to a few relatively common mutations associated with severe disease, others using a much broader panel with a corresponding increase in the number of unaffected carriers found. other programmes include a pap (pancreatitis associated protein) step in an attempt to maintain specificity but minimise or avoid carrier detection (12, 13). the uk protocol attempts to minimise carrier detection but by different means (figure 1, panel a) while also recognising that a mutation panel optimised for northern european populations has inherent disadvantages for those of other geographical origin (panel b). complicated protocols of this type place a considerable administrative burden on the laboratory. travelling in the opposite direction, studies of the potential of exome sequencing as a first-line method of newborn screening and the possibility that genomic data be made available as a resource for parents and doctors throughout infancy and childhood to inform health care there is usually less anxiety about genetic information obtained at the protein level even though, with haemoglobin variants for example, this may inform directly on the underlying dna sequence. indeed, when a baby has received a blood transfusion prior to the screening sample being taken some programmes use dna analysis rather than waiting several weeks for the infused erythrocytes to be removed. newborn screening for sickle cell diseases and related haemoglobin abnormalities detects a significant number of carriers: 1 baby in 71 born in england in 201112. there are different views on whether this is desirable despite the presence of an antenatal screening programme and, as far as possible, linkage between the two programmes. ms-ms methods capable of detecting individual haemoglobin variants without the corresponding carriers have been developed (15) and it remains to be seen whether this will affect practice. with improvements in analytical performance the definition of normal has tended to be drawn ever more tightly. however, the primary purpose of screening is prevention or amelioration of disease rather than the detection of a biochemical/genetic abnormality as such. thus, not all children with a persistently increased blood phenylalanine concentration will become mentally impaired: there is fairly good evidence for a threshold of about 600 this threshold has been adopted in france and germany but is ignored in many other countries. similarly with congenital hypothyroidism, improvements in assay sensitivity have resulted in many screening centres adopting lower cut-offs and consequently finding an increased number of cases. however, their gender distribution is markedly different from that in cases diagnosed clinically prior to the start of screening and there is no firm evidence that most of these additional cases benefit from their treatment. the biological level (gene, protein, enzymic activity, metabolic pathway, individual metabolite) at which screening and confirmatory investigations are performed also has an important impact on disease definition. thus, medium-chain acyl-coa dehydrogenase deficiency (mcadd) is readily detectable by increased blood-spot octanoylcarnitine on ms-ms screening. in the netherlands enzyme assay using phenylpropionyl-coa is regarded as the gold-standard confirmatory test. the current uk screening protocol defines mcadd in terms of having two mutations in the mcad (acadm) gene and genotyping is central to the clinical follow-up protocol. however, data from the pilot study (5) show that all babies except one with genotypes that have previously been reported from clinically-presenting cases showed urinary hexanoylglycine>5 micromol/mol creatinine on immediate follow-up. this would appear to be a more effective indicator of the risk of metabolic decompensation (which is what we are really wishing to screen for) than mutation analysis and avoids the complication of inadvertent carrier detection. viewed in the context of everything else that can go wrong in pregnancy, childbirth and afterwards, newborn screening seems a fairly insignificant thing to worry about but the importance of having suitable information available if required is universally recognized. the way that this is provided and the amount of detail given vary considerably. the uk national screening committee requires that evidence-based information, explaining the consequences of testing, investigation and treatment, should be made available to potential participants to assist them in making an informed choice. the emphasis on choice is in keeping with current political philosophy but it could be argued that if parents were truly informed then choosing to have their baby screened would be automatic. even carefully worded information can leave room for misunderstanding: we read the form and our understanding was that the conditions were hereditary. we did nt want to cause baby the pain of having a needle in her heel. come on then let s get on with it. we both looked at each other and were afraid to say anything so the baby screamed and we felt bad for doing it. she saved our daughter (with phenylketonuria) to grow up a lovely healthy bright little girl (17). should we really be presenting screening as an option which rational parents might like to consider ?. in some us states screening is compulsory, required by law, so that (in theory at least) every baby gets screened. the supreme court of ireland upheld the right of parents who already had a child with phenylketonuria not to have their newborn baby screened (18). the united nations convention on the rights of the child states that the best interests of the child must be a top priority in all actions. however, whether screening is truly in the interests of the affected child is sometimes debatable, particularly for disorders where treatment is invasive and the effect is to slow the progression of a debilitating disease rather than prevent it. most disorders show some degree of heterogeneity and this may make a clear answer impossible: for example, severe propionic acidaemia is almost untreatable, with an inexorable deterioration, but patients with milder variants can survive and go on to lead virtually normal lives if diagnosed and treated early. the current diversity of national policies relating to newborn screening reflects both practical limitations in health care organization and funding and widely different philosophical approaches to a variety of highly emotive issues. decisions will become more difficult still as screening technology advances, with the use of orbitrap mass spectrometry for example, and the number and variety of disorders readily accessible increases still further.

summarynewborn blood-spot screening to detect potentially treatable disorders is widely practiced across the globe. however, there are great variations in practice, both in terms of disorders covered, screening technologies, disease definition, information provision, parental informed consent, and storage and disposal of residual specimens, partly reflecting the degree to which screening is the subject of explicit legislation (and thus public and media pressure) or is embedded in a general health care system and managed at an executive level. it is generally accepted that disorders to be screened for should comply with the ten wilson and jungner criteria, but the way that compliance is assessed ranges from broadly-based opinion surveys to detailed analysis of quantitative data. consequently, even countries with comparable levels of economic development and health care show large differences in the number of disorders screened for. there are several areas on which there are no generally accepted guidelines: how should parents be informed about screening and to what extent should they be encouraged to regard screening as an option to choose to refuse? is dna mutation analysis acceptable as part of a screening protocol? how soon should the blood samples be destroyed once screening has been completed? as technology advances and the potential scope of screening expands at both the metabolite and genome level, challenging policy issues will have to be faced.

PMC4922336

pubmed-1357

it is a leading infective cause of mortality in children under 5 years of age. in 2013, bronchopneumonia caused death in 935,000 of children under 5 years. since pediatric population is vulnerable and specific, clinical features are often non-specific and conditioned by numerous factors. these factors include certain age group, presence of comorbidity, exposure to risk factors, carried out immunization etc. the most reliable way to diagnose bronchopneumonia is through chest x-ray, but that is not enough to determine the ethiological agent, so the treatment of bronchopneumonia is clinical rather than ethiological in most cases. since bronchopneumonia is an infectious disease, antimicrobic agents must be used in the treatment, along with additional supportive and symptomatic treatment. however, frequent use of antibiotics leads to a rise in bacterial resistance (1). bacterial resistance, along with limitations in establishment of timely diagnosis and difficult ethiological classification, often leads to severe clinical features and inadequate response to therapy, which results in increased number of treatment days, as well as increased consumption of antimicrobials. the aim of this study was to determine the most often proscribed antibiotics and supporting concomitant therapy in treatment for bronchopneumonia at the pediatric clinic in sarajevo, and to determine whether the treatment is in accordance with the british thoracic society guidelines. the study included patients under 18 with diagnosis of bronchopneumonia, patients with detailed history of the disease and detailed information on diagnostics and treatment carried out at the pediatric clinic, and patients who were hospitalized in the pulmonary department in the period from 1 july to 31 december 2014. analysis results are displayed in tables and graphs as per number of cases, percentage, and arithmetic mean (x) with standard deviation (sd), standard error (se) and a range of values (min-max.). testing of differences between age groups was performed using wilcoxon signed rank test and one-way analysis of variance (anova) with significance level of p<0.05 which was considered to be statistically significant., there was a higher number of male subjects (60 or 57.7%) than female patients (44 or 42.3%). according to the formed age groups, the highest number of patients was in the preschool and school age groups (39 patients each or 37.5%), followed by the age group of infants (22 or 21.2%). the youngest patient was 1 month old and the oldest was 192 months old (16 years). according to the data in patient s history, some of the symptoms were dominant in clinical features. cough was present in 88 or 84.6% of patients, with average body temperature of 38.70.9; 37-40.2 c. chest pain was experienced by 66 or 63.5% of patients, and vomiting was experienced by 64 or 64.5% of patients. duration of hospital stay (number of days of hospitalization) averaged 5.22.6 days, with shortest stay of 1 day and longest stay of 15 days. out of total number of subjects, 66 or 63.5% were immunized regularly. in the period of the study (july-december), the largest number of patients67 of them (64.2%)had three dominant symptoms in clinical features. in newborn and infant age group, the following symptoms prevailed: cough, increased body temperature, and vomiting. in preschool children age group, cough was present in 87.18%, increased body temperature in 97.44%, chest pain in 66.67%, and vomiting in 41.03% of the subjects. in school children age group, cough as a symptom was present in 79.49 %, increased body temperature in 94.87%, chest pain in 94.87 %, and vomiting in 7.69% of the subjects. in adolescent age group, cough, chest pain and increased body temperature were present in all subjects, while vomiting was not observed at all. upon admission, crp was elevated in 100% of newborns and adolescents, in 81.82% of infants, 79.49% of preschoolers, and in 92.31% of school-age children. upon admission, white blood cell count was elevated in 50% of newborns, 72.73% of infants, 71.79% of preschoolers, 58.97% of school-age children and in 100% of adolescents. used antibiotic therapy penicillin antibiotics were administered intravenously to 26 (25%) subjects. within this group, the most widely used medicine was ampicillin (17.68%), with an average dose of 1138.89 491 mg (450- 2000), and the average duration of therapy 3.56 1.42 days (table 1). analysis of use of penicillin group of antibiotics first-generation cephalosporins were administered to 42 patients (40.4%), intravenously in all cases. the only medicine in this group that was administered was cefazolin with average dose of 1,464.3 530 mg (300-3000) and average treatment duration of 4.3 1.6 days (2-7). analysis of use of the i generation cephalosporin third-generation cephalosporins were administered to 33 or 31.7% subjects (intravenously in all cases). the most often used drug in this group was ceftazidime with an average dose of 1568 585.34 mg (250-2400), and the average duration of therapy was 5.76 2.62 days (figure 1). analysis of use of the iii generation cephalosporins total duration of the antibiotic therapy averaged 4.5 1.9 days and ranged from 1 to 11 days. recommended therapy for treatment continuation medicines from the group of penicillins were recommended for continuation of treatment of 28 patients (26.9%). the most commonly recommended medication in the aforementioned group was ampicillin in two forms (suspension and tablets). the average dose of ampicillin was 11.2 4.59 ml in form of suspension and 1.160 634.35 mg as tablet. the most recommended medication in this group was cefixime, with an average recommended daily dose of 8.74 4.78 ml in the form of a suspension and 828.57 455.8 mg in the form of tablets (table 3). among other drugs recommended for treatment continuation at home, antipyretics were recommended in 54 or 51.9% of cases, methylprednisolone in 53 or 51% of the cases, and montelukast in 75 or 72.1% of cases. we present the results of 104 patients who were hospitalized at pulmology department of the pediatric clinic with diagnosis of bronchopneumonia. according to the results of our study we recommend oral administration of first-generation cephalosporins and penicillin antibiotics as effective treatment for bronchopneumonia in the pediatric population. in the last 30 years, a lot of research has been done with the purpose of achieving a more effective treatment of bronchopneumonia in the pediatric population and a reduction in bronchopneumonia-caused mortality. the turning point was year 1985 when the world health organization undertook activities to establish a unified strategy to combat pneumonia worldwide (1). the pediatric clinic of the university clinical center of sarajevo has also based its principles of treating bronchopneumonia on observing guidelines and protocols, as well as principles of good clinical practice. hence the usual empirical treatment is based on proven connection of certain causative agents with specific populations, while etiological treatment is very rare. a study done on 385 hospitalized children in africa in 2014 found that there is a very low risk of failure when using drugs mentioned in the guidelines and protocols relative to the targeted etiological treatment (0.37 (95% ci -0.84 to 0.51)(2). according to the research done by the world health organization, for quality management of bronchopneumonia, certain criteria must be met in order to refer a child to hospital treatment: a child with persistent high body temperature or fever should be considered as a potential pneumonia patient; if symptoms persist or if there is no response to treatment prescribed by pediatricians or family doctors, it is necessary to reassess and to consider the gravity of the clinical situation; children with less than 92% oxygen saturation or children who show severe signs of respiratory distress should be hospitalized; auscultatory absence of respiratory sounds and dull sound on percussion indicate the possibility of pneumonia with complications and can be used as an indication for hospital admission; children with elevated parameters of acute inflammation; children under 6 months of age with signs of disease and children with poor general health (3). treatment of bronchopneumonia involves administration of medicines and use of high calorie dietary regimes with adequate hydration. antimicrobials used in the treatment of bronchopneumonia are first and third generation of cephalosporins, as well as penicillin based antibiotics. in our study, antibiotic therapy lasted 4.5 1.9 days on average and ranged from 1 to 11 days. cefazolin in the group of first-generation cephalosporins was administered in 42 patients, or in 40.4% of all subjects. in all patients, cefazolin was administered intravenously at a dose of 1,464.3 530 mg (900-3,000) and the average duration of treatment was 4.3 1.6 days third-generation cephalosporins have been administered intravenously to 33 patients, or 31.7%. the most commonly used medicine in the group of third-generation cephalosporins was ceftazidime. a total of 17 subjects in the treatment received ceftazidime, the lowest dose was given in infants (900 mg) and the highest dose was given to school-aged children (2,400 mg). ampicillin as the most often used drug from the penicillin group was administered in 18 patients with an average dose of 1,173.1 500 mg (450-2,000) and the average treatment duration of 3.96 2 days. studies in india from 2013, conducted on a total of 1,116 children at the pediatric departments in 20 hospitals, showed that the treatment with penicillin antibiotics is more effective in comparison to treatment with other antibiotics (4). study showed that the second and third generation of cephalosporins were used in infants, but not in adolescents. in the treatment of children of preschool age, first-generation cephalosporins were most often used, while third-generation cephalosporins were most often used in school-aged children. in determination of difference in the use of antibiotic therapy in relation to age of patients, statistically significant difference was only demonstrated in the use of penicillin antibiotics (p<0.05). according to the dose of administered antibiotic, it has been shown that dose increases linearly with age, with the lowest dose administered in infants. a significant difference was observed only in patients to whom cefazolin and ceftriaxone were administered (p<0.05). there were no statistically significant differences in average duration of treatment in relation to age group (in all<0.05), but still there were some noticeable differences. duration of treatment with third-generation cephalosporins was longest in infants (7 days) and shortest in children of preschool age (4.7 days). according to the guidelines of the british thoracic society, certain guidelines every child with a clear diagnosis of pneumonia should receive antibiotic therapy since it is not possible to make immediate reliable differentiation of bacterial and viral pathogens (5). intravenous administration of antibiotics is recommended for children suffering from pneumonia in cases when a child can not tolerate oral intake of drugs or their absorption (i.e. due to vomiting), and also for hospitalized children with more severe clinical features (6). recommended intravenous antibiotics for treatment of severe bronchopneumonia are: amoxicillin, co-amoxiclav, cefuroxime and cefotaxime or ceftriaxone. use of these antibiotics can be rationalized if microbiological diagnostics is performed (7). it is advisable to consider oral administration of medicines in patients to whom antibiotics were administered intravenously and who subsequently experienced noticeable improvement in clinical features (8). american thoracic society recommends the so-called switch therapy, which implies switching from parenteral to oral antibiotics. the main problem is the lack of clear definition of the moment or conditions when the patient should make the switch to oral administration (9). orally administered antibiotics and concomitant therapy are recommended for continuation of treatment, and than can be considered as a variant of the switch therapy. studies conducted in italy in 2012, showed that intravenous administration of antimicrobials has several far-reaching effects on pediatric patients and treatment itself (10). in the opinion of child psychologists, parenteral route of administration is considered to be traumatic for the child, with more rapid appearance of adverse effects (11). studies of the american thoracic society from 2013 indicated that patients with respiratory disease should have a specific diet rich in minerals and vitamins with a moderate amount of easily digestible proteins, poor in carbohydrates and rich in fat (12). it is necessary to work on prevention in order to reduce the incidence of morbidity. studies conducted in uk in 2003 showed that introduction of vaccination revolutionized prevention of infectious diseases. it has been shown that introduction of vaccines against measles reduced incidence of mortality by 2.5 million a year. studies conducted in the united states in the period 2009-2013 have shown that introduction of the conjugate vaccine against streptococcus pneumoniae would make the biggest advance in prevention of pneumonia, since it is the most common etiologic agent of this type of pneumonia. controlled study with use of who standardization of radiographic definition of pneumonia included 37,868 children. vaccination effectiveness of 30.3% (95% ci 10.7% to 45.7%, p0.0043) has been observed in the study, taking into account age, sex and year of vaccination. during this four-year program implemented in the entire country, the incidence of disease was reduced by 39% (26 children) in children under 2 years of age. italian single-blind study from 2012 showed that there was a statistically significant difference in occurrence of bronchopneumonia in children who are not immunized compared to those who are (13). according to study conducted on patients hospitalized at the pulmonary department of the pediatric clinic, 38 patients (37%) did not receive immunization regularly. increase in use of third-generation cephalosporins, and aminopenicillins is a cause for concern. since such increase is also observed in vulnerable pediatric population, current situation must be analyzed and restrictive-educational measures ought to be recommended based on results of such analysis. study results showed that pediatric clinic has access to modern diagnostic tests, the treatment is carried out in accordance with the protocols and guidelines, which largely correspond to the guidelines of the british thoracic society. the study shows that the results of bronchopneumonia treatment at the pediatric clinic of the university clinical center of sarajevo are comparable to the results of other studies that were conducted at pediatric clinics. first and third generation cephalosporins (cephazolin and ceftriaxone, respectively) and penicillin antibiotics (ampicillin) were most commonly used antimicrobial agents with the average duration of antibiotic therapy of 4.3 days, all of which is consistent with the guidelines of the british thoracic society. concomitant therapy usually consisted of antipyretics (diclofenac and paracetamol), 2 adrenergic receptor agonist (salbutamol), leukotriene receptor antagonists (montelukast), and corticosteroids (methylprednisolone). availability and performance of diagnostic tests, as well as pharmacological measures conform to the guidelines of the british thoracic society. for the purpose of preventing bronchopneumonia in pediatric population, specific epidemiological measures ought to be taken, and they should involve all levels of health care. awareness of early signs and symptoms of bronchopneumonia should be raised in the population, and especially parents, in order to begin treatment a timely manner. to reduce the incidence of disease, introduction of the pneumococcal vaccination

introduction: bronchopneumonia is the most common clinical manifestation of pneumonia in pediatric population and leading infectious cause of mortality in children under 5 years. evaluation of treatment involves diagnostic procedures, assessment of disease severity and treatment for disease with an emphasis on vulnerability of the population.aim:to determine the most commonly used antibiotics at the pediatric clinic in sarajevo and concomitant therapy in the treatment of bronchopneumonia. patients and methods: the study was retrospective and included a total of 104 patients, hospitalized in pulmonary department of the pediatric clinic in the period from july to december 2014. the treatment of bronchopneumonia at the pediatric clinic was empirical and it conformed to the guidelines and recommendations of british thoracic society. results and discussion: first and third generation of cephalosporins and penicillin antibiotics were the most widely used antimicrobials, with parenteral route of administration and average duration of treatment of 4.3 days. concomitant therapy included antipyretics, corticosteroids, leukotriene antagonists, agonists of 2 adrenergic receptor. in addition to pharmacotherapy, hospitalized patients were subjected to a diet with controlled intake of sodium, which included probiotic-rich foods and adequate hydration. recommendations for further antimicrobial treatment include oral administration of first-generation cephalosporins and penicillin antibiotics. conclusion:results of the drug treatment of bronchopneumonia at the pediatric clinic of the university clinical center of sarajevo are comparable to the guidelines of the british thoracic society. it is necessary to establish a system for rational use of antimicrobial agents in order to reduce bacterial resistance.

PMC5010066

pubmed-1358

dendritic cells play a key role not only in asthma during the initiation of the allergic immune response but also in the effector phase of the allergic inflammation leading to typical clinical symptoms [1, 2]. basically, the dendritic cells can be divided into three groups: a small population of plasmacytoid dendritic cells, a predominant population of conventional dendritic cells, and, during inflammation, the monocyte-derived or inflammatory dendritic cells. the dendritic cells isolated and analysed in this study were the so-called conventional dendritic cells, which are positive for cd11c and mhcii. in addition, the expression of cd8 was used to separate cd8 from cd8 dendritic cells. upon comparison, fewer cd8 dendritic cells than cd8 ones were found in the lung tissue. the cd8 dendritic cells were more concentrated in the draining lymph nodes, making them a lymph node-resident dendritic cell population [4, 5]. furthermore, within lymph nodes the cd8 dendritic cells contribute to cytotoxic t cell responses via cross presentation of exogenous antigens [2, 4, 6]. cd8, but not the cd8, sorted dendritic cells from schistosoma-infected mice prevented allergic responses. cd8 and cd8 dendritic cells from bcg-infected mice suppressed allergic t cell responses in vitro and in vivo. in recent years, the expression of cd103 and cd11b has been introduced for phenotyping dendritic cells in asthma and elsewhere. the lymphoid resident dendritic cells are characterized as cd103 dendritic cells (cd11b, cd8, and cd8). in contrast the nonlymphoid residents are characterized as cd103 dendritic cells (cd11b, cd8, and cd8). our approach to the gene expression of conventional dendritic cells compared cd8 and cd8 conventional dendritic cells, revealing an interesting panel of regulated genes. since there is a close relation between dendritic cells in the tissue and the draining lymph nodes, both compartments were taken for analysis. the majority of dendritic cells pick up allergen not only in the bronchi but also in the alveoli and migrate to lymph nodes where the allergen is presented to b cell and t cells initiating and maintaining humoral and cellular lymphocyte responses. lymphocytes become activated and recirculate through the tissues including the lung where dendritic cell immigration and activation are mediated [1, 2]. the present study had the aim to compare the gene expression of distinct dendritic cells isolated from the lung tissue and the lung-draining lymph nodes in mice with induced asthmatic-like inflammation and controls. a further aim of the presented study was to compare lung tissue and lymph node-derived dendritic cells from control animals and animals suffering from allergic inflammation. the more the subsets that are defined using multiple markers, the more the difficult the harvesting of a sufficient number of dendritic cells. therefore, a strategy was chosen to obtain sufficient numbers of dendritic cells in a medium scale approach, using less than fifty animals each for the disease group and the control group. the classical combination of cd11c and mhcii defined the small numbers of conventional dendritic cells which yielded the draining mediastinal lymph nodes. for the bigger lung tissue yield of dendritic cells, the expression of cd8, which is relevant for pulmonary allergy, was additionally included. data from gene arrays in murine macrophages and dendritic cells from lung tissue is available, but to our knowledge there is no data on gene arrays in dendritic cells from allergic or asthmatic-like inflamed lungs. furthermore, no approach could be found including the analysis of dendritic cells from lung-draining lymph nodes. all experiments were carried out using c57bl/6 mice (812 weeks old, charles river, sulzfeld, germany). mice (n=38) were sensitized by intraperitoneal injection of 10 g ovalbumin (grade vi) emulsified in 1.5 mg aluminium hydroxide in a total volume of 150 l on days 1, 14, and 21. control mice (n=42) were sham-sensitized with 1.5 mg alum in pbs. ova provocation (1% ova grad v in pbs for 20 min) was performed on days 28 and 29 on all mice. lungs were obtained and cut into small fragments, digested with collagenase, and dnase and enriched by gradient centrifugation and magnetic depletion of granulocytes, lymphocytes, and erythrocytes. bronchial lymph nodes cells were isolated by passing the tissue through a metal mesh, directly followed by the magnetic depletion of granulocytes, lymphocytes, and erythrocytes. remaining cells isolated from lung tissue and bronchial lymph nodes were resuspended in pbs and stained for 30 minutes with -cd11c, -mhcii, and -cd8 (bd biosciences). after washing, the stained cells were analysed and dcs (cd11c and mhcii) were sorted by using a mo-flo-system (cytomation). the total rna was isolated using the rneasy mini kit (qiagen, hilden, germany) according to the manufacturer's recommendations. samples for microarray analysis were generated by applying an mrna-specific double linear amplification protocol (affymetrix). briefly, double-stranded cdna was generated in vitro in a reverse transcription using the t7dt23 primer (5-ggccagtgaattgtaatacgactcactatagggaggcgg(t)23-3; metabion, planegg, germany) and superscript ii reverse transcriptase (invitrogen, karlsruhe, germany), followed by a second-strand cdna synthesis involving dna polymerase i (invitrogen) and e. coli dna ligase (invitrogen). for the first amplification round the promega p1300 ribomax kit for t7 amplification (promega, mannheim, germany) was used to synthesize unlabeled crna from the purified cdna. a second amplification round was performed starting with the amplified crna and reverse transcription by using random hexamer primers (pharmacia, freiburg, germany) and superscript ii reverse transcriptase for the first-strand synthesis. the second-strand synthesis, again using t7dt23 primers and additional rnase h treatment, was performed as mentioned above. for the final amplification round, the genechip expression 3-amplification reagent kit for labeling (affymetrix, san francisco, ca, usa), producing biotinylated crna, was used. samples were fragmented and hybridized to a mouse genome 430 2.0 array (affymetrix). analysis of microarray data was performed using genespring gx 10.0 software (agilent technologies). the robust multiarray analysis (rma) the experiments and sorting procedures were performed with animal groups of 5 to 7 animals and repeated to finally reach the number of 42 control mice and 38 asthmatic-like mice for the cell isolation from lung tissue and the number of 15 control and 15 asthmatic-like mice for the cell isolation from lung-draining lymph nodes. the yield of dendritic cells from lung tissue was as follows. from 42 control mice in total 121.000 cd8 and 843.000 cd8 dendritic cells (cd11c/mhcii) were obtained from the tissue of whole lungs. from 38 asthmatic-like mice in total the yield of dendritic cells from the lung-draining lymph nodes was as follows. from 15 control mice in total 168.000 dendritic cells (cd11c/mhcii) were obtained and from 15 asthmatic-like mice 140.000 dendritic cells were obtained. the reanalysis in flow cytometry led to a purity of at least 95%, and in most experiments, the purity was 97 to 99%. in the cd8 subset of lung tissue-derived dendritic cells 871 transcripts were upregulated with a fold change>2 in the comparison between induced allergic inflammation and controls (figure 1). again this is in contrast to the cd8 subset in the same comparison, where only 19 transcripts were upregulated and 152 were downregulated (figure 1). the cd8 subpopulation is more constant and the few genes regulated are downregulated (figure 1). this does not exclude a distinct function of the cd8 dc subpopulation, like cross presentation of external antigen, for instance. since considerable lower numbers of dendritic cells could be obtained from the lung-draining lymph nodes, all dendritic cells were analyzed and again those from asthmatic-like lungs were compared to those of control lungs. here, one can speculate whether the cd8 dendritic cells, being part of the sorted lymph node dcs were again those with the most regulated transcripts. it is known that, during inflammation, there is a constant flow of migrating dendritic cells to the draining lymph nodes, which induces massive changes in the cellular microenvironment in the lymph node. this is interesting and may support a role of the lymph nodes in controlling the immune response and inflammation. a venn diagram (figure 2) was used to display the overlap between regulated transcripts in all three populations: 66 were shared between the cd8 and cd8 dendritic cell population and 4 were shared by all dendritic cells, including the lymph node-derived ones. the small overlap of regulated genes amongst the three dc populations may underline their phenotypical distinctness. moreover, the fact that the total lymph node dcs contain both cd8 and cd8 dc populations, but still show little overlap between the regulated genes found in asthmatic-lung-derived cd8 or cd8 dcs clearly shows that dc functions in asthma are dependent on the cellular environment. on the assumption that many cd8 dendritic cells had migrated to the lymph nodes, the intense interaction with the stimulated micromilieus might have changed their phenotype. in tables 1 and 2, gene enrichment analysis of cd8 dendritic cells is presented because cd8 dendritic cells seemed to be the most highly regulated during allergic inflammation. in the kegg pathway enrichment significantly regulated genes have been found, which are involved in chemokine signaling, cytokine/receptor-interaction, fcr-mediated phagocytosis, and tlr signaling (table 1). no significant kegg pathway enrichment was found in the comparison of cd8 dendritic cells (asthma versus control, see supplemental table 5 in supplementary materials available online at http://dx.doi.org/10.1155/2015/638032). in the go analysis, mainly changes of the plasma membrane and vesicles are prominent (table 2). in the cd8 dc go analysis, changes of the plasma membrane were prominent (supplemental table 5). in the dendritic cells from draining lymph nodes consisting of total dendritic cells (cd8 and cd8), only few components are overrepresented (supplemental table 6). no significant changes occurred on cell surfaces but rather intracellular compartments (table 3) seem to be modulated which might be related to the intense antigen processing and antigen presentation which is the core task of dendritic cells in the lymph node. from the abundant regulated genes in the cd8 dendritic cells highly upregulated genes were, amongst many others, distinct serpins, arl5b, and kif3b (supplemental material, excel sheet of regulated genes). since it was not the primary aim of this study, no candidate genes were selected to perform confirming pcr analyses. the most prominent transcriptional alterations could be observed in the cd8 dc compartment from asthmatic-like mice when compared to cd8 dc from control mice. in general genes that are being upregulated more than 10-fold under these conditions are, for example, ear11, abcd2, cd209e, fabp1, and slc7a2. ear11, officially known as rnase2a, encodes for the ribonuclease, rnase a family, 2a (liver, eosinophil-derived neurotoxin) protein. it has been previously reported to be asthma-induced in short-term, intermediate term, and long-term ovalbumin exposure of the lung. it encodes for the atp-binding cassette, subfamily d (ald), member 2, a protein belonging to the large group of atp-binding cassette (abc) transporters responsible for cross membrane transport of various substances. abcd2 is, for example, involved in the peroxisomal import of fatty acids. cd209e also known as signr4 is a mouse homologue to the human dc-sign protein. dc-sign is a type ii c-type lectin that functions as an adhesion molecule on the surface of dendritic cells. in the context of asthma, it has been reported that dc-sign on human mddcs mediates cellular responses to, for example, bermuda grass pollen antigens in vitro leading to the production of tnf-. fabp1 (fatty acid binding protein 1, liver) is upregulated+10.5-fold in cd8 dcs from asthmatic-like mice. that fatty acid binding proteins in principle are involved in dc function that has been demonstrated with regard to fabp4 (also known as ap2), another protein from the same family with at least nine members. mice lacking ap2 were shown to produce less il-12 and tnf and were less potent in inducing t cell proliferation. however, expression of fabp1 on dcs in an asthmatic-like context has not been described so far. slc7a2 stands for solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 and belongs to the amino acid-polyamine-organocation family of transport proteins. a comparably small group of genes in cd8 dcs were found to be downregulated in an asthma-dependent manner. for example, the two heat shock proteins 1a and 1b (hspa1a and hspa1b) show a drastic loss of expression (10.6- and 10.3-fold). hspa1a and hspa1b are also known as heat-shock 70-kd proteins 1a and 1b, respectively. they are involved in cellular stress responses and act as chaperons. of note, the toll-like receptor 3 (tlr3) tlr3 is a pattern recognition receptor sensing double-stranded rna typically associated with viral infections. as the cell surface is an important immunological interface of dcs, it is interesting to observe that genes whose protein products are associated with the cell surface are significantly enriched in the gene ontology analysis. in this category, the serotonin receptor 2c (htr2c) is highly upregulated (+ 7.1-fold) under asthmatic-like conditions. htr2c expression has been also demonstrated on epidermal dcs in context of a contact allergy model in mice. interestingly, cd2 human pdcs were shown to comprise a distinct dc population producing higher amounts of il-12p40 and expressing higher levels of costimulatory cd80 compared to cd2 pdcs following an influenza a virus infection of the lung. the cd200 receptor 4 (cd200r4) is upregulated+4.6-fold on cd8 dcs under allergic conditions and is a receptor for the ox-2 ligand (also known as cd200). this is of particular interest since it has been recently reported in a rat asthma model that local delivery of recombinant cd200 strongly reduces ova-induced lung accumulation of myeloid dcs in the lung. cd22 also known as siglec2 is upregulated 4-fold in cd8 dcs and belongs to the family of sialic-acid-binding lectins. originally cd22 is thought to be a b cell restricted protein inhibiting the b-cell antigen receptor (bcr) signaling. however, there are reports demonstrating its expression also on pdcs. on the other hand, the induction of asthma led in cd8 dcs also to the reduced expression of some surface proteins or at least their according transcripts. the killer cell lectin-like receptors klrb1b (5.6-fold), klrd1 (4.0-fold, also known as cd94) and klrk1 (2.1-fold, also known as nkg2d) are all downregulated in cd8 dcs compared to nonasthmatic-like conditions. interestingly, genes involved in antigen presentation like h2-oa and h2-ob which encodes for the histocompatibility 2 o region loci alpha and beta are slightly downregulated (h2-oa: 2.7-fold, h2-ob: 3.2-fold). of note, the costimulatory protein cd86, working in conjunction with mhc class ii proteins to activate cd4 t cells, is downregulated as well (2.2-fold). with regard to the lysosomal compartment genes like pla2g15, sort1, and several cathepsins phospholipase a2 group 15 (pla2g15,+5.3-fold) is involved in the eicosanoid synthesis, a substance class also containing prostaglandins and leukotrienes which have pro- and anti-inflammatory potential. sortilin 1 (sort1,+4.7-fold) very efficiently binds serum lipoproteins but can act as a multiligand type-1 receptor. the group of cathepsins (cathepsins a, b, d, f, k, and l) is upregulated in cd8 dcs from asthmatic-like lungs (ctsa:+3.2-fold, ctsb:+2.1-fold, ctsd:+3.0-fold, ctsf:+3.3-fold, ctsk:+2.4-fold, and ctsl:+3.3-fold). cathepsins represent a group of endoproteases typically abundant in the lysosomal compartment and hydrolytically degrade, for example, the extracellular matrix and basal membranes. in the functional group of chemokine and cytokine signaling, genes like cd24 (+ 6.1-fold), ccl24 (+ 6.1-fold), and pdgfc (+ 5.8-fold) were highly upregulated. cd24 is a protein that is able to provide costimulatory signals to t cells and has been described to occur in the context of dc differentiation from cd8 to cd8 dcs. interestingly, it was reported that cd24-deficient mice exhibit increased susceptibility to danger but not pathogen-associated molecular patterns. ccl24 is a chemokine also known as eotaxin-2 and is upregulated+6.1-fold in cd8 dcs. platelet-derived growth factor c polypeptide (pdgfc,+5.8-fold) is a potent mitogen for cells of mesenchymal origin. furthermore, genes like lepr (leptin receptor;+4.7-fold), adcy3 (adenylate cyclase 3;+3.7-fold), ccr1 (chemokine c-c motif receptor 1;+2.7-fold), ccl6 (chemokine c-c motif ligand 6;+2.4-fold), ccl8 (chemokine c-c motif ligand 6;+2.4-fold), and cxcr3 (chemokine c-x-c motif receptor 3; 4.5-fold) were differentially regulated to a smaller extend. dendritic cells and their subsets play a key role in initiating and maintaining allergic inflammation. dendritic cells are present in low numbers in lung tissue and in very low numbers in lung-draining lymph nodes. gene analysis requires the separation of dendritic cells from huge numbers of animals, limiting the analysis of very rare subsets. the present analysis showed a regulation, up and down, of many more transcripts in the cd8 conventional dendritic cells of the lung tissue as compared to the cd8 dcs supporting the pathophysiological predominance of the cd8 subset. surprisingly, the transcriptional reaction in the dendritic cells of the draining lymph nodes was moderate indicating the role of the lymph nodes more as a stabilizer or controller than as a booster of the allergic inflammation. conventional cd8 and cd8 dendritic cells are distinct subsets with differentiated roles in allergic inflammation. further investigations will investigate whether the sorting of alternative dendritic cell subsets will show an overlap to those analyzed in this study.

asthma is the consequence of allergic inflammation in the lung compartments and lung-draining lymph nodes. dendritic cells initiate and promote t cell response and drive it to immunity or allergy. however, their modes of action during asthma are poorly understood. in this study, an allergic inflammation with ovalbumin was induced in 38 mice versus 42 control animals. after ovalbumin aerosol challenge, conventional dendritic cells (cd11c/mhcii/cd8) were isolated from the lungs and the draining lymph nodes by means of magnetic cell sorting followed by fluorescence-activated cell sorting. a comparative transcriptional analysis was performed using gene arrays. in general, many transcripts are up- and downregulated in the cd8 dendritic cells of the allergic inflamed lung tissue, whereas few genes are regulated in cd8+dendritic cells. the dendritic cells of the lymph nodes also showed minor transcriptional changes. the data support the relevance of the cd8 conventional dendritic cells but do not exclude distinct functions of the small population of cd8+dendritic cells, such as cross presentation of external antigen. so far, this is the first approach performing gene arrays in dendritic cells obtained from lung tissue and lung-draining lymph nodes of asthmatic-like mice.

PMC4355561

pubmed-1359

approximately 795 000 strokes occur every year in the united states, 87% of which are ischemic, and 6.6 million living adults have experienced a stroke.1 it is the fifth leading cause of death, the leading cause of longterm disability, and costs $33 billion per year in health care expenses and lost productivity.1 effective healthcare delivery can prevent strokes through risk factor modification,1 and can improve longterm recovery and outcomes after stroke,2 but insurance status affects access to healthcare.3 in the united states, employersponsored private health insurance covers about half of the population.4 private insurance covers the majority of healthcare costs, with patients accountable for only a copay or deductible usually amounting to less than 10% of total costs. medicaid is a public insurance program for persons earning<138% of the federal poverty level and covers all services without any patient obligation. the uninsured receive no financial assistance with the costs of care and must pay for doctor visits, medications, procedures, and hospitalizations in full out of pocket. being uninsured increases mortality for cancer,5, 6 trauma,7, 8 sepsis,9 heart failure,10 and acute myocardial infarction.11 medicaid insurance status is also associated with the risk for death in several of these conditions.5, 6, 10 there is evidence that disability and mortality are also higher for medicaid and uninsured patients after stroke,12, 13 with the largest and most recent study being specific to hemorrhagic strokes.14 despite the affordable care act, in the united states the uninsured rate remained at 11.9% in the fourth quarter of 2015.15 therefore, the objective of this study was to determine whether patients without insurance or with governmentsponsored insurance had worse care or outcomes in acute ischemic stroke (is) than the privately insured, using a large, nationally representative stroke registry. we specifically sought to investigate differences in prevention, presentation, inhospital care, acute outcomes, and use of rehabilitation services after discharge. we used clinical data from the american hospital association's get with the guidelines (gwtg)stroke, a continuous stroke registry, details of which have been published previously.16 quintiles (cambridge, ma) is the data collection coordination center for the gwtg programs. duke clinical research institute (dcri) serves as the data analysis center for gwtg, and the reported analyses of aggregate deidentified data were approved by the duke university medical center institutional review board. participating hospitals nationwide use trained hospital personnel to enter deidentified patientlevel data into the database for all of their stroke hospitalizations. data collected include patient demographics, medical history, and comorbidities, inhospital treatment, discharge treatment and counseling, mortality, and discharge destination. the registry began in 2003, and this study presents data collected between october 1, 2012 and june 30, 2015 to minimize shifts in is care and insurance trends that have occurred over time. patients with stroke types other than ischemic, or with missing insurance or discharge information, were excluded from this study, leaving 589 320 is patients treated in 1604 us hospitals analyzed here (figure 1): 197 474 (33.5%) patients from 1534 hospital sites were aged<65 years of age (15.9% uninsured, 53.0% private, 12.0% medicare, 19.1% medicaid), and 391 846 (66.5%) patients from 1593 hospital sites were aged 65 years of age and older (1.0% uninsured, 39.4% private, 52.1% medicare, 7.4% medicaid). presenting characteristics include information on arrival to the hospital, initial nih stroke scale score, initial vital signs, and laboratory results on admission such as cholesterol and hemoglobin a1c levels. hospital characteristics include stroke center designation by the joint commission, teaching status, rural location, geographic region, number of beds, and annual volume of is admissions. quality achievement measures analyzed include use of iv tpa in patients arriving within the treatment window, early antithrombotics, and prophylaxis for venous thromboembolism (vte) acutely, as well as discharge with antithrombotics, anticoagulants in patients with atrial fibrillation or atrial flutter, statins in patients with ldl>100 mg/dl, and smoking cessation prior to discharge for smokers.17 the quality of care delivered is summarized by the defectfree care measure, which measures whether or not a patient's hospital treatment met all get with the guidelines quality measures.17, 18 outcomes analyzed include inhospital mortality (excluding patients who transferred out or left against medical advice), ambulatory status at discharge, modified rankin scale at discharge, and discharge destination. ambulatory status and discharge destination measures excluded those who died during hospitalization. patient and hospital characteristics and quality and outcomes measures were initially compared using chisquared tests for categorical variables and kruskalwallis tests for continuous variables. the associations among quality measures, stroke outcomes, and insurance status were evaluated using unadjusted and adjusted logistic regression models and with generalized estimating equations (gee) to account for hospital clustering. covariates in adjusted analyses included patient characteristics (demographics, comorbidities, initial nih stroke scale score, offhour arrival), and hospital characteristics (region, teaching status, size, rurality, annual stroke and tpa volumes, and joint commission stroke center designation). to handle missing data, multiple imputation with 25 independently created imputation data sets and the fully conditional specification approach, also known as multiple imputation by chained equations, were performed in sas (all variables had<5% missing data with the exception of initial nihss with ~18% missing). all analyses were stratified by age less than or 65 years because the majority of the population becomes eligible for medicare at age 65. all analyses were conducted using sas software version 9.4 (sas institute, cary, nc). patient and hospital characteristics for is patients under 65 are shown in table 1 and table s1. uninsured and medicaid patients were more likely to be black or hispanic than privately insured or medicare patients. uninsured is patients were less likely than privately insured, medicare, or medicaid patients to be taking strokepreventative medications including antiplatelets, anticoagulants, antihypertensives, cholesterolreducers, and diabetes mellitus medications, including those with preexisting conditions that would indicate need for such medications. patient and hospital characteristics for acute ischemic stroke in patients younger than 65 years old, overall and by insurance statusa mi indicates myocardial infarction; nih, national institutes of health; tia, transient ischemic attack; tjc, the joint commission. due to large sample size, all pvalues are statistically significant to<0.0001. continuous/ordinal row variables designated by*are presented as median (iqr). patients with is who were uninsured arrived to the hospital later after symptom onset, and fewer arrived by ambulance than medicaid or medicare patients (table 1). however, uninsured and privately insured patients were more likely to be able to ambulate independently when they arrived than medicare or medicaid patients. the private and uninsured is patients also had lesser stroke severity by nihss at presentation than medicare or medicaid patients. characteristics of is patients aged 65 and older are similar and can be found in table s2. medicaid patients under the age of 65 and uninsured and medicaid patients aged 65 and older were less likely to be treated in designated primary stroke centers, more likely to be treated in academic hospitals, and more frequently treated in hospitals with slightly lower annual ischemic stroke volumes (table 1 and tables s1 and s2). the majority of all is admissions in the data set occurred in the south, and the south had a larger share of uninsured is patients than other areas of the country. process of care, quality, and outcome measures by insurance status are shown in table 2 and tables s3 and s4. although nearly all of the differences were statistically significant due to the large sample size, there were few absolute differences in achievement or quality measures by insurance status that were large enough to have a clinical impact, including the provision of defectfree care that met all get with the guidelines quality measures and evaluation for rehabilitation. inhospital mortality was lower in privately insured patients under 65 and higher in uninsured patients aged 65 and older (tables s3 and s4), and uninsured patients were more likely to go home and less likely to go to rehab facilities after discharge. during their stroke hospitalization, uninsured patients were more likely to receive a new diagnosis of diabetes mellitus that had not been previously known. process of care measures and other outcomes of interest in acute ischemic stroke patients younger than 65 years old, overall and by insurance statusa ama indicates against medical advice; gwtg, get with the guidelines. due to large sample size, all pvalue are significant to<0.001 with the exception of iv tpa, arrive by 3.5 hours, and treated by 4.5 hours, p=0.0047. continuous/ordinal row variables designated by are presented as median (iqr). quality of care as measured by the provision of defectfree care was less likely in medicare and medicaid patients but slightly more likely in the uninsured than the privately insured (figure s1). uninsured patients were more likely to receive a statin when indicated by high ldl, and medicare and medicaid patients were less likely to receive antithrombotics, but there were no other differences in quality indicators. in patients aged 65 and older, medicaid patients were less likely to receive tpa when arriving within the window, and uninsured patients remained more likely to receive a statin, but other quality indicators and the provision of defectfree care were equal among insurance groups (figure s2). the uninsured had 30% higher odds of dying in hospital than the privately insured, and the odds of death increased to 1.5 for the uninsured aged 65 and older (figure 2; figure s2). forest plot for unadjusted and adjusted ors (95% ci) among patients with age<65 years. the first pvalue within each outcome tests if or differs by at least 2 insurance categories; all other pvalues test if or differs significantly from 1. covariates in adjusted model include patient age, sex, and race; patient medical history of atrial fibrillation or flutter, stroke/tia, cad/mi, carotid stenosis, diabetes mellitus, pvd, hypertension, dyslipidemia, or smoking; arrival offhours, nihss score; and hospital region, teaching status, number of beds, annual ischemic stroke volume, annual iv tpa volume, rurality, primary stroke center. compared with the privately insured, patients with medicaid were more likely to be considered for rehab but had 14% to 24% lower odds of being discharged to an inpatient rehabilitation facility. instead, patients on medicare or medicaid under age 65 had about 25% higher odds of being discharged to hospice and were twice as likely to be discharged to a skilled nursing facility as the privately insured. uninsured patients were equally likely to be considered for rehabilitation as the privately insured but had 37% lower odds when under 65 and 44% lower odds when aged 65 and over of being discharged to inpatient rehabilitation, 45% lower odds when aged 65 and older of being discharged to skilled nursing, and more than twice as high odds of being discharged home than the privately insured. we investigated whether insurance status affected prevention, presentation, inhospital care, acute outcomes, or use of rehabilitation services after discharge among patients with acute is. first, uninsured patients have poorer control of several risk factors for stroke when their is occurs. third, medicaid patients are less likely to receive iv tpa when arriving within the window time for is. finally, uninsured and medicaid patients have lower utilization of inpatient rehabilitation services after is, whereas medicaid and medicare patients have higher utilization of skilled nursing facilities and hospice after is than the privately insured. mortality was particularly increased in the uninsured is patients but also slightly increased in medicaid patients aged 65 and older and in both medicare and medicaid patients under 65. increased mortality in uninsured is patients under age 65 has previously been suggested in survey data13 and demonstrated for all uninsured patients with hemorrhagic stroke12, 14 and for all patients with is with any status other than privately insured19 in national registry data. this study confirms the increased risk of death and adds more information on presenting, prehospital, and inhospital factors that can be used for future investigations into the etiology of the mortality disparity. differences in prestroke preventative care and initial hospital arrival may partially explain the mortality difference for uninsured patients, particularly because stroke severity and provision of highquality processes of care did not differ. uninsured patients with risk factors for stroke such as diabetes mellitus, high blood pressure, or high cholesterol were less likely to be taking the appropriate medications for primary stroke prevention and had more poorly controlled cholesterol levels than other groups. they were also less likely to be aware of their risk factors for stroke, as evidenced by more frequent firsttime diagnosis of diabetes mellitus during the hospitalization for is. in addition, the uninsured arrived almost 45 minutes later on average after stroke onset than other is patients. these findings correlate with prior evidence that the uninsured delay needed care due to costs,20 have undiagnosed chronic disease,21 do not take appropriate control medications to decrease stroke risk,22, 23 and have poorer control of risk factors for stroke such as blood pressure.24, 25 in the united states the uninsured must pay out of pocket for a physician visit at least once a year at an average cost of $160 to renew prescriptions for preventative medications,26 not including any laboratory tests required to screen for or monitor risk factors such as diabetes mellitus or high cholesterol, and an average of $166 per month for 1 antihypertensive and 1 statin.27 the uninsured are mostly lowincome workers for whom $100 represents a large percentage of their monthly income and are more likely to work at hourlywage jobs without sick leave, limiting access to physician offices during weekday working hours.3 the uninsured are more likely to have low health literacy28 and may live in areas with limited access to primary care.29 all of these factors likely contribute to poorer preventative care and worse stroke outcomes in this group. although this study was unable to evaluate longterm outcomes such as recurrent stroke or longterm disability, if these patients remained uninsured after their is, evidence from this and prior studies30 suggests that they will also be less likely to achieve adequate secondary prevention for recurrent stroke. although some differences did exist in quality of care delivered to is patients by insurance status, they had minimal clinical impact, with less than 10% absolute difference with the exception of iv tpa for medicaid patients over the age of 65. medicaid patients were less likely to be treated at designated primary stroke centers, which could contribute to this disparity, but it persists after adjustment. medicaid patients had worse disability and were less likely to walk independently at discharge, but they were also less likely to walk independently at admission, so it is uncertain whether this poorer functional status relates to lack of iv tpa or to more severe prestroke disability. although is patients who were uninsured were equally evaluated for rehabilitation, they had more than twice the odds as the privately insured of going home after stroke, and 40% to 50% lower odds of going to inpatient rehabilitation or a skilled nursing facility. going home is typically viewed as a positive outcome after stroke, but in this group it may be inappropriate. medicaid patients had lower odds of going home but higher odds of going to hospice (if under 65 years of age) and higher odds of going to skilled nursing rather than inpatient rehabilitation. similar results have been found in previous national samples of stroke patients, but those have been limited to different subpopulations of stroke patients or to evaluation for rehabilitation without placement data.14, 31, 32 all comparisons were made to privately insured patients, who use inpatient rehabilitation more than the other groups, but ideal utilization levels are unknown, and it is not clear that utilization by privately insured patients represents appropriate utilization rather than overutilization. however, after suffering is, patients are at high risk for permanent disability, but evidence shows that rehabilitation improves functional outcomes, particularly when started early.2, 33, 34 lower utilization of postacute care has also been demonstrated in the uninsured after trauma and sepsis.8, 34 unfortunately, postacute care is expensive, and acceptance into such programs is highly dependent on ability to pay and insurance authorizations, which likely explains the differences observed in this study. limited access to rehabilitation after ischemic stroke for the uninsured and medicaid populations may increase the number of poststroke patients who become disabled, increasing their longterm costs. insurance status is correlated with sociodemographic variables, which may also play a role in the different stroke outcomes found in this study. for example, people on medicaid qualify due to poverty, and the uninsured are also a lowincome group.3 persons under the age of 65 on medicare are typically disabled, whereas almost all us citizens aged 65 and older qualify for medicare. in the group aged 65 and older, the overall uninsured rate is very low, and those who remain uninsured likely are either not us citizens or have poor social circumstances such as lack of personal identification due to homelessness that prevent them from obtaining medicare. persons on medicaid over the age of 65 are generally dually eligible for medicare and medicaid and are impoverished and elderly. however, some lowincome persons may have excellent employersponsored insurance, and some wealthy persons may be uninsured,3 suggesting the importance of insurance status as an independent adjustment factor for stroke population analyses, especially for the under65 population. the affordable care act has reduced the number of uninsured persons in the united states. however, over 30 million people remain uninsured, and medicaid enrollment has increased by 12 million.3, 36 therefore, the findings of this study remain relevant as we consider how to increase insurance coverage and how that coverage equates with access, affordability, and outcomes. future research should examine how highdeductible health plans, which are nearly ubiquitous in the marketplace plans that 10 million people have purchased through the aca and are increasing in prevalence in employersponsored plans, will affect stroke outcomes.36, 37 high deductibles may provide incentives similar to those the uninsured face in barriers to appropriate primary prevention medications prestroke and may or may not affect utilization of rehabilitation poststroke, depending on deductible amounts relative to the cost of stroke hospitalization. first, although get with the guidelines data represent a large number of stroke admissions across the country, they depend on the accuracy of data collection and entry by staff members at over 1500 different hospitals. as noted, 127 168 patients, or 18% of all acute ischemic strokes, were excluded from this study because they were missing key data, and some of the patients included were still missing other data points that, although predicted using multiple imputation, may introduce bias into the results. second, all included hospitals are participating in a program to improve stroke quality, and their results may not be generalizable to other hospitals not participating. third, this was a retrospective observational study, and some factors important to stroke severity or severity of comorbidities may not have been measured, introducing unmeasured or residual confounding. in particular, several socioeconomic factors that may interact with insurance status were not available, such as income level and employment status. retrospective studies also can not prove causality of insurance status on stroke outcomes. finally, although differences were found in acute outcomes and discharge destinations after is, this registry is limited to hospitalassociated outcomes, and longterm outcomes such as functional status and disability, recurrent stroke, and survival were not analyzed. preventative care prior to is, time to presentation for acute treatment, access to rehabilitation, and outcomes including mortality differ by patient insurance status. this study suggests that even highquality processes of stroke care in hospital can not overcome the prestroke health status, socioeconomic drivers, and choices of patients in how and when to seek care that vary by insurance status. because insurance status was independently associated with inhospital outcomes, expanded insurance coverage may help reduce death and disability due to is. the research analysis for this study was funded through a grant from the american heart association. dr fonarow receives research funding from patient centers outcome research center, is a member of the gwtg steering committee, is employed by ucla, and holds a patent on an endovascular device. dr bhatt discloses the following relationships advisory board: cardax, elsevier practice update cardiology, medscape cardiology, regado biosciences; board of directors: boston va research institute, society of cardiovascular patient care; chair: american heart association quality oversight committee; data monitoring committees: duke clinical research institute, harvard clinical research institute, mayo clinic, population health research institute; honoraria: american college of cardiology (senior associate editor, clinical trials and news, acc.org), belvoir publications (editorinchief, harvard heart letter), duke clinical research institute (clinical trial steering committees), harvard clinical research institute (clinical trial steering committee), hmp communications (editorinchief, journal of invasive cardiology), journal of the american college of cardiology (guest editor; associate editor), population health research institute (clinical trial steering committee), slack publications (chief medical editor, cardiology today's intervention), society of cardiovascular patient care (secretary/treasurer), webmd (cme steering committees); other: clinical cardiology (deputy editor), ncdraction registry steering committee (vicechair), va cart research and publications committee (chair); research funding: amarin, astrazeneca, bristolmyers squibb, eisai, ethicon, forest laboratories, ischemix, medtronic, pfizer, roche, sanofi aventis, the medicines company; royalties: elsevier (editor, cardiovascular intervention: a companion to braunwald's heart disease); site coinvestigator: biotronik, boston scientific, st. jude medical; trustee: american college of cardiology; unfunded research: flowco, plx pharma, takeda. dr peterson reports serving as principal investigator of the data analytic center for aha gwtg; receipt of research grants from johnson&johnson and janssen pharmaceuticals; and serving as a consultant to bayer, boehringer ingelheim, johnson&johnson, medscape, merck, novartis, orthomcneiljanssen, pfizer, valeant, westat, the cardiovascular research foundation, webmd, and united healthcare. dr schwamm receives research funding from patient centered outcome research center and ninds, is a member of the gwtg steering committee, and chair of the gtwtg stroke clinical workgroup. additional patient and hospital characteristics for acute ischemic stroke in patients younger than 65 years old, overall and by insurance status table s2. patient and hospital characteristics for acute ischemic stroke patients aged 65 years or older, overall and by insurance status table s3. additional stroke process of care measures and other outcomes of interest in acute ischemic stroke patients younger than 65 years old, overall and by insurance status table s4. process of care measures and other outcomes of interest in acute ischemic stroke patients aged 65 years or older, overall and by insurance status figure s1. forest plot for unadjusted and adjusted ors (95% ci) among patients with age<65 years. forest plot for unadjusted and adjusted ors (95% ci) among patients with age 65 years.

backgroundinsurance status affects access to care, which may affect health outcomes. the objective was to determine whether patients without insurance or with governmentsponsored insurance had worse quality of care or inhospital outcomes in acute ischemic stroke. methods and resultsmultivariable logistic regressions with generalized estimating equations stratified by age under or at least 65 years were adjusted for patient demographics and comorbidities, presenting factors, and hospital characteristics to determine differences in inhospital mortality and postdischarge destination. we included 589 320 ischemic stroke patients treated at 1604 us hospitals participating in the get with the guidelinesstroke program between 2012 and 2015. uninsured patients with hypertension, high cholesterol, or diabetes mellitus were less likely to be taking appropriate control medications prior to stroke, to use an ambulance to arrive to the ed, or to arrive early after symptom onset. even after adjustment, the uninsured were more likely than the privately insured to die in the hospital (< 65 years, or 1.33 [95% ci 1.221.45]; 65 years or 1.54 [95% ci 1.341.75]), and among survivors, were less likely to go to inpatient rehab (< 65 or 0.63 [95% ci 0.60.67]; 65 or 0.56 [95% ci 0.50.63]). in contrast, patients with medicare and medicaid were more likely to be discharged to a skilled nursing facility (< 65 years or 2.08 [ci 1.962.2]; or 2.01 [95% ci 1.912.13]; 65 years or 1.1 [95% ci 1.071.13]; or 1.41 [95% ci 1.351.46]). conclusionspreventative care prior to ischemic stroke, time to presentation for acute treatment, access to rehabilitation, and inhospital mortality differ by patient insurance status.

PMC5210352

pubmed-1360

these include evolution from fiberoptic imaging to generation of images using a charge-coupled device and high definition digital imaging. enhanced endoluminal imaging techniques have included chromoendoscopy and modalities that aspire to optical biopsy. among techniques for enhanced optical diagnosis, narrow band imaging (nbi) is a proprietary imaging modality in which the endoscope processor filters standard white light to specific wavelengths in the blue green spectrum (415 nm and 540 nm). nbi thereby capitalizes on the peak absorption of hemoglobin and has the ability to accentuate visualization of the mucosal vasculature 1. proposed clinical applications of nbi include endoscopic evaluation of barrett s esophagus and endoscopic diagnosis of colorectal polyps. studies of nbi in endoscopic inspection of barrett s esophagus have demonstrated high sensitivity of nbi in detection of barrett-associated high grade dysplasia 2, and the ability of nbi to detect dysplasia in a higher proportion of patients with fewer biopsy samples compared with standard white light endoscopy 3. with respect to colorectal polyps, an nbi-based classification scheme has been developed which may accurately distinguish adenomatous from hyperplastic polyps 4, although it is not certain that use of nbi improves polyp detection rates 5 6 7. despite data regarding potential gastrointestinal endoscopic applications of nbi, the degree to which nbi use has been adopted into clinical practice legitimate questions exist regarding the degree to which efficacy of nbi as demonstrated in clinical studies will translate into effective use of nbi in actual practice 8. the aim of this study, therefore, was to prospectively define the rate of nbi use among patients referred to a large group endoscopy practice for diagnostic endoscopy (esophagogastroduodenoscopy and colonoscopy), and to identify procedural factors associated with nbi use. approval to conduct this study as a quality assurance protocol was granted by the institutional review board at the study institution. this study was conducted at the endoscopy center of a tertiary care academic center, where both inpatient and outpatient procedures are performed in a hospital-based suite. each procedure room is equipped with nbi capability (180 series gastroscope or colonoscope, cv-180 evis exera ii video processor, and clv-180 light source; olympus medical systems, tokyo, japan). each consecutive esophagogastroduodenoscopy (egd) and colonoscopy was directly observed by in-room endoscopy technicians, who assist the endoscopist with equipment setup and when endoscopic accessory use (i. e. biopsy forceps, polypectomy snare, endoscopic hemostatic device, etc.) a technician is present in each procedure room for the entire duration of each procedure. for each eligible procedure, the technician documented whether or not nbi was used. procedures performed by the study author were not included; otherwise all faculty endoscopists were eligible for observation. the following endoscopic procedures were excluded from the analysis: egd or colonoscopy with therapeutic intent, specifically endoscopic dilation, endoluminal stent maneuvers, or delivery of endoscopic hemostatic therapy; egd for nasoenteral or percutaneous feeding tube placement; balloon- or spiral-assisted small-bowel enteroscopy; endoscopic retrograde cholangiopancreatography; egd with endoscopic ultrasound; ileoscopy and pouchoscopy. following completion of the 2-week observational study period, operative reports including reports generated by olympus endoworks software and dictated operative notes were manually reviewed by the study author, who was not blinded to study design or intent. the following data were extracted: procedure type, procedure indication, identification of attending endoscopist, trainee involvement, patient type (inpatient versus outpatient), procedure start time (a.m. vs. p.m.), sedation-type (endoscopist-directed conscious or deep sedation vs. monitored anesthesia care with anesthesia staff support); performance of endoscopic polypectomy, either by snare or forceps biopsy; performance of endoscopic tissue biopsy for intent other than polypectomy. extracted data were stored in a microsoft excel spreadsheet, and statistical analyses were performed using jmp 10.0.0 software (sas institute, cary, nc, usa). the chi-squared or fisher s exact test was used for comparison of categorical variables. two-sided p values of<0.05 were considered statistically significant. during the two-week observational study period in july august 2013, data regarding nbi use were recorded for 318 elective endoscopic procedures, consisting of 106 egds and 212 colonoscopies. the most common indications for egd were evaluation of abdominal pain/dyspepsia (22 %) and evaluation of gastroesophageal reflux disease (19%). additional indications included evaluation of weight loss, anemia or suspected gastrointestinal bleeding, and diarrhea. the most common indication for colonoscopy was screening/surveillance for colorectal cancer (56%). additional indications included evaluation of suspected or established inflammatory bowel disease, anemia or suspected gastrointestinal bleeding, and diarrhea. this included use of nbi in 4.7% (5/106) of egds and 7.5% (16/212) of colonoscopies (p=0.47 for comparison). no difference in rate of nbi use was found when comparing egd with or without biopsy (5.5% [3/55] vs. 3.9% [2/51]; p=1), or when comparing colonoscopy with or without biopsy (10.5% [8/76] vs. 5.9% [8/136]; p=0.28). nbi use was significantly higher in colonoscopy with polypectomy when compared with colonoscopy without polypectomy (13% [10/77] vs. 4.4% [6/135]; p=0.03) (fig. 1). rates of narrow band imaging (nbi) use for all study procedures, esophagoduodenoscopy (egd) with and without biopsy, colonoscopy with and without polypectomy., there was no association between patient type (outpatient vs. inpatient), procedure start time, sedation type, or trainee involvement and use/non-use of nbi. for both egd and colonoscopy, there was no association between procedure indication and use/non-use of nbi. nbi use was observed in zero of seven egds with a documented primary procedural indication of screening for or surveillance of barrett s esophagus. nbi use was observed in 2 of 9 (22 %) colonoscopies with a documented primary indication of surveillance in the setting of chronic ulcerative colitis. no use of an alternative imagine modality, such as methylene blue chromoendoscopy or high magnification endoscopy was described in any operative report. observed endoscopic procedures were performed by 23 faculty endoscopists, with a procedure volume ranging from 1 to 58 per endoscopist during the 2-week study period. the rate of nbi use ranged from 0 to 100% of procedures per endoscopist. after excludsion of endoscopists who performed fewer than five endoscopies during the study period, the rate of nbi use for the remaining 15 endoscopists ranged from 0 to 23 %. there was a significant difference in rate of nbi use (p<0.01 for overall comparison) amongst these 15 endoscopists. nbi use among the 5 highest volume endoscopists ranged from 0 to 16 %. among cases with observed nbi use, photodocumentation of nbi use was present in 24% (5/21) of operative reports. this observational study detected use of nbi in 6.6% of elective endoscopic procedures. the highest rate of nbi use was observed in colonoscopies with polypectomy (13%), and this rate was significantly higher than that observed in colonoscopies without polypectomy. this study also detected a significant difference in the rate of nbi use in global comparison of individual endoscopists. specific factors that influence the adoption of nbi use into routine practice by individual endoscopists were not examined in this study. endoscopists were not asked to self-report nbi use and were not informed of the study. the study was designed in this fashion to avoid the potential of a hawthorne effect, wherein performance may be influenced in subjects who are aware that they are being observed. the endoscopists were blinded to the study, making the study design and findings unique. a survey of european university hospitals reported use of nbi or alternative commercial enhanced-imaging technology in 67% of institutions in the evaluation of barrett s neoplasia 9, but did not report a per-case use rate. in the current study, there was no a priori hypothesis regarding the rate of nbi use expected in this observational study, as there are limited standard recommendations or guidelines for routine nbi use in diagnostic endoscopy, and existing guidelines are open to flexible interpretation. for instance, the american gastroenterological association medical position statement on barrett s esophagus suggests that chromoendoscopy or electronic chromoendoscopy is not necessary in the routine endoscopic surveillance of barrett s esophagus, but may be helpful in guiding biopsies for patients with dysplasia or visible mucosal abnormalities 10. the rate of nbi use in this study can therefore not be subjectively designated as high or low, but instead serves as a baseline metric in this study setting against which future nbi use can be measured. adoption of nbi use in routine diagnostic endoscopy may have implications for application of future enhanced-imaging technologies. in the hands of the endoscopist no additional equipment is required other than an existing video monitor and nbi-equipped scope and processor. an endoscopist can toggle between nbi and standard white light literally in seconds, without use of additional endoscopic devices, accessories, or medications. and while there may be a learning curve for interpretation of nbi images 11 12, endoscopist interpretation of endoluminal nbi images would seem less a departure from white light endoluminal images than interpretation of images generated by other optical techniques, such as endomicroscopy, that focus on cellular structures. based on these criteria, one would speculate that adoption and use of imaging technologies requiring additional equipment or capital investment, additional time with respect to procedure duration, and/or increasingly complex image analysis would be lower than that for nbi. finally, it is worthwhile to note that documentation of nbi use was uncommon. photodocumentation of nbi use was provided in 24% (5/21) of operative reports from cases in which nbi use was observed. future consideration may be warranted as to whether documentation guidelines should exist for procedural use of nbi or other adjunct imaging modalities. an american society of gastrointestinal endoscopy preservation and incorporation of valuable endoscopic innovations (pivi) statement on real-time endoscopic assessment of histology of colon polyps suggests that lesion photocumentation is necessary if a resect-and-discard strategy is to be implemented 13. while extending the study duration to increase sample size would increase statistical power and eliminate the possibility of type ii error in examining factors associated with nbi use, the study as completed is likely to have strong internal validity, in that the observed range of endoscopic procedures during the 2-week time period is likely to offer an accurate representation of the range of endoscopic practice in this setting. it may not be possible, however, to generalize the study findings to other institutions or endoscopy settings which may have differing practice patterns. instances of nbi use may not have been documented if not observed or not recognized by the endoscopy technician. an alternative would have been to video record the entire procedure for subsequent review; however maintenance of adequate blinding with this approach would be challenging. documented cases of nbi use can not distinguish between intentional and unintentional nbi use for instance, if the endoscopist had inadvertently pressed the button on the scope handle for application of nbi when he/she had intended instead to press the button for image capture. in addition, there was limited heterogeneity in some of the procedural variables (e. g. trainee involvement) to adequately assess for potential association with nbi use/non-use. a post hoc survey of participating endoscopists might offer insight as to whether their opinions on the role of nbi match their actual practice, but has no bearing on the aim of this study, which was to objectively document rate of nbi use. in summary, additional large-scale prospective data are needed to assess the magnitude of impact of nbi on routine endoscopic practice.

background and study aim: narrowband imaging (nbi) is an enhanced endoscopic optical technique which filters white light and accentuates imaging of the mucosal vasculature. potential clinical applications of nbi include endoscopic inspection of barrett s esophagus and endoscopic diagnosis of colorectal polyps. the degree to which nbi use has been adopted into clinical practice is unknown. the study objective was to identify the rate of nbi use in patients undergoing elective esophagoduodenoscopy (egd) and colonoscopy, and to identify procedural factors associated with nbi use. methods: elective endoscopic procedures were prospectively observed over a 2-week study period. nbi use during diagnostic egd or colonoscopy was recorded in blinded fashion. results: nbi use was observed in 6.6% (21/318) of procedures, including 4.7% (5/106) of egds and 7.5% (16/212) of colonoscopies. there was no difference in rate of nbi use when comparing egd with or without biopsy, or when comparing colonoscopy with or without biopsy. nbi use was significantly higher in colonoscopy with polypectomy compared with colonoscopy without polypectomy (13% [10/77] vs. 4.4% [6/135], p=0.03). nbi use varied significantly among endoscopists. there was no association between patient type (outpatient vs. inpatient), procedure start time, sedation type, or trainee involvement and use/non-use of nbi. procedural documentation of nbi use was limited. conclusions nbi use was observed in 6.6% of elective endoscopic procedures and was highest in colonoscopies with polypectomy. rate of nbi use varied significantly among endoscopists. additional studies are needed to assess the magnitude of impact of nbi on routine endoscopic practice.

PMC4440375

pubmed-1361

keratinocyte cancer (kc), including squamous cell carcinoma (scc) and basal cell carcinoma (bcc), is the most commonly diagnosed cancer in the united states. while ultraviolet radiation is an established risk factor for kc, growing evidence suggests infection with cutaneous human papillomavirus (hpv) may increase the risk of cutaneous scc [38]. we previously reported an increased risk of scc associated with the presence of four or more types of cutaneous hpv dna in eyebrow hairs. serological responses to genus beta hpv types were found to be associated with increased risk of scc. further, poor tanning ability was associated with 6.9 times increased risk of scc among those who were seropositive to beta hpv type. collectively, these findings suggest that cutaneous hpv may play a role in the pathogenesis of scc. however, it should be noted that some previous case-control studies reporting an association between beta hpv and scc included only primary cases of scc [6, 9], while others [3, 4] did not differentiate between primary and subsequent sccs. therefore, the role of cutaneous hpv infection in the development of subsequent scc is unknown. as compared to bccs, sccs are more likely to be clinically aggressive with a propensity to recur and metastasize [1, 10]. as reviewed previously, patients with metastatic scc have less than 20% survival rate over 10 years. varying recurrence rates for scc have been reported based on the site of involvement, metastasis, and grade. in a retrospective study, 30% sccs involving temporal bone others have reported lower scc recurrence rates of 915% [12, 13]. as summarized by alam and ratner, risk factors for recurrence of sccs include tumor size, site, tumor depth, perineural invasion, history of treatment for scc, and tumor differentiation. while these factors may aid in the identification of scc cases at high risk of recurrence, they provide limited scope for intervention beyond treatment or close surveillance to prevent the recurrence of scc. scc cases are also at a high risk of developing second primary sccs. in a large population based study of over 25,000 swedish scc cases, a significantly increased risk of second primary scc was observed (standardized incidence ratio=15.6). as cautioned by marcil and stern, some of the previous studies examining the risk of a subsequent scc included index scc cases with a history of>1 scc or did not provide such information. thus, the index sccs may have comprised both first and recurrent sccs. the high morbidity associated with development of subsequent sccs warrants further research. given the increased risk of scc observed in association with cutaneous hpv infection as described above, it is reasonable to hypothesize that patients with sccs associated with cutaneous hpv infection are more likely to develop subsequent sccs compared to patients with sccs that are not associated with hpv. using a subset of scc cases (n=150) from our previously conducted study [3, 4], we conducted a retrospective cohort study to examine the association between cutaneous hpv infection at the time of scc diagnosis in the parent study with the risk of subsequent scc. infection with cutaneous hpv was assessed using three biomarkers: serum antibodies to hpv, presence of hpv dna in eyebrow hairs, and presence of hpv dna in scc tumors. briefly, a clinic based case-control study was conducted at moffitt cancer center in tampa, florida, in 20072009. histologically confirmed cases of scc (n=173) were identified through the university of south florida (usf) dermatology clinic. both newly diagnosed primary sccs and cases with a prior history of scc were included in the case group. controls included patients without history of any type of cancer and who were negative for skin cancer based on a full-body screening exam at the time of enrollment. of the 173 scc cases enrolled in the parent case-control study, 150 scc cases were followed through 2014. the remaining cases either did not return to the clinic or were only referred to usf dermatology clinic for moh's surgery. cases without complete follow-up were similar in age and gender distribution to the cases with complete follow-up. the 150 index scc cases with complete follow-up through 2014 were included in the present analyses. serology data on 33 cutaneous hpv types, including hpv types in the alpha, beta, and gamma genera (n=150), as well as beta hpv dna (n=25 beta hpv types) status in eyebrow hairs (n=145) and scc tumor tissue (n=131), at the time of enrollment of index scc, were available from the parent study [3, 4]. self-reported data on demographics, smoking, alcohol consumption, and skin cancer risk factors, including history of blistering sunburns, sun exposure, and tanning ability were also available from the parent study. for the current retrospective cohort, subsequent scc was defined as the scc, including recurrent and second primary scc, detected after the index scc was diagnosed at the enrollment biopsy in the parent case-control study [3, 4]. medical charts of index sccs were reviewed, and data on the date of past history of scc prior to enrollment of an index scc case in the parent case-control study, date of subsequent scc diagnosed after the index scc enrollment, site and tumor dimensions of subsequent scc, other incident keratinocyte cancers and date of last contact or death were abstracted. all participants provided written informed consent, and the study protocol was approved by the institutional review board at usf. serum samples obtained from the index scc cases at the time of their enrollment in the parent study were examined for antibodies to major capsid protein l1 for 33 cutaneous hpv types including genera alpha (2, 3, 7, 10, 27, 57, and 77); beta [beta 1 (5, 8, 20, 24, 36), beta 2 (9, 15, 17, 23, 38, 107), beta 3 (49, 75, 76), beta 4 (92), and beta 5 (96)], gamma (4, 48, 50, 65, 88, 95, 101, 103), mu (1), and nu (41). as described previously, antibodies were detected using an enzyme linked immunosorbent assay [17, 18] and fluorescent bead-based multiplex serology. measurement of hpv dna in eyebrow hairs and fresh-frozen scc tumors has been described in detail previously [3, 4]. briefly, hpv dna was extracted from eyebrow hairs and scc tumor tissue with the qiagen ez1 dna tissue kit, and hpv genotyping was performed using a type specific multiplex assay detecting dna from 25 genus beta hpv types (5, 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96) [20, 21]. follow-up time was defined as the time interval between the date of index scc biopsy at enrollment in the parent study and date of diagnosis of subsequent scc or date of last contact. five-year cumulative incidence of subsequent scc was estimated using person-years of follow-up. demographic, lifestyle factors, and skin cancer risk factors were compared between those who developed a subsequent scc and those who did not, using chi square and fisher's exact tests, as appropriate. cox proportional hazards ratios (hr) and corresponding 95% confidence intervals (cis) were estimated for associations of seropositivity to cutaneous hpv, overall and by genera, with the risk of subsequent scc, adjusting for age and gender. seropositivity to any hpv was defined as proportions of scc cases that were positive for an antibody to at least one hpv type across all genera (overall cutaneous hpv seropositivity), within the specific genera (genus specific seropositivity) or within specific species (species-specific seropositivity). seropositivity to multiple hpv infections was defined as the presence of antibodies corresponding to 2 beta hpv types. any beta hpv infection in eyebrow hairs, overall and by species, was defined as the proportion of cases who had dna corresponding to at least one hpv type in genus beta or in a given species within genus beta, respectively. multiple hpv infections were defined as the presence of hpv dna corresponding to 2 beta hpv types. since scc tumors have lower viral dna load compared to eyebrow hairs, hpv dna status in tumors was categorized as cox proportional hazards ratios and corresponding 95% cis were estimated for associations of beta hpv dna positivity, overall and by species, with the risk of subsequent scc, adjusting for age and gender. associations of both beta hpv seropositivity and beta hpv dna positivity in eyebrow hairs with the risk of subsequent scc were stratified by scc tumor beta hpv dna status. as seen in table 1, scc cases included in this study were all white, with an average age at index scc diagnosis of 64.5 years, with 92.8% cases reporting a prior history of scc. of the 150 index scc cases, 105 (70%) were diagnosed with at least one kc during follow-up. ninety-one out of the 150 scc cases were found to have at least one subsequent scc since their enrollment in the previous case-control study [3, 4]. up to five scc tumors per case, the majority in the head and neck region, were detected at the time of diagnosis of subsequent scc. subsequent sccs were detected within an average of 1 year from the date of scc biopsy at enrollment visit. scc cases with a past history of scc or bcc or both were significantly more likely to have a subsequent scc (table 2). none of the other demographic or lifestyle characteristics were associated with subsequent scc (table 2). seropositivity to cutaneous hpv infections, overall, by genera and beta hpv species, was not associated with the risk of subsequent scc (table 3). presence of beta hpv dna in eyebrow hairs was not associated with the risk of subsequent scc, overall or by species (table 4). however, after stratification by tumor beta hpv dna, any beta hpv infection in eyebrow hairs was significantly associated with 70% reduced risk of subsequent scc among cases who were positive for any beta hpv dna in tumor tissue (table 5). further, species-specific analyses showed that any beta-2 hpv infection in eyebrow hairs was associated with 68% significantly reduced risk of subsequent scc among tumor dna positive index scc cases (table 5). beta hpv seropositivity was not associated with subsequent scc after stratification by tumor beta hpv dna status (table 5). cutaneous sccs contribute substantial morbidity due their tendency to recur and associated high risk of development of second primary malignances of the skin and other organs. with the rising incidence of scc, the prevalence of recurrent and second primary sccs will also increase contributing substantially to public health burden. it is important to identify factors predisposing to recurrent and second primary sccs to guide development of better follow up protocols, characterize high risk sccs, and inform preventive strategies aimed at reducing the morbidity associated with subsequent sccs. in this retrospective cohort study, while no association was observed between cutaneous hpv seropositivity and the risk of subsequent scc, a significantly inverse association was observed between cutaneous beta hpv infection in eyebrow hairs detected at the time of index scc diagnosis and subsequent scc development, among scc cases with hpv dna positive index scc tumors. these findings are in contrast to the positive association reported between cutaneous hpv infection, measured by hpv serology and presence of hpv in eyebrow hairs, and scc [3, 4]. however, our findings are consistent with findings from previous studies on mucosal hpv infection associated cancers. mucosal hpv infection has been previously associated with improved survival [2325] or reduced rate of recurrence in some cancers. for example, in a small study of 41 cases, recurrence rate of nasopharyngeal carcinoma was 75% in hpv tumor dna negative patients compared to 11% in tumor dna positive patients. reported that hpv positive oropharyngeal cancers had 70% significantly improved survival compared to hpv negative cases, with significant survival benefits for men versus women. as discussed previously, increased sensitivity to radiation treatment and reduced occurrence of p53 mutations in hpv tumor positive cases have been suggested as possible mechanisms of improved prognosis in association with mucosal hpv infection. in the context of cutaneous scc, which is mainly treated by excision, it is not clear whether the underlying mechanisms of improved prognosis of hpv positive sccs are similar to those suggested for prognosis of mucosal hpv associated cancers. cutaneous hpv infection is thought to be involved in the initiation, but not the maintenance of scc [27, 28]. one explanation of the observed inverse association between beta hpv infection and subsequent scc could be that beta hpv infection in index scc tumors may be immunogenic and could potentially stimulate host immune response that prevents the development of subsequent sccs. our findings may suggest different mechanisms driving tumor progression in hpv dna positive versus hpv dna negative scc cases. immunological and genetic factors that are associated with hpv dna negative tumors should be explored further. previously, we showed that, among a subset of scc cases included in the present study, single nucleotide polymorphisms (snps) in epidermodysplasia verruciformis genes, regulating immune response against hpv, were associated with reduced risk of beta hpv dna positive scc tumors. the findings from our study should be interpreted with caution. a majority (92.8%) of index scc cases had a history of scc prior to their enrollment in the parent case-control study. thus, external validity of our findings may be limited to those cases with a past history of scc. therefore, association of change in hpv infection status over time with the risk of subsequent sccs could not be evaluated. hpv dna status in subsequent scc tumors detected during follow-up was not measured. due to limited sample size, robust statistical analyses for hpv type specific association with subsequent scc development could not be conducted. the subsequent sccs detected in this study could be recurrent sccs, presenting at the same anatomic site as primary scc, or second primary sccs and the analyses stratified by these two outcomes were not conducted due to small sample. conversely, combining recurrent and second primary sccs as one outcome may have attenuated the true effect of hpv infection in index scc on the development of recurrent or second primary sccs. to our knowledge, this is the first prospective study reporting an association between cutaneous hpv infection in scc cases and subsequent sccs. of the 173 scc cases enrolled in the parent case-control study, 86.7% cases were included in the present analyses highlighting complete follow-up on a large proportion of the cases. since 92.8% scc cases reported a prior history of scc, the findings from our study may help further prognostic stratification of these sccs using a biomarker of infection. these cases may represent ideal candidates to direct infection related prevention measures due to the high morbidity associated with risk of developing subsequent sccs among sccs. based on the observed inverse association between hpv seropositivity and risk of subsequent scc (table 3), although not significant, immune response against hpv may protect against the development of subsequent sccs. thus, acquired immunity through vaccination against cutaneous hpv could potentially be explored among scc cases. additional strengths of this study include, examination of biomarkers of both, present hpv infection (eyebrow hair dna and hpv serology) and past hpv infection (hpv serology) and evaluation of hpv infection as a prognostic marker by hpv genera and species. in conclusion, the results from our prospective cohort study suggest that cutaneous hpv infection in eyebrow hairs is inversely associated with the development of subsequent sccs in a subset of scc cases with hpv dna positive tumors. the observed association supports previous reports of lack of a role for cutaneous hpv infection in progression of sccs. while cutaneous hpv may not be directly involved in the prognosis of scc, this line of research may eventually lead to discovery of underlying immunological factors involved in scc development and progression. cutaneous hpv infection status could potentially be used as a biomarker for prognostic stratification of sccs.

the role of cutaneous human papillomavirus (hpv) infection in the development of subsequent cutaneous squamous cell carcinoma (scc) is unknown. pathologically confirmed cases of scc (n=150) enrolled in a previously conducted case-control study were included in a retrospective cohort study to examine the association of cutaneous hpv at the time of scc diagnosis with the risk of subsequent scc development. data on hpv seropositivity, hpv dna in eyebrow hairs (eb) and scc tumors were available from the parent study. incidence of subsequent scc was estimated using person-years of follow up. cox proportional hazards ratios were estimated to evaluate the associations of both, hpv seropositivity and hpv dna positivity with subsequent scc. the five year cumulative incidence of subsequent scc was 72%. seropositivity to cutaneous hpv was not associated with the risk of subsequent scc (hr=0.83, 95% ci=0.411.67). any beta hpv infection in eb was associated with reduced risk (hr=0.30, 95% ci=0.110.78) of subsequent scc among cases who were positive for beta hpv dna in tumor tissue. infection with beta hpv type 2 (hr=0.32, 95% ci=0.120.86) in eb was associated with reduced risk of subsequent scc among hpv dna positive sccs. in conclusion, beta hpv infection was inversely associated with the risk of subsequent scc.

PMC5116506

pubmed-1362

in 2008, the greater london authority and the london health consortium, with funding provided by the big lottery, instigated a series of health interventions entitled well london. these activities were set up in 20 of london's most disadvantaged neighbourhoods, in an effort to promote healthier life-styles as a way of combating chronic disease (wall et al. an evaluation of the project was undertaken in which a series of in-depth interviews were conducted with 60 residents across three of the well london areas; a key component of the interview was to enquire about the environment and residents perceptions of their neighbourhood. a frequent and vociferous complaint that arose unprompted concerned the prevalence of dog excrement, or to use local terminology: dog poo. not only was the frequency of the complaint remarkable, but also its specificity and distinctive nature, making further detailed investigation necessary. apart from an element of antipathy, there was also considerable anger expressed in relation to: who was responsible; the lack of care demonstrated by its ongoing presence; and fear over its risk to human health. dog-poo was not the only form of pollution complained about in the three areas, but it was the most frequently cited. in terms of risk to human health, for example, since 2000 between one and eight laboratory confirmed reports of toxocarosis have been received annually by the health protection agency (hpa 2011). in addition, a keep britain tidy report into litter cites a 40% drop in the incidence of dog faeces in the uk over recent years (2007). despite this lack of evidence concerning risk or prevalence, canine faeces continues to be a significant cause for complaint, requiring further explication in order to understand its relevance for public health. in a study of urban environmental factors that impact on health, kennedy (2000) cites residents complaints of dog faeces as a blight that affected their sense of wellbeing. similarly, two separate studies by jones (1996) and cresswell (1992) report residents complaints of dog faeces in urban areas of the uk affected by poverty and environmental neglect. therefore, the presence of dog faeces has associations and implications that can be linked with more subjective responses. one key facet of the complaints raised by residents in the well london study was a sense of disgust evoked by therefore, the challenge was to explore not the grievance itself (whether dog poo existed or its potential as a disease carrying agent) but rather the symbolic this article critically engages with residents explicitly focused disgust with canine faecal matter and generates understanding of its symbolic value that helps clarify what has to date, been a poorly resolved public health issue. sixty qualitative interviews were conducted in total: 20 individuals in each of the three well london areas, which were located in the north, east and south of the city. all three areas were characterised by high levels of unemployment, poor health, environmental neglect and a mixed indigenous and global migrant population. quality assurance procedures were undertaken whereby three randomly selected interviews were recoded, blind to the initial coding, by two independent researchers. the three versions of each interview were then compared to identify new codes and establish the degree of consensus in applying a particular code to similar text. a major part of the interview focused on what residents thought about the area where they lived. recurrent and spontaneous reference was made to the prevalence of dog faeces in the area and, more subjectively, the feelings that were evoked by its presence. dog poo (most frequently cited among all its synonyms), over all other complaints. extended coding of the interviews revealed the contextual significance of these complaints. incivility emerged as a recurrent theme: a lack of respect for the neighbourhood environment by residents and negligence by local authorities whether the police or local council. this meant carrying out repairs, cleaning and ensuring personal safety, especially in relation to the presence of gangs, local drug dealers and other types of criminality. further, measurement of actual incidence of dog foul across london was undertaken: 40 areas were examined and divided into 211 were found to contain no dog faeces; 72 had a small amount and eight had a moderate amount. however, 117 coded references pertaining to dog poo (and its synonyms: dog shit/muck/litter/mess/fouling) were found in all 60 interviews. the disparity between high numbers of complaints and the limited presence of actual faecal matter required an alternative mode of analysis that would help locate, more specifically, residents concerns. interpreting how meaning is expressed through cultural symbols in this instance analysing the symbolic content and context in which the complaints occurred, highlighted compelling evidence of its link to social neglect, including criminality, dirt and the absence of cohesive community relations. the work of social anthropologist douglas (1966) is seminal in the symbolic interpretation of matters pertaining to pollution. also, studies by van der geest (1998, 2007, 2008), stallybrass and white (1986), laporte (2000), enzensberger (1972) and miller (1997) offer useful theoretical perspectives on dirt and disgust; especially regarding bodily substances that transgress boundaries in relation to social morality, inequality and taboo. the inherent value of examining cultural symbols shows how in this instance, they express not only fears of the transgression of boundaries, but also how dog poo is constructed as a metonym for the disgust felt by residents about their experience of incivility. this includes neglect of the environment, and a lack of respect by local authorities and fellow neighbours alike, as one resident implied: and the worst thing is they just, they poo everywhere in the buildings a month back there was dog poo in our own building. i do n't know whose poo it is whether it's human or dog poo, but i seen it. another described a similar sense of repulsion: the outside spaces, there's dog foul. i mean wherever you put your feet and it's unsafe because the poo of the dog and you put your foot oh no! the focus on canine excrement did not exclude other complaints: of drugs, gangs, noisy neighbours and the lack of intervention by either police or local authorities. however, complaining about dog poo conveyed a powerful message about these neighbourhoods blighted by multiple problems, and often by unknown perpetrators: like this drug dealing problem going on, on my doorstep. you do n't know who's done it, or why they done it, or where did they come from. and they're smoking, they've got needles there on the stairs, they wee do on the stairs, everything we ca n't say nothing. and my lift is still broken. (sita) the notion of incivility evoked through residents descriptions of their environment determined the appropriateness of symbolically linking complaints of dog poo, with the experiences of social neglect. as requests to improve living conditions and expressed worries about safety and cleanliness were ignored, a pervasive sense of hopelessness took over. right, the area is dangerous, dirty, very, very unkempt and has been basically forgotten by the powers that be for the past, i'd say, 15 years. in as much as one person who i spoke to at the local housing office who was at the time in charge of major works, did n't even know where i was living. did n't know where it was, so that's the it's just been a forgotten little area and it's been left to rot and decay. (robert) for stallybrass and white: what is socially peripheral, is so frequently symbolically central (1986: 5, authors italics). this resonates with the well london residents sense of social marginalisation that was marked by the absence of effective management of the neighbourhood, as explicitly articulated above. but, when these explicit complaints were neither heard nor acted upon, they became symbolically represented by excreta, which defined their experience of marginality. the path as you go up there, there's always glass up there or dogs poo, always there's dogs poo along there and it's, well, it's just disgusting. van der geest (1998, 2007, 2008) has written extensively on attitudes towards defecation, including an historical examination of the origins of our revulsion towards bodily functions and their management. he suggests that without the containment of bodily matter, there is nothing to prevent chaos and disorder (2007). therefore, if faeces, animal or human, is taken as an effective metonym that represents disorder, it follows that managing dirt has as its main function the restoration of order primarily, social order as part of a civilising process. dirt in its rightful place ordered and bounded within a set of social rules of behaviour. douglas ' (1966) work on symbolism and in particular matter out of place, states that dirt is defined by its place or context; in other words, where it is found and who is responsible for it. for example, faecal matter inside the body is less problematic than when it becomes external to it, where it is an object of revulsion. the attitude and management of excrement, both human and animal, is linked with the state as it intervenes in the management and consequent attitudes towards dirt; segregation of public and private means that all faecal matter is consigned to private rather than public space (enzensberger 1972, laporte 2000). faeces, now considered taboo in the public domain, contrasts with the pre-victorian era when horse manure covered the streets of london and running sewers were open to public senses (pike 2005). this segregation of public and private in relation to cleanliness was also acknowledged among residents: when we moved to this area, we saw dog poo now it's a little bit clean, my area is, because it's a private area. another spoke of her annoyance at her neighbour who pushed rubbish over to her side of the passageway, invading her private space and breaking boundaries. but, complaints also illustrated a more extreme form: the permeability of private space that could be infiltrated by bad smells and noise: yeah, the noise. my downstairs neighbours, they're singing like five o'clock in the morning and it wakes you up and you just ca n't do anything. and then they smoke marijuana and you know this is a converted building? so there's lots of holes in the you see those things? yeah, every time they smoke, every time they drink if they're vomiting in their toilet, it all comes up (here). dog poo has salience within their particular social and cultural milieu of urbanised, inner city london where neighbourhoods are unsafe, environmentally polluted and often without social cohesion. anger and blame is placed on those who seem to ignore the boundaries i.e dog owners, dirty neighbours, the police, or the local authorities responsible for the management of the neighbourhood. dog poo is therefore a transgression of not only spatial boundaries, but also social norms of what is considered civil behaviour; especially when focused on personal responsibility: and that's one thing as well, the dog fouling, they do n't pick up. they do n't pick up their dog fouling, do you know what i mean? and i'll tell you what, it would be a big sense of achievement if they actually put cameras in every direction to catch these people out, because it's terrible. you've got kids playing around and every time they're playing football, they stop in dog foul. as campkin and cox (2007) suggest theorising about dirt is constitutive of space and social relations. here, vociferous complaints of canine faeces in the absence of actual evidence of high incidence indicated a more hidden form of distress or grievance that was better understood by examining its symbolic content, as well as its context. by taking dog poo as a metonym for the disgust with which residents experienced their neighbourhoods, the authorities and each other it was possible to gain deeper insight into the problem. acknowledgement of this has implications for intractable public health problems, where not only the contextualisation of complaints is important, but also analysis of their deeper symbolic components; especially in the face of frequent, inexplicable complaints. conducting such an analysis uncovered the distress of residents living in some of london's more deprived neighbourhoods where grievances were frequently ignored, leading to frustration and anger. had the issue of dog faeces clean up operation alone, this would never have uncovered the scale of the problem or the depth of feeling in relation to the issues, which can be defined as poor social relations and inadequate or ineffective communication with local authorities. investigations and analyses of this kind lend themselves to the development of constructive, inclusive public health policies that can improve the wellbeing of marginalised populations by understanding how people articulate their concerns. the approach used here is effective in this, because it can: generate new insights into intractable public health problems; offer alternative and useful methods to understand and communicate complex social issues in relation to public health; advance theoretical knowledge; and broaden the scope of analytic tools with which to understand well-being and society.

during a well london study, residents were asked about their neighbourhood and its environment. above all other complaints, dog poo was mentioned as a key concern. despite low rates of infection and disease among the human population resulting from contact with canine faecal matter, the concerns of the public continue to rate it as a serious public health issue. most public health studies, therefore, seek to identify processes of transmission and disease pathology as a method of addressing the problem. this study approaches the issue through a contextualised analysis of residents complaints, using anthropological theory to examine the symbolic representation of dog poo. analysis of the interviews shows that these specific complaints were located among less easily defined or articulated experiences of social and environmental neglect, where neighbours were estranged from one another and local authorities seen as negligent. this approach has important implications for public health, as it provides not only a strong indicator of the level of dissatisfaction within some of london's more disadvantaged neighbourhoods, but also identifies a need for policies that are grounded in cross-disciplinary research into the relationship between health, wellbeing and experiences of marginalisation among urban populations.

PMC3545484

pubmed-1363

periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. prostaglandin e2 (pge2), interleukin- (il-)6, and il-8 are known to play important roles in inflammatory responses and tissue degradation. pge2 has several functions in vasodilation, the enhancement of vascular permeability and pain, and the induction of osteoclastogenesis and is believed to play important roles in inflammatory responses and alveolar bone resorption in periodontal disease. il-8 acts as a chemoattractant for neutrophils that produce proteases such as cathepsin, elastase, and matrix metalloproteinase- (mmp-)8, leading to tissue degradation. recently, we reported that several kampo medicines, shosaikoto, hangeshashinto, and orento, suppress lipopolysaccharide- (lps-) induced pge2 production by hgfs and suggested that these kampo medicines have anti-inflammatory effects in periodontal disease. another kampo medicine, kakkonto (tj-1), has been clinically used for various diseases such as the common cold, coryza, the initial stage of febrile diseases, and inflammatory diseases. there are several reports that kakkonto shows antiallergic effects [8, 9] and antiviral effects [1013] in animal and in vitro experimental models. for anti-inflammatory effects therefore, we considered the possibility that kakkonto decreases pge2 production by human gingival fibroblasts (hgfs) and has anti-inflammatory effects with respect to periodontal disease. moreover, lps-treated hgfs produce inflammatory chemical mediators such as pge2 and inflammatory cytokines such as il-6 and il-8 [2, 15, 16]. moreover, because hgfs have sustained production of pge2, il-6, and il-8 in the presence of lps, these mediators and cytokines in periodontal tissues are thought to be derived from hgfs. therefore, we believe that examining the effects of drugs on hgfs, as well as on monocytes and macrophages, is important in the study of periodontal disease. in the present study, we examined the effect of kakkonto on lps-induced pge2, il-6, and il-8 production using this in vitro model. kakkonto was purchased from tsumura&co. (tokyo, japan; lot number: d23122), and its components are listed in table 1. kakkonto was suspended in dulbecco's modified eagle's medium (d-mem, sigma, st. louis, mo, usa) containing 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (culture medium) and was rotated at 4c overnight. then, the suspension was centrifuged and the supernatant was filtrated through a 0.45 m-pore membrane. lps from porphyromonas gingivalis 381 was provided by professor nobuhiro hanada (school of dental medicine, tsurumi university, japan). hgfs were prepared as described previously. in brief, hgfs were prepared from free gingiva during the extraction of an impacted tooth with the informed consent of the subjects who consulted matsumoto dental university hospital. the free gingival tissues were cut into pieces and seeded onto 24-well plates (agc techno glass co., chiba, japan). hgfs were maintained in culture medium at 37c in a humidified atmosphere of 5% co. for passage, hgfs were trypsinized, suspended, and plated into new cultures in a 1: 3 dilution ratio. this study was approved by the ethical committee of matsumoto dental university (number 0063). the numbers of cells were measured using wst-8 (cell counting kit-8; dojindo, kumamoto, japan) according to the manufacturer's instructions. in brief, hgfs (10,000 cells/well) were seeded in 96-well plates (agc techno glass co., chiba, japan) and incubated in serum-containing medium at 37c overnight. then, the cells were treated with various concentrations of kakkonto (0, 0.5, 1, 2, 5, and 10 mg/ml) in the absence or presence of lps (10 ng/ml) for 24 h (200 l each well) in quadruplicate for each sample. then, the media were removed by aspiration and the cells were treated with 100 l of mixture of wst-8 with culture medium for 2 h at 37c in co incubator. optical density was measured (measured wavelength at 450 nm and reference wavelength at 655 nm) using an imark microplate reader (bio-rad, hercules, ca, usa), and the mean background value was subtracted from each value. hgfs (10,000 cells/well) were seeded in 96-well plates and incubated in serum-containing medium at 37c overnight. then, the cells were treated with various concentrations of kakkonto (0, 0.01, 0.03, 0.1, 0.3, and 1 mg/ml) in the absence or presence of lps (10 ng/ml) for 24 h (200 l each well) in triplicate for each sample. after the culture supernatants were collected, viable cell numbers were measured using wst-8 as described above. the concentrations of pge2, il-6, and il-8 in the culture supernatants were measured by elisa according to the manufacturer's instructions (pge2, cayman chemical, ann arbor, mi, usa; il-6 and il-8, biosource international inc., camarillo, ca, usa) and were adjusted by the number of viable cells. data are represented as ng or pg per 10,000 cells (mean sd, n=3). the effects of kakkonto on the activities of cyclooxygenase (cox)-1 and cox-2 were analyzed using a cox inhibitor screening assay kit (cayman chemical, ann arbor, mi, usa) according to the manufacturer's instructions. cox activities were evaluated by the measurement of prostaglandin produced from arachidonic acid by cox-1 or cox-2. these values were normalized to a relative value of 100% for cells without lps or kakkonto treatments, and are represented as means sd (n=3). hgfs were cultured in 60 mm dishes and treated with combinations of lps and kakkonto for the indicated times. then, cells were washed twice with tris-buffered saline, transferred into microcentrifuge tubes, and centrifuged at 6,000 g for 5 min at 4c. supernatants were aspirated and cells were lysed on ice in lysis buffer (50 mm tris-hcl, ph 7.4, 1% nonidet p-40, 0.25% sodium deoxycholate, 150 mm nacl, 1 mm ethyleneglycol bis(2-aminoethylether) tetraacetic acid (egta), 1 mm sodium orthovanadate, 10 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 5 g/ml leupeptin, and 1 g/ml pepstatin) for 30 min at 4c. then, samples were centrifuged at 12,000 gfor 15 min at 4c, and supernatants were collected. the protein concentration was measured using a bca protein assay reagent kit (pierce chemical co., rockford, il, usa). the samples (10 g of protein) were fractionated in a polyacrylamide gel under reducing conditions and transferred onto a polyvinylidene difluoride (pvdf) membrane (hybond-p; ge healthcare, uppsala, sweden). the membranes were blocked with 5% ovalbumin for 1 h at room temperature and incubated with primary antibody for an additional 1 h. the membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. antibodies against cox-2 (sc-1745, 1: 500 dilution), cytoplasmic phospholipase a2 (cpla2, sc-438, 1: 200 dilution), annexin1 (sc-11387, 1: 1,000 dilution), and actin (sc-1616, 1: 1,000 dilution), which detects a broad range of actin isoforms, were purchased from santa cruz biotechnology (santa cruz, ca, usa). antibodies against extracellular signal-regulated kinase (erk; p44/42 map kinase antibody, 1: 1,000 dilution) and phosphorylated erk (phospho-p44/42 mapk (thr202/tyr204) (e10) monoclonal antibody, 1: 2,000 dilution) were from cell signaling technology (danvers, ma, usa). horseradish peroxidase-conjugated anti-goat igg (sc-2020, 1: 20,000 dilution) was from santa cruz, and anti-rabbit igg (1: 20,000 dilution) and anti-mouse igg (1: 20,000 dilution) were from dakocytomation (glostrup, denmark). differences between groups were evaluated by the two-tailed pairwise comparison test with a pooled variance, followed by correction with the holm method (total 16 null hypotheses; 5 null hypotheses without kakkonto versus with kakkonto in the absence of lps, 5 null hypotheses without kakkonto versus with kakkonto in the presence of lps, and 6 null hypotheses without lps versus with lps) (figures 1 and 2). differences between the control group and experimental groups were evaluated by a two-tailed dunnett's test (figure 3). dunnett's test was performed using the glht function in the multcomp package. the viability of hgfs was approximately 90% at up to 1 mg/ml of kakkonto for a 24 h treatment in the absence or presence of lps (figure 1). the viabilities were approximately 70% and 20% at 5 mg/ml and 10 therefore, we used kakkonto at the concentrations of up to 1 mg/ml in further experiments. we examined whether kakkonto affects the production of pge2 and inflammatory cytokines (il-6 and il-8) by hgfs. because kakkonto affects cell viability, the concentrations of pge2, il-6, and il-8 needed to be adjusted according to viable cell number. when hgfs were treated with 10 ng/ml of lps, hgfs produced large amounts of pge2, il-6, and il-8. indomethacin decreased lps-induced pge2 production in a concentration-dependent manner but slightly decreased lps-induced il-6 and il-8 production (data not shown). kakkonto significantly decreased pge2 production in a concentration-dependent manner (figure 2(a)). in the absence of lps in contrast, kakkonto increased lps-induced il-6 and il-8 production (figures 2(b) and 2(c)). in the absence of lps, up to 0.1 mg/ml of kakkonto did not affect il-6 and il-8 production, but above 0.3 mg/ml of kakkonto, their concentrations were increased (figures 2(b) and 2(c)). similar results were obtained using human skin fibroblast tig-103 cells (data not shown). because pge2 production is regulated by cox enzymes and suppressed by acid nsaids such as aspirin and diclofenac sodium, which inhibit cox activities, we examined whether kakkonto inhibits cox-1 and cox-2 activities. kakkonto decreased cox-1 activity to approximately 70% at 1 mg/ml but did not affect cox-2 activity (figure 3). cpla2 is the most upstream enzyme in the arachidonic acid cascade and releases arachidonic acid from plasma membranes. kakkonto did not alter cpla2 expression in the absence or presence of lps (figure 4). however, kakkonto did not alter lps-induced cox-2 expression (figure 4). annexin1, also named lipocortin1, is an anti-inflammatory mediator produced by glucocorticoids that inhibit cpla2 activity [19, 20]. however, neither lps nor kakkonto showed an effect on annexin1 expression (figure 4). cpla2 is reported to be directly phosphorylated at ser505 by erk, resulting in cpla2 activation [21, 22]. erk phosphorylation was enhanced at 0.5 h after lps treatment and thereafter was attenuated. one mg/ml of kakkonto suppressed lps-induced erk phosphorylation at 0.5 h to 2 h (figure 5). in the present study, we examined the effect of kakkonto on lps-induced pge2, il-6, and il-8 production by hgfs. kakkonto concentration dependently decreased lps-induced pge2 production but did not affect pge2 production without lps treatment, similar to shosaikoto, hangeshashinto, and orento [57]. moreover, kakkonto suppressed lps-induced erk phosphorylation. in contrast, kakkonto increased lps-induced il-6 and il-8 production. it is widely known that pge2 leads to inflammatory responses such as vasodilation, enhanced vascular permeability, and pain generation. acid non-steroidal anti-inflammatory drugs nsaids show anti-inflammatory effects by suppression of pge2 production, even though they do not affect il-6 and il-8 production. our findings showing that kakkonto decreases lps-induced pge2 production suggest that kakkonto also has anti-inflammatory effects in periodontal disease and that its effects are mainly mediated by suppression of pge2 production even though kakkonto increased lps-induced il-6 and il-8 production. previously, we demonstrated that orento inhibits lps-induced erk phosphorylation and cpla2 activation, leading to the suppression of pge2 production in hgfs. therefore, we consider that kakkonto decreased lps-induced pge2 production through the suppression of erk phosphorylation in hgfs. although kakkonto increased cox-2 expression in the absence of lps, kakkonto did not alter pge2 production. we consider a likely reason to be the suppression of cpla2 activation through the inhibition of erk phosphorylation and/or the suppression of cox-1 activity. our results showed that kakkonto increased lps-induced il-6 and il-8 production by hgfs. previously, we reported that the activation of the protein kinase a (pka) pathway by adrenaline or aminophylline increases lps-induced il-6 and il-8 production in hgfs and that h-89, a pka inhibitor, decreases lps-induced il-6 and il-8 production [23, 24] in general, steroidal anti-inflammatory drugs (saids) suppress the expression of cpla2, cox-2, and inflammatory cytokines (such as il-6 and il-8) and induce the expression of annexin1. however, kakkonto did not affect cpla2, annexin1, or lps-induced cox-2 expression, and it increased il-6 and il-8 production. this therefore suggests that the mechanism by which kakkonto decreases pge2 production is different from that of saids. many studies have demonstrated that nsaid administration prevents gingival inflammation and several clinical studies have indicated that the concentration of pge2 in gingival crevicular fluid (gcf) is increased in periodontal disease and is decreased by oral administration or mouthwash with nsaids [27, 28]. considering that both nsaids and kakkonto suppress pge2 production, it is possible that administration of kakkonto also decreases the pge2 concentration in gcf and results in the improvement of gingival inflammation. therefore, kakkonto may be useful for the improvement of gingival inflammation in periodontal disease. importantly, kakkonto did not affect the basal level of pge2, although kakkonto decreased cox-1 activity to approximately 70%. because pge2 produced by cox-1 protects gastric mucosa, these results suggest that kakkonto may cause minimal gastrointestinal dysfunction. we demonstrated that kakkonto suppresses lps-induced erk phosphorylation, resulting in the suppression cpla2 activation and further pge2 production by hgfs. these results suggest that kakkonto is clinically useful for the improvement of inflammatory responses in periodontal disease.

periodontal disease is accompanied by inflammation of the gingiva and destruction of periodontal tissues, leading to alveolar bone loss in severe clinical cases. the chemical mediator prostaglandin e2 (pge2) and cytokines such as interleukin- (il-)6 and il-8 have been known to play important roles in inflammatory responses and tissue degradation. in the present study, we investigated the effects of a kampo medicine, kakkonto (tj-1), on the production of prostaglandin e2 (pge2), il-6, and il-8 by human gingival fibroblasts (hgfs) treated with lipopolysaccharide (lps) from porphyromonas gingivalis. kakkonto concentration dependently suppressed lps-induced pge2 production but did not alter basal pge2 levels. in contrast, kakkonto significantly increased lps-induced il-6 and il-8 production. kakkonto decreased cyclooxygenase- (cox-)1 activity to approximately 70% at 1 mg/ml but did not affect cox-2 activity. kakkonto did not affect cytoplasmic phospholipase a2 (cpla2), annexin1, or lps-induced cox-2 expression. kakkonto suppressed lps-induced extracellular signal-regulated kinase (erk) phosphorylation, which is known to lead to erk activation and cpla2 phosphorylation. these results suggest that kakkonto decreased pge2 production by inhibition of erk phosphorylation which leads to inhibition of cpla2 phosphorylation and its activation. therefore, kakkonto may be useful to improve gingival inflammation in periodontal disease.

PMC3945151

pubmed-1364

gliomas are the most frequent primary tumors of the central nervous system (cns) and represent approximately 2% of all malignant diseases. their annual incidence is about 11.5 new cases per 100.000 persons per year [1, 2]. the who classification of tumors of the central nervous system distinguishes the subtypes of glioma according to morphology and grade. high-grade gliomas (hgg, who-grade 3 and 4 glioma) are malignant tumors with a poor survival outcome. in a pivotal phase iii trial, where patients diagnosed with glioblastoma (gb, who-grade iv glioma) who were treated with postoperative radiation therapy (rt) and concomitant temozolomide (tmz) followed by six cycles of adjuvant tmz, the median survival was 14,6 months, while the overall survival (os) was 27.2% at 2 years, 16.0% at 3 years, 12.1% at 4 years, and 9.8% at 5 years. the prognosis of patients with who-grade iii glioma is superior to that of gb patients but much more heterogeneous and correlated with the histopathological and molecular-genetic subtype [5, 6]. following initial resection and postoperative rt, anaplastic gliomas recur after a median of 2-3 years. most often, recurrent grade iii glioma will have transformed into a more aggressive tumor at recurrence (a so-called secondary glioblastoma). the survival of patients with recurrent high-grade glioma following prior therapy with alkylating chemotherapy is grim and no treatment has demonstrated to improve the survival in a randomized clinical trial [6, 7]. hgg are among the most angiogenic tumors and typically express high amounts of vascular endothelial growth factor (vegf). vegf is a key molecular mediator of tumor-associated neoangiogenesis and its expression level has been correlated with tumor vascularisation, who-grade and prognosis [8, 9]. hgg also express the vegf-receptors and frequently carry an amplicon of chromosome 4q12 comprising the vegf-receptor-2 (vegfr2), pdgfr-alfa, and ckit genes in 2330% of cases [1012]. coexpression and/or amplification of both the vegf and vegfr2 constitute an autocrine/paracrine loop. bevacizumab (bev; avastin, roche, basel, switzerland) is a humanized immunoglobulin g1 monoclonal antibody that binds to and inhibits vegf. it has proven to be active in combination with cytotoxic agents and is registered by the fda and ema as part of a combination treatment regimen with chemotherapy for metastatic colorectal cancer, non-small-cell lung cancer, and breast cancer, and in combination with interferon-alfa in metastatic renal cell carcinoma [8, 1319]. on may 5, 2009, the fda granted accelerated approval to bevacizumab as a single agent for the treatment of patients with recurrent glioblastoma. the approval was based on the results of two phase ii clinical trials (avf3708 g and nci 06-c-0064e). the largest phase ii trial, involving a total of 167 patients with recurrent gb, randomized patients between treatment with bev (at a biweekly dose of 10 mg/kg) in one arm and the combination of bev (at the same dose) and irinotecan in a second arm. in the bevacizumab-alone and the bevacizumab-plus-irinotecan groups, the objective response rates were 28.2% and 37.8%, and the estimated 6-month progression-free survival rates were 42.6% and 50.3%, respectively. the number of adverse events in the bev plus irinotecan population was higher (65.8% versus 46.4% grade 3 adverse events), while median overall survival times was comparable between the two arms (9.2 months and 8.7 months, resp., compared to 7.5 months in a historical population). in a single-arm phase ii trial, investigating the sequential use of bev and the combination of bev and irinotecan, no activity was found for the combination after failure of bev as a single agent. bev has also demonstrated antitumor activity as a single agent in patients with recurrent anaplastic glioma [22, 23]; in combination with a variety of cytotoxic agents [2426], and when administered on a once every 3-week schedule (at a dose of 15 mg/kg every 3 weeks). mri-based tumor evaluation in patients treated for recurrent glioma have been characterized by a rapid regression of tumor-associated edema and restoration of the blood-brain/tumor barrier. progression of disease by diffuse, non-gadolinium-enhancing infiltration of the brain (= gliomatosis) may occur in patients that respond to bev. notwithstanding these atypical patterns of progression, updated overall survival of the patients treated in the brain (avf3708 g) study indicated that 16% of patients remained alive at 30 months of followup, a percentage that compares favorably with historical controls. besides the effect of bev on glioma-associated vasculature, responses documented by positron emission tomography (pet) using fluorothymidine (flt), an imaging marker of cell proliferation, were correlated with an improved overall survival in patients treated with irinotecan and bev [30, 31]. on 19 november 2009, the chmp (ema) refused to change the terms of the marketing authorization for bevacizumab in the eu to include recurrent glioblastoma. from april to november 2009, the belgian riziv/inami provided partial (60%) reimbursement for bevacizumab following an individual request to the bijzonder solidariteitsfonds/fonds spcial de solidarit. this paper reports the first experience with bevacizumab for recurrent glioma in patients treated at two brussels university hospitals. this observational (noninterventional) study was performed with the clinical data that were retrospectively retrieved from the medical files of all patients with recurrent high-grade glioma who initiated bev treatment between 9 january 2009 and 27 january 2010. these patients represent the first patients treated with bev for recurrent glioma at two belgian university hospitals, the uz brussel and ulb erasme. we collected data regarding the general clinical and neurological evolution during bev treatment, the bev treatment disposition (table 3), as well as laboratory tests performed during bev therapy. adverse events were classified according to the common terminology criteria for adverse events v3.0 (ctcae)., we made the sums of the maximal cross-sectional radii of the contrast enhancing tumor measured by consecutive contrast mr. a complete response (cr) was defined as a disappearance of all contrast enhancing tumor, with the patient neurologically improved or stable and off corticosteroids. a partial response (pr) was defined as a 50% or more decrease in the size of the contrast-enhancing tumor with the patient neurologically improved or stable and with the corticosteroid dose stable or decreased. progressive disease (pd) was defined as a 25% or more increase in the size of the contrast enhancing tumor or appearance of a separate tumor. stable disease (sd) was defined for all other situations. in addition, we assessed the abnormalities on sequential t2 and flair mri sequences in a similar fashion, and changes on pet scan of the brain for the subgroup of patients evaluated by this imaging modality. bev treatment disposition, bev-related adverse events, demographic and baseline patient, and disease characteristics were summarized using descriptive statistics. kaplan-meier statistics were used to estimate the probability of survival (spss inc., chicago, illinois 60606, usa). nineteen patients (11 men and 8 women) with recurrent supratentorial hgg were identified to have received treatment with bev for progressive disease following failure after prior treatment including surgery, radiation therapy, and chemotherapy. the median patient age at the initiation of bev treatment was 40 years (range 28 to 70). eighty-nine percent of the patients were younger than 50 years at the start of bev treatment. eight (53%) patients had a pathological diagnosis of primary gb, 4 (21%) of secondary gb, and 5 (26%) of recurrent grade iii glioma. in six patients, initial treatment consisted of surgery followed by radiotherapy. in 13 patients, the initial treatment consisted of surgery, followed by rt with concomitant tmz and adjuvant tmz (median number of adjuvant tmz cycles: 6, range 1 to 12). three patients had been treated with radiation therapy at recurrence (one patient was administered fractionated radiotherapy at a dose of 55,5 gy, two patients were treated using -knife radiosurgery). twelve patients underwent additional modalities of salvage therapy for recurrent disease (chemotherapy, dendritic cell vaccination) before initiating bev therapy. three patients initiated bev at a dose of 5 mg/kg every 2 weeks (in one of them, the dose was escalated to 10 mg/kg every 2 weeks after the first administration), 14 patients at a dose of 10 mg/kg every 2 weeks, and 2 patients at a dose of 15 mg/kg every 3 weeks, according to bev administration regimens published in the literature [20, 21, 24, 27, 3335]. a total number of 123 bev treatment cycles were analyzed in this study. a median number of 4 cycles were administered per individual patient (range 1 to 16). three patients were simultaneously treated with a cytotoxic drug (hydroxyurea, tmz, or ccnu). fourteen patients (74%) were treated with corticosteroids at the initiation of bev treatment. the dose of corticosteroids could be tapered in 4 patients and stopped in two of them. nine bev-related adverse events were encountered, of which none were grade 4 or 5 (table 2). two grade 3 adverse events (ulceration of skin striae and an abdominal pain syndrome) necessitated stopping bev administration in the absence of documented tumor progression. four patients (21%) experienced a rapid increase in disease-related symptoms after the initiation of bev. their clinical condition prohibited an objective tumor evaluation with mri after the initiation of bev. in all 4 patients, fifteen (79%) patients were assessable for tumor response on t1 gadolinium-enhanced mri-sequences (gd-t1). tumor regression was complete in 2 patients and more than 50% in an additional 3 patients. this correlates to an objective response rate of 33% according to the macdonald criteria for the 15 evaluable pts on mri and 26% for the intent to treat population. all of these patients had stabilization or improvement of disease-related symptoms and none of them had an increase in corticosteroid dose. seven patients (47%) obtained a stable disease, and 3 (20%) patients experienced immediate progression of disease during bev therapy (figures 1 and 2). assessment of tumor response by t2/flair mri-sequences was available for the same 15 patients who were evaluable by t1-weighted mri. complete disappearance of nonenhancing lesions was observed in 1 patient, and partial regression was observed in 6 additional patients (47%; for an objective tumor response according to the rano criteria of 37%). no change was observed in four patients (27%), and an increase of abnormalities at the first evaluation was observed in 4 patients (27%) (figures 1 and 3). four patients were evaluated by 2-(18f)-fluoromethyl-l-phenylalanine pet (fmp-pet) or 11c-methionine-pet of the brain before and during bev treatment. a reduced uptake of amino-acid tracer on pet-scan was documented in 3 out of 4 pts during bev treatment, in 2 patients with the most favorable progression-free survival, and a complete normalization of pet-tracer uptake was observed during bev therapy (figure 4). as of october 2010 (the time of this analysis), two patients (10,5%) remained free-from progression after 1 year of bev treatment. in one of these patients bev was stopped after 1-year of therapy in the absence of metabolic activity on methionine-pet and normalization of gadolinium enhancement on t1-mri. the second patient developed progression of disease following 18 months of remission on bev therapy. the six-month progression-free survival rate (6mpfs%) was 21% (95% ci 2.739.5), and the 6mos% was 47.4% (95% ci 24.869.9). the median pfs and overall survival (os) were 10 weeks (95% ci 225) and 25 weeks (95% ci 1732), respectively (figure 5). high-grade gliomas are highly aggressive and therapy resistant malignant tumors. with contemporary standard treatment options for patients diagnosed with gb, the prognosis remains grim and most patients do not survive for more than 2 years following the diagnosis. salvage therapies with cytotoxic agents are seldom successful (< 10% orr) [7, 36]. uncontrolled clinical studies with the vegf targeted igg1 monoclonal antibody bevacizumab have shown unprecedented tumor response rates and survival outcomes that compare favorably with historical control series. within the context of these prospective clinical trials, bev-associated toxicities have been acceptable and reflect a typical spectrum of side effects that are associated with vegf(r) targeted therapies. as most of the prospective trials have used quite stringent patient recruitment criteria, safety and activity of bev when used outside of a clinical trial merit consideration. we, therefore, retrospectively analyzed the clinical outcome of nineteen patients treated with bev for recurrent hgg during the first year that bev became available in this indication at two university hospitals in belgium. as expected, the baseline characteristics of the patients included in our analysis compared unfavorably with those of the patients treated in the pivotal phase ii trial. a larger proportion of patients treated in our series were treated at second or third relapse, and the baseline kps was less or equal to 60% in a significant proportion of patients. we, therefore, consider that the results from our study, although preliminary, indicate that the safety profile of bev for recurrent hgg outside the context of a prospective clinical trial is comparable to what has been reported in the literature. nevertheless, two patients needed to stop treatment because of bev-related side effects in the absence of documented progression. it, therefore, needs to be considered that frail patients might be at higher risk for bev-related adverse events. the objective tumor response, either analyzed by gadolinium-enhanced t1 mri and/or t2/flair imaging, was interestingly high in our patient population. reflecting the poor baseline prognostic characteristics of our population, both time to progression (ttp) and overall survival (os), in contrast, were low when compared to published series. however, two patients in our series experienced a durable complete response and progression-free survival for over 1 year following bev therapy for recurrence, indicating the potential for a durable therapeutic effect in a subgroup of patients. patterns of tumor response and progression during antiangiogenic therapy are a matter of controversy in the recent literature. in our small series, the tumor response pattern to bev was heterogeneous and could be divided in three distinct patterns. a first group of patients demonstrated no evidence of response (clinical or radiological) to bevacizumab therapy. such was the case in four patients, who deteriorated rapidly and could not be evaluated by mri, and three (20%) of the fifteen assessable patients on gadolinium-enhanced t1 mri. a second group of patients initially responded to therapy on gadolinium-enhanced t1 mri, but subsequently showed early (< 6 months) regrowth of the gadolinium-enhancing tumor mass (n=7; 47%) or deteriorated clinically without characteristic increase in the diameter of gadolinium-enhancing t1 mri abnormities (n=3, 20%). in these 3 patients, there was a marked progression of abnormalities on t2/flair mri, most likely representing vegfr-independent tumor cell infiltration of the brain. a small third group of patients (2 patients; 13%) experienced a very favorable and sustained tumor response to bev therapy, evident on both gd-t1 and t2/flair mri. further useful differentiation of response to bev may be obtained by metabolic tumor imaging using pet. pet-imaging has proven to be useful in assessing the response of recurrent glioma treated with a variety of modalities. likewise, flt-pet has been correlated with clinical outcome of patients treated with the combination of irinotecan and bevacizumab. in our series, normalization in pet tracer accumulation was observed in the 2 cases with the most favorable evolution on mri and survival. these case observations indicate that single agent bev can be associated with a reduction of pet-tracer uptake by the tumor, suggestive of a metabolic effect. these observations merit further study of pet as a tool for response assessment in patients with recurrent glioma treated by bev. pet response may be more predictive for survival as opposed to response assessment by mri. we conclude that our analysis of the first experience with bev for the treatment of patients with recurrent hgg is in line with the reported tolerability and activity of this new treatment from prospective clinical studies. our observations support the usefulness of bev as a new treatment option for patients with recurrent hgg taken into account the absence of alternative treatment options with proven activity. further observational study of the use of bev in this indication should be considered to optimize its use in daily practice. correlative studies between clinical, radiological, pet parameters with molecular-genetic features of the hgg should be conducted to provide predictive markers for response and survival benefit from bev.

background. bevacizumab (bev), a humanized immunoglobulin g1 monoclonal antibody that inhibits vegf has demonstrated activity against recurrent high-grade gliomas (hgg) in phase ii clinical trials. patients and methods. data were collected from patients with recurrent hgg who initiated treatment with bev outside a clinical trial protocol at two belgian university hospitals. results. 19 patients (11 m/8 f) were administered a total of 138 cycles of bev (median 4, range 131). tumor response assessment by mri was available for 15 patients; 2 complete responses and 3 partial responses for an objective response rate of 26% for the intent to treat population were observed on gadolinium-enhanced t1-weighted images; significant regressions on t2/flair were documented in 10 out of 15 patients (67%). a reduced uptake on pet was documented in 3 out of 4 evaluable patients. the six-month progression-free survival was 21% (95% ci 2.739.5). two patients had an ongoing tumor response and remained free from progression after 12 months of bev treatment. conclusions. the activity and tolerability of bev were comparable to results from previous prospective phase ii trials. reduced uptake on pet suggests a metabolic response in addition to an antiangiogenic effect in some cases with favorable clinical outcome.

PMC3317219

pubmed-1365

obsessive compulsive disorder (ocd) is the fourth most common psychiatric illness, with a lifetime prevalence of 1%3%.1 the world health organization rates ocd as one of the top 20 most disabling diseases.2 if untreated, the course is usually chronic, or waxing and waning. only approximately one-third of ocd patients receive appropriate pharmacotherapy, and fewer than 10% receive evidence-based psychotherapy.1 as ocd patients often acknowledge the senseless nature of their intrusive, recurrent thoughts and also the repetitive, unwanted behaviors, it may lead to shame, and reluctance to seek help.2 people with this disorder have long delays in accessing effective treatments; 17 years on average in one study.2 there is growing evidence that stigmatization and negative attitudes toward mental disorders are important factors that prevent these patients from seeking appropriate medical help.37 aversion to psychiatric treatment and ambivalence about mental health services because of the fear of labeling and stigma have been found throughout the world.8 in addition, medical professional attitudes might impact the quality of treatment as well as the outcome. various studies on doctors attitudes toward psychiatric patients, including psychiatrists attitudes, brought out some discordant findings.9 gateshill et al reported that the majority of mental health and nonmetal health professions felt sympathy for those with mental disorders, wanting to help them, and favoring their treatment in the community.4 however, some studies suggested that psychiatrists tended to have more positive attitudes toward mentally ill patients than nonmetal health professionals did.4,10,11 arvaniti et al found that familiarity with mental illness was associated with less negative attitudes, such as less social discrimination and less social restriction, in which case psychiatric staff had more positive attitudes than other staff.10 addison and thorpe studied the factors involved in the formation of attitudes toward mentally ill patients and found that people who had personal experience with mental illness sufferers were generally more positive in their attitudes than those who had no previous experience.11 in contrast, some studies revealed that psychiatrists had negative attitudes toward psychiatric patients, which resembles the attitude of other doctors and general public.1214 while there have been a variety of studies on the attitudes toward general psychiatric patients, and some studies on schizophrenia or major depression,12,13 rarely have the attitudes toward ocd been focused on. we found only one study by simonds and thorpe in 2003 about the attitudes toward the different subtypes of ocd symptoms, for which undergraduate students were used as subjects.15 vignettes of different subtypes of ocd symptoms were used, and it was found that the vignette describing a person with doubting, violent, and blasphemous obsessions and related compulsions received the more negative social evaluations when compared with the vignette describing a person with cleansing rituals and checking compulsions.15 to our knowledge, no studies focusing on the attitudes of psychiatrists toward ocd patients have been published before. therefore, our research aimed to specifically study psychiatrists attitudes toward ocd patients. this study was approved by the ethics committee of the faculty of medicine, ramathibodi hospital, bangkok. exclusion criteria were mainly directed to those psychiatrists who had close friends or relatives diagnosed with ocd, or had themselves been diagnosed with ocd before. the questionnaire was developed from a focus group interview of ten psychiatrists which centered on their attitudes and feelings toward ocd patients, and the different emotions or perceptions when compared with those prevalent when facing other psychiatric disorder sufferers. the data were then transformed into a self-reported questionnaire, which consisted of three parts. the first part is about the socio-demographic information of the psychiatrists, such as sex, age, duration of practice as a psychiatrist, workplace, and the estimated number of outpatients they have seen in one period (approximately 3 hours). the second part centers on their experience with ocd patients including the estimated number of ocd patients they used to have experience in treatment, the estimated time spent with ocd patients at their first visit and in follow-up sessions, their preferred mode of treatment for ocd, their experience and proficiency in exposure and response prevention (erp), and finally their confidence in treating ocd patients with various approach. the third part concerns their attitudes toward ocd patients, reflected in 16 items to reply to, according to the four-point likert scales ranging from strongly agree to strongly disagree. more specifically, there are seven items about the attitudes and feelings toward ocd patients, three items about their perceptions toward ocd patients compliance, and six items regarding the emotions and perceptions toward ocd patients comparing with patients with other psychiatric disorders. a statistical analysis was performed using the spss version 18 for windows xp (spss inc. psychiatrists characteristics and experience with ocd patients were reported by frequency; all attitude items were reported in percentage. pearson s chi-squared () test and fisher s exact test (fet) were used to analyze the association between psychiatrists characteristics and their attitudes toward ocd patients. twelve psychiatrists were excluded due to having relatives diagnosed with ocd; as a result, 91 psychiatrists remained in the study. most of the participating psychiatrists were female (63.7%), and approximately 80% were under the age of 45. almost half of them (44%) have been practicing as a psychiatrist for less than 5 years. they have been working in 32 different hospitals all over thailand, which included 15 psychiatrists from northern area, 9 psychiatrists from southern area, 5 psychiatrists from northeastern area, 4 psychiatrists from western area, and 48 psychiatrists from middle area of thailand. the remaining 10 psychiatrists reported only the type of hospitals they have been working for, but did not specify their hospital name. regarding the types of hospital, the largest group of participants (41.8%) has been practicing in 19 different general/provincial hospitals, followed by 29.7% in seven different medical university hospitals, 25.3% in four different mental hospitals, and 3.3% from private hospital or others. almost half of them (47.3%) estimated the number of outpatients they treated per period (3 hours) to be more than 30 (table 1). approximately 40% of the participating psychiatrists had experience in treatment for fewer than 10% ocd patients. about half of them spent approximately 1530 minutes for the first visit with ocd patients, and less than 15 minutes for follow-up sessions. approximately 70% of the psychiatrists chose medications combined with behavioral therapy as the most preferred mode of treatment. only 7.7% of them reported that they were proficient in erp, whereas almost 70% of the participants reported using erp for their patients but were not proficient in it. most of the psychiatrists (76.9%) had confidence in treating ocd patients, but the number of psychiatrists who had confidence in treating with medications (91.1%) was much higher than those expressing confidence in behavioral therapy (51.7%) and other psychotherapy (39.6%) (table 1). more than 80% of the participating psychiatrists agreed with the benevolent attitudes toward ocd patients such as pity, understanding, and empathy, but only 18.7% stated that these patients were admirable. in term of negative attitudes, 33% of psychiatrists felt tired when treating ocd patients, and 14.3% felt these patients were annoying. the number of psychiatrists who perceived that ocd patients had poor compliance with behavioral therapy (52.8%) and other psychotherapy (30%) was higher than those reporting poor compliance with medications (7.7%). when compared with other psychiatric disorders, approximately 30% of psychiatrists thought that ocd patients talk too much, ask too much, need more time and patience, and 14% reported that they do not really want to treat ocd patients. only 7.7% stated that building therapeutic relationship with these patients was more difficult than in the case of other psychiatric disorder sufferers (table 2). degree of association between all characteristics and attitudes and level of statistically significant difference are shown in tables s1s3. some psychiatrists characteristics are significantly associ-ated with the attitudes toward ocd patients, such as the duration of practice as a psychiatrist, which is associated with the feeling of annoyance (=16.657, df=6, p=0.011) and the perception that ocd patients have poor compliance with medications (=15.568, df=6, p=0.016). the group of psychiatrists who have practiced for 610 years felt annoyed and perceived that ocd patients had poor compli-ance, in the highest number. the workplace also associated with the feeling of annoyance (=12.764, df=6, p=0.047). psychiatrists in general/provincial hospitals felt that ocd patients were annoying in the highest numbers, followed by psychiatrists in mental hospitals, while psychiatrists in medi-cal university hospitals clearly least agreed with this attitude. the estimated number of outpatients psychiatrists treated in one period (approximately 3 hours) is related to feeling of admiration (=17.401, df=9, p=0.043); the group that has less than ten patients most agreed that ocd patients were admirable. the estimated number of outpatients was also evidently associated with the perception that ocd patients have poor compliance with behavioral therapy (=24.596, df=9, p=0.003), and the notion that ocd patients need more time when compared with other psychiatric disorder sufferers (=24.788, df=9, p=0.003). regarding psychiatrists experience with ocd patients, the estimated number of ocd patients they had treated is associated with the perception that ocd patients have poor compliance with behavioral therapy (=19.009, df=9, p=0.025). the group who has 1120 patients least agreed with this attitude. the estimated time psychiatrists spent dur-ing the first visit with ocd patients the group who spent less than 15 minutes with patients least agreed that these patients were pitiful, whereas psychiatrists who spent more than 45 minutes with them most agreed with this attitude. this characteristic is also related to the perception that ocd patients have poor compliance with behavioral therapy (=19.531, df=9, p=0.021). all of the psychiatrists who spent less than 15 minutes with patients agreed with this attitude, while only 33.3% of those who spent more than 45 minutes agreed. the estimated time psychiatrists spent during the follow-up session with ocd patients is related to the perception that building a therapeutic relationship with ocd patients is more effortful than other psychiatric disorder sufferers (=9.524, df=4, p=0.049). psychiatrists experience and proficiency in erp is associated with feelings of admiration (=18.279, df=9, p=0.032) and pity (=34.144, df=9, p=0.001); 57.2% of psychiatrists who were proficient in erp felt that ocd patients were admirable, while none of those who had never known erp held this attitude. likewise, all of erp-proficient psychiatrists felt that these patients were pitiful, but only 50% of the group who had never known erp felt in the same way. the proficiency in erp also related to feeling of tiredness (=17.591, df=9, p=0.04). only 14.3% of erp-proficient psychiatrists felt tired toward ocd patients, whereas 75% of those who had never known erp felt tired. the other significant associations of this characteristic were the perception that ocd patients have poor compliance with medications (=12.874, df=6, p=0.045). erp-proficient psychiatrists most agreed with this attitude, while none of those who had never known about erp agreed with it. this characteristic is also related to the perception that ocd patients need more time when compared with other psychiatric disorder sufferers (=20.758, df=9, p=0.014). the psychiatrists who never practiced or had a lack of proficiency in erp most agreed with this attitude, but none of erp-proficient psychiatrists agreed with this attitude. psychiatrists confidence in treating ocd patients is significantly associated with several items of specific attitudes. the number of confident psychiatrists (22.9%) felt tired toward ocd patients, which is much less than the psychiatrists without adequate confidence (66.7%) (=16.41, df=3, p=0.001). a lower number of confident psychiatrists (12.9%) felt annoyed toward these patients than was the case for those who lacked confidence (19%) as well (p=0.015, fet). most confident psychiatrists (72.9%) perceived that ocd patients were difficult to treat, while all of the psychiatrists who lacked confidence, held this attitude (=8.129, df=3, p=0.043). in a similar way, only 8.5% of confident psychiatrists agreed with the statement that i do nt want to treat ocd patients, when compared with other psychiatric disorder patients, whereas 33.3% of those psychiatrists who lacked confidence agreed (=13.698, df=3, p=0.003). finally, only 4.3% of confident psychiatrists perceived that building therapeutic relationship with ocd patients is more difficult than with other psychiatric disorder patients (p=0.012, fet) compared with 19% of the psychiatrists who lacked confidence. considering each treatment approach, the confidence in treating ocd with behavioral therapy is associated with several attitudes in a similar way with psychiatrists overall confidence, whereas the confidence in treating ocd with medications is significantly associated only with feelings of pity (=10.541, df=3, p=0.032) and tiredness (=8.384, df=3, p=0.039). other attitudes that have significant associations with confidence in treating ocd with behavioral therapy were the perception that ocd patients have poor compliance with other psychotherapy (=18.692, df=3, p=0.00) and the notion that ocd patients need more patience than other psychiatric disorder sufferers (=10.369, df=3, p=0.016). the psychiatrists sex, age, and their preferred treatment approach for ocd had no significant association with the attitudes toward ocd patients. it was found that more than 80% of the participating psychiatrists had positive feelings toward ocd patients, such as pity, understanding, and empathy. these results correlated with previous studies which found that psychiatrists tend to have a positive attitude toward mentally ill patients.4,10,11 some of the psychiatrists in our focus group mentioned that ocd patients were admirable because they had been very patient and worked hard to alleviate their symptoms. however, the results found that only 18% of the participants agreed with this attitude. therefore, the admiration may be a personal point of view, and not the common psychiatrists attitude toward ocd patients. one of the more noteworthy findings was that about one-third of the psychiatrists perceived that ocd patients talked and asked too much, needed more time and patience compared with other psychiatric disorder sufferers, and they felt tired while treating these patients. although not the majority of participating psychiatrists held these negative attitudes, there was still a significant number of negative attitudes, which might have a negative impact on their interaction with ocd patients including treatment process. moreover, almost 80% of the psychiatrists thought that ocd patients were difficult to treat. it might resulted from the nature of ocd which has a chronic, waxing, and waning course and rarely shows complete recovery for its patients. regarding attitudes toward patients compliance to treatment, more than half of the psychiatrists perceived that ocd patients have poor compliance with behavioral therapy. as we found in our study, despite no statistically significant difference, 58.6% of psychiatrists who lack proficiency in erp reported compliance problem with behavioral therapy, compared with 28.6% of erp-proficient psychiatrists. the reason maybe because of the psychiatrists who lack proficiency in this kind of therapeutic method might not be able to adequately support the patients to cope with their difficulties, which in turn may lead to this attitude. on the contrary, only 7.7% of psychiatrists reported about the compliance problem with medications. there was no assessment of how they evaluate the patients compliance; as a result, it might not be possible to conclude that this attitude reflects the truth about ocd patients compliance. it was found that some of psychiatrists characteristics were significantly associated with particular attitudes, such as the workplace. those psychiatrists in general/provincial hospitals, together with psychiatrists in mental hospitals, felt that ocd patients were more annoying than the perception of psychiatrists in medical university hospitals. this finding might result from the matter of workload, which was greater in general, provincial, and mental hospitals than in medical university hospitals. the estimated number of outpatients psychiatrists treated in one period (approximately 3 hours) reflected in a similar way. the psychiatrists who have the fewest patients felt most admired, whereas the group who has greatest number of outpatients most agreed with the perception that (a) ocd patients have poor compliance with behavioral therapy, and (b) these patients need more time when compared with other psychiatric disorder sufferers. these findings might reflect that the psychiatrist s burden of workload results in their negative attitudes. it correlates with previous study which found that high workload of health care organization lead to patient neglect.16 regarding the psychiatrists experience with ocd patients, there were several significant associations with the attitudes. the estimated time psychiatrists spent during the first visit with ocd patients was associated with feeling of pity and the perception about compliance problem with behavioral therapy. the psychiatrists who spent longest time felt that these patients were pitiful and disagreed with the compliance problem more than those who spent less time. it can be hypothesized that the psychiatrists who spend more time with the patients might be able to deeply understand them that results in the positive attitude. furthermore, they might create better therapeutic relationship and/or provide the patients behavioral therapy including psycho-education about etiology, course and treatment options for ocd, which leads to better compliance. however, we still need further investigation to prove whether psychiatrists who spend more time with ocd patients, especially on the first visit, will lead patients more easily to comply with behavioral therapy, and bring about better treatment outcomes.1719 psychiatrists experience and proficiency with erp had significant associations with several items of attitudes. the psychiatrists who were proficient in erp most agreed that ocd patients were admirable and pitiful. they also felt less tired and less agreed that these patients need more time than other psychiatric disorder sufferers than those who never practiced erp or lack proficiency in this therapeutic intervention. these findings can be hypothesized that psychiatrist s proficiency in erp might lead to their positive attitudes toward ocd patients, which need to be further proved. the confidence of psychiatrists in treating ocd with various methods was significantly associated with several kinds of attitudes. all of the associations manifested in agreement, psychiatrists who have confidence held more positive attitudes toward ocd patients than those who lack confidence. specifically, the confidence in treating ocd with behavioral therapy has shown more association with attitudes than other type of treatment approaches. the more they are confident in behavioral therapy, the more positive attitudes they have. so it can be suggested that more training in behavioral therapy for ocd may be needed to help psychiatrists to be more confident, which might improve not only the attitudes toward these patients but also the treatment outcomes. it was found that sex and age had no significant association with the attitudes toward ocd patients. this finding was correlated to a study by addison and thorpe,11 which found no difference about attitudes toward mentally ill patients between males and females. however, our finding differs from a study by arvaniti et al10 which reported that women and the older people had held negative attitudes toward mental illness, such as social discrimination. firstly, the sample was not a true representation of all thai psychiatrists because there was no randomization in the sample selection, so we had more female and younger psychiatrists who participated. however, it was found that psychiatrists sex and age had no association with their attitudes toward ocd patients. moreover, the distribution of their workplace was fairly consistent between the different types of hospitals, which is an important characteristic that might influence psychiatrists attitudes. secondly, the questionnaire used to assess attitudes toward ocd patients has never been standardized and has not been previously studied in pilot study. however, we developed new questionnaire from the focus group interview, which provided deeper and more specific questions about attitudes toward ocd patients rather than those questions toward general mental illness as in some standardized questionnaires. before distributing the questionnaire, thirdly, this study used questionnaire as a measure, so the results were subjective feelings of the psychiatrists, which might be different from the real situation. however, it still reflected the psychiatrists attitudes which usually have the impact on their patients. finally, there were limited studies in the past we can compare to because most of them focus on attitudes toward general mental illness, not specific to ocd, and also used different methodologies and questionnaires. even though the participating psychiatrists clearly held positive attitudes toward ocd patients more than negative attitudes, most of them reported that ocd patients were difficult to treat. the psychiatrists who have fewer workload and who spent more time with ocd patients during the first visit seemed to hold more positive attitudes. although three-fourth of the psychiatrists reported confidence in treating ocd patients, their confidence in treating with medications was higher than in the case of behavioral therapy and other psychotherapy. their confidence in treatment especially their proficiency in erp is significantly associated with more positive attitudes. thus, it might be beneficial to improve the psychiatrists competence in treating ocd with behavioral therapy, which may lead to better attitudes toward patients and treatment outcomes. to our knowledge, this is the first study about psychiatrists attitudes toward ocd patients; further studies are needed to affirm our results. abbreviations: bt, behavioral therapy; erp, exposure and response prevention; fet, fisher s exact test; ocd, obsessive compulsive disorder.

purposenegative attitudes from doctors and the resulting stigmatization have a strong impact on psychiatric patients poor access to treatment. there are various studies centering on doctors attitudes toward psychiatric patients, but rarely focusing on the attitudes to specific disorders, such as obsessive compulsive disorder (ocd). this research aimed to focus on psychiatrists attitudes toward ocd patients. patients and methodsthe participants were actual psychiatrists who signed a form of consent. the main tool used in this study was a questionnaire developed from a focus group interview of ten psychiatrists about their attitudes toward ocd patients. resultsmore than 80% of the participating psychiatrists reported a kindhearted attitude toward ocd patients in the form of pity, understanding, and empathy. approximately one-third of the respondents thought that ocd patients talk too much, waste a lot of time, and need more patience when compared with other psychiatric disorder sufferers. more than half of the respondents thought that ocd patients had poor compliance with behavioral therapy. the number of psychiatrists who had confidence in treating ocd patients with medications (90.1%) was much higher than those expressing confidence in behavioral therapy (51.7%), and approximately 80% perceived that ocd patients were difficult to treat. although 70% of the respondents chose medications combined with behavioral therapy as the most preferred mode of treatment, only 7.7% reported that they were proficient in exposure and response prevention. conclusioneven though most psychiatrists had a more positive than negative attitude toward ocd patients, they still thought ocd patients were difficult to treat and had poor compliance with behavioral therapy. only a small number of the participating psychiatrists reported proficiency in exposure and response prevention.

PMC4508082

pubmed-1366

autonomic dysreflexia (ad) is a well known condition of an uncontrolled sympathetic output, that generally occurs in patients with spinal cord injury (sci) who have a lesion above t6 spinal level1). ad is caused by spinal reflex mechanisms that remain intact despite the injury of the spinal cord. in sci patients, blood pressure levels in addition, the hypertensive attacts can be life-threatening by causing end organ damage14). central nervous system complications are uncommon but it may be the most common reason of morbidity and mortality. ad is an important clinical diagnosis that requires prompt recognition and treatment in order to avoid this devastating complications. most important aspect of the management of ad is prevention, early recognition and removing of the triggering factors. in particular, the urinary tract obstruction and fecal impaction, which are the most common triggers of ad, must be checked out urgently and regularly. here, we report a sci patient who had a rare hypertensive cerebral hemorrhage complication during an ad episode and we discuss the pathophysiology and the treatment methodes of the ad. the patient was a 43-year-old man who had a american spinal injury association impairment scale-b (asia-b) type incomplet sci at level c6 secondary to a traffic accident for three years (fig. he had been diagnosed with autonomic dysreflexia due to the hypertensive attacks which were followed with hypotension and bradicardia. his neurogenic bladder was initially managed with an indwelling foley catheter and then clean intermittent catheterisation for two years. because of persistant attacks, suprapubic catheterization with cystofix has been performed one year ago. the patient admited to our emergency department with letargy, left sided facial paresis and worsening in his paresis in the left upper extremity. his family described the history of cystofix dysfunction with abdominal distention and increasing in blood pressure up to 220/100 mm hg right before these symptoms. his cranial ct revealed right thalamic and inraventricular hematoma with 21 mm in volume (fig. he has been followed-up with medical treatment and his cystofix catheter had been replaced with a wider one. although it is common in sci, non-traumatic diseases such as multiple sclerosis and spinal cord tumor can also be the cause of ad2). ad mainly occurs in chronic stage of disease both in the complete and the incomplete injury group1). the pathophysiology of ad is related with the disconnection of the spinal sympathetic centers from supraspinal control. normal regulation of sympathetic output from the spinal cord is modulated by input from the higher centers. these changes are thought to occur because of the synaptic re-organisation and reduction in gliosis around the pre-synaptic sympathetic neurones2,5,14). as a result of this process, exaggerated reaction occurs within the sympathetic pre-ganglionic neurones as a response to the afferent stimulus. the reason that ad is a feature of lesions at or above the t6 level is related with splanchnic circulation response to this sympathetic overactivity. the excessive parasympathetic output (and lack of sympathetic tone) above the level of the lesion results with peripheral vasodilation. hence, patients experience sweating, nasal congestion, flushing and bradicardia. in brief, ad result from the parasympathetic excitation above the level of injury, and sympathetic excitation below the level of injury and clinically it is characterized by an acute increase in blood pressure, headache, sweating, facial flushing and bradicardia1,2,5,11). we must always be aware of and search these features during the sci patients follow up. this ad crisis may lead to injuries in cardiac, pulmonary, retinal and renal tissues16). uncommonly, this hypertansive crises may also effect central nervous system by causing cerebral hemorrhage10,12,13,14,15,16) and more rarely posterior reversible enchephalopathy syndrome (pres) or reversible cerebral vasoconstriction syndrome3,7,13). central nervous system involvement is very rare and if occurs, it is most commonly asymptomatic. but, it can also result in altered mental status, local neurologic deficits and seizures4,16). intracranial hemorraghe is an uncommon complication of ad but fatal cases with massive bleeding have also been reported6,8,10,13,17). here, we report a rare sypmtomatic cerebral hemorrahge which is result from an ad attack. physicians should repeatedly check and note the neurologic status of every sci patient even during chronic stage because it may be difficult to distinguish the new neurologic findings in this tetraparetic or tetraplegic high level sci patients. the main triggering factor of ad indwelling urinary catheters are one of the most common causes. choosing the safest and appropriate method for the patients and family education for controlling the function of the catheter are the important issues. the goal in all bladder treatment is the creation of a balanced bladder, with low intravesical pressure that is drained totally at regular intervals and is free of urinary tract infections. in patients with sci, clean intermittent catheterization has been recommended rather than indwelling urinary catheters if the patient and family can adjust9). there are also many other urological causes for ad like bladder distension, urinary tract infection, bladder or kidney stones, urologic procedures, detrusor sphincter dysinergia, epididymitis or scrotal compression. the second most common causes of ad due to sci are gastrointestinal problems, including faecal impaction and rectal distension also anorectal conditions like haemorrhoids and fissures. orthopedic conditions such as fractures, dislocation, heterotopic ossification and general surgical conditions of the abdominal organs, such as appendicitis, gastro-oesophageal reflux, and peptic ulceration may presipitate the ad. all of these triggering factors must be kept in mind during evaluation of a patient with ad. the first step of the treatment of ad, regardless of the cause, is to make patient sit in an upright position to maintain an orthostatic decrease in the blood pressure. if needed, the calcium antagonists or adrenoceptor blockers may be beneficial in the treatment of ad1,2,5). this life-threatening complication should be kept in mind in the patients, especially with upper sci. the medical team who are dealing with sci patients as well as the sci patients themselves and their relatives should be aware of this disorder and apropriate bladder care, bowel exercises and skin care should be provided. this case has been reported to emphasises the rare hypertensive cerebral hemorrhage complication during common ad episodes in sci. clinicians should always closely monitore the patients with sci, especially those in the high spinal levels. these patients can easily develop ad which is triggered by the stimulation of lower parts of the body. prompt removal of the triggers and choosing the safest urinary drainage method are very important to save the patients from the complications of ad.

autonomic dysreflexia is a clinical emergency syndrome of uncontrolled sympathetic output that can occur in patients who have a history of spinal cord injury. despite its frequency in spinal cord injury patients, central nervous system complications are very rare. we report a man with traumatic high level incomplete spinal cord injury who suffered hypertensive right thalamic hemorrhage secondary to an episode of autonomic dysreflexia. prompt recognition and removal of the triggering factor, the suprapubic catheter obstruction which led to hypertensive attack, the patient had a favorable functional outcome after the resorption of the hematoma and effective rehabilitation programme.

PMC4130954

pubmed-1367

laparotomy in goat is an invasive surgical procedure into the abdominal cavity that allows visual examination of abdominal organs and documentation and correction of certain pathologic abnormalities observed [1, 2]. generally, it constitutes the single largest group of surgical operations carried out in ruminants [3, 4]. laparotomy is indicated for exploration of abdominal and pelvic cavities and other surgical procedures involving abdominal and pelvic organs; other specific indications are caesarean section, embryo transfer to produce transgenic goats, ovariectomy, rumenotomy, abomasotomy, ventral abdominal herniorrhaphy, intestinal resection, anastomosis, and cystotomy [511]. two approaches (flank and midventral) have been recognized and are currently in use in both small and large animals surgery; however in ruminants flank approach is the most widely and frequently practiced [1, 2]; due to the fact that surgical site can be visualized and observed from a distance and access healing, it was also reported to have reduced potential risk for evisceration if wound dehiscence is to occur, and the overlapping arrangement of the oblique muscles in the flank helps maintain the integrity of the abdominal wall if wound complication occurs. the flank laparotomy approach is the most widely used among small ruminants surgeons for accessing abdominal and pelvic organs. however, the approach is associated with some challenges: animals tend to rub the surgical site during healing against available solid objects leading to loosening of sutures and subsequently formation of wound dehiscence, prolonged lateral recumbency in goats under anaesthesia is associated with decrease in rumen stasis thereby predisposing the animal to bloat and toxemic lactic acidosis, and the accessibility to the distant organs (far proximal or distal to the point of incision) is also limited. we hypothesized that midventral laparotomy approach could be an alternative to flank laparotomy approach without much intra- and postsurgical complications. to test this hypothesis we compare the surgical wound assessment, intra- and postsurgical assessment, haematological profile, and subjective healing interval of the two laparotomy approaches. the aim of the study was to compare and evaluate flank and midventral laparotomy approaches in goats. ten (n=10) apparently healthy goats free of any dermatological lesions with average age of 12 2.1 months (mean sd), male and female of different breeds, and average weight of 13.4 2 kilograms (mean sd) were used for the investigation. the goats were kept at the usmanu danfodiyo university veterinary teaching hospital facilities and were conditioned for two weeks during which they were evaluated and stabilized for surgery. during evaluation serial blood sampling was done for comprehensive haematology to ascertain that the goats are fit for surgery and fecal sample was also collected to ascertain the intestinal worms burden. the goats were maintained on daily ration comprising wheat bran, bean husks, ground nut hay, and water ad libitum. the goats were randomly grouped into flank (fa) and midventral (mva) approaches. feed and water were withdrawn from animals at least 12 hours prior to the surgery. the left flank region of each goat in the fa group was prepared for routine aseptic surgery by clipping the hairs around the proposed surgical site; the site was scrubbed with purit solution containing chlorhexidine gluconate b. p. 0.3% w/v (saro lifecare limited, lagos, nigeria) and rinsed with methylated spirit (binji pharmaceutical company, sokoto, nigeria). (sahib singh agencies, mumbai, india) at 4 mg kg through lumbosacral epidural anaesthesia as described by. the epidural space was identified by loss of resistance to injection of 1 ml of air after piercing the ligamentum flavum. mild sedation was achieved using xylazine 20 (xylazine hcl 20 mg ml, kepro holland) at 0.025 mg kg intramuscular and atropine sulphate 0.6 mg ml (laborate pharmaceuticals india) at 0.05 mg kg intramuscular as vagolytic agent. laparotomy was done according to standard procedure described by [1, 3, 14]. the laparotomy was routinely closed from within outward; muscle layers were closed using becton chromic catgut of the size of 1/0 and atraumatic 1/2 circle taper point needle (anhui kangning industrial groups, china) using interrupted horizontal mattress suture pattern with simple interrupted reinforcement. the subcutaneous layer was closed using becton chromic catgut of the size of 2/0 and atraumatic 1/2 circle taper point needle using simple continuous suture pattern. the skin was closed using ford interlocking pattern with agary nylon of the size of 0 and atraumatic 3/8 curved, cutting needle (agary pharmaceuticalsltd, xinghuai, china). in mva group, laparotomy was done through linea alba in all female goats with little paramedian incision at the level of prepuce in all the males according to standard procedure described by [1, 3, 4]. the incision was closed routinely in three layers from within outward (linea alba, subcutaneous layer, and skin) with the same suture materials as described in fa group. 5% acetaminophen injection 10 mg kg intramuscular (cadence pharmaceutical inc., ireland) was administered for 3 days after surgery to take care of postoperative pain. long acting 15% amoxicillin injection 20 the clinical appearance of the skin was assessed and scored twice: 1824 hours and 1014 days after surgery as described by using 4-point scoring scale, based on the following criteria: discharge, swelling, erythema, and dehiscence. blood samples were collected from each animal in the two groups through the jugular vein after thorough disinfection of the area with methylated spirit; the sample was collected using 5 ml syringe and needle into edta bottles. the samples were collected before surgery as baseline, 1824 hours after surgery, and subsequently on weekly interval till complete healing when sutures were removed. the samples were analyzed using digital automated haemoanalyser (full automated blood cell counter pce-210, erma inc., intra- and postsurgical complications were assessed using 3-point scoring system designed by ourselves; parameters considered were intraoperative haemorrhages, postsurgical seroma, incisional hernia, and wound fistula (table 1). subjective healing interval was determined by visual observation and taking notes of days of apparent surgical site healing according to. data generated from the four parameters (surgical wound scoring, haematology, surgical complications, and healing interval) were tabulated and mean and standard deviation were computed in each case. student's t-test was used to compare statistical significant difference between the flank and midventral variables of each parameter at 95% confident interval using graphpad instat statistical software package 2010. at 1824 hours after surgery, there was serous discharge in all groups; the mean discharge scores were (0.80 0.45 and 0.80 0.84) for flank and midventral approaches, respectively. there was no significant difference between the two groups when compared. at 1014 days after surgery midventral group had higher swelling score (2.00 00) in comparison with flank approach (1.8 0.45) and the overall swelling score was higher at 1824 hours after surgery compared to 1014 days after surgery (0.50 0.56 and 0.80 0.45) in flank and midventral, respectively (table 2). there was no significant difference between flank and midventral approach both at 1824 hrs and at 1014 days after surgery. the flank approach at 1824 hours had higher erythema score (1.40 0.55) when compared with midventral group (0.80 0.45) and there was significant difference (p<0.05) of erythema between the two approaches (table 2). at 1014 days after surgery, flank approach had higher erythema score (0.25 0.50) while midventral approach had no erythema record and there was significant difference (p<0.05) between the two approaches. dehiscence was not recorded at 1824 hours after surgery in all the groups; however, at 1014 days after surgery dehiscence was observed in flank approach with significant difference (p<0.05) between the two groups (table 2). intraoperative haemorrhage score was higher in flank approach (1.4 0.55) when compared with midventral approach (1.00 0.70); there was no significant difference (p>0.05) between the two groups (table 3). there were no postoperative complications of incisional hernia, seroma, and wound fistula recorded. there were variations of total white blood cells (wbc) count of the two approaches before surgery, at 1824 hours, and at the first and second week after surgery; the midventral group had higher wbc value at all the intervals with significant differences (p<0.05) at first and second week after surgery (table 4). there were slight variations of total granulocytes between the two groups with the midventral group having the higher values at all the intervals, but there is no significant difference between the two groups (table 4). the lymphocytes values of the two groups also varied and the midventral approache had the highest value. there were significant differences (p<0.05) recorded between the two approaches at first and second week interval: 21.33 8.22 flank approach against 28.32 11.98 midventral approach and 15.20 3.52 flank approach against 25.48 6.00 midventral approach (table 4). there were also slight variations of monocytes values between the flank and midventral approaches at different timing interval; the midventral had higher values when compared with flank approach but there were no significant differences between the two approaches at any given time interval (table 4). the values of the platelets varied slightly between the two approaches, with the midventral approach having a higher value when compared with flank approach, and there was no significant difference between the two approaches at all the timing intervals (table 5). the platelets critical values varied between the two approaches with the midventral having the higher values; there was significant difference (p<0.05) at second week interval between the flank and midventral approach (0.15 0.04 against 0.25 0.08), respectively (table 5). the mean platelets volumes also showed slight variations between the two groups, but there was no significant difference between the groups at any of the timing intervals; the midventral approach had higher values when compared with the flank approach (table 5). the platelets dimension width values were slightly higher in midventral approach compared to flank approach and a significant difference (p<0.05) was recorded between the two approaches at 1824-hour interval (table 5). the packed cells volume of the two approaches showed slight variations with the midventral approach having the higher pcv values when compared with the flank approach. there was significant difference (p<0.05) recorded at one week interval between the two approaches (table 6). there were no significant differences (p>0.05) between the two approaches in all other erythrocytic indices (red blood cells count, haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, and red blood cells distribution width). however, the values of midventral approach are higher at different timing intervals in all other erythrocytic indices (table 6). the mean subjective healing intervals were 13.0 1.14 and 12.4 0.5 for flank and midventral approach. there was no significant difference (p=0.643) between the two groups when compared (figure 1). laparotomy is commonly indicated either for exploratory purposes when clinical diagnosis is uncertain or for therapeutic surgical intervention when specific diagnosis has been made. flank approach is the most commonly practiced technique among large animal surgeons with the animal under local anaesthesia. ventral paramedian or midventral laparotomy approach is an alternative practice by few large animal surgeons that necessitates the animal placement in dorsal recumbency. the two main indications in bovine are ventral abomasopexy and cesarean section, in which it offers advantages in the delivery of oversized or emphysematous fetuses and in complicated deliveries, including uterine tears [12, 19]. surgical wound assessment showed significant difference of erythema both at 1824 and at 1014 days after surgery with flank approach having the highest erythema score and this could be due to surgical trauma elucidated by the traumatic surgical instruments on the soft tissue in the course of surgery; this is because the flank region has three layers of abdominal muscles that have to be passed through before getting access into the abdominal cavity in comparison with midventral approach through linea alba aponeurosis (ligament) which is passed through before gaining access to abdominal cavity; the ligament poorly responds to pressure of traumatic surgical instruments which brought about the less erythematous response. the high erythema score recorded in flank approach could also be a result of abdominal muscles tissue response to absorbable suture materials used for apposing the muscles mass which is more bulky than that of midventral approach. the overall scoring showed higher erythema earlier before surgery at 1824 hours and this finding is consistent with the studies conducted by [15, 17] where significant differences among the variables were observed. dehiscence was also observed in the flank approach at 1014 days after surgery with significant difference when compared with midventral approach; this could be a result of scratching the surgical site (flank) with available objects in the pen as a result of tissue irritation in the course of healing process. it could also be due to self-mutation with horn of hind limbs in response to tissue irritation. dehiscence score was by far less in midventral approach due to lesser chances of scratching and self-mutilation around the region. our finding was contrary to that of, which recorded no dehiscence in a similar study using canine species, and that of, which recorded mild dehiscence both at 1824 hours and at 1014 day after surgery but without significant difference in a similar study using caprine species. the intraoperative hemorrhage score recorded was higher in the flank approach compared with the midventral approach, though without significant difference; this could be a result of high vascular channels available in the abdominal muscle mass when compared to poor vasculatures associated with tendons and ligament in the linea alba. this could serve as one of the advantages of midventral approach particularly when dealing with nonelective laparotomy in which the patient hematocrit reading is below normal range. the packed cell volume (pcv) of the flank approach decreased significantly one week after surgery when compared with midventral approach; this could be due to high intraoperative hemorrhage recorded. this finding was in line with the finding of [20, 21], both in a study involving laparotomy with goat; they noted that remarkable hematocrit decreased after surgery with significant difference. higher values of total white blood cells count and lymphocytes count were recorded in midventral approach at the second week after surgery with significant difference when compared with the flank approach and this could be attributed to high persistent chronic inflammatory response in the course of tissue repair or it could be due to surgical stress because midventral approach is more stressful in relation to surgical positioning than lateral recumbency. our finding is also in line with those of [20, 21] who also recoded elevated values leukocytes count. but noticed an average total leukocytes value within normal physiologic range after abdominal surgery in dairy cows. percentage platelets critical value recorded was higher in midventral approach; this could be due to lesser whole blood loss observed intraoperatively as decrease in whole total blood volume leads to gross interference of the different components of the blood cells including platelets. this may also serve as an advantage in midventral approach because the higher the platelets critical values, the quicker the chances of blood clotting response. there were slight variations of means subjective healing interval of the two approaches but without significant difference (p=0.643), with the flank approach having higher means number of days (13 1.14) to complete surgical wound healing when compared with 12.4 0.5 mean days for midventral approach. the slight variation of days of healing interval might be due to surgical site interference with the object coming contact with the surgical wound as reported by [22, 23], as the chance of surgical site contact with surrounding object is higher in flank laparotomy site compared to midventral site. the variation could also be a result of other local factors that affect wound healing like oxygenation, foreign body contact with the surgical wound, and venous insufficiency as reported by. it was concluded that the midventral laparotomy approach can be safely and conveniently performed without fear of clinical complications in goats. we recommend the use of midventral laparotomy approach for routine abdominal surgery in goats as an alternative to flank approach. further study on pregnant goats to see whether midventral abdominal incisional closure can withstand pressure of gravid uterus also needs to be conducted.

the aim of the study was to compare two laparotomy approaches (flank and midventral). ten (n=10) apparently healthy goats of different breeds and sex, average age of 12 2.1 months, and average weight of 13.4 2 kg were used for the investigation. the goats were randomly divided into flank and midventral groups, each group comprising five goats (n=5). standard aseptic laparotomy was performed under lumbosacral epidural anaesthesia with mild sedation. postsurgical wound score showed significant difference (p<0.05) in erythema at 1824 hours and 1014 days after surgery between the two approaches; significant difference of dehiscence between the two groups was also recorded at 1014 days after surgery. total white blood cells (wbc) and lymphocytes counts were significantly different (p<0.05) at the first and second week after surgery. there was significant difference of platelets critical value and platelets dimension width at the first and second week after surgery. significant difference of packed cells volume between the two approaches was also recorded one week after surgery. it was concluded that midventral laparotomy approach can be conveniently and safely performed under aseptic precautions without fear of intra- and postoperative clinical problems.

PMC4590860

pubmed-1368

fine-needle aspiration (fna) is the standard method used to determine treatment plans for thyroid nodules. based on the bethesda system, the most generally accepted system for reporting thyroid cytology, the benign category implies a less than 3% risk of malignancy. follow-up ultrasound (us) is recommended when the nodule has benign cytologic result. repeat fna is recommended when a nodule shows significant growth or morphologic transformation with suspicious however, the practical risk of malignancy in nodules with benign cytology varies in each institute, ranging from 2% to 18%, and it has even been reported to have gone up to 62%. therefore, some investigators recommend routine repeat fna for thyroid nodules with benign cytology [7, 8]. considering cost-effectiveness and diagnostic value, repeat fna has been considered when the nodule shows any suspicious feature on the initial us [9, 10]. however, known us features associated with malignancy show an extremely variable probability of malignancy. microcalcifications, marked hypoechogenicity, and irregular or spiculated margin show a high risk of malignancy, while solid composition and hypoechogenicity show a relatively low positive predictive value (ppv) [1113]. based on these results, each suspicious us feature may not be considered as an equal risk factor for malignancy. radiologists specialized in breast imaging have been confronted with the same problem in the core needle biopsy of a breast lesion. correlation of pathologic results with sonographic findings has been used in some institutions to verify that the lesion was adequately sampled. this approach was suggested owing to the wide range of false negative rates of this category. based on different malignancy rates of suspicious us features in thyroid nodules and considering approaching steps in management of breast lesions, we conjecture that an imaging-cytology correlation can be a better diagnostic approach for patient management than initial us features in a thyroid nodule with benign cytology. therefore, we investigated the role of imaging-cytology correlation to reduce the false negative rates of cytology at thyroid nodules as compared with the use of initial us features. the institutional review board of the severance hospital approved of this retrospective study and required neither patient approval nor informed consent for our review of patients ' images and records. however, written informed consents were obtained from all patients for us-guided fnas (us-fnas) prior to each procedure as a daily practice. our institutional registry for the thyroid nodule was settled since 2006 including all patients with thyroid nodules who underwent us examinations and us-fnas at our institution. from march 2006 to december 2006, 3119 consecutive thyroid nodules in 2866 patients underwent us-fnas. among them, we included 667 nodules in 649 patients (men, 83; women, 566), which fulfilled the following criteria: (a) they had no history of prior fna on the same nodule; (b) they were reported as benign (category ii) in the initial fna. nondiagnostic, atypia or follicular lesions of undetermined significance, follicular neoplasm or suspicious for a follicular neoplasm, suspicious for malignancy, and malignant were excluded; (c) they were equal to or larger than 1 cm; (d) they underwent further evaluation such as follow-up us, follow-up fna, or thyroid surgery. in nodules which had not underwent operation, determinative cytologic reports (category ii or category vi) on follow-up us were used as standard reference. if a nodule decreased in size on follow-up us, the nodule was also included as a benign nodule; (e) there were available radiologic reports that included an additional radiologist's opinion about the concordance or discordance between imaging and cytologic results in postbiopsy correlation (figure 1). mean lesion size of the thyroid nodules was 20.7 mm (range, 1070 mm). median follow-up of 601 nodules in 584 patients which were included based on followup fna or us without surgical pathology was 1509 days from the date of initial fna to the last followup (iqr, 1522 days; range, 1722744 days). all us examinations were performed using a 7 to 15 mhz linear array transducer (hdi 5000; philips medical systems, bothell, wash) or a 5 to 12 mhz linear probe (iu22, philips medical systems) by 1 of 5 board-certified radiologists with 1 to 12 years of experience in thyroid imaging. all us-fnas were performed by the same radiologist who performed the us examinations. us features of all thyroid nodules that underwent us-fnas were prospectively recorded by the previously described methods. us features suspicious for malignancy were determined using previously published criteria from our institution: marked hypoechogenicity, microlobulated or irregular margin, microcalcifications, and taller than wider shape. when overall echogenicity of a nodule was darker than that of the surrounding strap muscle, it was defined as marked hypoechogenicity to differentiate it from hypoechogenicity based on the parenchymal echogenicity of the thyroid gland. only calcifications equal to or less than 1 mm in diameter were indicated. if microcalcifications were detected with macrocalcifications, the lesion was considered to have microcalcifications as a worrisome finding. if hyperechoic foci accompanied comet-tail artifacts on conventional us, they were considered as colloids. an anteroposterior to transverse dimension ratio greater than 1 was defined as taller than wider shape. us-fnas were performed on either thyroid nodules with suspicious assessment or the largest nodule among nodules with probably benign assessment on us. if there were multiple nodules with suspicious us findings in one patient or if the patient or physician requested a biopsy of a benign-looking nodule coexisting with a nodule showing suspicious us features, fnas were performed on multiple nodules in one patient. a free-hand biopsy technique was used with either a 23-gauge needle attached to a 20 ml disposable plastic syringe and an aspirator or a 23-gauge needle attached to a 2 ml disposable plastic syringe, depending on the performing radiologist's preference. each lesion was aspirated at least twice, and the aspirated materials were expelled onto a slide and immediately placed in 95% alcohol for papanicolaou staining. an inadequate specimen was defined as less than 6 groups of cells containing more than 10 cells. adequate specimens were categorized as benign, indeterminate, suspicious for malignancy, or malignant samples. the radiologist who performed fna routinely reviewed the initial us images within a week of the fna after the cytologic results were reported. for benign cytologic results, radiologists who performed the us-fnas decided and reported whether the cytology was concordant or discordant with the imaging findings. as researchers at our institution always try to assess lesions based on their most worrisome finding, the saved images should represent these worrisome us features. the final conclusion was not derived from the number of suspicious us features but from the subjective decision made by the radiologist who performed the us-fna. in our institution, concordant lesions included some nodules which had suspicious us features on the initial us but were acceptable for the benign cytology in postbiopsy image review as well as the nodules without features suspicious for malignancy on the initial us. concordant benign thyroid nodules were recommended for follow-up by us after one year. in contrast, discordant lesions included nodules which were initially suspected for malignancy on us and were still thought to be suspicious for cancer even after obtaining benign cytology. repeat fnas were usually recommended for discordant benign thyroid nodules after 612 months. among the 667 nodules that met all the inclusion criteria, 586 nodules (87.9%, 586 of 667) were reviewed by radiologists who had more than three years of experience in thyroid imaging and fna whereas the remaining nodules were managed by less experienced radiologist. we compared the clinical characteristics of patients between benign and malignant nodules by using the test for categorical variables and independent t-test for continuous variables. we also compared the risk of malignancy as well as the clinical characteristics between concordant and discordant nodules by using or fisher's exact test for categorical variables and independent t-test for continuous variables. the baseline characteristics were also compared between patients with included and excluded nodules among thyroid nodules equal to or larger than 1 cm with the same methods. the risk of malignancy was calculated for several subgroups classified according to initial us features and imaging-cytology concordance. using the generalized estimating equation, we compared the risk of malignancy in thyroid nodules with initially benign cytologic results with those of the remaining subgroups and also compared the risk of malignancy of thyroid nodules among subgroups. logistic regression analysis was performed to assess the odds ratio for the risk of malignancy. statistical analysis was performed using commercial statistical software (sas version 9.1, sas inc., cary, nc, usa). among 667 nodules with initially benign cytologic results, 656 nodules were benign (98.4%) and 11 nodules were malignant (1.6%) based on cytopathology (table 1). the mean age of patients with malignant nodules was not significantly different from that of patients with benign nodules (p=0.277). the mean size of malignant nodules (17.6 12.5 mm) was not significantly different from that of benign nodules (20.7 10.1 mm, p=0.315). there were 70 nodules with initial suspicious us features and 597 nodules without initial suspicious us features. the risk of malignancy was higher in nodules with initial suspicious us features (11.4%, 8 of 70) than in nodules without initial suspicious us features (0.5%, 3 of 597; p<0.001, table 1). when reviewing us images after initial fna results were reported, 40 out of 70 nodules which had suspicious features on initial us evaluation were finally concluded as concordant with benign cytology (figures 2 and 3). therefore, in 667 nodules with benign cytology, 637 nodules were concordant with cytology, whereas 30 nodules were discordant with benign cytology. the reasons that 40 nodules with revised radiologic diagnosis after imaging-cytologic correlation were initially classified as suspicious nodules were microcalcifications (n=16), microlobulated or irregular margin (n=9), taller than wider shape (n=3), or marked hypoechogenicity (n=1) in order of frequency, respectively, and more than one characteristic of the above features in 11 nodules. between the concordant and discordant group, gender of the patients was not significantly different (p=0.159). the patients with discordant nodules were significantly older than other patients with concordant nodules (53.5 10.5 years versus 48.9 12.0 years; p=0.038). the mean size of discordant nodules was significantly smaller than that of concordant nodules (16.0 6.6 mm versus 20.9 10.2 mm; p<0.001). 3%; 7 of 30) than in the concordant group (0.6%, 4 of 637; p about 44.5% (534 of 1201) of 1 cm or larger nodules with benign cytology in initial fna were excluded because they had neither standard reference, such as follow-up us, follow-up fna, or thyroid surgery, nor available radiologist's additional reports regarding imaging-cytologic correlation. the mean age of patients with included nodules was statistically different from the other patients (49.1 12.0 versus 50.7 13.1 years; p=0.033). patient gender (p=0.392) and mean nodule size (p=0.601) were not significantly different between included and excluded nodules. there were 60 nodules with suspicious findings in the initial us evaluation of excluded nodules (11.2%, 60 of 534), and the proportion was not significantly different from that of included nodules (10.5%, 70 of 667; table 2). when comparing the risk of malignancy between benign cytology alone and each subgroup by a combination of benign cytology with initial us findings or postbiopsy concordance, all combinations had significantly different risk values from cytology alone (table 3, figure 4). also, when comparing the risk of malignancy between discordant lesions and lesions with suspicious features on initial us, the former (23.3%, 7 of 30) was significantly higher than the latter (11.4%, 8 of 70). however, there was no significant difference in the risk of malignancy between concordant lesions (0.6%, 4 of 637) and lesions without suspicious features on initial us (0.5%, 3 of 597; p=0.438) (figure 4). although fna is a widely used tool for the diagnosis of thyroid nodules, the most significant problem it has is false negative results which bring out misses and delays in treatment of the cancer. errors in cytologic reports have arisen from the overinterpretation of nondiagnostic specimens as diagnostic ones [23, 24]. therefore, many reports discussed the differentiation of a nondiagnostic specimen from a diagnostic one in the cytologic interpretation of thyroid fna [1, 3]. diagnostic errors of thyroid fna can also be caused by the mistakes of cytopathologists and the inherent nature of thyroid nodules due to overlapping cytologic criteria among hyperplastic adenomatoid nodule in goiter, follicular adenoma, well-differentiated follicular carcinoma, and follicular variant of papillary carcinoma. moreover, reported false negative rates are variable among institutions and operators due to variable sampling skills [5, 6]. several guidelines recommend follow-up us in thyroid nodules with benign cytology unless the nodule shows significant growth or morphologic change in follow-up us [1, 3, 4]. however, it has been argued that follow-up might be not enough in some nodules because of the inevitable false negative diagnosis and the possible risk of delayed treatment [6, 22]. to reduce false negative results of thyroid fnas, there have been two suggested approaches; first, routine repeat fna in thyroid nodules with benign cytology [26, 27] and, second, selective repeat fna [8, 10, 28, 29]. in the aspect of cost-effectiveness, it is more rational to consider performing follow-up fna selectively for nodules with a high-risk of malignancy rather than performing a total inspection of cytologically benign nodules in initial fna. based on several reports, the rate of malignancy in benign thyroid nodules with suspicious us features was 3.747.1% which was significantly higher than that of benign thyroid nodules without suspicious us features (table 4) [9, 1518]. although the us criteria applied to each study had subtle differences, initial us features may be reliable factors in determining whether to repeat fna or not. going one step further from simply matching cytologic results against imaging findings evaluated before biopsy, the postbiopsy correlation of us features with cytologic results could be an alternative in determining whether the nodule should be reaspirated to confirm its cytology or not. imaging-pathologic correlation after biopsy has been found to be useful in validating biopsy results of breast lesions, and discordance has been suggested as an indication for excision because of its higher upgrade rate than that of concordant lesions [3032]. however, there has been no organized study that applies imaging-cytology correlation to patient management and considers how to accept results of postbiopsy correlation in regard to reducing false negative diagnosis in thyroid nodules. in this study, 1.6% of nodules with benign cytology in initial fna were finally proven to be malignant. as expected, the malignancy rate of thyroid nodules (11.4%) with suspicious features on initial us was significantly higher than that of nodules (0.5%) without suspicious features on initial us, and the malignancy rate of nodules (23.3%) with discordant imaging findings was also significantly higher compared to concordant nodules (0.6%) in postbiopsy imaging-cytologic correlations. furthermore, the rate of malignancy was higher in the nodules showing imaging-cytology discordance compared to nodules showing suspicious feature on the initial us. however, there was no significant difference in the risk of malignancy between concordant nodules in postbiopsy correlation and nodules without suspicious features on initial us. this result lets us conclude that imaging-cytology correlation is a more effective approach than using initial us features alone when deciding follow-up management in patients with cytologically benign thyroid nodules without a statistical increase in missing malignancy. in this study, 40 of 70 nodules with suspicious features on initial us were determined as concordant with benign cytology after postbiopsy imaging-cytology correlation. although many descriptions of each suspicious us feature are present, interobserver and intraobserver variability still exist for the us assessment of thyroid nodules. among us characteristics, margin and calcification showed relatively less consistency between observers and nodules in most patients whose radiologic assessments were changed after obtaining benign cytology were initially assumed as suspicious nodule due to calcification (16 of 40), margin (9 of 40), or multiple features (11 of 40) including them in our study. also, there have been difficulties in deciding whether a thyroid nodule shows echogenic spots on us. echogenic spots can be due to microcalcifications related to cancer or crystals related to colloids. therefore, postbiopsy imaging-cytology correlation can be a good diagnostic approach in deciding whether to repeat fna or not at a thyroid nodule with benign cytology. first, some nodules were excluded in analysis despite having benign cytologic results due to loss of follow-up and absence of additional reports. however, the initial us assessment was not significantly different between included nodules and excluded nodules which were 1 cm or larger with benign cytology in the initial fna. second, interobserver and intraobserver variability among radiologists are possible in the interpretation of us images and among cytologists, especially when reviewing follicular lesions. third, there might be a bias arising from the postbiopsy review process itself which was based on saved images instead of on a review in real-time us. although we always tried to save any images showing worrisome us findings and the postbiopsy review was preferably done within a week of biopsy by the performer, an observer bias might not have been completely removed from the final results. fourth, suspicious us features such as calcification, margin, vascularity, and echogenicity have been differently applied to thyroid nodules by various guidelines and different institutions. fifth, most (87.9%) of the nodules in this study were reviewed by highly experienced radiologists in thyroid imaging. repeat fna can be effectively limited to patients with cytologically benign thyroid nodules showing discordance in imaging-cytology correlation, which reduces unnecessary repeat aspirations as well as decreasing false negative results.

objective. to determine the role of imaging-cytology correlation in reducing false negative results of fine-needle aspiration (fna) at thyroid nodules. methods. this retrospective study included 667 nodules 1 cm or larger in 649 patients diagnosed as benign at initial cytologic evaluation and that underwent follow-up ultrasound (us) or fna following a radiologist's opinion on concordance between imaging and cytologic results. we compared the risk of malignancy of nodules classified into subgroups according to the initial us features and imaging-cytology correlation. results. among included nodules, 11 nodules were proven to be malignant (1.6%) in follow-up fna or surgery. the malignancy rate was higher in nodules with suspicious us features (11.4%) than in nodules without suspicious us features (0.5%, p<0.001). when a thyroid nodule had discordant us findings on image review after having benign fna results, malignancy rate increased to 23.3%, significantly higher than that of nodules with suspicious us features (p<0.001). however, no significant difference was found in the risk of malignancy between the nodules without suspicious us features (0.5%) and imaging-cytology concordant nodules (0.6%, p=0.438). conclusions. repeat fna can be effectively limited to patients with cytologically benign thyroid nodules showing discordance in imaging-cytology correlation after initial biopsy, which reduces unnecessary repeat aspirations.

PMC4211172

pubmed-1369

the patient was a healthy 29-year-old multigravida. in week 28, the fetus was found to have an anterior neck mass and polyhydramnios was also detected by prenatal ultrasound. during the last four weeks prior to delivery, the neck mass grew substantially, had a broad base, and was 7 cm in diameter. an exit procedure was performed at week 38 of gestation by a multidisciplinary team of experts including a pediatric otorhinolaryngologist, neonatologist, obstetrician, two anesthesiologists, and nurses. the patient was placed in a supine position with a left lateral tilt to avoid aortocaval compression. an arterial line for continuous arterial pressure monitoring and two peripheral intravenous lines (16-gauge) were placed before induction. endotracheal intubation (6.5 cuffed) was done after thiopental sodium (3 mg/kg) and succinylcholine (1.5 mg/kg) administration by a rapid sequence induction. general anesthesia was maintained with sevoflurane 2-3 vol% and nitrous oxide in oxygen (50-50) combined with an additional dose of remifentanil (0.1-0.5 g/min/kg) and rocuronium (50 mg in total). an end-tidal (et) concentration of sevoflurane was maintained at 1-1.5 minimum alveolar concentration (mac) for proper uterine relaxation until the umbilical cord was clamped. prior to uterine incision, the patient was given two boluses of intravenous nitroglycerine (30 g), followed by an infusion at 0.5-1 g/kg/min to ensure uterine relaxation. to maintain a maternal systolic blood pressure of 100-120 mmhg, the following treatments were required: four boluses of intravenous phenylephrine (50 g), followed by an infusion at 0.05-0.1 g/kg/min, an infusion of dopamine at 5 g/kg/min, and rapid infusion of volume expander (500 ml)., the obstetrician confirmed that the uterus was fully relaxed and inserted an angiocatheter (20-gauge) into the uterus under the guidance of ultrasound for decompression by slow reduction of the amniotic fluid volume. then, the fetal head and the part of the torso just above the nipple were partially delivered. a sterile pulse oxymetry was applied at the left ear lobe of the fetus after removal of amniotic fluid, but reliable readings could not be obtained. ultrasound-guided monitoring was done by an obstetrician for the early detection of placental detachment until the fetal airway was secured. the fetus was intubated with a 2.5 endotracheal tube by a pediatric anesthesiologist at the second attempt (fig. the stylet of the endotracheal tube was not removed by an assistant due to the slipperiness of the vernix caseosa. the position of the endotracheal tube was confirmed by a sterile end-tidal co2 monitor. once the fetal airway was established and the positive-pressure ventilation was satisfactory, the umbilical cord was severed and the neonate was fully delivered from the uterus. the 1-min apgar score of the neonate was 7; the 5-min score was 9. after delivery, the infusions of nitroglycerine, dopamine, and phenylephrine were all discontinued, and the et concentration of sevoflurane was also lowered to 0.8-1.0 vol%. then, five units of oxytocin were injected intravenously by bolus, and 20 units/l was infused continuously. the estimated blood loss was 1,100 ml. the total intake of fluid during the operation was 1,800 ml. three days later, the neck mass of the neonate was completely excised by a pediatric otorhinolaryngologist. the excised mass was confirmed to be a 5 5 5 cm benign teratoma (fig. the aim of the exit procedure is to manage life-threatening neonatal emergencies and thereby increase the survival rate at delivery. the exit procedure therefore requires a multidisciplinary team approach, including at least two anesthesiologists for both the mother and the fetus, a pediatric otorhinolaryngologist, an obstetric surgeon, a neonatologist, and a team of nurses. during the exit procedure, anesthesiologists have to maintain the uteroplacental circulation during cesarean section to increase the time available to secure a potentially obstructed fetal airway. in conventional cesarean section, the major goals are to minimize the time from anesthetic induction to umbilical cord clamping, thereby minimizing fetal exposure to inhalational anesthetics. in fact, during the exit procedure, even intramuscular administration of drug cocktails and/or inhalational anesthetics (1-1.5 mac) are recommended to ensure a motionless, well-anesthetized fetus. furthermore, there is no need to limit the time of anesthesia induction or the skin incision to delivery time. the anesthetic goals and principles for an exit procedure differ markedly from those for a conventional cesarean section with respect to maintaining the uteroplacental circulation until the time of secured delivery. first, deep inhalational anesthetics (1.5-3 mac) are often used to relax the uterine muscles and to maintain the uteroplacental circulation. inhalational anesthetic concentrations of at least 2 mac are usually recommended for proper relaxation of the uterine muscles and to prevent early placenta detachment. for rapid onset and offset of tocolytic action during the exit procedure, sevoflurane or desflurane are recommended. these drugs include nitroglycerine and magnesium sulfate. however, nitroglycerin is a better choice than magnesium sulfate because nitroglycerin is easily titratable and short acting. the onset of uterine relaxation using nitroglycerin is 30-60 s, and a loading bolus of 100 g with continuous dosing of 0.5-1 g/kg/min is recommended, as needed, to maintain effective uterine relaxation. lastly, dopamine (5 g/kg/min), phenylephrine (0.05-0.1 g/kg/min), and ephedrine bolus (5-10 mg) can be utilized to maintain the maternal hemodynamics. many of the suggested anesthetic maneuvers used during the exit procedure to enhance uterine relaxation, such as the use of higher mac inhalational anesthetics combined with short acting tocolytics, potentially decrease maternal blood pressure and uteroplacental perfusion before delivery, and may lead to uterine atony and continued hemorrhage post-delivery. meticulous on- and off-techniques are critical to achieve full relaxation of the uterus before delivery and to ensure proper contraction to prevent uterine atony and massive hemorrhage post-delivery. sevoflurane (1-1.5 mac) and remifentanil (0.1-0.5 g/kg/min) were used to maintain anesthesia. the et concentration of sevoflurane was less than 2 mac before the uterine incision, even though the concentration was set from the beginning of anesthetic induction. therefore, an additional bolus of nitroglycerine (30 g) was given and infused continuously at 0.5-1 g/kg/min. the uteroplacental circulation was maintained for 7 min (from uterine incision to cord clamping) without maternal or fetal accident until the fetus was intubated and the endotracheal tube was confirmed to be in the correct position. usually, during the exit procedure, the dose of nitroglycerine used in the combined spinal epidural anesthesia is more than 100 g in a bolus, and the et concentration of inhalational anesthetics used in general anesthesia is above 2 mac. in our case, we performed general anesthesia using a lower bolus dose (two consecutive doses of 30 g) of nitroglycerine and a lower et concentration of inhalational anesthetics (1-1.5 mac) to maintain maternal hemodynamic stability and ensure fetal airway safety. the exit procedure is a relatively new procedure and holds promise for newborns with airway obstruction. anesthesia for the exit procedure can be performed in various ways, as long as uterine relaxation and uteroplacental circulation are maintained until the fetal airway is secured. preoperative planning and communication with regard to anesthesia are the most important factors for success.

the ex utero intrapartum treatment (exit) procedure is a very rare technique performed in cases of fetal congenital malformations. the exit procedure increases the rate of survival at delivery by maintaining the uteroplacental circulation until the airway of the fetus is secured. to maintain the uteroplacental circulation, a higher dose of inhalational anesthetics and/or intravenous nitroglycerin can be used as compared to conventional cesarean section. the aim of this report is to share our anesthetic experience during the exit procedure with members of the korean society of anesthesiology for the first time, and to highlight the maternal implications of the use of inhalational anesthetics and nitroglycerin during cesarean section for the exit procedure.

PMC3030024

pubmed-1370

the family reoviridae accommodates a wide range of members that infect protists, fungi, plants, invertebrates, and vertebrates, and are characterized by 912 segmented double-stranded rna (dsrna) genomes, multi-layered virion structures, and particle-associated enzymes for rna synthesis. the family now consists of 15 genera divided into two subfamilies spinareovirinae (turreted or spiked reoviruses) and sedoreovirinae (non-spiked reoviruses) (attoui et al., 2012). among the 15 genera is a relatively recently established genus mycoreovirus containing three members (mycoreovirus 13, myrv1-3) that were isolated in two phytopathogenic fungi, specifically, the chestnut blight fungus (myrv1 and myrv2) and the white root rot fungus (myrv3) by the groups of drs. bradley i. hillman, william macdonald, and naoyuki matsumoto (enebak et al., 1994; hillman and suzuki, 2004; hillman et al., 2004 myrv1 and myrv2 have 11-segmented genomes (figure 1; s1s11, termed with an increasing order of mobility in sds-polyacrylamide gel electrophoresis), while myrv3 has a 12-segmented genome (s1s12). the mycoreovirus belongs to the spinareovirinae subfamily and its members retain conserved ntp binding motif and di-histidine motif specifically conserved in the subfamily (suzuki et al., 2004; supyani et al., 2007; spear et al., 2012). each segment has a large single orf corresponding between 42 and 98% of its entire size. light gray boxes refer to orfs and the sizes of their protein products are shown. the locations and rdrp, guanylyltransferase, and ntp binding protein are assigned to s1, s3, and s6. vp4 and the plus-sense strand of each dsrna segment possesses the strictly conserved 5 pentanucleotide (5-gauca), single orf and 3 heptanucleotide (gcaguca-3). myrv1 has a double-shelled particle structure of approximately 80 nm in diameter.. both could involve large extents of genome segment alterations, but they are different in the generation mechanism. reassortment events, involving exchange of genome segments between two viruses, occur at varying rates during genome packaging in coinfected cells. genome rearrangements, defined as alterations of considerable tracts of sequence within single genome segments often in the form of deletions and extensions (taniguchi and urasawa, 1995), are a common phenomenon in all major genera of the family reoviridae (table 1). they occur under natural conditions (murao et al., 1996; schnepf et al., 2008) and laboratory conditions, for example, by serial passage of rotaviruses at high multiplicities of infection (mois; hundley et al, 1985; kojima et al., 2000) and exclusive maintenance in one of their two hosts (plant, vector insect) in the case of plant reoviruses (nuss, 1984). rearrangements can be regarded as non-homologous, intramolecular rna recombination and exclude intermolecular rna recombinations that are documented frequently in plus-sense (+), single-stranded rna (ssrna) viruses but are infrequently reported for reoviruses (taniguchi and urasawa, 1995; desselberger, 1996). rearrangements are hypothesized to happen by copy choice (template switch) mechanism during rna synthesis mediated by the rna-dependent rna polymerase (rdrp) complex, as is rna recombination in ssrna viruses. nsp, non-structural protein; del, deletion; dup+, duplication with the original orf extended; dup, duplication with the original orf unaltered; inter, intergenic recombination; wt, wild-type virus; lab, laboratory; nat, natural.*we overview genome alterations, with a focus on unusual genome rearrangements observed in mycoreoviruses and discuss differences between other reovirus rearrangements in type of genome segment alterations and possible mechanisms underlying their occurrence. readers are referred to excellent review articles on rna recombination (lai, 1992; nagy and simon, 1997; simon-loriere and holmes, 2011; sztuba-solinska et al., 2011). three types of very unusual genome alterations were reported to occur in mycoreoviruses that differ from those reported for other reoviruses. the first example is complete loss of one of the segment s8 reported for myrv3, a very rare event for a reovirus. an entire set of genome segments are believed to be incorporated and packaged into single core particles during replication (plus-sense strand synthesis). therefore, all reovirus strains with rearranged segments still contain a set of all genome segments whether intact or altered. to our knowledge, (2004) is the only example of reovirus that lacks a genome segment and is still viable. these observations allowed the authors to conclude that myrv3 s8 is dispensable for maintenance under laboratory conditions. the second type of mutations found in myrv1 is unique among reovirus examples in their dependence on a distinct virus or a viral protein. specifically, myrv1 rearrangements are observed at extremely high rates in the fungal host either coinfected with the prototype hypovirus cryphonectria hypovirus 1/ep713 (chv1-ep713) or expressing a multifunctional protein p29 encoded by chv1-ep713 (sun and suzuki, 2008). after repeated subculturing of those fungal strains for one to two months, several dozen percent of resulting myrv1 isolates carry genome rearrangements. from doubly infected fungal mycelia, a myrv1 variant, myrv1/s10ss is isolated from 22.4% of subcultures, which harbors an altered segment s10ss, a deleted form of s10, in place of an intact s10. s10ss lacks approximately 75% of the internal orf region while retaining the 5 and 3 terminal regions (sun and suzuki, 2008). in transformant fungal strains with the chv1 p29 coding domain, a variety of rearrangements are generated in addition to s10ss that include s1l, s2l, s3l, and s6l (figure 2). in contrast to s10ss, these rearranged segments involve orf extensions, but are distinct from many previously reported animal reovirus examples. extensions have been reported mostly from rotaviruses and those entail all head-to-tail tandem partial duplication occurring downstream of the termination codon of the authentic orf. thus, unaltered protein products are being synthesized from their cognate transcripts in infected cells. however, altered myrv1 genome segments, i.e., s1l, s2l, s3l, and s6l are all with duplicated orfs in-frame with their preceding orfs, resulting in extension of orfs by 1.41.9 fold. importantly, the products of the expected sizes are detectable in mycelia infected with myrv1 variants although s1l-encoded product remains to be detected (sun and suzuki, 2008; tanaka et al., 2011). these extended segments s1l, s2l, s3l, and s6l are frequently concomitant with s10ss although its significance is unclear. s1, s3, and s6-encoded proteins carry sequence motifs characteristic of rdrp (hillman et al., 2004), guanylyltransferase (supyani et al., 2007), and ntp binding proteins (suzuki et al., 2004). it should be noted that these sequence motifs are duplicated in the expected protein products. the maximum size expansion of an orthoreovirus was predicted to be 10% of the entire size (approximately 2.0 kb) (roner and steele, 2007). congruent with this notion, myrv1/s1l+s10ss variant, with the largest extension per segment (approximately 2.3 kb), has a genome-based extension of 1.8 kb after deduction of the deleted s10 sequence in s10ss. these extensions correspond to approximately an 8% increase on a genome-size (23,433 bp) basis. there are two types of myrv1 rearrangements reported: extensions (s1l, s2l, s3l, and s6l) and deletions (s4ss and s10ss) that could happen by single events. s1l, s2l, and s3l result from in-frame orf fusion but s4ss and s10ss do not always involve in-frame orf deletions. genetic organizations of the normal (top) and rearranged forms (bottom) of genome segments is shown. for extended segments s1l (a), s2l (b), s3l (c), and s6l (d), three fragments colored differently (blue, red, and green) are separated by break points. the red portions refer to duplicated sequence while red color gradation is to indicate orientation (head-to-tail for all rearrangements). in internal deletion mutations s4ss and s10ss (e, f), only 1122% (for s4ss) and 1043% (for s10ss) of the intact orfs are retained. note that s4ss and s10ss each have at least 4 (s4ss1s4ss4) and 2 variants (s10ss1 and 2) and one each of them (s4ss1 and s10ss1) are shown in (e) and (f), respectively. solid lines denote the 5 and 3 utrs, while blue and green boxes refer to the n and c terminal portions of orf retained after each rearrangement event. nucleotide positions for the start and stop codons, and rearrangement breakpoints are shown above the diagrams of each normal segments. nucleotide and aa sequences adjacent to the altered sites on normal and rearranged segments are also shown below the diagrams of each altered segment. neither sequence heterogeneity nor mismatch was detected at the junction sites for s1l, s2l, and s3l, while mismatches are found at or close to breakpoints of s4ss and s10ss (sun and suzuki, 2008; eusebio-cope et al. myrv1 s4 (eusebio-cope et al., 2010) and s10 (suzuki, unpublished results), albeit very infrequently, are rearranged in a chv1 p29 independent fashion. myrv1/s4ss variants carry deletion mutations on s4 (s4ss1, s4ss2, s4ss3, and s4ss4) lacking approximately 80% of the s4 orf but retaining its ability to be transcribed and replicated. the genome segments including s4ss, s4ss1s4ss4 are different from each other in deletion endpoint. a reassortant variant of myrv1 that contains substantial deletions in both s4 and s10 (myrv1/s4ss1+s10ss2) was generated in fungal colonies coinfected with myrv1/s4ss1 and myrv1/s10ss2. surprisingly, myrv1/s4ss+s10ss shows comparable replication levels to the wild-type myrv1. this is the first example of a reovirus that is replication-competent without two viral proteins. unlike members of the genera orthoreovirus (kobayashi et al., 2007), orbivirus (boyce et al., 2008; matsuo et al., 2010), and rotavirus (komoto et al., 2006), no reverse genetics system has been developed for mycoreoviruses. however, the myrv1 rearrangements (shown in figure 2) provide insights into the functional roles of the genome segments and their protein products. common to all these altered rearrangements, they are competent to be packaged and transcribed given the comparable accumulation levels of their transcripts in infected cells. these results show that signals for packaging, transcription, and replication reside within the remaining genome sequence. the viability of myrv1/s4ss+s10ss indicates that neither myrv1 non-structural protein encoded by s4 (vp4) nor s10 (vp10) is required for virus replication. 2010) reported that vp4 contributes to mycelial growth and efficient vertical transmission through conidia. vp10 contributes inversely to the reduction of aerial mycelial growth induced by the virus (sun and suzuki, 2008). recombination events altering protein sequences are often deleterious to its function and purified from the population (simon-loriere and holmes, 2011; sztuba-solinska et al., 2011). in fact, most extensions reported previously for reoviruses occur downstream of the termination codon and thus result in no change of protein sequences (kojima et al., 1996). in this sense, the fact that several myrv1 variants with extension rearrangements are viable is surprising. interpretation of functional roles for extended segments (s1l, s2l, s3l, and s6l) in virus replication is complex due to coexistence of the extended and normal unaltered genome segments in fungal colonies infected by myrv1 variants. therefore, heterocomplementation of rearranged segments by the normal segments can not be ruled out. in this regard (2011), that fungal colonies infected with myrv1/s1l+s10ss2, myrv1/s2l+s10ss2, and myrv1/s3l+s10ss2 are similarly restored in aerial mycelial growth to some extent (figure 3). the restoration levels of aerial mycelial growth increase as the ratio of transcript levels of rearranged s1l to normal segment s1 rises (tanaka et al., 2011) fungal colonies were grown on pda for 8 days under the bench top conditions approximately 24c. c. parasitica strain ep155 was infected with wild-type myrv1 (myrv1), myrv1/s10ss2 (myrv1/s10ss), myrv1/s1l+s10ss2 (myrv1/s1l+s10ss), myrv1/s2l+s10ss2 (myrv1/s2l+s10ss), or myrv1/s3l+s10ss2 (myrv1/s3l+s10ss). as in other reoviruses, rearrangements are considered to contribute to evolution and molecular diversity of mycoreoviruses, while mycoreoviruses with such rearrangements including the myrv3 s8 loss are found under laboratory conditions, but not in the natural environment. myrv1 variants with large deletions may have selective advantages in replication or packaging over wild-type myrv1. myrv1 variants with large extensions require chv1 p29 for their maintenance, suggesting no selective advantage of these variants in the absence of p29. maintenance of animal viruses in cultured host cells frequently induces mutations that confer selective advantages under laboratory conditions and lead to virulence attenuation in host animals. in this regard, myrv1 with genome rearrangements are different because they retain the ability to induce symptoms, while distinct from those induced by wild-type myrv1 (figure 3), including the reduction of host virulence to plants. rather, myrv1 is reminiscent of plant reoviruses which undergo various mutations when they are maintained exclusively in one of their hosts, plant and insect vectors in laboratory conditions. examples include those of would tumor virus (wtv) and rice dwarf virus (rdv) that are vectored by different species of leafhoppers (table 1). in fact, transmission defective isolates of wtv emerge after being maintained for a long period of time in cultured insect cells that carry internal deletions on segments encoding structural and non-structural proteins (nuss, 1984; anzola et al., 1987). in the case of rdv maintained only in plants, point mutations rather than internal deletions accumulate resulting in little expression of protein products encoded by the mutated segments.. however, one single passage in cultured insect vector cell lines resulted in inverse population structures (pu et al., 2011). two major mechanisms are proposed for it, specifically, replication independent breakage-rejoining recombination, and replication dependent rdrp-mediated recombination (template switch or copy choice). the non-replicative, breaking-joining mechanism entails dsrna or ssrna fragmentation and re-ligation for which several variations are proposed such as self-recombination requiring no proteins, and rnase-catalyzed cleavage and ligation (chetverin, 1999). the second model, replicase-mediated mechanism is more widely accepted (lai, 1992; nagy and simon, 1997; sztuba-solinska et al., 2011). in this model, the nascent rna and rdrp complex jumps after proceeding to a certain point from one template to another (intergenic or intermolecular recombination) or to another point of the same template (intragenic or intramolecular recombination). template switches could occur in homologous and non-homologous (illegitimate) manners such that their crossover regions are of similar and unrelated sequences respectively. non-homologous recombination is sometimes considered to be facilitated by sequences features of template and acceptor strands, such as direct, inverted repeats and/or secondary structures. a number of viral rna and protein elements as well as host factors are involved in rna recombinations in different single-stranded, plus-sense rna plant virus/host systems (serviene et al., 2005; sztuba-solinska et al., recently, rna silencing was shown to contribute to the generation of defective-interfering (di) rnas of the prototype hypovirus (chv1; zhang and nuss, 2008), associated with the picornavirus superfamily (koonin et al., 1991), that infects the chestnut blight fungus and confers hypovirulence. rearrangements reported for members of the family reoviridae could be accounted for by an intramolecular template switch, in which the replicase complex jumps backward (for extension) or forward (for deletions) on the same template molecule. for example, intragenic rearrangements of human rotavirus segment s11 are hypothesized to be generated in the process of plus-sense strand synthesis (transcription) mediated by short direct repeats of 46 nucleotides (kojima et al. (2001) inferred that duplications of human rotavirus genes 7 and 11 were presumed to occur by template switch mediated by an inverted repeat-involving secondary structure during the replication step (gault et al. a rare example is intergenic recombination with a crossover of two rotavirus segments encoding nsp2 and nsp5,6 (cao et al., 2008; recent structural studies led to 3d modeling of rdrps from two reoviruses, human orthoreovirus and rotavirus. they are commonly characterized by 4-window structures which allow entry of substrates (rear window) and templates (left window), and exit of template rna (front window) and nascent rna (bottom window; tao et al., 2002; lu et al., 2008 based on this rdrp structural prediction, a loop model was proposed to explain how reovirus extension rearrangements occur (matthijnssens et al., 2006). according to this model, extension rearrangements occur during the transcription step via intramolecular template switch from the transcribed region of the minus-strand to upstream regions of the same molecule re-entering from the left channel. it remains unknown how the mechanism of intergenic recombination or large deletion rearrangements fits into the rdrp 3d model. addressing how p29 is involved in the enhanced emergence of myrv1 rearrangements is an interesting challenge. hypovirus rna are replicated in golgi-derived vesicles of 80 nm whose production is enhanced in the cytoplasm upon viral infection (fahima et al., 1994; hillman and suzuki, 2004; jacob-wilk et al., 2006). reovirus rnas are synthesized in viroplasms or virus factories formed in the cytoplasm of infected cells by viral and host components (patton et al., 2006; cheung et al., 2010). chv1 p29 is a multifunctional protein acting as a symptom determinant (choi and nuss, 1992; craven et al., 1993; suzuki et al., 1999, 2003), protease (choi et al, 1991a, b), an rna silencing suppressor (segers et al., 2006), and an enhancer of heterologous and homologous viruses (suzuki et al., 2003; sun et al., 2006). some functional domains of p29 are mapped such as the n terminal aa 124 essential for virus viability, adjacent 2574 for the symptom determinant and the c terminal half for the proteolytic catalytic domain (suzuki and nuss, 2002; hillman et al., 2004). firstly, p29 may interact the rna synthesis machinery to perturb transcription (plus-sense rna synthesis) and/or replication (minus-sense rna synthesis), leading to enhanced rate of template switch of rdrp-mediated via rna sequence features such as inverted or direct repeats. another possibility is that p29 alters the physiological state of infected cells, which enhances selection of pre-existing mutant viruses with rearranged segments by unknown means., it remains unknown whether chv1 p29 reside in myrv1 core particles viewed as a nano-scale rna synthesis factory (guglielmi et al., 2010) to perturb myrv1 rna synthesis. sun and suzuki (2008) showed that chv1 p29 interacts with myrv1 vp9, considered to be a non-structural protein, in vitro and in vivo, providing a clue to the mechanism. as in cells infected with other reoviruses, viroplasm is produced in myrv1-infected cells (sun and suzuki, unpublished data). one or two viral non-structural proteins are required as matrix proteins for production of reoviral viroplasms which are believed to be the site of virus replication where core particle assembly and rna synthesis occur. consistent with this notion, knocking-down of the genes responsible for functional viroplasm formation is detrimental for virus replication (campagna et al. as discussed above, a myrv1 variant shows comparable replication competency without vp4 and vp10. therefore, vp4 and vp10 are unlikely to be matrix proteins of viroplasms given the essentiality of viroplasm. one of key points to be addressed is what the possible role of vp9 in myrv1 replication is. it is of interest to infer that vp9 is a viroplasm matrix protein that is remodeled upon interaction with p29. moreover, to explore whether chv1 p29 is incorporated into myrv1 core particles is a prerequisite. the other possibility may be associated with the reason why almost a half of the myrv1 segments, whether encoding structural or non-structural proteins, are generated at high rates. chv1 p29, as a symptom determinant, slightly enhances mycelial growth and suppresses asexual sporulation and orange pigmentation, which involve complex regulatory pathways. additionally, p29 is an rna silencing suppressor which may potentially be able to perturb the cellular physiological state. the p29 protein might be involved in selection and maintenance of myrv1 variants with rearrangements. apparent reversion of s1l, s2l, s3l, and s6l to respective normal segments (sun and suzuki, 2008; tanaka et al., 2011) in the absence of p29 suggests a role for the protein in the maintenance of the extended segments. three unusual types of genome alterations are found in the genus mycoreovirus; a s8-deficient myrv3 mutant, a myrv1 variant with two rearranged segments (s4ss and s10ss) each lacking three fourths of the orf, and myrv1 variants with genome rearrangements (s1l, s2l, s3l, s6l, and s10ss) induced by a multifunctional protein p29 of an unrelated virus, chv1. while s8-lacking myrv3 strains have properties indistinguishable from wild-type myrv3, most myrv1 variants with rearranged segments induced different symptoms in the chestnut blight fungus than the wild-type virus. therefore, these genome segment alterations are useful for gaining insights into functional roles of genome segments of mycoreoviruses for which a reverse genetics system is unavailable like many other reoviridae members. the chv1 p29-dependent rearrangements of myrv1 are a novel type of intriguing virus/virus interactions, and accordingly there are a few important unanswered questions. what is the biological significance of the interactions between the two viruses? what function of chv1 p29 is related to rearrangements? does p29 contribute to generate de novo genome rearrangements or to select pre-existing rearrangements by altering the cellular state, while it is poorly defined at present? does chv1 p29 induce genome rearrangements on other reoviruses such as myrv2 and myrv3? experiments to address these issues are underway. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

the family reoviridae is one of the largest virus families with genomes composed of 912 double-stranded rna segments. it includes members infecting organisms from protists to humans. it is well known that reovirus genomes are prone to various types of genome alterations including intragenic rearrangement and reassortment under laboratory and natural conditions. recently distinct genetic alterations were reported for members of the genus mycoreovirus, mycoreovirus 1 (myrv1), and myrv3 with 11 (s1s11) and 12 genome segments (s1s12), respectively. while myrv3 s8 is lost during subculturing of infected host fungal strains, myrv1 rearrangements undergo alterations spontaneously and inducibly. the inducible myrv1 rearrangements are different from any other previous examples of reovirus rearrangements in their dependence on an unrelated virus factor, a multifunctional protein, p29, encoded by a distinct virus cryphonectria parasitica hypovirus 1 (chv1). a total of 5 myrv1 variants with genome rearranged segments (s1s3, s6 and s10) are generated in the background of a single viral strain in the presence of chv1 p29 supplied either transgenically or by coinfection. myrv1 s4 and s10 are rearranged, albeit very infrequently, in a chv1 p29 independent fashion. a variant of myrv1 with substantial deletions in both s4 and s10, generated through a combined reassortment and rearrangement approach, shows comparable replication levels to the wild-type myrv1. in vivo and in vitro interactions of chv1 p29 and myrv1 vp9 are implicated in the induction of myrv1 rearrangements. however, the mechanism underlying p29-mediated rearrangements remains largely unknown. myrv1 s4 rearrangements spontaneously occurred independently of chv1 p29. in the absence of reverse genetics systems for mycoreoviruses, molecular and biological characterization of these myrv1 and myrv3 variants contribute to functional analyses of the protein products encoded by those rearranged segments.

PMC3365852

pubmed-1371

metastatic lesions comprise approximately 1% of the tumors in the sellar/parasellar (sps) area for which patients undergo transsphenoidal surgery (tss) [1, 2]; however, it has been reported in autopsy series that the rate of metastasis to these areas could be as high as 28%. breast and lung cancer are the two most common types of malignant tumors that metastasize to the sps region, with respective rates of 40% and 33%. metastases of prostate, renal cell, gastrointestinal, thyroid [8, 9], and pancreatic cancers, and lymphoma, leukemia, melanoma, and plasmocytoma have also been reported. despite the advancement in the imaging modalities, tumors that have metastasized to the sps areas may still be difficult to differentiate from pituitary adenoma on radiographic studies [2, 14, 15]. thickening of the pituitary stalk and invasion of the cavernous sinus may be suggestive of such lesions, but invasion of the cavernous sinus may commonly occur with pituitary adenomas. this distinction is also clinically challenging, although there are very few symptoms that suggest a metastatic lesion. management options are multimodal and vary depending on whether a primary source is known or on the likely differential diagnoses based on the clinical and radiological findings. multimodal options include radiation therapy, chemotherapy, and/or surgery [16, 17], although the tumor invasiveness renders surgical resection limited. although surgical series have not shown any survival benefits, the patient's quality of life may be improved [10, 14]. in this paper, we review the clinical, endocrine, and radiological features of the metastatic sps tumors with currently accepted therapeutic options based on the pertinent literature. in addition, we report six cases from our institution and discuss their management with long-term clinical outcome. a systematic review of the literature was performed using pubmed and the bibliographies of reviewed articles. the medical records of six patients admitted to the university of utah health sciences center between 2001 and 2011 were reviewed retrospectively. clinical presentation, radiographic studies, treatment, histopathological confirmation, outcome, and prognosis were recorded (table 1). a 77-year-old man with known prostate cancer presented with a four-month history of left retro-orbital pain followed by left eye ptosis. at presentation, he had complete left third nerve palsy. his visual acuity was intact in both eyes, with full visual fields to confrontation. brain magnetic resonance (mr) imaging (figure 1) showed a heterogeneously enhancing mass lesion that measured 29 17 29 mm involving the sella, with invasion of the left cavernous sinus and the upper clivus. the lesion extended to the inferior orbital fissure and was centered in the sella turcica and the cavernous sinus. he subsequently underwent chemotherapy and focused radiation to the sellar region and was noted to have stable neurological examination findings two months after surgery, with no change in his ophthalmoplegia. an 82-year-old woman with known history of breast cancer presented with several weeks ' complaint of progressive left-sided hearing loss as well as facial pain and numbness in the first and the second divisions of the trigeminal nerve, respectively. on neurological examination, left-sided hearing loss and facial numbness along the v1 and v2 distributions were confirmed. mr imaging showed evidence of a heterogeneously enhancing mass in the left petrous apex that extended to involve meckel's cave, the lateral cavernous sinus, and the internal auditory canal (figure 2). she was discharged home on postoperative day three in stable condition for followup with oncology. a 79-year-old man with known history of prostate cancer presented with several weeks ' history of progressively worsening double vision and eventual right eye ptosis. mr imaging disclosed an enhancing soft tissue mass involving the clivus, pituitary fossa, cavernous sinus, and posterior nasal cavity (figure 3) that surrounded both internal carotid arteries in the cavernous sinuses. an endonasal transsphenoidal approach to the sphenoid sinus was carried out to obtain a biopsy of the lesion. a histopathological diagnosis of metastatic melanoma was made, and evaluation was undertaken by the oncology team.) a 21-year-old man with a remote history of osteosarcoma and newly diagnosed metastatic renal cell carcinoma had complaints of worsening vision and facial pain. because there was a discrepancy in the pupillary size between his eyes, he underwent a computed tomography (ct) scan of the head, followed by craniofacial mr imaging, which revealed an enhancing mass in the right sphenoid sinus with adjacent extension. on neurological examination, he was noted to have complete hemifacial numbness and horner's syndrome on the right side. mr imaging of the face demonstrated a homogeneously contrast-enhancing lesion centered within the right sphenoid sinus measuring 33 20 27 mm (figure 4). the lesion extended into the carotid canal, pterygopalatine fossa, and optic nerve canal with destruction of the vidian canal and foramen rotundum on the right. a 42-year-old woman with several weeks of frontal headaches initially presumed to be secondary to a sinus infection underwent mr imaging after antibiotic medications failed to alleviate her symptoms. brain mr imaging demonstrated a lobulated, 25 20-mm sellar and suprasellar lesion with extension into the right cavernous sinus and encasement of the right internal carotid artery. this lesion was isointense on t1- and t2-weighted images and heterogeneously enhancing with gadolinium administration. a ct scan was consistent with a lytic lesion involving the central skull base extending laterally and posteriorly to involve the bilateral medial sphenoid wings and the clivus, respectively. histopathologic evaluation was consistent with a diagnosis of diffuse large b-cell lymphoma (figure 5). a 53-year-old woman presented with a known diagnosis of breast cancer and a one-year history of worsening left eye vision with a more precipitous decline in the last month. mr imaging of the brain demonstrated a skull base lesion involving the left sphenoid bone, the anterior clinoidal process, and the cavernous sinus. on neurological assessment, the patient was noted to have an acutely diminished visual acuity in her left eye to a level of finger counting with left temporal visual field cut. mr imaging (not shown) showed a homogenously contrast-enhancing lesion of the skull base involving the greater sphenoid wing and the anterior clinoidal process with encasement of the optic nerve on the left. a left frontotemporal craniotomy was performed for biopsy of the lesion and to decompress the optic nerve and the cavernous sinus with a subtotal resection. one year after surgery, her neurological findings were unchanged, and her systemic disease was under control. neoplasms originating from a multitude of sites have been reported to metastasize to the sps region. breast and lung cancer account for approximately two-thirds of sps metastases, being the most common sources in women and men, respectively, [11, 1821]. histological examinations of the tissue samples obtained during palliative hypophysectomy performed in patients with end-stage breast cancer and from autopsy series have documented metastasis to the sps region in 6% to 29% of cases [3, 2225]. one hypothesis put forth to explain this prevalence is that the prolactin-rich environment of the pituitary enhances the proliferation of breast tumor cells. after carcinoma of breast and lung, lymphoma and prostate cancer have been reported to be the most common sources of metastasis to sps region. liver, renal cell, colon, and thyroid cancers and melanoma are rare sources of distant metastases to this region. the relatively rarer occurrence of our other cases, prostate, melanoma, renal cell, and lymphoma, is consistent with the literature. most cases are found in the sixth or the seventh decade of life as a part of a generalized metastatic spread, commonly associated with multiple, particularly osseous metastases [18, 20]; however, metastases can occur in young patients. the age of presentation ranged from 21 to 82 years in our small series, with an average age of 60 years. very occasionally, these lesions are the first manifestation of an occult cancer or the only site of metastasis [14, 20]. thus, in a patient without any prior history of cancer, an sps lesion can not be assumed to be an adenoma, just as in a patient with a known primary cancer, it is not always metastatic. clinically, metastasis is generally suspected in patients with rapid onset and progressive symptoms, irrespective of a history of malignancy. the possible metastatic pathways to the pituitary and parasellar region include direct blood-borne metastasis to the posterior pituitary lobe, pituitary stalk, clivus, dorsum sellae, or cavernous sinus or leptomeningeal spread with involvement of the pituitary capsule [10, 26, 27]. there has been some controversy regarding the most common location of metastasis within the pituitary gland. authors of early series have reported that the majority of pituitary metastasis occurs in the posterior pituitary, but some dispute this claim. teears and silverman reported that 57% of the lesions localized to the posterior pituitary alone, 13% to the anterior pituitary alone, 12% to both lobes, and the remaining 18% to the capsule or stalk. they hypothesized that the posterior pituitary, by receiving direct arterial supply, is more likely to develop metastases than the adenohypophysis, which receives its blood supply from the hypophyseal portal system. the posterior lobe has a larger area of contact with the adjacent dura, which may be another contributing factor [4, 18]. metastatic inoculation in the anterior lobe is usually the result of contiguous spread from the posterior lobe. these may be isolated, such as diplopia or ptosis, with the third (oculomotor) and the sixth (abducens) nerves being the most commonly involved, followed by the fourth (trochlear) nerve [14, 2931], or they may appear in a constellation of symptoms characterized by unilateral, rapidly progressive ophthalmoplegia with retroorbital pain. this latter presentation is the usual presentation of the cavernous sinus syndrome, also known as parasellar syndrome. if the branches of the trigeminal nerve are affected, alteration in the facial sensation, facial pain, or dysesthesia occurs. headache has been reported as a rather common symptom, with an incidence as high as 70% [14, 34, 35]; however, the majority of pituitary metastases are clinically silent. in the autopsy study by teears and silverman, these metastases are often seen in patients with terminal malignancy who present with malaise, generalized pain, central nervous system involvement, or treatment-associated symptoms, although symptoms of pituitary insufficiency may be masked. several studies have indicated that diabetes insipidus (di) was the most common symptom [10, 20, 24, 36, 37]. in the series of mccormick et al., di developed in 70% of patients; however, if the anterior pituitary function is compromised, di may be concealed by reduced mineralocorticoid function. in some recent series, di has not been reported, which is likely because modern imaging techniques are able to detect abnormalities earlier than the timeframe required for di development. other rarer hormonal findings may be hypothyroidism and hypoadrenalism, hypogonadism, or overproduction of adrenocorticotropic hormone (acth), growth hormone (gh), or prolactin [10, 3840]. because of their invasiveness, pituitary metastatic lesions have a high potential to cause visual deficits from suprasellar extension, with an incidence as high as 50% reported by branch jr. and laws jr. and others [10, 34, 41]. in both the series by chiang et al. and that of sioutos et al cranial nerve palsies involving the third and the sixth nerves and facial pain with numbness (trigeminal origin) were the most common presentation (33%) in our cases with cavernous sinus involvement. retro-orbital pain, vision compromise, gait instability, horner's syndrome, and hearing loss were infrequent and associated with petroclival and sphenoorbital extension of the lesions. all these symptoms were experienced with a relatively rapid onset from a few weeks to a few months that suggested the aggressive character of the lesions. symptoms strongly suggesting metastasis in the parasellar or sellar space include painful ophthalmoplegia in association with the sudden onset of di [10, 26, 34, 42]. the pain may be retro-orbital or may be due to trigeminal dysfunction [4345]. in our series, 5 of the 6 (83%) patients had a previous history of malignant disease. one of the patients that had prior history developed a malignancy (melanoma) other than the original one. only one patient without a prior history of malignancy first presented with lymphoma metastasis to the sps region. because pituitary adenomas also present with invasion of the sellar floor, cavernous sinus, or clivus, no specific neuroimaging criteria to define metastatic lesions in sps region have been reported. the diagnostic imaging tools for sps metastasis mainly include high-resolution ct and mr imaging. although ct is superior to mr imaging in detecting the bone involvement, the latter is preferable to determine the relationship of the lesion to the surrounding neurovascular structures. although nonspecific, the characteristics of these lesions on mr imaging are an iso- or hypointense mass on t1-weighted imaging with a usually hyperintense signal on t2-weighted imaging, and homogeneously enhancing mass in images obtained after the administration of contrast agent. invasion of the cavernous sinus, sclerotic changes around the sella turcica and clivus, isointense signal on both t1- and t2-weighted imaging, and loss of high-intensity signal in the posterior pituitary have been reported to be helpful in differentiating metastatic lesions from benign ones [15, 30, 48].. found that thickening or enhancement of the infundibulum was the most characteristic ct or mr imaging feature. schubiger and halter reported that the invasion of the infundibular recess by a suprasellar mass is suggestive of metastasis. because of the rapid growth of metastatic lesions, a dumbbell-shaped intra- and suprasellar tumor with indentation at the diaphragm level is generally indicative for these cases [20, 37, 49]. the radiodiagnostic findings that suggested a malignant/metastatic process in our cases were the involvement of multiple compartments in the anterior, middle, posterior cranial fossae, extension to the infratemporal and pterygopalatine fossae, sphenoid sinus, and nasal cavity with bony destruction in the cranial base and asymmetric or bilateral invasion into the cavernous sinus. the management of sps metastases is multimodal, including surgical resection, radiation therapy, and chemotherapy. treatment is mainly palliative and depends on the symptoms and the extent of systemic disease [10, 51]. because of the invasiveness and the high vascularity of the tumor, total surgical resection is generally not undertaken [1, 20]. therefore, surgical treatment should aim for symptomatic relief and the preservation of visual function, even in patients with widespread primary disease, and should be followed by local radiation treatment and/or chemotherapy [10, 51]. the body of evidence is inconclusive on the effect of the latter two modalities on survival [14, 52]. laws jr. reported improvement in symptoms, especially in pain and visual field defects, with no difference in survival after complete resection compared with subtotal or partial resection [10, 14]. on the other hand, others have supported the concept of improvement in survival after surgical resection of the lesion [5355]. surgical exploration is also essential if tissue diagnosis is likely to affect therapy in patients with no known primary malignancy. resection is most commonly done via transsphenoidal route, although subfrontal or pterional approaches are also options depending on the location and the extension of the lesion. four out of six patients in our series underwent surgical biopsy through either transsphenoidal or transcranial route and then underwent subsequent radiation/chemotherapy. one patient who had visual compromise had subtotal resection for palliation followed by radiation/chemotherapy. one patient who had renal cell carcinoma was directly referred to radiation oncology for immediate radiation and subsequent chemotherapy. besides its role as an adjunct after surgery [14, 42, 56], radiosurgery or conventional radiation is recommended as the initial course of treatment in patients with systemic disease out of control, recurrence in the systemic disease with concomitant sps metastasis, or medical comorbidities that put the patient at risk for a surgical intervention [2, 7, 18, 41, 5762]. radiosurgery, which is considered less invasive than conventional radiation, has been reported to achieve good tumor control [63, 64]. in a series of 23 patients by iwai et al., the rates of tumor control and symptom improvement were 67% and 53%, respectively; however, radiosurgery to the sps region is limited by its potential to cause radiation injury to the surrounding neurovascular structures such as optic apparatus, pituitary gland, or cranial nerves coursing in cavernous sinus. doses reported in the literature for these structures range from 8 to 40 gy [63, 6568], and the optimal dose may be quite variable depending on the proximity of the lesion. furthermore, the debate about whether the radiation should be directed to the sps region alone or to the entire brain continues. chemotherapy is commonly used alone or along with radiation therapy mostly for palliation in the treatment of metastatic disease in sps region. its value has not been adequately studied and reported in the literature. the prognosis of patients with metastases to the sps region is grim in a majority of cases because of the aggressive character of the primary disease. even in patients with no other metastasis at the initial evaluation, the prognosis remains poor because of radiologically undetectable microscopic metastases; however, it has been suggested that the extent of systemic disease affects survival in these patients. patients with a single sps region metastasis may have a better outcome [10, 20]. median survival is less than 2 years independent of the management strategy [10, 14, 70]. the records on the long-term follow-up of the majority of our patients are lacking because the patients were not monitored for surgical outcome, or the follow-up periods were too short. the only patient that had palliative surgery for vision compromise was seen at one year after the surgery with stable neurological findings. suggestive symptoms include rapid onset of progressive ophthalmoplegia with retro-orbital or facial pain, visual impairment, and/or di. management varies depending on whether a primary source is identified, the symptomatology, the location and extent of the lesion, the stage of the primary disease, and the medical comorbidities. subtotal or partial surgical resection is aimed mainly for symptom relief. a multimodal approach involving subtotal resection of the lesion followed by radiation and/or chemotherapy is widely accepted, especially in symptomatic patients whose primary disease is under control. radiation with or without chemotherapy is generally recommended as first-line treatment in patients with advanced primary disease or in those with high-risk medical comorbidities. a biopsy usually precedes radiation therapy if the primary source of the metastasis is unknown. the prognosis for patients is generally poor, independent of the therapeutic modality, and the overall survival is less than two years.

the sellar and parasellar (sps) region is a complex area rich in vital neurovascular structures and as such may be the location of first manifestation of a systemic malignancy. metastases to this region are rare; breast cancer is the most common source among those that metastasize to the sps region. ophthalmoplegia, headache, retroorbital or facial pain, diabetes insipidus, and visual field defects are the most commonly reported symptoms. lack of specific clinical and radiological features renders sps metastases difficult to differentiate from the other frequently encountered lesions in this area, especially when there is no known history of a primary disease. currently accepted management is multimodality therapy that includes biopsy and/or palliative surgical resection, radiation, and chemotherapy. although no significant survival benefits have been shown by the surgical series, surgical resection may improve quality of life. here we review the relevant literature and present six illustrative cases from our own institution.

PMC3263702

pubmed-1372

understanding information transmission within neuronal circuits, and the factors underlying long-range axonal signaling malfunction, relies on identifying the axonal ion channels that are key regulators of node of ranvier (nor) excitability. nors are highly specialized zones in myelinated axons containing high densities of voltage-gated sodium channels (navs) and associated cytoskeletal complexes, creating active zones essential for saltatory conduction of action potentials (aps) (debanne et al., 2011). the repolarizing currents required to sustain nav availability at nors for reliable ap transmission is less clear cut, particularly in the mammalian brain. both low- and high-voltage-activated potassium (k) channel subunits (kv7 and kv3.1/3) have been anatomically localized to nors (devaux et al., 2003, 2004; pan et al., 2006), and although their presence can vary between brain regions and axon types (debanne et al. 2003, 2004), recent experiments provide direct evidence that kv7 channels stabilize nor membrane potential (vm) in cortical l5 pyramidal cells (battefeld et al., 2014) consistent with findings in peripheral nerve (schwarz et al., 2006). the influence of delayed rectifier (dr) k channels (kv1.1 and kv1.2), which are widely observed in the juxtaparanodal (jp) zone (devaux et al., 2003; ogawa et al., 2010; rasband, 2010; zhou et al., 1998), has, however, been difficult to assess without demyelination (rper and schwarz, 1989; wilson and chiu, 1990), and whether local vm becomes sufficiently depolarized to recruit jp kv1 channels during saltatory conduction is disputed (arancibia-carcamo and attwell, 2014). voltage-gated ca channels (cavs), which could provide an additional source of depolarization as well as gating ca-dependent processes including recruitment of ca-dependent k channels (kca), have been described in central myelinated axons and shown to influence excitability at the axon initial segment (ais) (bender and trussell, 2009; bender et al., 2010; yu et al., 2010). however, although cavs have been proposed to influence nor formation during development (alix et al., 2008), their presence at mature nors in the brain is not established (zhang et al., 2006). purkinje cells (pcs) in the cerebellum fire at high rates, both spontaneously and in response to synaptic input, and thus require fast recovery of sodium channels at their nors. we have obtained direct evidence that, rather than solely relying on voltage-gated potassium channels, activity-dependent, spatially localized ca influx at nors of pc axons recruits an intermediate-type kca (ik, or kca3.1) to provide a node-specific repolarizing conductance crucial for axonal spike propagation. using simultaneous somatic and axonal patch-clamp recordings, local pharmacology, and two-photon ca imaging of cerebellar pc axons, we directly investigated which ion channels are engaged at nors during ap propagation. we visualized pc axons in cerebellar slices by dye filling via the somatic recording pipette and recorded axonal aps downstream of nors (see the experimental procedures; figure 1a) identified by virtue of their presence at axonal branchpoints (clark et al., aps are securely transmitted by pc axons at high firing rates, with failures occurring above 250 hz (khaliq and raman, 2005; monsivais et al., 2005). this propagation reliability is retained across axonal branchpoints, with equal limiting frequency in both the main projection axon (257 17 hz; also monsivais et al., 2005) and in recurrent axon collaterals (figures 1b and 1c, 253 14 hz, 0.05% differentially propagated spikes, n =6 cells, see also foust et al., 2010). we used local application (figure s2a) of various ion channel antagonists to test their impact on ap propagation at nors in spontaneously firing pcs (firing rates 2080 hz). ttx (10 m) completely blocked ap propagation, confirming the presence of a nor at branchpoints (figure s1d; see also khaliq and raman, 2005). in contrast, application of tea at a concentration (10 mm) that should block a wide variety of k channels including kv1, kv3, and kv7 types (grissmer et al., 1994 ;, 2000) did not affect ap propagation, having no impact on axonal capacitive current amplitude, firing rate, or conduction velocity (figures s1b and s1c). this lack of effect was similar at high firing rates, and the limiting frequency for spike propagation was unchanged (figure s1d). these results are unexpected given that tea-sensitive k-channels are thought to contribute to axonal ap repolarization (devaux et al., 2003; hille, 1967; rper and schwarz, 1989), to stabilize nodal vm as well as preventing antidromic spike reflection (goldstein and rall, 1974), and kv3.3 subunits have been localized to pc axons (chang et al., 2007). potential explanations for these results might be that first, tea-sensitive k channels are absent from pc axons and/or their nors and repolarization is mediated by an alternative mechanism. second, they may be present but at insufficient density to be primary regulators of node excitability. third, tea-sensitive k channels might be located behind tight myelin junctions in the juxtaparanodes (jps) (wang et al., 1993) and contribute to repolarization of the axonal membrane while being inaccessible to tea, as observed in peripheral nerve (chiu and ritchie 1980; kocsis and waxman 1980) (however, see mierzwa et al., 2010). fourth, these channels may be present in the jps but weakly activated by the limited vm changes that are calculated to occur in healthy myelinated axon segments (arancibia-carcamo and attwell, 2014). recently, an intermediate-type kca conductance (ik, kca3.1), not previously observed in the cns and with a lower sensitivity to tea (ic50 =24 mm) (wei et al., 2005), was described in cerebellar pcs and shown to contribute to postsynaptic integration (engbers et al., 2012). we examined whether ik might provide an alternative mechanism for regulating excitability at nors and found that local application of the selective ik channel antagonist tram-34 (engbers et al., 2012; wulff et al., 2000) to axonal branchpoints led to a concentration-dependent block of axonal spike propagation during spontaneous firing (2080 hz; figures 1d1 g), with no observed dependence on firing rate (r =0.1). clotrimazole (1 m), another ik-selective blocker, had similar effects (figure 1 g), while apamin (1 m) and iberiotoxin (1 m), which block small (sk) and large (bk) kca channels, respectively, had no impact (98% 0.44% [n =5] and 97.3% 1.2% [n =4] of control axonal spike amplitude). ik gating depends solely on intracellular [ca] (kd: 0.10.3 m) (wei et al., 2005; 1997), and, since both local removal of extracellular ca and application of ni (100 m) similarly suppressed action potential propagation (figures 1d1 g), the activation of ik is likely initiated by ca influx via cavs. we confirmed that pc axons are immunopositive for the kca3.1 subunit, the sole molecular entity that constitutes the ik channel, but found, surprisingly, that no hotspots were seen at nors and that kca3.1 labeling was instead relatively uniform along the axon (figure 2). activity-dependent ca influx has been observed in optic nerve (lev-ram and grinvald, 1987; zhang et al., 2006), but the resulting ca transients are spatially uniform along both nors and internodes (zhang et al., 2006), and the route of ca entry is uncertain. as yet, there is no direct evidence for ca influx localized to nors in the brain. using two-photon ca-imaging, we investigated whether the spatial distribution of calcium signals in pc axons might confer nor specificity of ik recruitment. we detected prominent activity-dependent increases in intracellular ca concentration ([ ca]i) at nors (identified by their location at branchpoints) and at the ais (see bender and trussell, 2009) during trains of evoked aps (figures 3a3c; 204 21 hz). ca signals were restricted to approximately 5 m from the center of nors (figures 3c and 3d), and there were no detectable changes in internodal [ca]i (figure 3b), consistent with the lack of effect on spike propagation of 0 mm ca, 10 mm bapta application to the internodes (figure s2b). [ca]i at nors required axonal aps, were suppressed when spontaneous firing was arrested by somatic hyperpolarization (figure 3 g), and could not be driven by somatic depolarization when spikes were blocked with bath-applied ttx (figures 3e and 3f). increases in [ca]i were spike rate dependent (figure s3), slow, and cumulative and lead to sustained [ca]i during continuous activity (figures 3 g and 3h). [ca]i increase was also suppressed by removal of extracellular ca (16% 6% of control n =4; figure 3i) and bath application of mibefradil (figures 3i and 5 m, 43% 14% of control, p =0.013) but was not sensitive to agatoxin iva, which blocked ca transients in pc synaptic boutons as expected (hillman et al., 1991) (figures 3i; 9% 3% of control, p <0.0001). this implies that t-type and not p-type cavs are the primary source of ca entry at nors. additionally, although prior depolarization of the soma reduced [ca]i at the ais, ap-triggered nodal ca signals were unaffected (figures s3c and s3d), demonstrating that, in pcs, nodal [ca]i is independent of somatic vm. to understand the major impact of ik block on axonal ap propagation, we used a multicompartmental model of a pc (clark et al., 2005) and tested whether ik, as the sole repolarizing conductance at the nors, can support reliable propagation of aps in a continuously firing axon (see the experimental procedures for details). at a minimum ik density of 0.05 s/cm, with an associated t-type cav with a maximum permeability of 0.002 cm/s (anwar et al., 2012) and an accompanying, low-density, juxtaparanodal dr k conductance (0.002 s/cm, clark et al., 2005) to mimic the kv1.1/kv1.2 channels commonly observed in immunohistochemical studies (rasband 2010), ap propagation along the model axon was highly reliable. complete removal of the ik conductance alone caused nor depolarization, trapping of sodium channels in inactivated and blocked (khaliq et al., 2003) states, and ap propagation block (figure 4a). ik may therefore act to stabilize the nor vm during continuous firing and application of the ik channel inhibitor tram-34 (figure 1) could cause depolarization block. our observed lack of effect of tea application to nors (figure s1) implies that other k channel types previously shown to be present in pc axons (kv3.1, chang et al., 2007) and nors (kv7.3, pan et al., 2006) may not, in the absence of ik, be sufficient to support ap propagation. a direct test of the hypothesis that ik stabilizes nodal vm requires quantitative measurements of axonal vm during ik block. since pc nors are too small for patch-clamp recording and voltage-sensitive dye methods can not be used to detect absolute changes in vm, we used the ais as a proxy for nors while recording spontaneous firing at the soma, presuming that some ik might be present there, as suggested by our immunohistochemistry data (figure 2). during tram-34 application to the ais, we observed a reduction in somatic firing rate of 25% 5.2% (p <0.001, n =15 cells, figure 4c), accompanied by a small somatic depolarization (1.5 0.2 mv, p <0.0001) and an increase in ap threshold (1.8 0.16 mv, p <0.0001). in a minority of cells (4/15), tram-34 application to the ais caused sufficient depolarization (vm =2.56 1.28 mv, mean vm =48.4 1.9 mv) to reversibly but completely block firing in some trials. reduction of firing rate correlated with initial firing rate (r =0.53, p <0.05) so that cells with higher baseline rates showed a smaller reduction in spike rate on tram-34 application. this may reflect the presence of rate-dependent recruitment of bk and sk currents shown to regulate pc somatic vm during spontaneous firing (raman and bean, 1999). analysis of the second derivative of the somatic ap revealed a reduction in the component driven by current flow from the ais (figure 4d) as well as the local somatic component of the action potential during tram-34 application, indicating a reduced recruitment of sodium current at both locations. kca channels have been implicated in control of excitability in both unmyelinated (lscher et al., 1996) and myelinated (lev-ram and grinvald, 1987) mammalian axons, although the evidence for the latter is indirect and the physiological impact unknown. our results provide the first direct evidence for local, activity-dependent cav-mediated ca influx at nors and for the recruitment of an intermediate kca current (ik, kca3.1) that is an essential component for spike propagation. our model indicates that ik alone can sustain the nav availability required for propagation security, although it is conceivable that other conductances might also play a role, for example, in setting the nodal vm in the absence of firing. the sensitivity of ap conduction to low concentrations of ni and the suppression of the ca transients by mibefradil is consistent with the involvement of a t-type cav, as seen in association with dendritic ik in pcs (engbers et al., 2012). during sustained firing, this channel type is expected to be largely inactivated but could provide a small window current for ca entry. this might be of advantage since, due to the higher affinity of ik for ca (wei et al., 2005), a small ca influx can be effective but less expensive for an energetically demanding cellular compartment. ik may provide advantages over other k channel types in axons that continuously transmit aps, often at high rates. ik lacks intrinsic voltage dependence and does not inactivate, but, by virtue of the activity dependence of ca influx, it could be recruited and sustained at levels controlled by mean spike rate. its localized recruitment by ca may also be amplified by ca-induced ca release (llano et al. it has recently been shown that pcs also express bk channels under the myelin in the paranode region (hirono et al., 2015). these channels appear to become functionally relevant at firing frequencies above 100 hz, suggesting that they may act as a complementary partner to ik under conditions when [ca]i is sufficient for their activation. in the absence of fast voltage-gated k currents, aps at nors may also be broader, as generated by the model, than the exceptionally narrow somatic aps in pcs, potentially further facilitating ca entry at these sites. besides its role in axonal electrogenesis, nodal ca influx could also be important in regulating nor structure and functional properties. for example, activity-dependent local ca levels might be crucial to target and maintain cavs (forti et al., 2000) and navs (hund et al., 2010) at nors (wang et al., 2007) and to preserve or modify nodal architecture and myelin distribution (alix et al., 2008; einheber et al., 1997). ca signaling is thought to underlie the activity-dependent changes in mitochondrial motility observed in mammalian axons (chiu, 2011), notably in pcs (ohno et al., 2011). importantly, given their spike frequency dependence, nor ca signals could provide a readout of neuronal circuit activity, which could result in ca-dependent axonal plasticity beyond the ais (grubb and burrone, 2010; grndemann and husser, 2010; kole, 2011; kuba et al., 2010). in providing direct evidence for activity-dependent ca entry at nors and its recruitment of ik, our findings show that ca may play both short- and long-term roles in regulating axonal excitability, crucial for long-range signaling in neuronal circuits. methods used for preparing and recording from pcs and pc axons in cerebellar slices (200250 m) from p18p43 c57bl/6 mice were as previously described (monsivais et al., 2005) and carried out under institutional and national approval. pc axons visualized by dye-filling and ap-associated cell-attached axonal capacitive currents were recorded in voltage clamp mode at 34c 1c. amplitude of capacitive currents reflects the rate of rise of the axonal action potential and therefore pharmacological reduction in the amplitude reflects reduced sodium channel availability. drugs were diluted in acsf and bath applied or diluted in hepes-buffered or standard acsf and pressure-applied locally via patch electrodes using a picospritzer (0.510 psi, parker). 30 m alexa fluor 488 or 594 was included in the solution to help adjust pressure and pulse duration to target localized drug ejection. two-photon ca imaging and simultaneous electrophysiological recordings were performed using a custom-built dual galvanometer-based laser-scanning microscope (prairie technologies). a ti: sapphire pulsed laser (maitai, spectra physics) tuned to 810 nm was used for two-photon excitation. 200 m oregon green 488 bapta-1 (ogb-1, invitrogen) replaced egta in the pipette solution, which also contained 50 m alexa fluor 594 hydrazide (sigma-aldrich) to visualize morphology. pcs were dialyzed for at least 15 min after establishing whole-cell mode before imaging. data were digitized (itc-18, instrutech, heka) at 50100 khz and acquired using axograph x (http://www.axographx.com/). analysis was performed using custom-written routines in matlab (mathworks) or igor pro 6 (wavemetrics) in combination with neuromatic (http://www.thinkrandom.com/). two-photon imaging data were acquired with custom-written matlab software either as line scans (2 ms/line, 500 hz) or as xyt frame scans for a chosen region of interest (520 frames/s), digitized with a bnc-2090 board (national instruments), and analyzed with custom-written scripts in matlab, imagej, and igor pro 6. line scan and xyt frame-scan data were filtered using a boxcar running average or 2d-averaging filter, respectively. relative changes in ca-sensitive fluorescence (ogb-1, f/f) were calculated as raw fluorescence fraw minus baseline fluorescence f0 normalized to the background (fb) subtracted baseline fluorescence (yasuda et al., 2004): data are displayed as raw ogb-1 fluorescence values or relative changes in ogb-1 fluorescence normalized to the alexa fluor 594 red fluorescence (f/r, figure s3). significance is tested using student s t test unless otherwise stated, and a p value<0.05 is regarded as significantly different (instat, graphpad software). mice were perfused with ice-cold pbs for 1 min and then with 4% paraformaldehyde (pfa) in pbs (10 min). brains were dissected and 80 m slices of cerebellar vermis were cut using a vibratome (leica). slices were washed in pbs and blocked in 10% goat serum for 2 hr before 48 hr antibody incubation in 2% goat serum (kca3.1, mouse monoclonal, santa cruz biotechnology, calbindin d-28k, rabbit, swant) followed by second antibody incubation for 24 hr (633 goat anti-rabbit, 488 as well as 633 goat anti-mouse, invitrogen). confocal image stacks were acquired using a 63 objective (na 1.4, z-step: 130 nm, lsm700, zeiss). ap propagation along pc axons was simulated in neuron using a previously published multicompartmental pc model (clark et al., 2005), which included a resurgent sodium conductance typical of pcs (khaliq et al., 2003) additionally, at nors the model included a low-threshold ca conductance (cavt, 0.002 cm/s) (anwar et al., 2012) as well as an intermediate type kca (ik, kca3.1). nodal kca3.1 was based on a previously published bk conductance (solinas et al., 2007), and its kinetics were adjusted to a four-state model to match previously published experimental recordings of ik (bailey et al., 2010; hirschberg et al., 1998). the simulations were run for 500 ms, and values shown in figure 4 were measured at the center of the compartment.

summaryfunctional connectivity between brain regions relies on long-range signaling by myelinated axons. this is secured by saltatory action potential propagation that depends fundamentally on sodium channel availability at nodes of ranvier. although various potassium channel types have been anatomically localized to myelinated axons in the brain, direct evidence for their functional recruitment in maintaining node excitability is scarce. cerebellar purkinje cells provide continuous input to their targets in the cerebellar nuclei, reliably transmitting axonal spikes over a wide range of rates, requiring a constantly available pool of nodal sodium channels. we show that the recruitment of calcium-activated potassium channels (ik, kca3.1) by local, activity-dependent calcium (ca2 +) influx at nodes of ranvier via a t-type voltage-gated ca2+current provides a powerful mechanism that likely opposes depolarizing block at the nodes and is thus pivotal to securing continuous axonal spike propagation in spontaneously firing purkinje cells.

PMC4590545

pubmed-1373

despite many years of research efforts and impressive progress in knowledge of mechanisms of endometriosis development, the etiopathogenesis of the disease and exact cause of infertility in patients suffering from endometriosis still remain poorly understood. none of the theories and models of endometriosis pathogenesis provide definitive explanation of the disease development, considering its different manifestations and various localizations. recently published studies present new data on potential role of free radicals in endometriosis pathophysiology. although the origin of the oxidative stress occurring in the peritoneal cavity in endometriotic patients is unknown, accumulating data suggest that increased iron levels, together with apoptotic endometrial fragments and activated macrophages, may promote prooxidant environment. in addition, oxidative stress in endometriotic patients may potentially be induced by environmental factors, including dioxins or heavy metals [1, 2]. in our preliminary work we found significantly increased levels of ox-ldl in peritoneal fluid of women with stage iii/iv endometriosis compared to patients with follicle ovarian cysts. however, peritoneal fluid oxldl concentrations did not differ significantly between patients with minimal/mild endometriosis and women from the reference group. murphy et al. showed increased low-density lipoprotein (ldl) oxidation in peritoneal fluid of patients with endometriosis, which may be a result of peritoneal cavity macrophages hyperactivity. it was also proved that oxidized ldls stimulate monocyte chemotactic protein-1 (mcp-1) expression in mesothelial and endometrial cells which provides direct evidence of oxidative stress role in etiopathogenesis of the disease. increased concentrations of lipid peroxidation end products, malondialdehyde (mda), 8-isoprostane, and 25-hydroxycholesterol, were found in peritoneal fluid of infertile women with endometriosis [610]. serum of patients with endometriosis, compared to healthy women, contains also significantly higher 8-isoprostane levels. murphy et al. showed that peritoneal fluid of patients with endometriosis contains increased concentration of lysophosphatidylcholine, another lipid peroxidation product with confirmed chemotactic properties for monocytes. mda and 7-hydroxynonenal (hne-7) expression were increased in endometriosis implants tissue; however, both lipid proteins are also expressed in eutopic endometrium. concentrations of antibodies against lipid peroxidation products were found to be increased in serum of women with endometriosis, with no immunoglobulins detected in their peritoneal fluid. serum of women with endometriosis contains also elevated concentration of lipid hydroperoxide (looh), and its levels correlate positively with the stage of the disease according to revised american fertility society classification. peritoneal fluid of endometriotic patients contains oxidatively modified protein-lipid complexes, showing both chemotactic properties and ability to stimulate selected cytokines production. however, there are relevant data published in the literature according to which the concentrations of mda and mda-cu complexes demonstrate no significant differences, being comparable in peritoneal fluid of patients with and without endometriosis, showing also no significant correlation with the stage of the disease [1618]. no differences were also found in peritoneal fluid concentration of another lipid peroxidation product, cholest-3,5-dien-7-one. the objective of the study was to assess concentrations of oxidized low-density lipoproteins (oxldl) in peritoneal fluid (pf) of women with endometriosis. clinically and histologically confirmed diagnosis established the following groups: women with endometriosis (e, n=110) and as the reference groups: patients with simple serous (r1, n=78) and dermoid (r2, n=41) ovarian cysts. in each case the disease was found to be minimal (e1) in 23 cases, mild (e2) in 25 patients, moderate (e3) in 39 women, and severe (e4) in 23 cases. subjects were not given hormonal therapy and/or anti-inflammatory medications for at least 3 months before laparoscopy. medical history of the patients and basic clinical examination showed no general chronic diseases, except for the condition, which was the indication for laparoscopy. similarly, no significant difference was found in the phase of menstrual cycle of the time of laparoscopic procedures between women in all study groups. all patients signed an informed consent, and the lublin medical university ethics committee approval was obtained for the study. all visible pfs were aspirated during laparoscopy from the anterior and posterior cul-de-sacs, under direct vision to avoid blood contamination. samples were immediately centrifuged at 500 g for 5 minutes, and the supernatants were aspirated and stored at 70c until analysis. oxldl concentration in the pf was measured in duplicate using a commercially available enzyme-linked immunoassay kit (immundiagnostik ag, cat. all data were tested with the shapiro-wilk test for normality. because data were not normally distributed, statistical significance between e and r groups was determined with the mann-whitney u test. data are presented as medians (me), minima (min), maxima (max), and lower and upper quartiles. concentrations of oxldl in pf of patients with endometriosis were significantly higher compared to women with serous ovarian cysts (p=0.03). however, no significant difference in the pf oxldl levels was found between patients with endometriosis and women with dermoid ovarian cysts (p=0.4). levels of oxldl in pf of women with serous ovarian cysts were similar to those noted in patients with dermoid cysts (figure 1, table 1). by analyzing concentrations of oxldl in pf of women with different stages of the disease, it was noted that they were higher only in the subgroup of patients with stage iv endometriosis as compared to women with ovarian serous cysts. no significant differences were found between concentrations of oxldl in pf of women with different stages of the disease (table 2). pf oxldl concentration did not differ significantly between the subgroups of women in the follicular and the luteal phase of the menstrual cycle (me, range: 71.5, 1.21470 ng/ml versus 80.3, 16.71870 ng/ml, p=0.5). in our work, we demonstrated that peritoneal fluid oxldl concentration was significantly higher in patients with endometriosis than in women with serous ovarian cysts; however, it did not differ significantly as compared to subjects with dermoid cysts. after analysis of data obtained in women with different stages of the disease, it was noted that these results are found only in patients with severe endometriosis. in case of minimal, mild, and moderate disease, to our knowledge, only murphy and colleagues investigated the possible role of oxldl in the pathogenesis of endometriosis. based on a small number of cases, they found increased oxidation of low-density lipoprotein in women with pelvic endometriosis and increased levels of oxldl in the pf of patients with this disease. our results agree with these findings. however, our data suggest that only the severe stage of endometriosis is associated with increased oxidation of low-density lipoprotein in the peritoneal cavity, probably as the result of an imbalance in prooxidant/antioxidant pf systems. shanti et al. demonstrated that women with endometriosis had increased serum concentrations of autoantibodies to markers of oxidative stress including oxldl. data from our work indirectly confirm these results. based on many findings, there is an emerging concept of treating endometriosis as an autoimmune disease. an increased incidence of endometriosis was observed in the group of women with autoimmune diseases such as multiple sclerosis, lupus erythematosus, psoriasis, crohn's disease, hypothyroidism, hyperthyroidism, and rheumatoid arthritis. endometriosis shares many similarities with other autoimmune diseases including elevated levels of cytokines, decreased cell apoptosis, and t- and b-cell abnormalities. a variety of autoantibodies have been detected in patients with endometriosis patients, which suggests a polyclonal activation of b cells. the most commonly reported types are antiendometrial, antiovarian antibodies, and autoantibodies against phospholipids, histones, and nucleotides. other similarities between endometriosis and autoimmune diseases include familial occurrence, tissue damage, preponderance of females, and multiorgan involvement [21, 22]. lipid peroxidation processes are closely associated with the pathophysiology of autoimmune diseases. therefore, increased levels of oxidized ldl in pf of women with endometriosis support the theory of treating endometriosis as an autoimmune disease. oxidized ldl induces secretion of numerous proinflammatory cytokines including macrophage colony-stimulating factor (m-csf), interleukin-6 (il-6), and tumor necrosis factor (tnf-). concentrations of these cytokines were found to be elevated in the pf of patients with endometriosis. we can speculate that increased levels of oxidized ldl in the peritoneal cavity of women with severe endometriosis may be one of the factors responsible for increased levels of m-csf, il-6, and tnf- in pf. elevated pf concentrations of these cytokines promote adhesion, invasion, proliferation, and angiogenesis of ectopic endometrium and create an inflammatory environment in the peritoneal cavity in women with endometriosis.. demonstrated that oxldl caused an increase in accumulation of monocyte chemotactic factor-1 (mcp-1) in the medium of cultured mesothelial and endometrial cells. they also found that cells cultured in the presence of pf from endometriosis patients secreted more mcp-1 than those cultured with pf from subjects without the disease. therefore, we hypothesize that increased concentration of oxldl may be responsible for, as demonstrated in other studies, higher concentrations of mcp-1 in the pf of women with endometriosis. stimulation of mcp-1 production by increased pf levels of oxidized ldl may hypothetically be another factor responsible for the creation of proinflammatory environment in the peritoneal cavity of patients with endometriosis. unfavorable changes in lipid profile are present not only in the pf of women with endometriosis. it has been recently shown that plasma of patients with this disease contains higher concentrations of total cholesterol, low-density lipoproteins, high-density lipoproteins (hdl) and triglycerides as compared to healthy women. although all lipoproteins were significantly elevated in endometriosis patients, the difference was most substantial for ldl levels, which were 38% higher in women with endometriosis. verit et al. found that patients with endometriosis displayed significantly lower serum levels of hdl and higher levels of triglycerides, total cholesterol, and ldl than women without the disease. they also demonstrated that serum of women with endometriosis is characterized by significantly lower activity of paraoxonase-1 (pon-1), which negatively correlated with the progression of the disease. this hdl-associated antioxidant enzyme with paraoxonase activity prevents ldl and hdl oxidation and is also responsible for the antioxidant effect of hdl. therefore, authors speculated that unfavorable lipid profile combined with lower pon-1 activity in women with endometriosis may contribute to the increased susceptibility for the development of atherosclerosis. in conclusion, oxldl levels were significantly higher in the peritoneal fluid of patients with severe endometriosis than in women with serous ovarian cysts. this suggests that disrupted oxidative status in the peritoneal cavity of women with endometriosis may play a role in the etiopathogenesis of the more advanced stages of the disease. however, it can not be excluded that high oxldl levels are the result of more oxidant environment in the peritoneal cavity of patients with severe endometriosis than with less advanced disease.

the etiopathogenesis of endometriosis still remains unknown. recent data provide new valuable information concerning the role of oxidative stress in the pathophysiology of the disease. it has been proved that levels of different lipid peroxidation end products are increased in both peritoneal fluid (pf) and serum of endometriotic patients. we assessed the concentration of oxidized low-density lipoproteins (oxldl) in pf of 110 women with different stages of endometriosis and 119 women with serous (n=78) or dermoid (n=41) ovarian cysts, as the reference groups. pf oxldl levels were evaluated by elisa. we found that concentrations of oxldl in pf of endometriotic women were significantly higher compared to women with serous but not dermoid ovarian cysts. interestingly, by analyzing concentrations of oxldl in women with different stages of the disease, it was noted that they are significantly higher only in the subgroup of patients with stage iv endometriosis as compared to women with ovarian serous cysts. in case of minimal, mild, and moderate disease, pf oxldl levels were similar to those noted in reference groups. our results indicate that disrupted oxidative status in the peritoneal cavity of women with endometriosis may play a role in the pathogenesis of advanced stages of the disease.

PMC3703791

pubmed-1374

idiopathic demyelinating polyneuropathies are not common in the context of acute kidney injury (aki). poems syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes) is a rare disease characterized by the presence of proliferation of plasma cells with mainly lambda-chain restriction and diverse systemic symptoms occurring over several years, meaning that patients typically visit several clinical departments before obtaining the final diagnosis1). kidney dysfunction is not included in the diagnostic criteria of poems syndrome, but aki can be present with other systemic symptoms2). a 52-year-old korean man was admitted to this hospital because of abdominal distension and diarrhea. he had been well until 4 years ago, when he was diagnosed with cerebral infarct at another hospital. he was a businessman who rarely drank alcohol and had no previous history of medication. when he was transferred to our hospital 2 years ago, he had the symptom of mild tingling sensation in the left hand. one and a half years ago, paresthesia of right arm and intermittent difficulties with handwriting developed. brain magnetic resonance imaging (mri) with mr angiography showed neither infarct nor vascular stenosis. nine months ago, after undergoing health checkups, he was referred to the nephrology, endocrinology, and ophthalmology clinics because of high scr (1.52mg/dl), elevated thyroid stimulating hormone, and papilledema in both eyes, respectively. since then, he suffered from gradually increasing paresthesia of both feet, progressing to both ankles and to both lower legs. he did not feel muscle weakness, but had some difficulties maintaining balance while walking as well as morning stiffness of both arms. nerve conduction studies showed absent bilateral tibial nerve responses, slow motor responses on bilateral peroneal nerves, decreased sensory responses on bilateral sural nerves on the lower extremities, and slow motor and sensory nerve conduction velocities over the right median nerve. on cutaneous examination, red papules with diameters of 2 to 6 mm, which developed in the previous year, were scattered on the truncal areas. hypertrichosis with hyperpigmentation was observed in both hands and lower arms and finger clubbing was present. the pathologic diagnosis of the papules was glomerular hemangioma. computed tomography (ct) of the chest and abdomen ct of abdomen showed multiple small lymph node enlargements around the aorta as well as a small amount of ascites. a diagnosis of chronic inflammatory demyelinating polyneuropathy was made and therapy with oral prednisolone was initiated. during the next 2 months, weakness in the patient's legs increased and he developed a limping gait. he could not walk on his toes and heels and his perception of vibration decreased in both knees. the test results for syphilis, hepatitis b and c, and hiv were negative. tests for autoantibodies such as rheumatoid factor, anti-nuclear antibody, anti-ganglioside antibody, and anti-myelin-associated glycoprotein antibody, were all negative. tests for vasculitis and anti-phospholipid syndrome were also negative. the levels of serum complements c3, c4 and immunoglobulins ig g, a, and m were within normal limits. a small m-peak was noted (0.1 g/dl) in serum protein electrophoresis (pep) and no light chain was detected in urine pep. six months before admission, he was referred to the oncologist for this finding and monoclonal gammopathy of undetermined significance (mgus) was a plausible diagnosis. three months after steroid pulse therapy, his muscle weakness and gait improved, but burning paresthesia persisted. after 4 months, the steroid dose had been tapered and stopped. after stopping steroid treatment, he noted rapid progression of muscle weakness (he could not stand from the floor) and difficulties with walking and balancing. although his appetite markedly decreased, his body weight increased by 5 kg and leg edema and abdominal girth increased. he had not taken any nephrotoxic agents including nonsteroidal anti-inflammatory drugs (nsaids). laboratory results showed an increase in scr to 3.21mg/dl and fena and feurea was 0.24% and 12.3%, respectively. as abdominal distension increased with ascites he needed therapeutic paracentesis every 4 to 5 days. light microscopic examination revealed renal cortex and medulla containing about 33 glomeruli, one of which was globally sclerotic. most glomeruli showed increase in size and cellularity with mild to moderate endocapillary proliferations with diffuse double contours of capillary walls. there were diffuse interstitial edema and mild interstitial lymphocytes infiltrations with focal tubular atrophy containing hyaline casts destructing of tubular cells. immunofluorescence study did not show any immunoglobulin or complement deposition or staining for kappa and lambda light chain. electron microscopic examination revealed that most glomerular capillary lumina were compromised with diffuse duplications of capillary walls and subendothelial widenings with flocculent materials and mesangial interposition. visceral epithelial cells showed hyperplasia and focal effacement of foot process about 20% of external capillary surfaces. bone marrow biopsy showed normal bone marrow cellularity (60%) and a normal ratio of the plasma cells (2.3%). megakaryocyte hyperplasia was observed and a lymphoid follicle was seen in the bone marrow biopsy. immunohistochemical staining showed that the majority of plasma cells were positive for lambda light chain (fig. the patient's diagnosis was almost certainly poems syndrome accompanied by recurrent aki; however, an additional major criterion was required. upon retrospective review of his previous chest and abdomen ct, we found osteosclerotic bone lesions in thoracic vertebral bones in the chest ct scan (fig. 3). therefore, poems syndrome was diagnosed based on polyneuropathy, monoclonal plasma cell disorder with iga lambda, sclerotic bone lesion, extravascular volume overload, endocrinopathy, hepatomegaly and skin changes, consistent with proposed diagnostic criteria. once the diagnosis of poems had been established, the patient was treated with a 28 days cycle of bortezomib and dexamethasone combination followed by autologous hematopoietic stem cell transplantation (hsct) at another hospital. two months after the procedure, the patient showed a notable clinical improvement including polyneuropathy, normal scr (0.80mg/dl) and mobilization was possible with the aid of a walker. poems syndrome is a rare paraneoplastic syndrome resulting from a proliferative monoclonal plasma cell disorder1). the diagnosis of poems syndrome must include two mandatory major criteria of (1) demyelinating polyneuropathy and (2) monoclonal plasma cell-proliferation (almost always type). in addition, one other major criterion must be present (any one of the following three features: osteosclerotic bone lesions [almost always present], elevated levels of vascular endothelial growth factor [vegf], or castleman disease). in addition, at least one of the following seven minor criteria should be met: organomegaly, extravascular volume overload, endocrinopathy (excluding diabetes mellitus or hypothyroidism), typical skin changes, papilledema, or thrombocytosis, polycythemia1). making a diagnosis can be a challenge as the syndrome is rare and can be mistaken for other neurologic disorders. the most prominent symptom of this patient was his progressive sensory neuropathy, followed by the development of motor weakness over a period of 4 years. possible differential diagnoses of polyneuropathy and kidney injury include nutritional deficiency such as pyridoxine or thiamine, human immunodeficiency virus-related neuropathy, amyloid, vasculitis, and paraneoplastic syndrome3). his laboratory results for viruses and vasculitis revealed no positive findings and his aki had no definite cause. during the clinical course of this patient, there was no event of renal ischemia caused by nephrotoxic drugs or other systemic hemodynamic change. in the kidney, continuous endothelial injury caused mesangial interposition, mesangial proliferation and finally mesangial sclerosis. in addition to the glomerular change, ischemic damage caused by obliteration of arterioles and small arteries was proposed to induce recurrent acute kidney injury2). vegf is a potent microvascular permeability-enhancing mediator and a selective mitogen for vascular endothelial cell8). therefore, the vegf produced by plasma cells leads to a decrease in intravascular volume by increasing vascular permeability, which may contribute to prerenal aki5). we might discern from this case that serum and urine pep should be obtained in patients with polyneuropathy and unexplained kidney injury to detect monoclonal gammopathy. however, serum and urine pep of this patient were normal 8 and 6 months ago (table 1). in poems syndrome, the median percent of plasma cells in the bone marrow is less than 5%, which is reflected by the small amount of m-protein, and serum pep often can not detect the m-peak1). instead, serum and urine ife should be performed to identify the isotype, as these techniques are three times more sensitive in detecting monoclonal immunoglobulin than serum pep4). in kidney biopsy, immunofluorescence staining showed no deposition of -light chain and electron microscopy showed no electron-dense deposits. these are quite different findings from other plasma cell disorders that showed kidney damage with deposition of a monoclonal immunoglobulin4). instead, renal pathology of this patient showed extensive endothelial cell injury and evidence of increased extravascular permeability. secretion of vegf from the monoclonal plasma cells in patients with poems syndrome has been suspected to trigger these changes25), but the mechanism remains unknown and must be investigated6). in contrast to the pathologic lesions, proteinuria was absent and microscopic hematuria was minimal in this patient, which may reflect a relatively intact glomerular basement membrane structure and podocyte functions. treatment of poems syndrome is directed to target the underlying monoclonal plasma cell disease7) and therapeutic strategies for the treatment of multiple myeloma have been reported to show successful patient and renal outcomes and an improvement in morbidities related to treatment67). as this patient showed bone marrow involvement, he received systemic chemotherapy and had autologous blood stem cell transplantation.

poems syndrome is a rare paraneoplastic syndrome and there are few reports of polyneuropathy and monoclonal gammopathy associated with kidney dysfunction. here, we report a case of poems syndrome with recurrent acute kidney injury (aki). a 52-year-old man presented with bilateral aggravating paresthesia and latermotor weakness of the lower extremities accompanied by repeated elevation of serum creatinine. the patient was finally diagnosed with poems syndrome on the basis of fulfilling the two mandatory major criteria (polyneuropathy and monoclonal gammopathy), one other major criterion (sclerotic bone lesion), and several minor criteria. a renal biopsy was performed to clarify the cause of aki and showed membranoproliferative glomerulonephritis-like lesions with mesangiolysis and endothelial cell injury. this case illustrates that renal manifestations, not included in the diagnostic criteria for poems, can be apparent before various other systemic symptoms.

PMC4949204

pubmed-1375

the main goal of endodontic treatment is the complete apical and coronal seal of root canal system to prevent the bacterial leakage and percolation many studies have confirmed the importance of coronal leakage as a possible cause of failure of root canal treatment (rct). malone and donelly considered coronal restoration as it replaces missing tooth structure, protects remaining tooth from fracture, and prevents canals recontamination as the first protective barrier for the periapical tissues after rct. torabinejad et al., confirmed the results of swanson and madison 1978 that root canal treatment failure could be assumed to the delayed final restoration placement or when the temporary filling partially or completely lost. hence, a great attention should be paid for the immediate placement of coronal restoration. some studies suggested the placement of a barrier made of different materials on the root canal orifices for complete coronal seal. this barrier is indicated even in cases with post retained restorations. in 1995, ray&trope published a radiographic study about the correlation between the periapical pathosis and endodontically treated teeth. their results indicated that the adequacy of the coronal restoration is more important than the quality of the root canal filling. they also showed that the periapical pathosis related clearly to the quality of coronal restoration and not to the root canal filling. kirkevang et al. gained similar results. on the contrary, many studies, showed the adequacy of root canal filling is the most important preventive measure for the periapical tissues (although there was no negligence on the coronal restoration importance). moreover, ricucci and bergenholtz reported that exposure of root canal fillings to the saliva (teeth with missing coronal restorations) was not necessarily associated with apical pathosis in cases with adequate endodontic treatment. many epidemiological studies from different countries: brazil, france, turkey, belgium and australia gave conflicting results about the impact of the final coronal restoration on the status of periapical tissues. 2012 made a comprehensive review to studies related to this subject and could nt resolve this controversy. the aim of this study was to assess the influence of the quality of coronal restorations and root canal treatment (separately and in concomitant) on the periapical status of endodontically treated teeth from an adult syrian subpopulation. we randomly selected panoramic x-rays for patients attended dental school of damascus university who fulfill the selection criteria: patient's age was set to be minimum of 19 years old, they had neither received any dental treatment for more than one year, nor had previously visited dental school clinics (selected patients are private clinic patients). our sample contained 200 high qualities panoramic x-rays which had been examined under good illumination and magnification by two endodontists with minimum 5 years experience individually. in cases when disagreement occurred, the number of examines teeth in panoramic x-rays (except third molars) were 5331 teeth, 784 teeth (361 teeth for males and 423 teeth for females) received endodontic treatments (there was a radioopaque material in the root canals or pulp chambers). we recorded the type of the final fillings as following: type of coronal restoration no filling in 39 teeth (5%)filling in 320 teeth (42.1%)prefabricated post&filling in 26 teeth (3.3%)crown in 304 teeth (38.8%)post&crown in 85 teeth (10.8%). no filling in 39 teeth (5%) filling in 320 teeth (42.1%) prefabricated post&filling in 26 teeth (3.3%) crown in 304 teeth (38.8%) post&crown in 85 teeth (10.8%). the quality of the final restorations and root canal fillings had been classified as: adequate or inadequate according to tronstad et al. inadequate coronal restoration: final restoration appears radiographically with overhang, open margin, recurrent caries, temporary filling or no filling. adequate root canal filling: all canals obturated with dense fillings ending about 2 mm shorter than the radiographic apex. inadequate root canal filling: root canal fillings end more than 2 mm shorter than the radiographic apex or grossly overfilled. periapical status was assessed by periapical index (pai) proposed by rstavik et al. 1986, who scored the periapical area of the radiographic images as follows: normal periapical structures.small changes in bone structure.changes in the bone structure with little mineral loss.periodontitis with well-defined radiolucent area.severe periodontitis with exacerbating features. normal periapical structures. according to this index we classified periapical tissue as: normal or healthy periapex: absence of radiographic evidence of diseased periapical tissues. the worst score of all canals was taken to represent the pai score for multicanal teeth. spss software was used for statistical analysis (spss inc, chicago, il). out of 784 endodontically treated teeth, the mandibular molars were the most frequent treated teeth. 459 cases (58.54%) had been classified as having adequate coronal restoration, and 325 (41.46%) cases as having inadequate one [table 1]. illustrate the type of the teeth in the study and the adequacy of the final coronal restoration in the 459 teeth with adequate final restoration, the status of periapical tissues was healthy in 353 teeth (76.9%) and diseased in 106 teeth (23.1%). in the 325 teeth with inadequate coronal restoration, the status of periapical tissues was healthy in 169 teeth (52%) and diseased in 156 teeth (48%) [table 2]. illustrate the status of periapical tissues according to the adequacy of final coronal restoration chi-squared test was used to evaluate the major differences in the status of periapical tissues according to the adequacy of final coronal restoration as illustrated in table 3. evaluation of the major differences between studied groups statistical analysis revealed that the healthiness of periapical tissues affected by quality of coronal p<0.01 [table 4]. the effect of coronal restoration type on the status of periapical tissues the study showed that 72% of the cases with final restoration absence accompanied with a clear radiographic radiolucency. teeth with prefabricated post and filling or cast post and crown accompanied with periapical pathosis (38.5% and 31.8%) respectively. overall, the post presented in 111 cases (14.1%), 74 cases accompanied with healthy periapical tissues and 37 cases with diseased tissues. endodontic treatments had been evaluated as adequate in 145 teeth (18.5%), and inadequate in 639 teeth (81.5%). when we compared the status of periapical tissues according to the adequacy of root canals fillings we noticed that 95.2% of the teeth were with adequate root canal fillings, and about 60.1% of the inadequate endodontically treated teeth were with healthy periapical tissues (score one and two rstavik et al. periradicualr disease (pai>2) had been found in 262 teeth (33.4%). 7 cases of there were with adequate treatment and 255 cases were inadequate [table 5]. the status of periapical tissues according to the adequacy of the root canal filling the difference in the number of teeth with healthy periapical tissues and adequate root canal filling were significant when compared to teeth with inadequate root canal filling. (p<0.001). the impact of the adequacy of the coronal restoration and root canal filling on the status of periapical tissues had been studied (separately and in concomitant). classification of teeth according to the adequacy of the final restoration and root canal filling in this study the status of periapical tissue were healthy in 96.6% of cases when both final coronal restoration and root canal filling were adequate. but the status of periapical tissues was healthy in 88.5% when only the root canal fillings were adequate and 70% when only the coronal restoration was adequate. the status of periapical tissue was healthy in only 48.8% of cases when both the endodontic treatment and the final restoration were inadequate [table 7]. the effect of endodontic treatment and the final coronal restoration on the status of periapex for cases with both adequate restorations and adequate root canal filling, the success rate was 96.6%. healthy periapex was observed in 88.5% of the teeth with adequate root canal filling and inadequate restoration. when only the final restoration was adequate (root canal treatment inadequate) the success rate was 70% (p=0.04). the difference when both the final restoration and the root canal filling were inadequate was significant when compared to the other three groups. this epidemiological study used x-rays to evaluate the presence of periapical radiolucencies, the quality of the root canal fillings and restorations with all limits of the 2 dimensional images. one of this study disadvantages is the inability to collect detailed information about the endodontic treatment: when it was done? if the periapical pathosis is pre or post treatment disease? whether the periapical periodontitis is healing or expanding? in fact, radiographic image represents a snapshot of a continuous process of a dynamic disease. according to the study of petersson et al. the number of healed lesions equals the number of developed ones after 10 years of follow up, indicating the reliability of epidemological studies for recording the long-term success of endodontic treatments. this type of studies gave us valuable information about the level and development of dental practice in different periods, the common endodontic errors and the prevalence of periapical pathosis in subpopulations. to overcome the disadvantages of this type of studies, the sample was expanded and radiographs had been evaluated by using good illumination and magnification. all panoramic x-rays in this study were taken in dental school clinics of damascus university. many studies did not find significant differences between periapical and panoramic x-rays in evaluation of periapical pathosis. one of the most important questions is whether the study represents the syrian subpopulation? to come up to an answer we should mention that dental school clinics of damascus university is one of the biggest dental clinics in damascus that offers consultations and treatments to so many people from damascus and its province. the criteria of sample selection was: the patient should have received his previous treatment outside dental school clinics (by general practice dentists), did not receive any dental treatment for at least one year of attendance to give a chance for any radiographic radiolucency to be healed by that time. according to rstavik d (1996) 89% of lesions would heal after one year of endodontic therapy. the number of endodontically treated teeth was 784 teeth (half of them were upper and lower molars). the prevalence of periapical lesions was about 33.42%, 58.5% of the coronal restorations were evaluated as adequate with different type of restorations, while 5% of the sample lost its restorations. the incidence of apical periodontitis associated with adequate coronal restorations was lower with significant difference than inadequate ones. the effect of the type of the coronal restorations on the status of periapical tissues was not crucial. in 111 cases out of 784 cases, some studies indicated that the presence of canal posts accompanied with higher percentage of periapical pathosis due to leakage during post space preparation or infrequent irrigation or weakening the remaining gutta percha. root canal filling was adequate in 18.5% of cases which is so low when compared to other studies that consider root canal therapy as predictable treatment. in this study, the correlation between periapical pathosis and inadequate root canal treatment was highly significant, other studies confirmed these results. when each factor (root canal filling or coronal restoration) was evaluated separately, it had a significant influence on periapical health; in fact, these two factors are un separated. to evaluate which factor had the greater impact on outcome, we statistically analyzed the results of combination of these two factors. success rate (the presence of healthy periapex) scored in 96.6% of cases with adequacy of both factors and decreased to 88.5% in cases with adequate root canal fillings and inadequate coronal restorations but with non-significant difference (it means that root canal treatment exert greater impact on outcome). in cases with adequate coronal restorations and inadequate root canal fillings success rate was recorded in 70%, which again indicates the importance of root canal filling in the success of the endodontic treatment. the worst results were obtained when both factors are inadequate which accompanied with lower successful results and high significant difference compared to all studied groups. that indicates the importance of a complete treatment (adequate root canal filling and placement of adequate final coronal restoration). overall results indicate that the factor that plays the key role in the success of a treatment is the quality of root canal filling without neglecting the effect of the coronal restoration. the prevalence of periapical lesion in syrian subpopulation is high which indicate poor dental practice. thus, considerable efforts should be spent to improve the level of endodontic treatment and promote periradicular health.

background: the purpose of this study was to determine the status of periapical tissues of endodontically treated teeth according to coronal restorations and root canal fillings separately and in concomitant in adult syrian subpopulation. methods:784 endodontically treated teeth from two hundred randomly selected syrian adult patients were radiographically evaluated. according to predetermined criteria, the quality of coronal restorations and root canal filling of each tooth was scored as adequate or inadequate. the status of periapical tissues was also classified as healthy or diseased. results were analyzed using chi-squared test. results:adequate coronal restorations were determined in 58.54% of cases which was accompanied with less periapical pathosis than that in teeth with inadequate restorations (p<0.01). 14% of teeth were restored by posts which showed no significant impact on the periapical tissues health. 18.5% of endodontic treatments were evaluated as adequate with less number of periapical radiolucencies than that of inadequate root canal fillings (p<0.01). absence of periapical pathosis was 96.6% in cases with both adequate coronal restorations and root canals fillings. the rate was 88.5% in cases with only adequate root canals fillings, and about 70% in cases with only adequate coronal restorations. when the treatment was inadequate in both coronal and root canals fillings, success rate was only observed in 48.8%. conclusion: the most important factor with regard to the periradicular tissue health is the quality of root canal filling without neglecting the influence of coronal restoration (regardless of its type). there is a high prevalence rate of periapical pathosis in syrian subpopulation due to poor dental practice.

PMC4184332

pubmed-1376

three series have reported the prevalence of autoimmune pancreatitis as between 5% and 6% of all patients with chronic pancreatitis. it is very important to distinguish between these two entities regarding to differences in treatment and prognosis. aip appears to be a disease of the elderly, but vary widely in age, as most patients are more than 50 years old at diagnosis. it is reported to be at least twice as common in men as in women. although diffuse swelling of the pancreatic parenchyma can be morphologically characteristic of aip, a focal type of this clinical entity has been recently recognized and is still difficult to establish. however, in this day and age, even with heightened awareness of aip and appropriate preoperative workup (including serum immunoglobulin g fraction 4 (igg4) measurement and, in very select cases, a short steroid trial), some patients with aip are likely to be resected for the suspicion of malignancy. a 56-year-old patient was referred to our unit for upper abdominal pain since two week and lost of 6 kg in two months. in his past medical history the serum carcinoembyonic antigen and cancer antigen 19-9 (ca) level was normal. transabdominal ultrasonography showed a hepatic steatosis and 5 angiomas. magnetic resonance imaging (mri) showed the angiomas and a lesion of 2020 mm of the pancreatic tail with decreased signal intensity on t1-weighted mr images, increased signal intensity on t2-weighted mr images, and discreetly hypovascularized (figure 1). due to concerns of pancreatic malignancy (pancreatic adenocarcinoma), we found a tumor about 5 cm in diameter lacking clear margins in the tail of the pancreas, which was removed via an open distal splenopancreatectomy. histological analysis of the resected specimen revealed no malignancy, but dense fibrosis with lymphoplasmositic infiltration and acinar atrophy (figure 2). postoperatively, autoantibodies and immunoglobulin were normal but igg4 was slightly above of the upper limit of the normal range. patient was treated by 40 mg/j of corticotherapy with a remarkable improvement. in the follow up, because of the past medical history of mellitus diabetes, the young age of the patient, the normalization of the serum igg4 level, the absence of malignancy in histology, and the clinical improvement after corticoid therapy, the diagnosis of aip was made. figure 1magnetic resonance imaging shows the affected pancreatic lesion involving tail with decreased intensity on the t1-weighted image (a) and increased intensity on the t2-weighted image (b) compared with the signal intensity in the liver. magnetic resonance imaging shows the affected pancreatic lesion involving tail with decreased intensity on the t1-weighted image (a) and increased intensity on the t2-weighted image (b) compared with the signal intensity in the liver. figure 2the histologic findings of the resected specimen of the pancreas include: dense fibrosis (black arrow), lymphoplasmacytic infiltration, and acinar atrophy (white arrow). hematoxylin and eosin (he, 250). the histologic findings of the resected specimen of the pancreas include: dense fibrosis (black arrow), lymphoplasmacytic infiltration, and acinar atrophy (white arrow). although diagnostic criteria were established for aip, there remains no practical strategy to differentiate pc from aip. making a correct differential diagnosis between the two conditions is of paramount importance as the treatment approaches are different. the common presenting symptoms of the pseudo tumoral autoimmune pancreatitis are mild abdominal pain, jaundice, and weight loss. cholestatis was seen in 75100% of a japanese series, mild pancreatitis, acute recurrent pancreatitis, biliary duct strictures are a various clinical forms of aip. it is made by radiologic computed tomography scan finding of a narrowing of the pancreatic duct and parenchymal edema of the pancreas (a sausage shape). isolated focal pancreatic mass is difficult to be diagnosing without doubt as a focal aip. it can show focal aip as a low-attenuation or an iso-attenuation mass. on the other hand, features highly suggestive of pancreatic cancer were a pancreatic low density mass, main pancreatic duct obstruction, distal pancreatic atrophy, and metastases. endoscopic ultrasonography provides the opportunity for fine needle aspiration, but does not have pathognomonic findings on its own. immunological abnormalities include hypergammaglobulinaemia, elevated serum igg4 levels and the presence of autoantibodies including antinuclear antibody, anti smooth muscle antibody, rheumatoid factor, antilactoferrin antibody and anticarbonic anhydrase antibody ii. the specificity and sensitivity of a high serum igg4 level in the diagnosis of aip are>90%. after resection, histological changes in aip show predominantly periductal inflammation consisting of a dense interstitial lymphoplasmacytic infiltrate, thus causing duct obstruction with acinar tissue fibrosis. in addition, aip has been seen in association with retroperitoneal fibrosis, kidney involvement and lung nodules. these features are considered as extrapancreatic signs of aip and might be helpful to diagnose especially in focal form. our patient had no extrapancreatics features. because of the difficulty of the diagnosis, several types of diagnosis criteria have been proposed. since there is currently no diagnostic serological marker for aip, and approach to the pancreas of histological examination is generally difficult, aip is currently diagnosed on the basis of presence of a combination of abnormalities unique to aip. for practical purposes, the revise japanese clinical diagnostic criteria (2006) and hisort criteria from the mayo clinic and in 2011 international consensus diagnostic criteria for aip were proposed. in this set of diagnostic criteria, the diagnosis of aip is made using one or more positive criteria on: i) imaging criteria: diffuse enlargement of the pancreas and diffuse or segmental irregular narrowing of the main pancreatic duct; ii) laboratory criteria: elevated levels of igg and/or igg4 or the presence of autoantibodies; iii) histopathologic criteria: fibrosis and lymphoplasmocytic infiltration; iv) association with other autoimmune diseases; and v) response to steroid therapy. despite all these investigations procedures and the existence of these criteria, 35% of patients undergoing pancreatic resection for presumed pc in fact has aip, as was the case of our patient. reported that 7 of 37 (18.9%) aip patients had surgery because they were misdiagnosed as having pc or bile duct cancer. this treatment may represent a diagnosis test, considered as one of the main diagnosis criteria. extrapancreatic features, igg4 level, endoscopic ultrasound guided fine needle aspiration and corticotherapy are helpful to evoke aip. the preoperative diagnosis should prevent unnecessary surgery causing a high morbidity comparing to medical treatment (corticosteroid) of aip.

autoimmune pancreatitis (aip) is a rare disorder, although the exact prevalence is still unkown. it is a type of pancreatitis that is presumed to have an autoimmune aetiology, and is currently diagnosed based on a combination of 5 criteria. however, in this day and age, some patients with aip are likely to be resected for the suspicion of malignancy. the authors report a case of pseudo-tumoral autoimmune pancreatitis, reviewing some literature about it and underlining the difficulty in the diagnosis. a 56-year-old patient was referred to our unit for upper abdominal pain. in his past medical history we note mellitus diabetes. the clinical examination was unremarkable. laboratory data showed no abnormal values. upper endoscopy showed antral gastritis. transabdominal ultrasonography showed a hepatic steatosis and 5 angiomas. no computed tomography scan was made. magnetic resonance imaging (mri) showed 5 angiomas and a lesion of 2020 mm of the pancreatic tail with decreased signal intensity on t1-weighted mr images, increased signal intensity on t2-weighted mr images. due to concerns of pancreatic malignancy, the patient underwent open distal spleno-pancreatectomy. histolo gical analysis of the resected specimen revealed no malignancy. postoperatively, immunoglobulin g fraction 4 was slightly above of the upper limit of the normal range. after corticotherapy the patient is getting better. this case underlines the difficulties still encountered in the diagnosis of aip. it has been frequently misdiagnosed as pancreatic cancer and caused unnecessary resection. in order to avoid unnecessary resections for an otherwise benign and easily treatable condition, it is urgent to refine diagnostic criteria and to reach an international consensus.

PMC3981193

pubmed-1377

in most cells, proteostasis involves continuous regulation of protein synthesis and degradation, but the eye lens is a notable exception. during the final stages of maturation, lens cells eject all organelles, preventing normal protein turnover. the lens therefore provides a unique opportunity to observe the effects of aging in relation to damage accumulation in long-lived proteins, which are increasingly a subject of interest. they constitute approximately 3540% of the total soluble mass of the lens and are composed of a 3:1 ratio of a to b. while a is found almost exclusively in the lens, b has been found throughout the body including heart, glia, muscle, kidney, lung, and skin cells. gene knockout studies revealed that a mouse lens can develop normally without b but not without a, which led to prompt cataract formation. nevertheless, the chaperone activity of heteropolymers formed from a 3:1 ratio of a to b is higher than either homopolymer, suggesting that the combination of both proteins is optimal. beta and gamma crystallins are the two other major subgroups of crystallins in the lens. they act in conjunction with the -crystallins to maintain lens structure and transparency and to achieve a suitable refractive index. to behave properly as chaperones, -crystallin monomers must interact with each other to form polydisperse oligomers. a and b form dimers that then assemble into larger complexes ranging from 15 to 50 monomers, with the subunits dynamically intermixing and exchanging. removing the n- and c-terminal extensions allows for determination of partial crystal structures. when crystallins do not interact with each other properly, they become water-insoluble and begin to aggregate. the influence of many post-translational modifications (ptms) on this process has been investigated, including deamidation, oxidation, disulfide formation, and truncation. importantly, all of these ptms lead to mass shifts that are easily detectable by mass spectrometry (ms). more subtle modifications that also increase with age, such as isomerization and epimerization, do not lead to easily detectable mass shifts and have been significantly less studied. methods for detecting isomerization in peptides (meaning either inversion of the chiral center of an amino acid to produce an epimer or conversion of aspartic acid to isoaspartic acid) include stereoselective enzymatic digestion, ion mobility, and ms. previous studies have shown that aspartic acid and serine are the two amino acids most susceptible to spontaneous isomerization. deamidation of asparagine residues generates aspartic acid, and this transformation can also be accompanied by isomerization. the mechanism by which l-asp converts into l-isoasp, d-isoasp, and d-asp has been studied previously and is known to proceed via nonenzymatic formation of a succinimide ring, which can yield four isomers, as shown in scheme 1. enol tautomerism and then hydrolyzed to form d-asp or d-isoasp. the mechanism by which l-ser epimerizes to d-ser in proteins is not well-established, although proposals have been made. alpha and beta carbons are labeled to highlight the difference between l-asp and l-isoasp, respectively. although isomerization might appear to be a ptm, studies have shown it can cause major perturbations in protein structure. noguchi and coworkers successfully crystallized hen egg-white lysozyme with an isoaspartic acid substitution at asp101 that caused backbone deflection of nearly 90 relative to the native structure. crystal structures of a modified ribonuclease revealed that isoasp-32 induces conversion of an -helix to a u-shaped loop. isomerization not only perturbs 3d structure but also can affect physical properties such as solubility and bioactivity. for example, isomerization of asp92 in immunoglobulin 2 (igg2) leads to deactivation of the antigen-binding region. large-scale (i.e., proteomic) identification of single amino acid isomerism within peptides is challenging because there is no change in mass. however, differences in fragment intensities following ms/ms analysis can be used to detect isomers. this method was first applied to stereoisomers by tao and coworkers, who reported differences as a ratio of relative abundances of a pair of fragment ions differing most between l- and d- enantiomers (rchiral=rd/rl). it has been shown that radical-directed dissociation (rdd) yields the best chiral discrimination for analysis of peptide epimers. previous work on the detection of isomerization and epimerization in the sheep lens using tandem lc ms revealed novel sites of isomerization in the crystallins. the present study focuses on changes in isomerization and epimerization for a, b, and b3 crystallin between water-soluble (ws) and water-insoluble (wi) protein fractions from cow, sheep, and pig eye lenses. to determine which regions of these proteins are most susceptible to isomerization, enzymatic digestion into peptides ms/ms analysis using both collision-induced dissociation (cid) and rdd. importantly, we found several regions in the well-ordered crystallin domain that are disproportionately isomerized in the wi fractions. specific isomerization hotspots were identified and correspond to regions with serine or aspartic acid repeats. potential explanations for the isomerization of these sites are offered, and comparison with other long-lived and problematic proteins reveals that these sequence motifs are common. cow, pig, and sheep eye lenses were acquired as discarded tissue from corona cattle, inc. the approximate ages for each of the animals were 1218 months for the cow, 56 months for the pig, and 68 months for the sheep. the supernatant was separated from the precipitate following centrifugation at 15,100 g for 20 min at 4 c. the precipitate (wi) was solubilized in 6 m urea and purified by dialysis against 6 m urea. for ws digestion, the protein was dissolved in 50 mm nh4hco3 buffer, ph 7.8; disulfide bonds were then reduced with 100 mm dithiothreitol (dtt) at 95 c for 5 min. after returning to room temperature, reduced cysteine residues were capped using 100 mm iodoacetamide in the dark for 20 min. finally, the proteins were digested with trypsin overnight at 37 c using a 50:1 protein to enzyme ratio. for the wi digestion, the proteins were dissolved using 6 m 50 mm tris-hcl, ph 8.0. disulfide bonds were cleaved using 200 mm dtt in tris-hcl, ph 8.0 at 37 c for 20 min. following this, 200 mm iodoacetamide in tris-hcl, ph 8.0 was added and the mixture was incubated in the dark for 1 h. to consume unreacted iodoacetamide, 200 mm dtt was added and incubated for 1 h in the dark. next, the urea concentration was diluted to<0.6 m using 50 mm tris-hcl, 1 mm cacl2, ph 7.6. the proteins were digested using trypsin with a 50:1 protein to enzyme ratio for 16 h at 37 c. for the iodobenzoic acid modification, the digested peptides were desalted and cleaned using a peptide trap (michrom bioresource). approximately 5 nmoles of the digestion mixture, 15 l of 15 mm 4-iodobenzoic acid nhs-activated ester in dioxane, and 5 l of borate buffer (ph 8.6) were combined and incubated for 1 h at 37 c. important: dimethyl sulfoxide should not be substituted for dioxane in this step because it can cause aspartic acid isomerization. the modification side products at arginine and tyrosine side chains were removed by incubating the reaction mixture in 1 m hydroxylamine, ph 8.5. these procedures have been previously determined not to yield any detectable isomerization/epimerization in control experiments. to quantify isomer identification, an rchiral value approach,, risomer represents the ratios of the relative intensities of a pair of fragments that varies the most between two isomers (ra/rb). if risomer=1, then there the two tandem ms spectra are indistinguishable and the species are likely not isomers. if risomer>1, then a larger number indicates a higher probability that two unique molecules are represented. to confidently identify each of these isomers by ms/ms, we use a threshold that was determined by performing a t test on the risomer values obtained by performing cid and rdd on a mixture of synthetic peptides separated by lc ms. using 99% confidence intervals, the risomer threshold for cid is>1.9 and for rdd it is>2.4. we have previously described a modified bottom-up strategy for detecting sites of isomerization, including epimerization, in proteins. in brief, proteolytically digested peptides are separated by reverse-phase column chromatography and analyzed by ms with minimal time exclusion windows to favor repeated analysis of the same m/z. for example, the n-terminal fragment of a crystallin from the wi protein fraction of sheep lens, acetyl-mdiaiqhpwfk, elutes at four different times, each separated by 2 min, as shown in figure 1a. to establish that these peaks represent isomers, repeated ms/ms analysis of each eluting peak is required (both cid and rdd are used in separate runs).figure 1b shows cid spectra from each of the lc peaks in figure 1a. although the spectra are similar, suggesting that they originate from the same peptide sequence, the relative intensities of certain peaks change noticeably. for example, the intensities of y9, b7, and y10 ions vary considerably between lc peaks 1 and 2. these differences can be quantified into risomer values, which are provided in figure 1c. for cid analysis, risomer values above 1.9 represent statistically significant differences, meaning that all four peaks in figure 1a are different isomers of acetyl-mdiaiqhpwfk. mass spectra alone do not provide information about which isomer corresponds to the native l-asp peptide, but in this case, the largest peak constitutes>90% of the total relative abundance and likely represents the unmodified l-asp isomer. if the wi fraction is highly isomerized, in which case identification of the l-asp isomer becomes ambiguous, then comparison with the less isomerized ws digest or an authentic standard is used to identify the l-asp isomer. (a) lc chromatogram for the separation of the four isomers of ac-mdiaiqhpwfk in the wi sheep lens digest. (b) cid spectra from each of the lc peaks. labeled fragments are those used to determine risomer values. the degree of peptide isomerization from the ws and wi protein fractions of sheep a crystallin is summarized in figure 2. all isomers were confirmed by comparison of either cid or rdd ms/ms spectra, as described above. for example, the two isomers of aipvsr were indistinguishable by cid but could be confidently detected using rdd (see si figure s1). additionally, some isomers coelute, preventing quantitation of the relative abundance of each form. the first downward bar on the left side of figure 2a (111) represents the data from figure 1. this peptide, acetyl-mdiaiqhpwk, was found to be 9% isomerized, with error bars representing standard deviations from three technical replicates. the color coding of the bars corresponds to the three distinct structural regions present in -crystallins, the n-terminal disordered region, the crystallin-ordered region, and the disordered c-terminal extension. these regions are illustrated relative to a partial crystal structure on the right side of figure 2. the full protein sequence is provided at the bottom of figure 2, color coded and with residues of interest marked for easy location. the trends in figure 2 illustrate that the average amount of isomerization is significantly higher in the n and c termini than in the well-ordered crystallin domain for both the ws and wi fractions. these dynamic regions play important roles in the assembly of -crystallins into higher-order structures, but this flexibility may also enable isomerization by facilitating more frequent access to the pathways outlined in scheme 1. percent isomerization of water-soluble (ws) a sheep versus water-insoluble (wi) a sheep. orange, disordered n-terminus; blue, structured -crystallin domain; purple, disordered c-terminus. peptide 164173 does not contain error bars because it only appeared baseline-resolved in one digest. the full protein sequence is given below the plot, with aspartic acid residues in bolded/black and serine residues in underlined/black. asp105 and asp106 are in bold red text in the amino acid sequence and are shown explicitly in the crystal structure (pdb 3l1f) to highlight an important region of isomerization. stars indicate isomerized regions where isomerization was identified, but quantitation was not possible due to incomplete chromatographic separation. interestingly, the degree of isomerization in the disordered regions, although slightly more abundant in the wi fraction, is similar in both the ws and wi fractions. this suggests that these modifications do not significantly drive aggregation and loss of solubility, as previously reported. differences in degree of isomerization are more notable within the crystallin region, where isomerization is significantly more abundant in the wi fraction (except for the dramatically modified peptide 104112 that will be discussed further below). cumulatively, these results imply that modifications to flexible regions may be more facile but also less consequential in terms of altered functionality. fewer peptides are isomerized in b, and the average degree of isomerization is less in both the ws and wi fractions compared with a. the flexible n-terminal domain is highly isomerized, similar to a. interestingly, the c-terminal extension is not isomerized, which contrasts with a and is largely due to the fact that the c-terminal extension of b lacks any aspartic acid residues and contains only a single serine. the degree of isomerization observed in the ws versus wi fractions also varies more dramatically compared with a, with greater isomerization being observed in the wi fraction for all peptides. a and b share similar functionality and freely intermix to form higher order structures, yet comparison of figures 2 and 3 illustrates significant differences in propensity and effects of isomerization. the sequence alignment of the two proteins is<60% (figure s2), but the tertiary structures of a and b are very similar, as illustrated by the crystal structure of truncated bovine b on the right side of figure 3. despite the similarity, sequence variation appears to have a significant effect on the ability of b to accommodate isomerization and retain solubility, in agreement with previous observations. another interesting difference between the two -crystallins is the overall abundance of acidic amino acids. aspartic acid in a comprises 8.8% of residues and glutamic acid contributes another 5.8%, whereas b contains 6.3% aspartic acid and 8.0% glutamic acid. although isomerization of glutamic acid is possible, the formation of the glutarimide intermediate is much slower compared with the succinimide equivalent in aspartic acid. therefore, even though both proteins contain a similar percentage of acidic residues, a contains more residues prone to isomerization, and these residues often reside in regions where isomerization is facile. isomerization of b from sheep, which differs significantly from what was observed for a (see figure 2a, formatting is identical). asp109 in the amino acid sequence is in red bold text and shown explicitly in the crystal structure (pdb 3l1 g) to highlight an important site of isomerization. the same analysis that was performed on the crystallins was also conducted on one of the crystallins. b3 is composed of 211 amino acids, with two large structured domains connected by a flexible linker. the disordered n-terminal and c-terminal regions are much smaller than those observed in the -crystallins. the total amount of isomerization detected in the ws and wi fractions of b3 is lower than that in a and b, which is likely due to the greater fraction of highly structured regions. similar to b, b3 is characterized by significant disparity between the degree of isomerization in the ws versus wi fractions, with greater isomerization being observed in the latter. this suggests again that structural perturbations in b3 are consequential and lead to rapid loss of solubility. the isomerized versions of several peptides, including slrplhidgpdhk and kmeivdddvpslw, are only detectable in the wi fractions (see figure s3). slrplhidgpdhk is part of the structurally critical connecting peptide that joins the two domains of the monomer, denoted by the green bar in figure 4. kmeivdddvpslw contains aspartic acid residues that form ion pairs during assembly into higher order oligomers. sequence alignment for human, pig, cow, and sheep b3 shows that each contains this aspartic acid repeat motif (figure s4). the results in figure 4 suggest that isomerization of either of these peptides leads to dramatic loss of solubility and, by extension, function. formatting is identical to figure 2, except for the linker of the structured domains, shown in green. asp110, asp113, asp133, asp134, and asp135 are all in red bold text in the amino acid sequence and are shown explicitly in the human crystal structure (pdb 3qk3) to highlight important regions of isomerization. his113 from human b3 was mutated to asp113*. the same analysis was carried out on pig and cow lenses, and the results are summarized with the sheep results in figure 5. the percent isomerization per protein was determined by taking the sum of the percent of isomerization per peptide and dividing it by the total number of peptides, including a value of zero for those that were not isomerized. peptides where the degree of isomerization could not be determined were omitted for all samples of the same protein. for example, tryptic digestion of 173 residue a yielded 12 peptides that cover 92% of the sequence. contributions from these same peptides were used to calculate the numbers for each of the ws and wi a digests for each animal. the data were then normalized to the a digest that was most isomerized, which was the wi sheep digest. the same approach was applied to b and b3. although there is some variation between species, in general, the degree of isomerization is, on average, significantly greater in the wi fractions for all species. several of the peptides in figures 24 appear to exhibit an unusual degree of isomerization relative to their peers. for example, qddhgyisr from a is not only is the most isomerized site in the protein but also is significantly more isomerized than any other peptide in the structured -crystallin domain. quantitatively, the average amount of isomerization per peptide in the structured domain of a is 17.9% for the ws digest and 27.3% in the wi digest. at 76.7 and 89.3% isomerization in the ws and wi fractions, qddhgyisr is isomerized 4 the average rate in the ordered crystallin region. the analogous sequence in b, qdehgfisr, is also the most isomerized peptide extracted from the wi fraction, although full quantitative comparisons can not be made due to lack of separation of some isomers for other peptides in the ordered region (see figure s5 for details). finally, kmeivdddvpslw is the most isomerized peptide in the wi fraction from b3. each of these peptides shares a common feature, sequential repeats of acidic residues. in two cases, these observations suggest that sequential acidic residues represent sites of greater propensity for isomerization. there are at least two possible explanations that could account for increased isomerization at acid residue repeats. first, if there are multiple aspartic acid residues, then isomerization at multiple sites is possible. structural perturbation engendered by the first modification could easily lead to increased local backbone flexibility, facilitating isomerization at additional sites. indeed, examination of the elution profiles for these peptides reveals an abundance of isomers, indicating modification at more than one residue. for example, the elution profiles for the ws and wi fractions from pig for pqddhgysir reveal the presence of seven isomers; see figure 6. the lowercase p at the n-terminus of the sequence denotes pyroglutamate, which is a common product during trypsin digestion. comparison with synthetically prepared all l-asp isomer was used to confirm the peak corresponding to the canonical peptide. risomer values (4.8, 5.5, 3.7, 4.6, 4.5, 1.2, and 3.4) relative to the synthetic version confirm that the sixth peak in the digest is the all l-asp isomer (see figure s6 for details). the ws/wi degree of isomerization for pqddhgysir from sheep is shown in figure 6c, d. a similar elution profile is seen between wi pig and sheep; however, the amount of l-asp in the ws pig lens is nearly twice as great as it is in ws sheep. (a) ws chromatogram and (b) wi chromatogram for pqddhgysir from pig. (d) wi sheep. lower case p indicates the pyroglutamate that forms during tryptic digestion of n-terminal glutamine residues. isomers were confirmed by ms/ms analysis and are labeled with percent abundance. another factor contributing to isomerization at sites with multiple acidic residues, which may be more important than increased backbone flexibility, is inhibited repair by protein isoaspartyl methyltransferase (pimt). pimt is the only known repair enzyme that targets protein damage caused by aging. it is present and active in bovine lenses and reverts l-isoasp back to l-asp.d-asp is also repaired, although much less efficiently, but d-isoasp is not a substrate. importantly, sequence effects for substrate recognition have been identified and revealed that pimt has substantially lower affinity for isoasp if the n+1, n+2, or n+3 residues are negatively charged or if the n+1 site is proline. therefore, the sequence regions with multiple acidic residues that we have observed to be highly modified are unlikely to be repaired by pimt, and our results confirm the importance of this enzyme in the repair of long-lived proteins. acidic residue repeat sites may therefore be prone to sequential isomerization and difficult to repair, a potent combination with important consequences for protein aging. indeed, examination of other long-lived proteins associated with age-related diseases reveals the presence of numerous acidic acid repeat sites that would similarly be susceptible to isomerization. for example, amyloid precursor protein associated with alzheimer s disease contains 27 acidic residue repeat sites that would be poor substrates for pimt. table 1 lists long-lived proteins that contain aspartic acid repeats, are aggregation-prone, and are associated with diseases. it is clear that the results obtained here may be relevant on a much broader scale for any system composed of long-lived proteins. number of asp residues with an acidic residue in the n+1, n+2, or n+3 position. we have also identified isomerized regions with multiple serine residues, such as eekpssapss. in the ws and wi fraction of the sheep lens this peptide elutes in six different peaks (figure s7), and comparison of resulting cid spectra confirms that they represent different isomers. it is interesting to note that the total amount of isomerization in the wi sheep fraction for eekpssapss is much lower than what was observed for qddhgyisr, 27.3% compared with 90.4%, respectively. it is possible that isomerization of aspartic acid may have a larger influence on local structure than epimerization of serine, inducing isomerization of nearby residues more efficiently. alternatively, there is no known repair enzyme for epimerization of serine, which may suggest that the failure of pimt to repair isomerization may be the most important factor influencing isomerization at acidic residue repeats. however, the number of peptides with unambiguous epimerization at serine, that is, peptides that are isomerized and do not contain aspartic acid, is relatively small. therefore, more data will need to be acquired before strong conclusions about serine epimerization can be drawn. peptide epimerization and isomerization are difficult to detect and remain among the least studied ptms despite their potentially important role in numerous diseases. we have evaluated these modifications in crystallin proteins from the eye lenses of several organisms, and our results suggest that isomerization and epimerization lead to reduced protein solubility in many cases. although there is variation between species, several common themes emerge from analysis of the data. for example this finding suggests that many other long-lived proteins with known disordered regions, such as -synuclein, tau, and -amyloid, may also exhibit pathology related to isomerization or epimerization. although less prone to modification, isomerization within well-structured regions of proteins leads to more drastic changes in behavior, including unchecked aggregation. it was previously known that serine and aspartic acid are the most easily isomerized among the natural amino acids, but we have further demonstrated that when multiple acidic residues are close in sequence, the propensity for modification is enhanced further. we postulate that increased flexibility following an initial modification and failure of pimt to repair damaged residues contribute to the dramatic isomerization of sites with multiple acidic residues. the degree of isomerization at these sites also confirms the importance of pimt for long-term maintenance of protein structure. proteins associated with parkinson s disease, alzheimer s disease, amyotrophic lateral sclerosis, and huntington s disease all contain regions with multiple aspartic acid or serine residues in close proximity, suggesting that these proteins may also exhibit hotspots of isomerization. both a and b exhibit similar structural and functional behavior, yet a is found only in the eye lens. the isomerization behavior of a and b are quite distinct, with a being more prone to isomerization, but with b suffering greater loss of solubility following isomerization. the ability of a to sustain isomerization without loss of solubility may make it uniquely suited to the zero-turnover environment of the eye lens. it is clear that further, detailed examination of isomerization and epimerization in other tissues is needed and will expand our understanding of the role of long-lived proteins in aging-related diseases.

the eye lens crystallins represent an ideal target for studying the effects of aging on protein structure. herein we examine separately the water-soluble (ws) and water-insoluble (wi) crystallin fractions and identify sites of isomerization and epimerization. both collision-induced dissociation and radical-directed dissociation are needed for detection of these non-mass-shifting post-translational modifications. isomerization levels differ significantly between the ws and the wi fractions from sheep, pig, and cow eye lenses. residues that are most susceptible to isomerization are identified site-specifically and are found to reside in structurally disordered regions. however, isomerization in structured domains, although less common, often yields more dramatic effects on solubility. numerous isomerization hotspots were also identified and occur in regions with aspartic acid and serine repeats. for example, 128kmeivdddvpslw140 in b3 crystallin contains three sequential aspartic acid residues and is isomerized heavily in the wi fractions, while it is not modified at all in the ws fractions. potential causes for enhanced isomerization at sites with acidic residue repeats are presented. the importance of acidic residue repeats extends beyond the lens, as they are found in many other long-lived proteins associated with disease.

PMC5387677

pubmed-1378

since prostate-specific antigen (psa) was demonstrated in prostatic tissue in 1970, measurement of the serum psa level has become widely used for the early diagnosis and management of prostate cancer. however, it is well known that an increased serum psa level does not always indicate the existence of prostate cancer. the serum psa level can also be increased in patients with benign prostatic hyperplasia (bph), in patients with prostatitis, and after interventions such as prostate biopsy and transurethral resection of the prostate (turp). especially in patients with mildly increased serum psa (4-10 ng/ml), the single most common cause of serum psa elevation is known to be bph, rather than prostate cancer. other benign prostatic diseases that can cause elevation of the serum psa level include acute urinary retention, acute prostatitis, and prostatic ischemia. moreover, it has been reported that the serum psa level has a tendency to increase temporarily for 20 days after turp and will decrease afterwards. therefore, it is difficult to distinguish prostate cancer from benign prostatic conditions in patients who have undergone interventions on the prostate. one report on this issue suggested that 3% of patients who have undergone surgeries for bph will actually develop prostate cancer during the follow-up period; thus, establishment of clinical guidelines for follow-up strategies for such patients is necessary. it is now well understood from several reports on changes in the serum psa level after turp that the serum psa level decreases after turp. by contrast, reports on changes in the serum psa level after potassium-titanyl-phosphate (ktp) laser vaporization of the prostate based on the analysis of large populations are rare. treatment of patients with bph by 80 w high-power ktp laser vaporization was first introduced in clinical practice in 2000 and is now accepted as an effective minimally invasive treatment modality. this method eliminates prostatic tissues that cause obstruction through vaporization, which occurs when hemoglobin selectively absorbs the ktp laser that generates heat energy. ktp laser vaporization has gained considerable attention among many urologists recently because this treatment modality is less invasive while offering treatment results similar to those of conventional turp. hwang et al reported that patients who underwent ktp laser vaporization demonstrated significant improvements in international prostate symptom score (ipss), maximal flow rate (qmax), quality of life (qol), and residual urine 3 months after the operation. because the use of ktp laser vaporization is expected to increase, a need exists for establishing patterns in changes in the serum psa level after the operation. the elucidation of such patterns might offer useful clinical information on appropriate management and follow-up strategies. a total of 662 patients underwent ktp laser vaporization of the prostate for bph between october 2004 and august 2008. among these patients, those with prostate cancer, prostatitis, a history of urinary retention, or use of antiandrogen medication, all of which can influence serum psa levels, also excluded were patients for whom insufficient data were available or who were lost to follow-up. therefore, 278 patients were included in the prospective analysis of serial serum psa levels. careful history taking, digital rectal examination (dre), ipss, urinalysis/urine culture, transrectal ultrasound (trus), qmax, postvoiding residual urine (pvr), and serum psa levels were checked in every enrolled patient and evaluated to determine whether to perform ktp laser vaporization. patients with abnormal dre findings or a serum psa value of 4.0 ng/ml or higher underwent trus-guided 12-core prostate biopsy to detect and exclude prostate cancer. prostate volume was estimated by trus, first by measuring length (l), width (w), and height (h) and then by calculating volume by use of a prolate ellipsoid formula (lwh/6). either epidural or subarachnoid anesthesia was used for the operations, and the vaporization was carried out by use of an 80 w ktp laser system (greenlight pvtm; laserscope, san jose, ca) that uses a 6 fr side-deflecting optical fiber emitting laser at a wavelength of 532 nm, which is delivered through a 23 fr, 30 continuous flow cystoscope connected to videoendoscopy. the distance from the probe to the prostatic tissue was kept closer than 2 mm, and normal saline was used as the irrigation fluid. the vaporization was performed until the tissues that caused the obstruction were completely removed, resulting in the formation of an appropriately sized cavity. a foley catheter (16 or 18 fr) was inserted right after the procedure and was removed one day later. serum psa levels were measured in every patient before the operation and 1, 3, 6, and 12 months after the operation to analyze patterns of change. in addition, serum psa levels at 24 months after the operation were also checked in 183 patients. the mixed linear model in spss (version 12.0, spss inc) was used for statistical analysis of the changes in the serum psa level, and statistical significance was defined as a p-value of less than 0.05. the mean age of the patients was 69.0 years (range, 50-91 years). the mean serum psa value was 2.722.93 ng/ml, and mean prostate volume estimated by trus was 35.715.5 cc (table 1). the serum psa level temporarily increased and reached 3.183.23 ng/ml at 1 month after the operation (p=0.032). however, levels began to decrease continuously afterward and decreased to 1.922.26 ng/ml in 3 months (p<0.001) and 1.791.82 ng/ml in 6 months (p<0.001). within 12 months, the serum psa level was 1.702.25 ng/ml (p<0.001), thus demonstrating a decreasing pattern of stabilization (table 2, fig. the decreasing pattern showed its greatest value between 1 month and 6 months (fig. 1). the serum psa level at 24 months after the operation was not checked in all patients, but it also was shown to decrease and stabilize (table 2). as a result, the serum psa level had decreased by 37.5% by 12 months after the operation compared with the preoperative level. bph is a common disease in older men; 40% to 70% of men over the age of 60 years are suspected of having bph, and it is still on the rise along with increasing life expectancy and westernized diets. turp is accepted as the standard surgical method for treating the lower urinary tract symptoms of bph, although several other treatment options have been developed to date. however, complications of turp such as bleeding, tur syndrome, urethral stricture, retrograde ejaculation, and urinary incontinence are still among the side effects of this procedure and have not decreased significantly despite developments in techniques and instruments. ktp laser vaporization of the prostate was introduced in the 1990s but was not widely accepted as an effective treatment option because it was performed with a low-power (20 w, 34 w) ktp laser in the early years and required a relatively longer surgical time than conventional turp. more recently, however, the 80 w high-power ktp laser was developed to improve vaporization speed, and as a result, ktp laser vaporization has become an effective treatment modality. malek et al proved its efficacy by reporting that patients with prostates over 45 cc who underwent 80 w ktp laser vaporization showed significant improvements in ipss, qol, qmax, and pvr within 5 years of follow-up. therefore, ktp vaporization is now widely practiced in many institutes because of its efficacy and reduced invasiveness, even though some controversy remains concerning its complication rates compared with conventional turp [13-15]. ktp laser vaporization can not eradicate prostate cancer that originates in the peripheral zone, however, because it only eliminates tissues at the transitional zone of the prostate. marks et al reported that among 82 patients who underwent turp to treat lower urinary tract symptoms, prostate cancer had developed in 6 patients within 5 years of follow-up. with ktp laser vaporization in particular, possible pathologic diagnosis of prostate cancer could be missed because it is impossible to obtain a prostate specimen from the operation, which is possible in conventional turp. therefore, it would be useful to establish clinical guidelines by serum psa follow-up after the operation to detect coexisting prostate malignancy with bph. to date, however, only limited reports on serum psa changes after ktp laser vaporization of the prostate were available. it is widely known that serum psa levels generally increase after interventions on the prostate for a certain period of time and decrease afterward. temporary elevation of the serum psa level occurs as a result of psa leakage to the systemic circulation due to damage to the blood-prostate barrier during manipulations. in addition, prostatic inflammation after the manipulation can also facilitate increases in the serum psa level. there are several different reports on the length of time it takes for the serum psa level to decrease after a procedure. volkan et al reported that the serum psa level increased immediately after ktp laser vaporization, but eventually decreased and returned to its preoperative level within 15 days after the operation. in our series, the serum psa level increased for up to 1 month and then decreased significantly over the next 3 months. it is not possible to clearly explain this variability in the length of time for which the serum psa level is increased. further investigation of the probable responsible factors, such as differences in prostate volume, the preoperative serum psa level, operating time, and devices, are necessary to clarify this matter. aus et al analyzed the serum psa level of 190 patients who underwent turp and reported that the serum psa level was reduced by 70% compared with its preoperative level. they also reported that the serum psa level became lower than 4 ng/ml in 90% of those patients. wolff et al also reported that the serum psa level became stabilized at 2 ng/ml or higher in patients who were diagnosed with prostate cancer after turp. therefore, similar to patients who undergo turp, the possible existence of prostate cancer should also be considered in patients who demonstrate a serum psa level that remains high or increases constantly after ktp laser vaporization. in our study, the serum psa level decreased significantly within 3 months after the operation and showed only a slight decrease from 6 to 12 months, which might mean that the levels stabilize after 6 months. in the present study, we were able to accurately analyze the changes in the serum psa level after ktp laser vaporization by frequently checking the postoperative serum psa level in a relatively large population for more than 1 year. the serum psa level may be temporarily increased after ktp laser vaporization for a certain period of time, but values will show a decreasing pattern within 3 months and will eventually become stabilized between 6 and 12 months. therefore, it may be appropriate to wait for 3 months if the serum psa level rises after the procedure. however, if the serum psa level remains high even after 6 months, close follow-up or further investigation should be considered.

purposethe prostate-specific antigen (psa) level decreases after transurethral resection of the prostate (turp). however, changes in the psa level after potassium-titanyl-phosphate (ktp) laser vaporization of the prostate are not well known. the aim of this study was to investigate the effect of ktp laser vaporization of the prostate on psa levels in patients with benign prostatic hyperplasia (bph). materials and methodsserum psa levels were checked before and 1, 3, 6, and 12 months after the procedure in patients who underwent ktp laser vaporization between october 2004 and august 2008. patients with prostate cancer, a history of urinary retention, or prostatitis during the follow-up period were excluded. the results for 278 patients were studied. resultsthe mean age of the patients was 69.06.7 years (range, 50-91 years) and the mean preoperative psa level was 2.722.93 ng/ml. the psa level tended to be increased at 1 month after the operation (3.183.23 ng/ml, p=0.032) but decreased within 3 months and became stabilized after 6 months at 1.791.82 ng/ml (p<0.001). conclusionspsa levels may increase after ktp laser vaporization for a certain period of time, but eventually decrease and become stabilized after 6 months. therefore, it may be appropriate to wait up to 3 months if the psa level rises after the procedure, and further investigation should be considered if the psa level still remains high after 6 months.

PMC2855487

pubmed-1379

typically, we study experience with health care conditions, including health care costs and quality, as if these conditions occur in isolation, one at a time. the vast majority of extant clinical guidelines and disease management programs focus on a single condition, although the experience of multiple chronic illnesses is the reality for many patients particularly among the elderly and near elderly. the institute of medicine s report crossing the quality chasm highlights the problem with health system fragmentation and stresses the need for health care systems that promote continuity of care and integration of services.1 realigning the focus of health services research to be more in line with the complex experience of patients is central to developing solutions that work. this paper provides information on what we know about multiple chronic conditions, specifically the prevalence and health challenges of multiple chronic conditions, and the ramifications of specific combinations of chronic conditions on quality, patient management, and costs. we performed a semistructured literature review to identify relevant articles. specifically, we queried medline for peer-reviewed publications that examined the prevalence, outcomes, costs, and patient management challenges associated with multiple chronic conditions. chronic disease and comorbidity, and limited our search to articles on adults published in english between january 2000 and march 2007 (n=643). the first strategy used a set of specific mesh terms related to prevalence, quality, access, delivery of care, patterns of care, morbidity, mortality, and expenditures. to ensure that we did not overlook any important articles in the original set the final set of 123 articles was the union of abstracts gained from these 2 approaches. the remaining abstracts were reviewed by the first author and abstracts that did not mention at least 1 specific somatic chronic illness, abstracts that did not examine specific comorbidities, and articles that focused on an acute illness or procedure were removed. information summarized in this review stem from the remaining articles and prior publications cited by these articles. the number of persons in the united states who have not just a single chronic condition, but multiple co-occurring chronic conditions is large and growing. in 2005, 21% or roughly 63 million americans had more than 1 chronic condition, or multiple illnesses or impairments expected to last a year or longer. a persons risk of having more than 1 chronic condition, henceforth referred to as multiple chronic conditions or mcc, increases with age: 62% of americans over 65 have mcc. with the aging of the us population, the number of americans with mcc is projected to be 81 million by 2020.2 the institute of medicine s seminal report crossing the quality chasm noted that 23% of medicare beneficiaries have 5 or more chronic conditions.1 prior research has documented the prevalence of individual conditions in the u.s. for example, based on data from the medicare current beneficiary survey (mcbs), the most prevalent individual conditions among the over-65 population include: arthritis (57%), hypertension (55%), pulmonary disease (38%), diabetes (17%), cancer (17%) and osteoporosis (16%).2 however, there has been very little research to date exploring the prevalence of particular combinations or clusters of chronic conditions, and almost all studies examining specific comorbidities do so from the perspective of a specific index disease rather than examining all co-occurring chronic conditions.3 only a fragmentary portrait of the prevalence of mcc emerges from studies examining comorbidities among patients with specific index conditions. a case-control study of asthmatics found that diabetes was more common in concert with asthma, but obesity was more common in patients without asthma4 of patients with alzheimer s disease, 28% also have congestive heart failure, 27% chronic obstructive pulmonary disease, 22% diabetes mellitus, and 20% cancer.2 in comparison to the general population, persons receiving care for schizophrenia or affective disorders in community-based treatment centers were more likely to suffer from asthma, chronic bronchitis, diabetes, and liver problems.5 finally, persons suffering from epilepsy have higher rates of a host of chronic conditions, including bowel disorders, bronchitis/emphysema, heart disease, and stroke in comparison to the general population.6 fewer studies have explored the natural clustering of chronic conditions. using cross-sectional medicare claims data, wolff and colleagues grouped a national sample of medicare patients into major diagnostic categories (mdc) based on a well-validated grouping algorithm. they found that the tendency of patients to have comorbid conditions varied from 80% among individuals in the mdc myeloproliferative disorders to 32% in the circulatory disorders mdc. these investigators found that specific combinations of chronic conditions occurred more frequently than expected, and proposed that perhaps underlying biological vulnerabilities may help explain the clustering of diseases within individuals.7 a subsequent more limited study used cluster analysis to identify conditions that tend to co-occur among elderly american indians. the specific conditions in this group include heart disease, stroke, hypertension, diabetes, urinary or bladder conditions, and tuberculosis. interestingly, although arthritis is 1 of the 2 most common chronic conditions in this population, it does not commonly occur in concert with other conditions.8 perhaps the most widely known example of the clustering of chronic conditions in a biologically and clinically meaningful way is the so-called metabolic syndrome. population,9 the metabolic syndrome is present when patients have at least 3 of 5 chronic conditions: obesity, hypertriglyceridemia, low-serum high-density lipoprotein (hdl), hypertension, and glucose intolerance.10,11 the metabolic syndrome is associated with increased risk of cardiovascular disease and all cause mortality12,13 and may reflect underlying genetic predispositions to this combination of chronic conditions.14,15 overall, specific chronic conditions have a stronger relationship with functional impairment than others, and persons with more chronic conditions become more functionally impaired sooner than persons with fewer chronic conditions.16 the picture, however, appears to be more complex. one of the most revealing studies to date found that after controlling for the presence of individual conditions, specific mcc were associated with disability far greater than expected based on the disability observed for each disease in isolation. the authors suggested that some diseases may be associated with disability only in the presence of other specific diseases, and that a new, potentially effective strategy for prevention or amelioration of disability would be to decrease targeted disease-disease interactions.17 fultz et al.18 also found synergistic interactions between some pairings of mental and physical conditions, but not others. for example, persons with stroke and cognitive impairment had a higher level of impairment in activities of daily living than predicted by the presence of stroke and cognitive impairment alone. other combinations, such as stroke and depression, did not have the same synergistic effect on activities of daily living impairments.18 a recent analysis of near-elderly veterans found that in general the risk of 5-year mortality increased with the number of co-occurring chronic conditions; however, osteoarthritis in combination with any other chronic condition actually lowered the risk of 5-year mortality.19 persons with multiple chronic conditions are particularly vulnerable to suboptimal quality care.2 they tend to use services more frequently and to use a greater array of services than other consumers of care. this makes coordination of care more difficult for individuals with multiple chronic conditions. the number of different physicians seen annually by the average medicare patient with a chronic condition ranges from 4 with 1 condition to 14 with 5 or more. as the number of providers involved in patients care increases, patients are likely to find it increasingly challenging to understand, remember, and reconcile the instructions of those providers.20 because patients with more than 1 chronic condition take on average more medications, they are more likely to suffer adverse drug events (ades), including ades that result from drug-drug interactions,2124 or in the specific case of heart failure coupled with chronic obstructive pulmonary disease, present challenges to appropriate pharmacological management.25 having multiple chronic conditions also makes it more challenging for patients to participate effectively in their own care.26 surveys of physicians confirm that they believe quality problems are increased among their patients with multiple chronic conditions.27 however, the link between co-occurring chronic conditions and poor quality is far from clear. an assessment of quality among canadians over 65 with specific combinations of chronic conditions found deficiencies in care associated with some combinations of conditions, but not all. for example, patients with hyperlipidemia and chronic obstructive pulmonary disease were less likely than patients with hyperlipidemia alone to receive lipid-lowering medications. individuals with psychoses and arthritis were less likely to receive arthritis medications than individuals with arthritis alone. however, glaucoma patients with breast cancer are no less likely to receive glaucoma medications than those without breast cancer.28 a well-known study of the predictors of initiating psychiatric treatment found that competing demands from physical problems hindered the initiation of psychiatric care.29 however, other studies have found that co-occurring chronic conditions are actually associated with more appropriate care. contrary to prior expectations, researchers examining a cohort of diabetic patients enrolled in a heart failure disease management program found that despite their targeted heart failure care, these patients also received comprehensive diabetes care.30 patients with somatic chronic conditions may actually receive more appropriate care for depression or other psychiatric disorders. among elderly persons, depression care was more likely to be adequate among elderly persons with co-occurring diabetes than without,31 and neither the number nor specific comorbid conditions were found to impact the effectiveness of interventions aimed at improving depression care.32 more general measures of quality also yielded mixed results. braunstein et al.33 found that the occurrence of hospitalizations for ambulatory care sensitive conditions increased among elderly heart failure patients when they suffered from other comorbidities. hospitalizations for ambulatory care sensitive conditions are considered preventable by good primary care.34 the odds of experiencing these so-called preventable hospitalizations were largest when heart failure occurred in combination with hypertension or chronic renal insufficiency. however, for unknown reasons, certain comorbidities, such as hypercholesterolemia or dementia, seemed to protect heart failure patients against hospitalization for ambulatory care sensitive conditions.33 among vulnerable persons age 65 and over, min et al.35 found that overall, persons with more chronic conditions had higher (better) risk-adjusted quality scores. however, specific combinations such as diabetes and cardiovascular disease were associated with worse quality of care as measured by a composite of up to 207 quality indicators. it is reasonable to hypothesize that clinicians systematically vary in the provision of indicated services when caring for patients with particular combinations of conditions, just as they systematically overlook certain issues in caring for single illnesses.36 for example, systematic differences in colon cancer screening rates among elderly persons with chronic conditions may reflect conscious decisions to concentrate screening on patients whose life expectancy can be improved through cancer treatment.37 several observers have argued that current strategies including disease-specific health guidelines may not be suitable in many cases to optimizing care of individuals with mcc.21,24 instead, it is argued, guidelines need to be tailored to clusters of illnesses in ways that acknowledge not only the biology of those clusters, but also the special challenges and threats to quality of care associated with mcc in general and specific clusters in particular. moreover, single-disease-oriented disease management programs, which frequently offer services provided outside traditional health care facilities (call centers and coaches, for example), have the potential to further fragment care. even wagner s chronic care model, which emphasizes coordination of care around chronic illness, focuses primarily on single illnesses, not multiple chronic conditions,38 so that its relevance and effects on multiple chronic conditions remain to be explored. the intrinsic challenges to optimizing quality and value of care among individuals with multiple chronic conditions, the evidence that quality may be suboptimal for some individuals with multiple chronic conditions, and indications that quality may vary with the specific clusters of chronic conditions, all suggest the need to explore more systematically the relationship between quality of care and clusters of chronic conditions. in a country with health care expenditures exceeding $1.7 trillion and 15% of gross domestic product,39 controlling costs of care has become an overwhelming concern among public and private policy makers and managers. from this perspective the care of individuals with chronic conditions is estimated to account for 78% of health expenditures in the united states. patients with more than 1 chronic condition are estimated to account for 95% of all medicare spending; those with more than 5 account for two thirds.2 the congressional budget office reports that among high-cost medicare beneficiaries (e.g., the 25% of beneficiaries accounting for 85% of programmatic costs), about 30% had 4 co-occurring chronic illnesses: coronary artery disease, diabetes, congestive heart failure, and chronic obstructive pulmonary disease.40 the likelihood that patients with a particular condition such as heart failure or diabetes will use expensive health care resources such as hospital care increases substantially with the presence of other comorbidities.33,41 for example, the likelihood that a medicare patient with a chronic medical condition will use emergency department services doubles when depression is present as a comorbidity.42 medicare beneficiaries with heart failure who have comorbidities are more likely to be readmitted for heart failure than patients without comorbidities.43 the concentration of health care expenditures in subpopulations with chronic conditions has led to the widespread proliferation of disease management programs. in 2004, 97% of private health plans had disease management programs for diabetes, 86% for asthma, 83% for heart failure, and 70% for ischemic heart disease.44 state medicaid programs have also begun implementing similar disease management programs.45,46 under provisions of the medicare modernization act, congress instructed the centers for medicare and medicaid services to undertake a variety of initiatives to improve care for high-cost, chronically ill patients, including the chronic care improvement program (ccip), a large national experiment with applying disease management programs to patients in the traditional medicare program.47,48 the ccip will target more than 30,000 beneficiaries with 3 conditions (diabetes, heart failure, and chronic obstructive pulmonary disease) in 10 regions of the country. despite the conceptual attractiveness of the disease management approach, evidence of clinical and cost-effectiveness remain limited.49,50 recent analyses have found that cost savings and return on investments varied by diagnosis.51,52 most disease management programs focus on management of a single chronic condition. this raises concerns about whether they may undermine coordination of care for patients with mcc,53 thereby introducing new inefficiencies and potential threats to quality of care.21,24,54 furthermore, by focusing on a single illness, programs fail to account for the synergistic impact of chronic conditions occurring in combination. the medicare program has begun experimenting with improving management of patients with mcc under other demonstrations including its medicare coordinated care demonstration and its care management for high-cost beneficiaries demonstration. more recently, some private health plans have also shifted toward intensive case management programs aimed at high-risk patients with multiple complex conditions,50 often using predictive modeling applications to identify members whose past utilization suggests they are likely to generate high health care costs in the future.55 however, even these initiatives may suffer from the fact that they lack information necessary to take into account the potential variation in costs and quality associated with particular mcc and information on the most efficacious treatments for specific disease combinations. understanding how to care effectively for persons with multiple chronic conditions is among the most important challenges our health care system faces. despite the depth of research into specific chronic conditions, there is little information about the prevalence of mcc, and the health and cost impacts of specific combinations of chronic conditions. the small amount we do know suggests that specific chronic conditions combine and impact health and costs in unpredictable ways, and that specific combinations have particularly large impacts of health or costs of care. currently, there are a number of methodological challenges to research on mcc, including, fundamentally, the need for large and preferably longitudinal, clinically meaningful data that can be used to identify the natural history of disease, and control for the severity of individual conditions when assessing outcomes for mcc. the increasing adoption of health information technology has the potential to greatly improve the level of clinical detail of widely available data,56 and may help accelerate clinically meaningful research. such research should help illuminate why certain clusters of comorbid illness may be more prone to quality lapses or be associated with significant but unexpected clinical outcomes, and lead to the development of targeted strategies, including tailored mcc-specific clinical guidelines, to improve the management of patients with key mcc. similarly, research on more clinically detailed data may be used to develop computerized decision support that incorporates new knowledge regarding the additive clinical impact of specific comorbidities co-occurring within a patient and anticipate the tendency of clinicians to overlook or overprescribe certain elements in the process of care. although payers have begun to target high cost combinations, far more research is needed to understand the clinical impact of the clustering of chronic illness and to incorporate this more refined understanding into targeted quality improvement and clinical management strategies. with the aging of population ,

persons with multiple chronic conditions are a large and growing segment of the us population. however, little is known about how chronic conditions cluster, and the ramifications of having specific combinations of chronic conditions. clinical guidelines and disease management programs focus on single conditions, and clinical research often excludes persons with multiple chronic conditions. understanding how conditions in combination impact the burden of disease and the costs and quality of care received is critical to improving care for the 1 in 5 americans with multiple chronic conditions. this medline review of publications examining somatic chronic conditions co-occurring with 1 or more additional specific chronic illness between january 2000 and march 2007 summarizes the state of our understanding of the prevalence and health challenges of multiple chronic conditions and the implications for quality, care management, and costs.

PMC2150598