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Neuropathic pain is a chronic pain defined as a pain caused by damages to or dysfunction of somatosensory system and can expresses itself in the following forms: allodynia, hyperalgesia, and spontaneous pain (1). Along with causing changes to nervous system, neuropathic pain can decrease physical activity levels (2, 3). Furthermore, neuropathic pain can affect structure and function of muscles through muscular atrophy (4, 5). Many studies have proven that models of neuropathic pains will be followed with muscle atrophy (5 - 8). However, exact cellular mechanisms, which cause changes after nervous damages are still unknown (8). Cyclin - dependent kinases (cdks) are serine / threonine protein kinases that play key roles in the regulation of cell cycle, initiation of transcription, and control of certain metabolic cascades in mammalian cells (9, 10). Cdks activity has a vital role in different functions of nerve cells such as neuronal growth and migration, cell secretion, dopamine signaling, and cytoskeletal dynamics (10). Glycogen synthase kinase-3 (gsk3) is also a serine / threonine kinase that exists in two isoforms of alpha and beta (11) and participate in the modulation of various functions such as cell signaling, growth metabolism, and many other transcriptional regulating survival and death factors in organisms (12). In general, cdk5 and gsk-3 are two of the most important protein kinases involved in neuropathic pain signaling (13, 14). Furthermore, the role of these two proteins in the regulation of muscle plasticity has been clearly proved . For example, it has been shown that gsk-3 is necessary in myofibrillar protein loss system and its reduction causes preserving of contractile protein against proteolysis (15). Cdk5 also has a key role in skeletal muscle s structure and its function so that it is effective in changing muscle phonotype and essentially these changes come from its effects on cytoskeleton proteins structure such as actin and microtubules and regulation of myogenesis (16, 17). On the other hand, changes to expression of cdk5 and gsk-3 in muscular and nervous systems after numerous interferences infer such an insight that related systems change through following conditions: chronic state of motor unit activity, afferent activity level toward motor neurons, number of innervated muscle fibers by motor neurons, and metabolic level of target tissue . We set the goal of this study on considering the effects of decreased activity in the form of neuropathic pain caused by ligation of the l5 spinal nerve on cdk5 and gsk-3 gene expression in soleus muscle of wistar male rats . In the present study, 12 mature 10-week - old wistar male rats, weighted 200 - 240 g were provided by animal maintenance unit of razi research center (razi institute animal center, karaj, iran) and transferred to animal laboratory of tarbiat modares university . All rats were kept under a controlled environment condition with mean temperature of 22 3c, dark - light cycle of 12:12 hours, relative humidity of 40% and free access to food and water ad libitum . The experimental protocols to perform this study were approved by the ethics committee on the use of animals of tarbiat modares university, tehran, iran . All efforts were made to minimize discomfort of the animals and reduce the number of experimental animals . All procedures conformed to the ethical guidelines regarding the care and use of laboratory animals, published by the international association for the study of pain and the national institutes of health . After two weeks of acclimatization of the animals with new environment, experimental protocols were initiated and the rats were randomly (simple randomization) allocated in 3 groups (4 rats in each group): (1) tight ligation of the l5 spinal nerve (snl: n = 4); (2) sham surgery (sham: n = 4), and (3) control (c: n = 4). Calculated sample size by the following equation resulted resulted in 3 animals in each group: where z = 1.96, z = 0.84, sd = 0.18 and d = 0.4 . Then, l5 spinal nerve was tightly ligated according to the method of kim and chung (1992) (18). Briefly, the left paraspinal muscles were separated at l5-s2 level and the left transverse process of l6 vertebra was removed . The left l5 spinal nerve was identified and gently separated from adjacent l4 spinal nerve . Then, l5 spinal nerve was tightly ligated using silk threads (6/0) and was transected just distal to ligature to ensure that all fibers were interrupted . Next, the wound was closed with silk threads (3/0). Sham group, the surgical procedure was identical to that described above, except for the left l5 spinal nerve that was not ligated and transected . Only animals showing no signs of motor deficiencies were considered to be used for further experimentations, those animals without neuropathic pains in their behavioral tests . Then the rats were divided in 3 groups with 5 members in each: non - operated, sham, and neuropathic pain (snl) groups . After 4 weeks, rats were anesthetized by injecting intraperitoneal ketamine (90 ml / kg) and xylazine (10 ml / kg) and the muscle tissue samples were separated from the left soleus muscle and situated in -80c nitrogen for future analysis . Radiation heat apparatus was used to measure hyperalgesia in which the middle part of animal s paw from plexiglas level was exposed to thermal constant radiations and paw withdraw threshold time (pwts) was calculated . Heat excitations repeated 3 times, with 5 to 10 minutes intervals . To measure the mechanical allodynia, animal was located on the wired network and inside the plexiglas capsule with 20 20 30 cm dimensions . After acclimatization with new environment, von frey fibers with weight range of 2 to 60 g (2, 4, 6, 8, 15, 26, and 60 g) (manufactured by stolting inc .) Were used . The experiment started with the lightest fiber and in the non - response cases, heavier fibers were gradually employed . For excitation initiation, each fiber was inserted into animal s paw in 3 consecutive times for 1 s with 5 s intervals . If we had positive response (animal raising its foot) in 2 consecutive performances, that weight of fiber would have been selected as the response threshold . If animal had no response to all of the fibers, number 60 would have been considered as the response threshold (19). Soleus muscles from the left leg were quantitatively dissected from the bone, immediately weighed, and frozen at 20c . Later, these muscles were lyophilized and weighed for their dry weight . After excision of muscles, the left tibia was removed and freed from connective tissue, and the maximal length was measured . (20) showed that expressing muscle mass per unit of tibial length is a valid way to normalize mass when body weight differs between experimental groups . Rna extraction was done by qlazol lysis reagent (germany, qiagen) and chloroform (germany, qiagen) according to manufacturer s instructions . So, about 50 mg of the muscle tissue was separately homogenized in 1 to 10 portions in qlazol lysis reagent for total rna extraction and removing protein components . Then, it mixed with chloroform in 1 to 5 portions and was shaken severely for 15 s. then the supernatant was centrifuged at 12000 g for 10 minutes at 4c and its water and mineral part were removed . Finally, its rna contained portion was removed and mixed with isopropanol in 1 to 5 portions . It was left for 10 minutes at room temperature and then centrifuged at 12000 g at 4c for 10 minutes . Rna concentration was measured by uv spectrophotometry method (eppendorf, germany), and 260 to 280 portions in 1.8 - 2 were determined as the desired purification . Cdna synthesis was done by using quanti tect reverse transcription kit (qiagen, germany) in accordance to the manufacturer s manual . Real - time pcr quantity method was used by premix sybr green ii (qiagen, germany) for measuring cdk5 and gsk-3 mrna expression levels (applied biosystems stepone, america). Reaction mixture was done in final volume in 20 l (includes 1 l of cdna, 1 l of forward primer, 1 l of reverse primer, 7 l of depc water and 10 l of syber green) and each reaction in duplicate . Designing of primers was done according to cdk5, gsk-3 and gapdh genes in gene bank of ncbi and by german company, qiagen . Furthermore, gapdh was used as the reference gene . Thermal program used in real time - pcr included: 95c for 10 minutes, 95c for 15 s, 60c for 1 min (40 cycle repetitions). Melt curve and standard curve were drawn and considered for evaluating data authenticity and optimization experiment conditions, respectively and cdk5 and gsk-3 expression data were normalized using gapdh (reference gene). Abbreviations: for, forward; cdk5, cyclin - dependent kinase 5; gapdh, glyceraldehyde-3-phosphate dehydrogenase; gsk-3, glycogen synthase kinase-3; rev, reverse . All statistical analyses were done by using spss software (version 19, spss inc ., one - way repeated measures anova and independent t tests were used to compare groups regarding under study variables and followed by tukey hsd post hoc . In the present study, 12 mature 10-week - old wistar male rats, weighted 200 - 240 g were provided by animal maintenance unit of razi research center (razi institute animal center, karaj, iran) and transferred to animal laboratory of tarbiat modares university . All rats were kept under a controlled environment condition with mean temperature of 22 3c, dark - light cycle of 12:12 hours, relative humidity of 40% and free access to food and water ad libitum . The experimental protocols to perform this study were approved by the ethics committee on the use of animals of tarbiat modares university, tehran, iran . All efforts were made to minimize discomfort of the animals and reduce the number of experimental animals . All procedures conformed to the ethical guidelines regarding the care and use of laboratory animals, published by the international association for the study of pain and the national institutes of health . After two weeks of acclimatization of the animals with new environment, experimental protocols were initiated and the rats were randomly (simple randomization) allocated in 3 groups (4 rats in each group): (1) tight ligation of the l5 spinal nerve (snl: n = 4); (2) sham surgery (sham: n = 4), and (3) control (c: n = 4). Calculated sample size by the following equation resulted resulted in 3 animals in each group: where z = 1.96, z = 0.84, sd = 0.18 and d = 0.4 . Then, l5 spinal nerve was tightly ligated according to the method of kim and chung (1992) (18). Briefly, the left paraspinal muscles were separated at l5-s2 level and the left transverse process of l6 vertebra was removed . The left l5 spinal nerve was identified and gently separated from adjacent l4 spinal nerve . Then, l5 spinal nerve was tightly ligated using silk threads (6/0) and was transected just distal to ligature to ensure that all fibers were interrupted . Next sham group, the surgical procedure was identical to that described above, except for the left l5 spinal nerve that was not ligated and transected . Only animals showing no signs of motor deficiencies were considered to be used for further experimentations, those animals without neuropathic pains in their behavioral tests . Then the rats were divided in 3 groups with 5 members in each: non - operated, sham, and neuropathic pain (snl) groups . After 4 weeks, rats were anesthetized by injecting intraperitoneal ketamine (90 ml / kg) and xylazine (10 ml / kg) and the muscle tissue samples were separated from the left soleus muscle and situated in -80c nitrogen for future analysis . Radiation heat apparatus was used to measure hyperalgesia in which the middle part of animal s paw from plexiglas level was exposed to thermal constant radiations and paw withdraw threshold time (pwts) was calculated . Heat excitations repeated 3 times, with 5 to 10 minutes intervals . To measure the mechanical allodynia, animal was located on the wired network and inside the plexiglas capsule with 20 20 30 cm dimensions . After acclimatization with new environment, von frey fibers with weight range of 2 to 60 g (2, 4, 6, 8, 15, 26, and 60 g) (manufactured by stolting inc .) Were used . The experiment started with the lightest fiber and in the non - response cases, heavier fibers were gradually employed . For excitation initiation, each fiber was inserted into animal s paw in 3 consecutive times for 1 s with 5 s intervals . If we had positive response (animal raising its foot) in 2 consecutive performances, that weight of fiber would have been selected as the response threshold . If animal had no response to all of the fibers, number 60 would have been considered as the response threshold (19). Soleus muscles from the left leg were quantitatively dissected from the bone, immediately weighed, and frozen at 20c . After excision of muscles, the left tibia was removed and freed from connective tissue, and the maximal length was measured . (20) showed that expressing muscle mass per unit of tibial length is a valid way to normalize mass when body weight differs between experimental groups . Rna extraction was done by qlazol lysis reagent (germany, qiagen) and chloroform (germany, qiagen) according to manufacturer s instructions . So, about 50 mg of the muscle tissue was separately homogenized in 1 to 10 portions in qlazol lysis reagent for total rna extraction and removing protein components . Then, it mixed with chloroform in 1 to 5 portions and was shaken severely for 15 s. then the supernatant was centrifuged at 12000 g for 10 minutes at 4c and its water and mineral part were removed . Finally, its rna contained portion was removed and mixed with isopropanol in 1 to 5 portions . It was left for 10 minutes at room temperature and then centrifuged at 12000 g at 4c for 10 minutes . Rna concentration was measured by uv spectrophotometry method (eppendorf, germany), and 260 to 280 portions in 1.8 - 2 were determined as the desired purification . Cdna synthesis was done by using quanti tect reverse transcription kit (qiagen, germany) in accordance to the manufacturer s manual . Real - time pcr quantity method was used by premix sybr green ii (qiagen, germany) for measuring cdk5 and gsk-3 mrna expression levels (applied biosystems stepone, america). Reaction mixture was done in final volume in 20 l (includes 1 l of cdna, 1 l of forward primer, 1 l of reverse primer, 7 l of depc water and 10 l of syber green) and each reaction in duplicate . Designing of primers was done according to cdk5, gsk-3 and gapdh genes in gene bank of ncbi and by german company, qiagen . Furthermore, gapdh was used as the reference gene . Thermal program used in real time - pcr included: 95c for 10 minutes, 95c for 15 s, 60c for 1 min (40 cycle repetitions). Melt curve and standard curve were drawn and considered for evaluating data authenticity and optimization experiment conditions, respectively and cdk5 and gsk-3 expression data were normalized using gapdh (reference gene). Abbreviations: for, forward; cdk5, cyclin - dependent kinase 5; gapdh, glyceraldehyde-3-phosphate dehydrogenase; gsk-3, glycogen synthase kinase-3; rev, reverse . All statistical analyses were done by using spss software (version 19, spss inc ., one - way repeated measures anova and independent t tests were used to compare groups regarding under study variables and followed by tukey hsd post hoc . All rats that received l5 snl developed mechanical and heat hypersensitivity on the ipsilateral hind paw . As shown in figure 1 and 2, pwt as well as pwl in the snl group were lower postoperatively on the first, second, third and fourth weeks (p <0.05 versus sham group), indicating that mechanical allodynia and thermal hyperalgesia has been induced by snl operation . Pwt decreased in the snl rats postoperatively on the first, second, third and fourth weeks (p <0.05 versus sham group). Pwt decreased in the snl rats postoperatively on the first, second, third, and fourth weeks (p <0.05 versus sham group). Data are presented as mean sd, which indicate significant differences with other groups (p <0.05). For the gapdh (186 bp), cdk5 (210 bp), and gsk-3 (206 bp) genes, m = 100-bp dna ladder . Data are presented as mean sd, which indicate significant differences with other groups (p <0.05). Data are presented as mean sd, which indicate significant differences with other groups (p <0.05). Weight of the soleus muscle decreased in snl rats and this result indicates muscular atrophy as a consequence of tight ligation of the l5 spinal nerve (figure 3). To elucidate the possible regulation of cdk5 and gsk-3 at mrna level in rat models with neuropathic pain, we examined cdk5 and gsk-3 at mrna level in soleus muscle after 4 weeks of tight ligation of l5 spinal nerves . Agarose gel electrophoresis presented a single band with the expected size (figure 4) and as indicated in figure 5 and 6 cdk5 mrna levels were down regulated and gsk-3 mrna levels were up regulated . All rats that received l5 snl developed mechanical and heat hypersensitivity on the ipsilateral hind paw . As shown in figure 1 and 2, pwt as well as pwl in the snl group were lower postoperatively on the first, second, third and fourth weeks (p <0.05 versus sham group), indicating that mechanical allodynia and thermal hyperalgesia has been induced by snl operation . Pwt decreased in the snl rats postoperatively on the first, second, third and fourth weeks (p <0.05 versus sham group). Pwt decreased in the snl rats postoperatively on the first, second, third, and fourth weeks (p <0.05 versus sham group). Data are presented as mean sd, which indicate significant differences with other groups (p <0.05). For the gapdh (186 bp), cdk5 (210 bp), and gsk-3 (206 bp) genes, m = 100-bp dna ladder . Data are presented as mean sd, which indicate significant differences with other groups (p <0.05). Data are presented as mean sd, which indicate significant differences with other groups (p <0.05). Weight of the soleus muscle decreased in snl rats and this result indicates muscular atrophy as a consequence of tight ligation of the l5 spinal nerve (figure 3). To elucidate the possible regulation of cdk5 and gsk-3 at mrna level in rat models with neuropathic pain, we examined cdk5 and gsk-3 at mrna level in soleus muscle after 4 weeks of tight ligation of l5 spinal nerves . Agarose gel electrophoresis presented a single band with the expected size (figure 4) and as indicated in figure 5 and 6 cdk5 mrna levels were down regulated and gsk-3 mrna levels were up regulated . Candidate gene selection followed by decreased activity in neuropathic pain form studies is greatly influenced by the current knowledge of the pathogenic mechanisms involved in this disease . In the present study, it was observed that decreased activity in form of tight ligation of the l5 spinal nerve causes decreased cdk5 mrna gene expression in soleus muscle, while amount of gsk-3 mrna gene expression increases . Moreover, we show that tight ligation of the l5 spinal nerve causes soleus muscle atrophy that could be attributed to this changes . Intracellular signaling cascades, which result in immediate early gene induction and maintenance, can control widespread changes in gene and protein expression . Careful study in the relationship of different gene regulations is important for further understanding of pathogenesis of neuropathic pain . Previous studies showed that neuropathic pain increased cdk5 protein (22) and decreased gsk-3 protein (23). However, they did not show whether this increase in cdk5 protein levels and decrease in gsk-3 protein levels are due to the changes in their mrna or degradation . The present data led us to conclude that down regulation of cdk5 and up regulation of gsk-3 may cause decrease and increase in their protein level, respectively, a conclusion that previously reported by others . Such decreases in the activity cause inability in physical fitness, increase in blood pressure, and decrease in musculoskeletal functions . Improper physical activity leads to structural and functional reduction of skeletal muscle (24). Besides, damages to motor neurons lead to many morphological and biochemical changes in muscles (25, 26). Spinal cord injury is related to atrophy and decrease in the size of muscle mass, which pathologists related it to nerve damages and decrease in activity and movement because of pain existence (27). Decreased activity because of hyperalgesia and allodynia increases proteolysis and decreases protein synthesis in muscles (28). Furthermore, existing evidence shows that tight ligation of the l5 spinal nerve causes limitation but not complete inhibition of muscle electrical activity . (1997) (29) showed that by exciting motor neurons of l4 segment, only 30% of maximum generated force of soleus muscle was recruited; however, by exciting motor neuron of l5 segment, 70% of maximum generated force of the muscle was recruited . Denervation causes effective intracellular environment changes, decrease in muscle mass, number and volume of mitochondria, and increase in apoptosis proteins (30). Cdk5 activity and its receptor (p35) in afferent neurons are very important in understanding the reason for its acute pain (14). In muscles, cdk5 expression is very necessary in its natural amount and unnatural cdk5 expression in non - nervous cells . Both expressions are done mainly through its direct or indirect effects on organizing cytoskeleton structures such as actin and microtubules, which seems to cause such structure changes and these changes on cytoskeleton structure leads to cellular disorders and disease (16). (2002) (26) reported cdk5 expression in skeletal muscles of rats after nervous damages showed an unnatural increase, which followed by increase in acetilcoline receptors in neuromuscular junction . Differences in the findings of the present study to that of fu and his colleagues can be due to different denervation methods . However, both unnatural decrease and increase in cdk5 expression can be related to some complications and diseases (17, 31). On the other hand, gsk-3 is an enzyme with the ability to do several tasks and involved in numerous cellular processes . Along with its metabolic role, gsk-3 is involved in maintenance and plasticity of skeletal muscle (15). It has been shown that in pain receptors, gsk-3 has a role in morphine bearing and modulation of its pain relieving effects (13). In muscular tissue, gsk-3 has a key role in regulation of muscle atrophy (33, 34). Also it has been shown that inhibition of gsk-3 causes decrease in muscular atrophy (33) and in absence of gsk-3, myofibrils are protected against atrophy (15). Gsk-3 controls expression of atrogin-1 and murf1 (muscle ring finger-1) due to effects of atrophy activators . Expression of atrogin-1 and murf1 in response to igf-1 (insulin - like growth factor-1) or insulin, which are protein synthesis activators, decreases (35). (2005) (33) reported that atrogin-1 and murf1 levels decrease by inhibiting gsk-3 drugs while in absence of gsk-3, dexamethasone injection (which is an industrial glucocorticoid that its injection causes induction of proteolysis) modulates amount of myofibrillar atrophy . Activation of signaling cascades of phosphatidylinositol 3-kinase (pi-3k)/protein kinase b (akt) is due to insulin or igf - i effects (the main regulators of gsk-3 activity), which causes akt activation / protein kinase b (pkb) directly through gsk-3 phosphorylation (36, 37). Akt blockage causes increase in gsk-3 activity and obvious increase in atrogin-1 and murf1 levels (35, 38). Phosphorylated - akt activates (mammalian target of rapamycin) mtor 1 complex and inactivates gsk-3 simultaneously that leads to increase in mrna transcription and protein synthesis capacity (39, 40). Akt decreases gsk-3 activity through phosphorylation and leads to increased expression of mrna derived from eif2b (eukaryotic translation initiation factor 2b) increased activity because of decreased inhibition of eif2b through gsk-3 (41). Gsk-3 has an important role in regulation of protein synthesis and muscular hypertrophy (42). However, acute decrease in gsk-3 of contractile proteins maintains proteolysis of contractile proteins more than excitation of protein synthesis . Initiation of protein transcription is apparently the time - limiting stage in muscular protein synthesis and controlled mainly by eukaryote initiation factors (eifs). Activated gsk-3 is responsible for eif2b inhibition, which eventually causes decrease in the initiation of protein synthesis . On the other hand, inactivation of gsk-3 by upstream kinase of it, akt, causes decrease of eif2b phosphorylation and facilitation of transcription initiation of mrna (41). It was shown that akt is the critical point in signaling cascade of hypertrophy and atrophy (38). Akt was activated after activation of a series of intracellular signaling cascades like igf-1 and pi3k (phosphatidylinositol-4, 5-bisphosphate 3-kinase) (43). Downstream target of akt is gsk-3, which is phosphorylated and inactivated (44) and as a consequence, eif2b would be exited from its inhibitive state (45). Akt may take part in muscular atrophy involvement processes; it is also probable that muscular atrophy will be followed by phosphorylated akt reduction (46). In the present study, it was shown that decreased activity and tight ligation of the l5 spinal nerve in neuropathic pain cause decrease and increase in cdk5 and gsk-3 mrna levels, respectively and these changes are followed by pain - related disorders and soleus muscle atrophy . Generally increase in gsk-3 may be related to muscular atrophy and decrease in protein synthesis, while decreases in cdk5 expression is related to structural disorders and function of skeletal muscle . Thus, it is likely that increased activity in the form of strength and endurance training can contradict unnatural expression of these two proteins in this kind of neuropathic pain . (2013) (47) showed that abovementioned types of training can lead to optimization of cdk5 expression in brain . However, leger et al . (2006) (46) reported increased activity in form of 8 weeks strength training causes muscular hypertrophy by increasing phosphorylated state of akt, gsk-3, mtor proteins.
The majority of cellular proteins exist as symmetric oligomers with distinct biochemical and biophysical properties, which often provide the means for additional regulation of their function at the post - translational level . The study of oligomeric systems in solution is frequently hindered by their large molecular weight, which limits the resolution of nmr spectra due to fast transverse spinspin relaxation rates, and complicates the application of noe methods for the derivation of interface restraints . Moreover, the symmetry inherent to such protein complexes gives rise to spectral degeneracy, as the equivalent spin sites experience the same chemical environment among the protein subunits . Despite the spectral simplification resulting from symmetry, analysis of corresponding noe spectra while more laborious isotopic filtering schemes can be used to distinguish between intra and inter subunit noes, such measurements intrinsically offer lower sensitivity and are often not carried out . Previous methods for the determination of the solution structure of dimeric proteins and protein complexes have often relied upon the availability of interface noes(6) or highly ambiguous distance restraints, such as those obtained from chemical shift mapping experiments . Residual dipolar couplings (rdcs) and small - angle x - ray scattering (saxs) data also have been used as supplementary refinement restraints to improve the convergence of the nmr ensemble . In the absence of distance restraints, in several recent studies, information obtained from fitting of rdc data to previously available structural models of the monomeric subunits was used to limit the degrees of freedom in a rigid - body search, according to the orientation of the alignment tensor . In systems with internal symmetry one of the axes of the alignment tensor must be collinear with the symmetry axis of the system and hence the rigid body degrees of freedom need only be sampled in the plane that is perpendicular to the symmetry axis . Although this method can successfully identify a binding interface that is consistent with the crystal structure, it is inherently limited by inaccuracies in the protein orientation due to lack of precision in the atomic coordinates of the models used to fit the experimental rdc data . Analysis has shown that these inaccuracies can lead to errors in the orientation of the alignment tensor on the order of 510,(16) which can dramatically alter the results of docking calculations . Moreover, these methods rely upon the availability of a previously determined structural model from either x - ray crystallography or conventional nmr methods. (17) the difficulty in interpreting rdc data in the absence of an accurate structural model limits their use in determining the structure of dimers with unknown monomer structure, thus reducing the range of targets that can be studied in solution using rdcs as the only type of experimental data that report on the arrangement of the monomeric subunits . Recent work from our group has shown that the structure of symmetrical assemblies of considerable size can be predicted using modeling methods as implemented in the program rosetta . Symmetric docking in rosetta(18) can provide accurate structures of oligomers with various sizes and topologies, for which the structure of the monomeric subunit has been previously determined using x - ray crystallography . Rosetta can also provide high - resolution structures of multichain, symmetric oligomers with interleaved topologies using a protocol (fold - and - dock) in which the folding and docking degrees of freedom are explored simultaneously. (19) in the current work, we extend these approaches to allow the high accuracy oligomer structure determination from chemical shifts, limited noes, and rdcs . We initially assume the oligomer is nonintertwined and begin by calculating the structure of the monomeric state using sparse nmr data, taking advantage of recent advances in the cs - rosetta modeling methodology . We next use backbone rdc data as domain orientation restraints to dock the monomeric subunits . With such data, the rosetta symmetric docking algorithm can effectively identify the native oligomer structure (provided that the number of monomers in the oligomer is known from experiments), without need for interface noe restraints which are the main source of convergence in previously published protocols addressing this task . If the oligomeric structures produced are not converged, we restart the modeling calculations allowing for the possibility that the oligomer is intertwined by using the rosetta fold - and - dock protocol guided by the chemical shift and rdc information . The method produces accurate oligomer models using backbone chemical shifts and rdcs from 1 to 2 alignment media for all of the benchmark cases studied . We have generalized both the rosetta symmetric docking protocol(18) and the rosetta fold - and - dock protocol(19) to take full advantage of nmr data to guide the conformational search . For noninterleaved oligomers, the nmr data allow fuller sampling of variations in monomer structure and guide homo - oligomer assembly . For interleaved structures, the nmr data better define the local structure and guide formation of the correct dimer interface . In the first step, models of the free monomer are generated using rosetta supplemented with either chemical shifts alone(21) or chemical shifts and a small number of backbone noes, depending on available data . Previous work has shown that by using chemical shifts alone, convergence to the correct structure is generally achieved for proteins of 100 amino acids, while larger structures of up to 150 amino acids often can be determined using a very limited number of noes . At this point having sampled a diversity of low - energy conformations at the monomer stage (figure 2a), we then proceed to rosetta symmetric docking(18) supplemented with domain orientation restraints from rdc data . The rigid body orientation of the monomers and the side chain conformations are optimized by monte carlo minimization, as described in materials and methods. (24) the lowest energy dimers sampled are then subjected to simultaneous refinement of backbone, side chain, and rigid body degrees of freedom to allow for restricted (within 1 rmsd), local adaptations of the backbone to the final docked orientation of the dimer . The low - energy refined structures from independent docking runs starting from different monomers are pooled, and convergence is then assessed by structural clustering and by evaluation of pair wise rmsds within the low - energy pool . The structural ensemble is considered converged if the 10 lowest - energy structures can be clustered within 3 backbone rmsd to the center of the cluster (a larger rmsd threshold could be useful for oligomers with more disordered loops). If there is clear convergence in the low energy population, the converged ensemble is taken as the computed model of the complex . Otherwise, if convergence is not observed, the oligomeric complex may be intertwined, and the rosetta fold - and - dock protocol(19) supplemented with rdcs is carried out as described below . This pipeline enables the structure determination of dimers showing a variety of interface types (figure 1). Cs - rosetta uses backbone chemical shift assignments to derive an ensemble of models for the monomer . Convergence at this step is used as an indication of a noninterleaved interface, in which case symmetric docking is performed for each monomer seed from the low - energy ensemble . If convergence is not observed, the complex may be intertwined and the internal and rigid body degrees of freedom are optimized simultaneously using the fold and dock protocol guided by the nmr data . (a) in the first step, the cs - rosetta protocol produces a low - energy ensemble of full - atom conformations, showing a high degree of convergence to the monomer in the nmr structure (pdb i d 2jwk). An overlay of the 10 lowest - energy structures (color) on the native structure (gray) is shown in the structure diagram on the bottom . Only backbone chemical shift data are sufficient to produce this result (no rdcs were used at this step). (b) in a second step employing rdc data, the low - energy monomer conformations from a, in this case produced in docking calculations from monomers 10 and 5, are docked in a symmetric manner to produce homodimeric structures that are within 1.5 from the previously reported nmr structure of the dimer, obtained using rdcs and a full set of interface noes. (10) the structures generated by the protocol map the energy landscape of the complex, subject to the experimental constraints . The funneling of the energy toward the native structure shows that among the diversity of backbone structures obtained at the monomer stage there exist conformations that can provide the correct backbone scaffold for convergence toward the native structure of the dimer . In this case the low energy docked conformations generated with different monomer seeds converge on the native structure of the dimer . The use of rdcs biases sampling of the rigid body degrees of freedom toward experimentally relevant regions of the conformational space and further helps to discriminate against low - energy, non - native conformations . We compare the results of our method to docking results previously obtained using rdcs as the only type of interdomain restraints (table 1). The structure of the homodimer ykuj has been previously determined by both x - ray crystallography as well as a protocol based on a fixed symmetry axis forced to coincide with one of the principal axes of the alignment tensor. (15) using our new method we find a low - energy, converged ensemble that agrees with rdc data collected in two alignment media, as indicated by rdc q - factors of 0.26 and 0.18 respectively, and which falls very close (0.9 backbone rmsd) to the x - ray structure (figure 3a). For the side chains of most interface residues, there also is a high degree of convergence to the rotamers observed in the crystal structure (0.4 rmsd for all interfacial atoms), which reflects the use of rosetta s full - atom energy function and the fact that all backbone and side chain degrees of freedom are optimized to accommodate structural changes arising from interactions between the monomeric subunits (figure 3b). Such a degree of atomic detail in the absence of interface distance restraints could not be obtained using the rigid body search with the coarse energy terms presented in the earlier study. (15) using a similar protocol to fix the symmetry axis, in addition to employing paramagnetic surface mapping, lee and co - workers reported a structural model of the weakly associated homodimer ser13. (14) we applied our protocol to compute the dimer structure using only backbone chemical shifts and rdc data from two alignment media . The resulting structural ensemble is in excellent agreement with the measured rdcs (q - factors of 0.26 and 0.24 respectively) and shows a high degree of convergence to the structural model of lee et al . (figure 4c). (14) rdc (number of alignment media, number of rdcs per medium per monomer). Typical estimated rdc errors are 0.51 hz . The rmsd calculated here for backbone atoms in the monomer / dimer / interface (defined here according to a 3.5 distance cutoff). Q - factors calculated according to cornilescu and co - workers(52) for alignment media a / b (if available). When using saxs data in addition to rdcs, the dimer structure can be determined using a limited data set of 68 nh rdcs . Indicating comparison to a low - resolution structural model or structure of a homologous protein . Indicating a dimer with an interleaved interface . Structural convergence of the method for bacterial protein ykuj . (a) backbone of the 10 lowest energy conformations generated using rosettaoligomers (blue) superimposed on the crystal structure of the dimer in red (pdb i d 2ffg). (b) detailed view of the side chains at the interface of the dimer, presented in a view that is perpendicular to the interface . Good convergence of the low energy conformations to the pdb structure is indicated by backbone rmsd values for the interface residues of ca . 0.4 (here the interface is defined as all residues within 3.5 from the other subunit). (c) without the use of rdcs as orientation restraints during the search the docking algorithm converges to an alternative structural ensemble that uses a -sheet interface to form the dimer . The lowest - energy dimer conformations obtained using rosettaoligomers are superimposed on the previously determined structures (in red). (a) arabidopsis thaliana hypothetical protein and structural genomics target at5g22580, showing good agreement to the previously published x - ray structure (pdb i d 1rjj). (b) periplasmic protein tolr (pdb i d 2jwk), previously determined using a docking protocol in cns(25) using rdcs and a set of 65 interface noes . (c) weakly associating homodimer ser13, previously modeled using a rigid body search of monomer orientations around a fixed axis of symmetry (pdb i d 2k1h). (d) catalytic core domain of the hiv-1 integrase dimer (residues 50212), showing 1.3 backbone coordinate rmsd relative to the previously determined x - ray structure (pdb i d 1bis). Taken together, these results show that our method can reproduce the results obtained using a fixed symmetry axis and that the produced structural ensembles show a high degree of resolution and convergence to the native structure . Moreover, in the case of c2 symmetry considered here, by searching all four independent rigid body degrees of freedom simultaneously rather than fixing the symmetry axis of the system according to the rdc alignment tensor, the present method is robust to errors in determining the axis of symmetry from the rdc data due to structural noise present in the monomer models . We have also evaluated the performance of the new method relative to established protocols that make use of interface noes as well as rdcs and other data types . For the periplasmic protein tolr, previously determined with the program cns(25) using a full set of distance restraints, including 65 interface noes, rdcs, and saxs data,(10) our method converged to a solution within 1.5 rmsd for the backbone and 0.6 rmsd for interface atoms from the published structural ensemble (figure 4b). This was achieved using only backbone chemical shifts and rdcs, far less data than the cns calculation . Convergence of the symmetric docking calculations improved when increasing amounts of rdc information was utilized; best results were obtained using all four internuclear vectors (nh, cc, cn, ch). Similar results were obtained for the protein at5g22580, a 101-residue structural genomics target from arabidopsis thaliana . The structure of the at5g22580 dimer was previously determined using rdcs, dihedral and hydrogen bond restraints, and to a set of 2117 assigned noes of which 31 were intermolecular. (26) in contrast, using only rdcs to dock the dimer, the current protocol converged to a 1.7 rmsd for the interface atoms (defined according to a 3.5 distance threshold between any pair of atoms on the monomeric subunits) relative to the nmr ensemble (figure 4a). This illustrates the merit of our approach for solving the solution structure of larger - size homodimers (> 100 residues), for which obtaining interface noes can become challenging and labor intensive . The approach used in the above examples assumes only modest structural adaptations (within 1 rmsd) of the backbone due to the interactions between the monomers . However, the assumption that the monomers can fold as independent chains does not necessarily hold for all homo - oligomeric structures . A concern regarding the general applicability of the current strategy is its performance in the case of homo - oligomers with a significant degree of interaction between the two subunits, such as found in domain - swapped multimeric systems. (27) we have tested whether we can diagnose such cases without any a priori knowledge on the degree of interaction between the monomeric subunits . We considered the homodimer yiif from shigella flexneri with interleaved backbone topology (pdb i d 2k5j), involving the formation of a -sheet using strands from the two monomers . As expected, cs - rosetta calculations of the individual monomers failed to converge (figure 5a); the native state cannot be energetically distinguished by considering only interactions within the monomer . If nevertheless the low energy, partially unfolded monomers are used as starting points in the symmetric docking protocol we obtain a converged structural ensemble, which shows a significant degree of interaction between the chains (figure 5b and d) that further suggests an interleaved dimer . The previously published fold - and - dock protocol(19) is a more suitable treatment for this system . When improved with the use of rdc data, this method converges to a 0.5 interface rmsd structure relative to the previously reported nmr ensemble (figure 5c and e). The converged low - energy structures resulting from this protocol (figure 5e) are consistently lower in energy than the partially unfolded dimers obtained with symmetric docking (figure 5d), providing further indication that the structure is interleaved . Results for the yiif interleaved dimer using the two different modes of the rosettaoligomers structure determination pipeline . Results from the application of cs - rosetta followed by rdc - assisted symmetric docking (a, b) are contrasted to results obtained using the fold - and - dock protocol supplemented with rdcs,(19) specifically designed to predict the structure of interleaved dimers (c). This does not converge to a single structure, due to the fact that interactions within the dimer are essential for folding the monomeric subunit . (b) converged docking solutions . Using the nonconverged low - energy conformations from a, converged docking solutions are obtained (d) which contain a major part of the interaction interface but are missing the intersubunit -strand pairing . (c) correct native structure of the dimer . Using the previously published fold - and - dock protocol, all structure diagrams the native structure (pdb i d 2k5j) is shown in red, and the lowest - scoring rosetta structure, in blue: x - axis backbone rmsd relative to the crystal structure (); y - axis rosetta full - atom energy, supplemented with an rdc energy term . We have further tested this approach for the protein atu0232 from agrobacterium tumefaciens (pdb i d 2k7i). The previously determined solution structure shows a complicated interleaved interface with an intermolecular 5-strand -sheet in which subunit forms strands 24, while subunit forms strands 1,5 . Again, the monomer cs - rosetta calculations and symmetric docking calculations starting from the cs - rosetta monomers do not show convergence, indicating an interleaved dimer interface . The fold - and - dock protocol, supplemented by 45 rdcs, converges to a 2.5 structure, which shows the correct interleaved backbone topology (supporting information figure 1). Very similar results were obtained for the structural genomics target kr150 with remote homology to the protein sp_0782 from streptococcus pneumoniae for which a crystal structure of the dimer (pdb i d 3obh) shows an interaction interface containing an exposed -helix . Using the rosetta fold - and - dock branch of the protocol together with the backbone chemical shifts, a set of 67 manually assigned backbone noes, and rdcs, we obtain a dimer structure that is within 2.6 backbone rmsd from the homologous structure (supporting information figure 2). Furthermore, the structures are very similar in fold to the structures obtained using conventional structure calculations and a full set of noes . Good agreement with the rdcs is indicated by a q - factor of 0.32 (see materials and methods for definition of the q - factor). Again, the lack of convergence for the monomer cs - rosetta calculations are an indication of a potentially interleaved dimer and the user is guided to use the fold - and - dock branch of the protocol (figure 1). An intermediate case of a dimer with a semi - interleaved interface topology is the hiv-1 capsid protein (ca) c - terminal domain (ctd). Dimerization of the ctd results in the crosslinking of individual 5mer and 6mer rings formed through interactions of the n - terminal domain (ntd), which promotes the assembly of the virus capsid . The solution structure of the protein has been previously determined using rdcs, talos dihedral angle and hydrogen bond restraints, and a large set of noes, of which 210 were intermolecular. (28) the structure determined in solution fits well into the cryo - em density map and shows a semi - interleaved dimer interface in which the n - terminus of the ctd (residues 145151 in the full - length protein) fit into a helical groove on the symmetric subunit, as confirmed by the observation of several intersubunit noes (pdb i d 2kod). Here, we have used the chemical shift assignments from solid - state nmr experiments reported in reference (29) and backbone nh and ch rdcs measured in solution(28) as the only source of experimental information to dock the dimer into a structure that is very close (1.2 interface rmsd) to the previously reported nmr ensemble (supporting information figure 3). The side chains of residues trp 184 and met 185, shown previously to be crucial for dimerization,(30) are found in the core of the interface forming packing interactions in the dimeric structure . Moreover, the monomeric subunit shows a kink in helix 9, which forms the core of the dimer interface, in agreement with the nmr structure and cryo - em density. (28) in this case, we used the fold - and - dock protocol followed by symmetric docking optimization of the obtained low - energy conformations using perturbation runs (see materials and methods). Although the low - energy conformations obtained from the fold - and - dock protocol showed moderate convergence, the use of symmetric docking optimization of the low - energy conformations resulted in improved convergence and recovered a docking funnel towards the native structure of the dimer . We also evaluated the capability of rosettaoligomers to determine the structure of dimers built from larger monomers, using as a test case the 36 kda homodimeric catalytic core domain (residues 50212) of the hiv-1 integrase enzyme (in), whose structure was originally solved by x - ray crystallography . Solution studies of a soluble variant of the wild - type sequence containing five point mutations have shown that it exists in a conformation for which the monomeric unit is very similar to that seen in the crystal structure; however, the data collected in solution were insufficient to determine the structure of the dimer. (33) the same study found that this variant of in also exists predominantly as a symmetric dimer . We measured rdcs in two different alignment media (see materials and methods for data collection details). With the use of backbone chemical shift - derived fragments, a converged structural ensemble is obtained for the monomeric subunit that falls very close to the monomeric subunit observed in the crystal structure (within 1 backbone rmsd calculated for residues in the well - ordered regions of the structure, secondary structure elements and structured loops). Consistent with earlier nmr data, the final ensemble shows a high degree of structural variability in the loop connecting the two c - terminal helices, spanning residues 185195. (33) moreover, the catalytic loop spanning residues 140153, previously shown to be conformationally dynamic by n relaxation analysis, is found to be structurally variable in the low - energy monomer ensemble derived by cs - rosetta (supporting information figure 4). Starting from the ensemble of monomers, using symmetric docking and the rdcs obtained in two alignment media, a converged dimer structural ensemble is obtained (figure 6 inset). This ensemble is in good agreement with the crystal structure of the wild - type sequence, with a 1.3 backbone rmsd over the regions with well - defined electron density (excluding the disordered active site loop at residues 140153). The c - terminal helix spanning residues 197209, which forms part of the dimer interface, is in a very similar orientation as that in the crystal structure . The rmsd of the interface atoms is 0.7, and there is a high degree of convergence of the interface side chains to the rotamers observed in the crystal structure . In the final dimer ensemble, the lack of structural variability for the catalytic loop is due to the fact that a single monomer conformation seed happened to provide most of the low - energy solutions at the symmetric docking stage . Solution structure of the hiv integrase dimer determined with rosettaoligomers using backbone chemical shifts and rdc data . The structure of the catalytic core domain of the hiv integrase homodimer (residues 50212) was determined using exclusively solution nmr data . Backbone chemical shifts were used to solve the structure of the monomer, which was then docked in a symmetric manner with the use of nh rdcs measured in two alignment media. (56) two types of docking calculations, starting from the native dimer orientation (red) and starting from a completely randomized orientation (green) both converge to the same energy minimum, indicating global convergence of the method to the x - ray structure (pdb i d 1bis), as shown in the structure diagram (inset). The rmsd is computed for the backbone atoms of the well - ordered regions of the molecule, as described in the main text . An ensemble of the 10 lowest - energy conformations (shown in color) is superimposed on the x - ray structure (shown in gray). An rdc q - factor of 0.3 for both alignment media indicates good agreement of the final ensemble to the rdc data . These results are consistent with the observation of a single, ensemble - averaged resonance for each atom in the nmr spectra and show that the solution data are consistent with the dimeric structure observed in the crystal state, as also indicated by rdc quality factors of 0.3 and 0.32 for rdcs collected in liquid crystalline phage and peg media, respectively . The high degree of consistency between experimental rdcs and the ones calculated from the dimer models presented here suggest that our method offers a reliable way to interpret limited solution data in deriving structural models of a quality that approaches high - resolution x - ray structures . We have further tested the practical usefulness of our approach for the symmetric modeling of larger - size oligomers by applying it to determine the structure of the equine infectious anemia matrix virus protein homotrimer using previously published nmr data. (34) previous solution studies have shown that the protein exists in equilibrium between a monomer and a trimer; however the structure of the trimer has not been previously determined in solution . Using rdcs and backbone chemical shifts alone, our approach converges to a trimer structure that is in agreement with the rdc data (q - factor of 0.4). Moreover, the structure determined here is in qualitative agreement with chemical shift mapping results from titration experiments that report on the residues that form the trimer interface (shown as red spheres in supporting information figure 7). On the basis of the structure of a remote homologue, a different trimer organization was previously suggested;(34) the two alternative models should be distinguishable in future work using additional solution data, such as small - angle x - ray scattering and intermolecular noes . The p53 oligomerization domain was the oligomer with the largest number of subunits evaluated here . Previous experimentally determined structures by both solution - state nmr and x - ray crystallography(38) have shown that it exists as a tetramer of d2 symmetry (a dimer of dimers), in which the basic dimer has an interface with an interleaved topology . Deriving the correct topology for such a system out of the large possible number of arrangements that would be compatible with identical resonance positions for the four components of the tetramer presents a difficult challenge . This problem was solved correctly by a detailed analysis of multiple isotope - edited and isotope - filtered nmr spectra . Modest differences between the original nmr structures and the subsequent 1.7 resolution x - ray structure in terms of backbone rmsd (1.2 and 1.9 to the mean coordinates of the two nmr ensembles, respectively) in part reflect the technical challenge in obtaining a high accuracy solution structure from nmr data . Subsequent refinement of the nmr structure(39) with the addition of multiple interdomain noes (through a more exhaustive peak - picking in the noesy spectra) resulted in a more compact interface between the dimers and interhelical angles that are closer to those seen in the x - ray structure (backbone rmsd of 0.6). (38) all these solution studies employed 2d, 3d, and 4d heteronuclear - separated and isotope - filtered noesy techniques using samples of both uniformly labeled (n, c) and mixed heterotetramers with equal amounts of labeled and unlabeled proteins, to distinguish between noes arising from interactions between the different subunit combinations . Together with other types of experimental information, such as chemical shifts, j - couplings, and hydrogendeuterium exchange, numerous restraints (e.g., 4472 restraints in reference (39) of which 3752 were distance noes) were used to derive converged structural ensembles . The number of intersubunit restraints used in reference (35) was 864, including 840 noes and 24 hydrogen bond restraints . Using our method and backbone chemical shifts alone, we obtain a converged structural ensemble that falls very close to the x - ray structure in terms of backbone rmsd (1.1 for all backbone atoms, 0.3 for all interface atoms, defined according to a 3.5 distance cutoff). A comparison with the crystal(38) and conventional nmr structures(35) (figure 7) illustrates that the structure determined here is of comparable quality to the one determined using standard (and more laborious) nmr structure determination protocols, using a much more limited set of data (only n, h, c, c, and co assignments were sufficient to obtain a converged structural ensemble), which is typically the starting point in data collection for nmr structure determination . Although we assumed d2 symmetry to obtain this result, c4 symmetry (the only other alternative for a 4-subunit protein) was excluded on the basis of separate calculations: with this type of symmetry, the calculations do not converge to a single structure and result in average energies that are far greater than when using d2 symmetry . This shows that our method also has the potential to dinstinguish between different point groups in simple cases . Taken together, these results indicate the practical use of our approach for determining the structures of symmetric oligomers of various numbers of subunits and symmetry groups . The structures of the p53 oligomerization domain tetramer (d2 symmetry) determined using x - ray crystallography(38) (pdb i d 1c26) (red), solution - state nmr(35) (pdb i d 1olg) (green), and our method (blue) are superimposed on a same reference frame . Only backbone chemical shifts were used here to determine a highly similar structure, otherwise obtained using a full set of assigned intersubunit noes . We have evaluated the use of small - angle x - ray scattering (saxs) data in our approach for the protein tolr using previously published data. (10) to calculate saxs curves from the coordinates of the sampled conformations we have implemented a method that uses a coarse - grained representation of the protein with residue - specific form factors that have been parametrized using a database of high - resolution protein structures. (40) a score term that is proportional to the rms from the experimental data is used in both the low - resolution search and full - atom refinement stages of the symmetric docking calculation (supporting information figure 6c). When supplemented by saxs data alone, the symmetric docking calculations converge to two local minima, showing that the use of saxs data effectively eliminates the search in many additional false minima of the docking energy landscape otherwise observed in an unbiased calculation (supporting information figure 6b, green versus red points). Inspection of representative dimer structures from each minimum shows that one corresponds to the native structure, while the other is a dimer in which one of the monomeric subunits is inverted relative to its native orientation (supporting information figure 6d, e). This results in very similar saxs profiles (supporting information figure 6a) and rosetta energies, suggesting that additional data types, such as rdcs, are needed for full convergence to a single structure . In fact, with the use of rdcs for the nh bond vectors alone in addition to the saxs data, our method converges to the native dimer structure (supporting information figure 6b, blue points). This indicates that the use of saxs data can complement rdcs in dimer structure determination, by reducing the amount of rdc data required to achieve convergence (68 vs 261 rdcs required to achieve convergence in the absence of saxs data). The strategy presented here enables the determination of the higher - order structure of protein dimers using exclusively nmr data, such as backbone chemical shifts and amide nh rdcs, without the need for any prior structures of the monomeric subunits . In all cases tested here, the method converges on structures similar to previously published high - resolution dimer structures obtained by x - ray crystallography or by conventional nmr structure determination protocols making use of interface noes . Moreover, the computed structures show details in terms of side chain orientations at the interface that are very similar to those determined using high - resolution methods . It is perhaps surprising that accurate models of oligomers can be generated from chemical shift and rdc data alone . The success of our approach illustrates the power of molecular symmetry in confining the search space and making modeling more tractable . Even with the constraints provided by symmetry, it is expected that for larger systems, inaccuracies in determining the monomeric structure from chemical shifts alone and the existence of many local minima in the docking energy landscape would make additional data necessary to unambiguously converge on the native structure of the oligomeric complex . Rosettaoligomers provides an automated pipeline for deriving accurate dimer structures by nmr that can be readily applied in high - throughput structural genomics initiatives . The approach described here for homodimers can be readily extended to trimers and other size homo - oligomers of various symmetry groups, as illustrated for the equine anemia virus matrix protein trimer and the p53 tetramer . However, it is expected that in larger systems, inaccuracies in determining the monomeric structure from chemical shifts alone and the existence of many local minima in the docking energy landscape would require additional data to unambiguously converge on the native structure of the oligomeric complex . It is anticipated that incorporation of additional data types that report on the interface (sparse noes) and shape (saxs) of the protein complexes will enable the structures of much larger oligomeric systems with internal symmetry to be solved, further expanding the range of biologically important systems amenable to solution nmr . Our method is ready to support such data types, thus providing a powerful tool for determining the solution structure of symmetric protein assemblies . We have used the cs - rosetta method as described previously to determine the ensemble of the monomeric subunit . All protocols used here can be downloaded as part of the standard rosetta 3.0 distribution(41) (svn version 39640 can be obtained at http://www.rosettacommons.org/). To run cs - rosetta starting from an extended polypeptide sequence, we first select backbone conformations of all possible overlapping residue fragments of three and nine residue lengths that are consistent with the recorded backbone chemical shifts . Also, 200 fragments are selected for each residue position from a chemical shift - annotated database . To perform this task, we are using a new fragment search method (manuscript in preparation), with information such as backbone secondary structure prediction using the talos+ program(43) and sequence profile information provided by the program psi - blast. (44) the weights for the different types of selection criteria are given in a separate weights parameter file (see the supporting information). This method is robust to incomplete assignments to as low as one atom type per residue,(45) and, prior to running the structure calculations in cs - rosetta, can be executed as a stand - alone application, using the command line options shown in the supporting information . In all cases attempted here, good convergence of the cs - rosetta protocol was obtained by running 10 00020 000 calculations on a linux - based cluster . In all cases used here to benchmark the method, homologues present in the fragment database were excluded from our analysis according to a sequence similarity criterion (psi - blast score of 0.05 or less) and by manual exclusion from the fragment database of the structures that were highly represented in the selected fragments (present in more than 10% of all sequence positions) and showed structural similarity to the target proteins . Having obtained fragments of lengths three and nine residues using chemical shift information, we proceed to the cs - rosetta monomer calculation using the recently implemented minirosetta application,(41) which also supports the inclusion of any available noe and rdc constraints . The symmetric docking protocol,(18) that was adapted to use rdc data, was then used to dock the low - scoring monomers extracted from the cs - rosetta runs . In this protocol, the individual subunits are assumed to be perfectly symmetric about a user - defined axis . A detailed description of the implementation of symmetry used here is included in the original publication. (18) for the c2 symmetry used here, or any type of cyclic symmetry, the orientation of the symmetry axis is defined in an input symmetry definition file . By default alternatively, the program can take an arbitrary symmetry axis as the axis of symmetry, which can be extracted in the form of a symmetry definition file from a pdb input file containing the coordinates of the dimer chains (a, b) using an in - house script, which is part of the standard rosetta svn distribution (an example of running the script is shown in the supporting information). Using the symmetry definition file prepared in this manner and the input pdb files for the monomer conformations, we then perform symmetric docking calculations using the symdock application (svn version 39640 can be obtained at http://www.rosettacommons.org/) as described in the supporting information . In all cases attempted here, good convergence of the symmetric docking protocol was obtained by running 10 000 calculations on a linux - based cluster . Starting from a completely randomized orientation between the monomeric subunits around the symmetry axis, the symmetric docking protocol performs iterations of monte carlo - based optimization of the rigid body and side chain degrees of freedom in two steps: in a first, low resolution step using a coarse energy function, the rigid body orientation of the monomeric subunits is randomly perturbed and the two subunits are translated into contact along an axis that is perpendicular to the symmetry axis . At this step, side chains are represented using a single, residue - specific pseudoatom, positioned at the c carbon . Monte carlo trials of the total energy of the system are used to find a local energy minimum of the rigid - body orientation of the symmetric subunits . In the second, more time - consuming high - resolution step, rosetta s full - atom energy function is used with a soft - repulsive term for van der waals interactions . During this stage, the side chains are combinatorially optimized and the rigid body and side chain degrees of freedom are subjected to quasi - newton minimization after which the trial is accepted or rejected according to a metropolis criterion. (46) up to this point, the backbone is kept fixed to that of any of the lowest energy conformers, obtained by the cs - rosetta structure determination for the monomer . A final relaxation step of all degrees of freedom, including the backbone dihedrals and side chains, was implemented in this study to account for local structural changes due to the interactions between the monomeric subunits, according to the algorithms described previously. (47) the adaptation in backbone rmsd during this step was found to be less than 1 for all proteins tested here . This step also allows for improved discrimination of the native docking funnel in rosetta s full - atom energy . To evaluate the robustness of our docking approach and to test the presence of a clear energetic signature of the native state in the rosetta full - atom energy, for all test cases, we performed independent docking perturbation studies as previously described in reference (24). Starting from a symmetry definition file prepared using the native structure of the dimer as input, the orientation of the monomeric subunits was randomly perturbed by a displacement and a rotation around each one of the three axes drawn from gaussians centered at 3 and 5, respectively . The correct orientation between the monomeric subunits is consistently recovered in docking calculations using a small perturbation of the native dimer structure (red scatter plot in figure 6) as well as using a completely randomized orientation of the two monomers (green scatter plot in figure 6). This indicates a high degree of convergence of the docking algorithm to the lowest - energy structure and further shows that rosetta s all - atom energy function enhanced by the rdc energy term is able to discriminate the native structure from the many non - native local energy minima . In the cases of dimers with interleaved interfaces, the fold - and - dock protocol simultaneously explores the folding and docking degrees of freedom, as described previously. (19) the protocol consists of four low - resolution stages of increasing complexity in the energy function, in which symmetric fragment insertions are interleaved with symmetric rigid - body trials . Finally, symmetric repacking of the side chains and gradient - based minimization of the side chain, rigid body, and backbone degrees of freedom are applied . In the current implementation first, a symmetry definition file is constructed in the same manner as described previously for the symmetric docking protocol . Overlapping residue backbone fragments are selected according to the same methods used in cs - rosetta . This application is included in the rosetta 3.0 software suite(41) and can be run as described in the supporting information . In one case (see ca dimer), the use of symmetric docking runs starting from the low - energy conformations obtained from the fold - and - dock protocol by perturbing the orientation of the individual subunits in the dimer was found to greatly improve convergence and native fold - discrimination . To perform such perturbation runs, a symmetric definition file is prepared using as input a conformation from the fold - and - dock low - energy ensemble, followed by symmetric docking as previously described . In all cases attempted here, good convergence of the fold - and - dock protocol was obtained by running 20 00030 000 calculations on a linux - based cluster . During the symmetric docking or fold - and - dock protocols, rdc - based restraints were constructed by duplicating the measured rdc values for each subunit of the dimer . The singular value decomposition method as described by losonczi and co - workers(48) was used to determine the elements of the alignment tensor that best fit the experimental data in the least - squares sense and to calculate rdc values given a structural model . The jacobi method was implemented to calculate the eigenvalues of the order matrix for subsequent analysis. (49) using this treatment, one of the axes of the order matrix is collinear with the symmetry axis of the system . Finally, a term that is proportional to the rmsd between experimental and calculated rdcs was used during the monte carlo trials and gradient - based minimization, according to the implementation previously described by hess and scheek, which allows for gradient - based optimization of the rdc target function. (50) initial estimates for the magnitude of the alignment tensor, for the purpose of rescaling data sets from multiple alignment media, were obtained from a powder pattern distribution of the rdc data. (51) for the purpose of validation of final structural models, we have calculated q - factors, defined asafter cornilescu and co - workers. (52) the catalytic core domain (residues 50212) of hiv-1 integrase (strain nl4 - 3) was expressed recombinantly and purified as described previously. (33) the soluble q53e c56s w131e f185k q209e variant was used for all experiments . Perdeuterated, n-, c - labeled protein was concentrated by centrifugal ultrafiltration to 500 m monomer concentration (250 m dimer) in 100 mm nacl, 20 mm pipes buffer (ph 6.5), 40 mm mgcl2, 0.5 mm tris(2-carboxyethyl) phosphine (tcep), 0.02% (w / v) nan3, 6% (v / v) d2o . The sample was separately aligned in two media, bacteriophage pf1 obtained from asla biotech (riga, latvia) and 4% (w / v) c12e5 polyethylene glycol (peg)/n - hexanol. (55) for the pf1-aligned sample, pf1 was added to a final concentration of 12 mg / ml, and the nacl concentration was increased to 200 mm to reduce nonspecific interactions between the protein and the phage . The h quadrupolar splittings were 8.3 and 19.6 hz in the pf1 and peg - aligned samples, respectively . Rdcs were measured at 25 c on a bruker avance - iii 900 mhz spectrometer, equipped with a triple - resonance cryogenic probe . Couplings were obtained from 2d nh trosy - hsqc spectra using the artsy technique. (56) the n acquisition time was 80 ms (250 complex points), and the h acquisition time was 110 ms (1784 complex points). Nmr sample preparation and backbone assignments of kr150 using standard triple resonance experiments were performed as described previously. (57) rdcs were measured in 4% (w / v) c12e5 polyethylene glycol (peg)/n - hexanol using a j - modulated experiment.
Creutzfeldt - jakob disease(cjd) is a subacutely progressive neurological disorder caused by deposition of prion proteins in brain . It is a very rare entity and the world - wide incidence is 0.5 - 1.0 cases per million per year . A majority of cases are sporadic, some cases are familial, carrying a prion protein mutation and it is usually fatal within 1 year . We report the significance of mri findings to correlate and support the clinical diagnosis of cjd . A 75 year - old lady presented with rapid cognitive decline of three months duration . A patient was evaluated as a case of dementia and had frontal, parietal, temporal, and occipital deficit signs on neuro - psychiatry evaluation . Mri was done as a part of her investigation on a 1.5 t ge (signa, usa) scanner, which showed diffuse cortical t2 and flair hyperintensities in bilateral frontal, parietal, temporal, and occipital locations [figures 1 and 2]. The cortical hyperintensities showed significant restriction on dwi [figure 3], giving rise to the strikingly conspicuous lesions compared to the normal cortex signal in the above locations . Axial t2 w mr images showing faint and equivocal cortical hyperintensities (arrows) in bilateral frontal, parietal, and temporal lobes . Axial flair mr images shows the signal abnormalities slightly better compared to figure 1, although cortical hyperintensities (arrows) are not very striking . Diffusion weighted mr images clearly shows increased visibility of high signal from restricted diffusion (arrows) in the bilateral frontal, parietal, temporal, and occipital regions along the cortical locations . Later in the course of the disease, she developed myoclonic jerks, eeg changes which were consistent with the who criteria of probable cjd . According to the national cjd registry at nimhans, bangalore, india, there are only 85 recorded cases of cjd till september 2005 . The diagnostic triad of cjd i.e., of a progressive dementia, myoclonus and periodic sharp wave eeg activity may not be evident in about 25% of the cases . Mri with dw and flair sequences is an invaluable modality in supporting the diagnosis of cjd . Shiga et al in his study concluded that diffusion - weighted mri(92%) had higher sensitivity in the detection of cjd than flair sequences (41 - 59%), t2 (36 - 50%), eeg (50 - 78%), csf protein 14 - 3 - 3(84%) or neuron specific enolase (73%). Diffusion - weighted mri is more resistant for motion artifacts than t2w and flair images . The abnormalities on dw - mri could be detected as early as at 3 weeks of symptom duration and even before the appearance of periodic triphasic waves on eeg . Matoba et al noted that the hyperintensity in the basal ganglia and cortex during the early stages was more extensive and conspicuous while in the later stages there was disappearance of the abnormal signals in the cortex . The cause for restricted diffusion is attributed to accumulation of abnormal vacuoles in the cytoplasm and microvacuolation of neuritic process heralding spongiform degeneration . Sporadic cjd shows t2 prolongation and restricted diffusion in the corpus striatum followed by the neocortex and posterior and medial thalami . In variant - cjd, the t2 hyperintensities are commonly located in the posterior (pulvinar) and medial thalami followed by periaqueductal grey matter, striatum, and less commonly in the neo - cortex . The increased signal intensity in the pulvinar relative to the anterior putamen is called the pulvinar sign is the most sensitive marker for variant cjd . The bilateral pulvinar sign has a sensitivity of 78% and correlates with the histological gliosis . The combination of flair and dw imaging has a sensitivity, specificity and accuracy of over 90% in differentiating cjd from other dementias . The multifocal cortical and subcortical hyperintensities in the grey matter showing restricted diffusion on mri may be more useful than the csf protein 14 - 3 - 3 analysis . 14 - 3 - 3 protein analysis was not performed because of the lack of the availability of the test at our institute . Csf protein 14 - 3 - 3 analysis availability is limited only to a few cities and is not done routinely in all the hospitals . However, diagnosis can only be confirmed by histological examination of brain tissue obtained either by the brain biopsy or after autopsy . These are not performed by many institutions due to the transmissible nature of the disease . This case highlights the role of dw - mri as practical modality in the early diagnosis of cjd . Mri radiologist can give the first clue to an unsuspected case of cjd, as in this case and can make it easy for the clinician who has to do an elaborate work - up of dementia cases . Radiologists and neurologists should be familiar with the characteristic appearance of dw imaging as a clue to the diagnosis of this dreaded disease, for which no treatment is available till date . In a patient with rapid dementia, mri should be done as it is accurate, noninvasive, and cost - effective (as compared to csf-14 - 3 - 3 analysis) and if findings are suggestive of cjd, the clinician should be alarmed and appropriate infection control measures are to be taken.
Forearm fractures are the most common childhood fracture that presents to the emergency room in the united states . Despite improvements in many childhood conditions, the incidence of forearm fractures in pediatric patients has increased throughout the last few decades . More concerning, children with a history of a forearm fracture have a higher risk of entering adulthood with decreased peak bone mass . Although most research efforts have focused on fractures in late adulthood, there is emerging evidence that fractures in childhood may be the initial insult that leads to lifelong bone fragility . In neonates and children, vitamin d deficiency has been directly linked to suboptimal bone mineralization and adverse conditions such as rickets and short stature [46]. Specifically, vitamin d is an important regulator of calcium (ca) and phosphorus (p) homeostasis, the minerals necessary for bone formation . Through its interactions in the kidney and intestine, activation of vitamin d stimulates bone mineralization [8, 9]. Furthermore, by the time children enter adolescence 40% of their peak bone mass has been accrued . Dual - energy x - ray absorptiometry (dxa) imaging is a widely available clinical tool used in adults to diagnose osteoporosis, predict fracture risk, and monitor response to therapy . With increased concern for future bone health, investigations using dxa in the pediatric population are becoming an area of high research interest [12, 13]. Similar to adults, dxa is the preferred imaging modal to measure bone mineral density (bmd) and bone mineral content in children due to the precise results, minimally invasive approach, and little exposure to radiation . Multiple studies have demonstrated an association between vitamin d deficiency and fracture risk in children [1417]. However, few studies have correlated vitamin d deficiency with bmd, fracture risk, and laboratory markers of bone health . To our knowledge, no study has evaluated vitamin d status, bmd, and serologic markers of bone health in children from a developing country presenting to the emergency room with a forearm fracture . Therefore, the aim of this study is to determine the prevalence of vitamin d deficiency and its effects on bmd in egyptian children presenting with forearm fractures . We hypothesize that egyptian children with forearm fractures have lower serum 25-hydroxy - vitamin d levels and bmd z - scores compared to children without forearm fractures . This study was conducted at ain shams university hospital (asuh) in cairo, egypt . The ethical committee of asuh approved the study which was conducted between december 2012 and june 2013 which corresponds to winter and spring in egypt . Children between the ages of 3 and 10 years who presented to the emergency room at asuh and had a confirmed radiologic fracture to the radius, ulna, or both were included in the study as cases . Controls were children who had forearm pain or trauma but did not have radiographic evidence of forearm fracture . Children were excluded from the study if they had a previous history of fractures, prolonged immobilization, or chronic use of antiepileptic drugs or glucocorticoids . Children with chronic illnesses that may interfere with bone health such as kidney disease or intestinal malabsorption were also excluded . This was a prospective case control study in children with or without forearm fractures to determine their vitamin d status and its impact on bmd . Intake data was categorized into adequate, suboptimal, and low intake using the us department of agriculture nutrient database for standard reference (table 1). Extent of sun exposure was also categorized into daily, weekly, or no sun exposure . All total body bmd measurements were performed using a hologic discovery ci / wi bone densitometer (hologic, bedford, massachusetts, usa) by a certified radiology technologist . Measurement results were obtained using hologic's internal pediatric data software which takes into account patient's height, weight, and gender . A z - score of zero was equivalent to the mean, while a z - score between 1 and + 1.5 was equivalent to values of one standard deviation below and 1.5 standard deviations above the mean, respectively . Per the international society for clinical densitometry, a z - score less than or equal to 2 is defined as low bone mass or bone mineral density . The following laboratory tests were obtained in all subjects: serum ca was measured via colorimetric assay (o - cresolphthalein complexone method) using sp120 autochemistry analyzer (spectrum diagnostics, cairo, egypt). Normative values were provided by the manufacturer and were between 9.2 and 11 mg / dl in children of 416 years and 7.2 and 11.2 mg / dl in children of 4 weeks3 years.serum p was measured by one - step colorimetric endpoint method without deproteinization by vitro scient company, cairo, egypt . Expected values were 4.45.5 mg / dl in children of 216 years.serum alkaline phosphatase (alp) was measured by one - step colorimetric endpoint (teco diagnostics, anaheim, ca, usa). Normal values stated by the manufacturer include 3292 iu / l.serum vitamin d measurements were analyzed using 25-hydroxy - vitamin d (i ria kit by diasorin company, still water, mn, usa). Serum ca was measured via colorimetric assay (o - cresolphthalein complexone method) using sp120 autochemistry analyzer (spectrum diagnostics, cairo, egypt). Normative values were provided by the manufacturer and were between 9.2 and 11 mg / dl in children of 416 years and 7.2 and 11.2 mg / dl in children of 4 weeks3 years . Serum p was measured by one - step colorimetric endpoint method without deproteinization by vitro scient company, cairo, egypt . Serum alkaline phosphatase (alp) was measured by one - step colorimetric endpoint (teco diagnostics, anaheim, ca, usa). Serum vitamin d measurements were analyzed using 25-hydroxy - vitamin d (i ria kit by diasorin company, still water, mn, usa). Categorization of 25-oh - vitamin d results followed the recommendations from the american academy of pediatrics: sufficiency> 20100 ng / ml.insufficiency> 1520 ng / ml.deficiency 15 ng / ml.severe deficiency 5 ng / ml . Sufficiency> 20100 ng / ml . Insufficiency> 1520 ng / ml . Deficiency 15 ng / ml . Severe deficiency 5 ng / ml . Continuous data was analyzed using student's t - test, and categorical data was analyzed using chi - squared analysis, or fisher's exact test where appropriate . Stata v.13 (microsoft corporation, college station, texas, usa) was used to analyze data . The mean age was 7.2 2.0 years, and 61% of subjects were boys . Overall, half of the participants in the study had suboptimal or low intake of vitamin d and ca . However, 30% of the subjects in the cases group reported low vitamin d and ca intake compared to 4% in the controls group (p 0.05). Values for all serum ca labs drawn ranged from 5.5 to 11.3 mg / dl with a mean of 8.8 mg / dl . Serum alp levels drawn ranged from 160 to 400 iu / l, with a mean of 245 children with fractures had statistically significant lower serum ca values (p 0.05) and higher serum alp levels when compared with the controls group (p 0.05). Vitamin d measurements ranged from 1.3 to 100 ng / ml with a mean of 38 ng / ml . Our study had one patient that was vitamin d deficient and eight patients with severe vitamin d deficiency . The four subjects in the cases group with no reported sun exposure all had severe vitamin d deficiency . The mean z - score in cases was 0.19 1.99 and 0.3 1.04 in the controls group (p = 0.3). The 7 cases with severe vitamin d deficiency had a z - score 2 (low bone mineral density). The single patient in the controls group that had severe vitamin d deficiency had a z - score of 1.9 . Graphic representation is seen in figure 1 . As shown in figure 2, serum ca had correlations with serum 25-oh - vitamin d levels (p 0.01). There were no significant correlations found between serum 25-oh - vitamin d and phosphorus or alkaline phosphatase . No association was found between vitamin d and gender, age, or body mass index (bmi). This study found a high percentage of severe vitamin d deficiency in egyptian children with forearm fractures . Furthermore, this study demonstrates that 25-oh - vitamin d is an important marker of bone mineralization in children with fractures . [17, 18] who found that a significant proportion of children with forearm fractures were vitamin d insufficient [21, 22]. In our study, 33% of children had vitamin d insufficiency . Similarly, their studies describe a direct association between 25-oh - vitamin d levels and bmd z - scores . The patients in our study had overall lower z - scores compared to the subjects in their study (.06 z - score versus 0.8 z - score). Collecting dietary calcium and vitamin d intake and sunlight exposure allows us to infer that the differences in z - scores may be secondary to the suboptimal / low nutritional status (50%) of the egyptian children, as well as the lack of sun exposure [24, 25]. Our study included four individuals with no sun exposure who were found to have vitamin d deficiency as well as low bmd measurements . This was recently emphasized by studies comparing indoor and outdoor training in athletes and found significant lower vitamin d levels in those with indoor training . Our study adds to the growing literature that supports the importance of outdoor sunlight exposure for children . Studies as early as the industrial revolution in europe have demonstrated bone deformities in children living in the inner cities exposed to minimal sun exposure . This study showed a significant correlation between serum calcium levels and vitamin d. vitamin d plays an important role in calcium homeostasis through its absorption in the intestine and kidney . In cases of poor calcium intake or absorption, 1,25-hydroxyvitamin d mobilizes ca from bone thereby inhibiting mineral deposition into the osteoid matrix [29, 30]. In postmenopausal women, low levels of serum calcium furthermore, poor calcium intake and distal forearm fractures during childhood are strong predictors of skeletal fragility in adulthood [32, 33]. In agreement with our results michaus et al . Found lower mean values of bmd z - scores with higher alkaline phosphatase levels in children who had multiple bone fractures . In our study, although serum alp levels were within normal reference values, they were significantly higher in those with fractures . The importance of serum calcium and alkaline phosphatase as markers for abnormal bone health and osteomalacia has been described in the study by peach et al . . In their study, they found elevated plasma alkaline phosphatase and hypocalcemia in 48% of patients diagnosed histologically as having osteomalacia . Moreover, alkaline phosphatase measurements have recently been recommended as a screening tool for osteomalacia . Also, alp has been described as a screening tool for preterm newborns at risk for metabolic bone disease, especially when accompanied with low serum phosphate . We found that cases have significant higher body mass index (bmi) than controls, p = 0.01 . This supports other studies which found that high adiposity is associated with increased risk of forearm fractures in children . This study is important because children who develop vitamin d deficiency can potentially have life - long complications . Considering that peak bone mass is reached by the end of the second decade of life, children with early fractures have a high probability of future fracture and more concerning osteoporosis later in life [39, 40]. Thus improvement in awareness of bone health in the pediatric population can directly impact on quality of life . Although pubertal status is important in evaluating bmd, our subjects were in the age between 3 and 10 years which is less than the optimal age of puberty in egyptian children . A major limitation of this study is the small number of subjects included in the study . This study demonstrates that vitamin d insufficiency / deficiency is common among middle eastern children.
The nonvenereal dermatoses can be classified into five groups based on pathogenesis: inflammatory diseases (psoriasis, seborrheic dermatitis, lichen planus), infections and infestations (scabies, dermatophytosis), congenital disorders (median raphe cyst), benign abnormalities (angiokeratoma of fordyce, sebaceous cyst), premalignant and malignant lesions (eryrthroplasia of queyrat, squamous cell carcinoma). As these groups includes various types of disorders, the identification of diseases is quite challenging . These nonvenereal disorders are the cause of considerable concern to patients causing mental distress and guilt feeling in them . Nonvenereal dermatoses are quiet often a diagnostic dilemma to the treating physician, who has to effectively manage the condition and also allay the associated anxiety . Determining any causal or aggravating factor can save the patient from the agony of persistent discomfort and restrict social life, thereby considerably improving the dermatology - specific quality of life . A comprehensive understanding of various presentation, there cause and appropriate management options is, therefore, essential . The study was to find the pattern of nonvenereal dermatoses presenting with genital lesions and to correlate its various parameters . A total of consecutive 100 male patients with genital lesions of nonvenereal origin, attending the dermatology opd at j a group of hospitals constituted the study group . All male patients> 18 years of age who presented with genital complaints were screened for nonvenereal dermatoses . A detailed history including demographic data, chief complaints related to skin, onset and duration of disease and associated medical or skin disorders was elicited and recorded . The external genitalia was examined, and findings were noted . A detailed physical examination was done to see any associated lesions elsewhere in the body . Investigations such as gram - stain, koh mount, venereal disease research laboratory test, hiv test and histopathological examination were done as and when required establishing the diagnosis . A proforma was prepared to record the relevant details of patient, examination, investigations and diagnosis . A total of 100 male patients with nonvenereal dermatoses of external genitalia were included in the study . The age of the patients ranged from 18 years to 65 years, with the mean age of 32.2 years . Most patients belong to the age group of 21 - 30 years (40%), followed by the age group of 31 - 40 years (20%). Seventy - four patients (74%) were from the urban area while twenty - six patients (26%) belong to rural background . Fifty - two (52%) patients were married and the remaining forty - eight (48%) patients were unmarried . Scrotum was involved in 60% and penis in 30% while both scrotum and penis were affected in 10% cases . A total of sixteen types of nonvenereal dermatoses were noted in this study [table 1]. The most common disorder was vitiligo [figure 1] present in 18 cases, followed by pearly penile papule [figure 2], which accounted for 16 cases . The other disorder encountered included fixed drug eruption (fde) in 12; scabies [figure 3] in 10, scrotal dermatitis [figure 4] and lichen planus [figure 5] in 9 cases each etc ., [table 1]. Genital vitiligo - depigmented macule over glans and prepuce pearly penile papule- asymptomatic discrete papules present over corona sulcus scabies - multiple nodules over glans lichen planus - violaceous annular plaques over glans penis the common presenting features were itchy genitalia, de - pigmentation . Other complaints were pain, burning sensation, redness, exfoliation of the skin, raised lesions over the skin, oozing, ulceration, erosions and thickening of the skin . As venereal dermatoses are of primary concern to the patient and causes mental stress and guilt feeling among patients, it is, therefore, utmost important to distinguish between venereal and nonvenereal dermatoses . Male patients with nonvenereal dermatoses usually present to genitor - urinary experts or physicians, where the training and expertise are not oriented to adequate dermatological diagnosis and treatment . Disorders of genitalia have proved confusing to various specialists involved in the diagnosis and treatment . The problem is confounded by the fact that the normal characteristics of common diseases at flexural sites are lost or modified, making the diagnosis difficult for even an experienced dermatologist . The nonvenereal dermatoses of male external genitalia include wide spectrum of disease with varied etiology . There are very few comprehensive study on the pattern of nonvenereal dermatoses in males from our country . Also, our study is first of its kind from this part of the country . Had done a study on the pattern of nonvenereal dermatoses of male external genitalia from south india . The age ranged from 18 to 65 years in the present study with the mean age of 32.2 years whereas the age ranged from 9 to 70 years with a mean age 33.7 years in a study by karthikeyan et al . Most of the patients belong to the age group of 2130 years (40%) in the present study which is similar to karthikeyan et al . A total of 16 different nonvenereal dermatoses were observed in this study [table 1]. The most common disorder was genital vitiligo [figure 1], which accounted for 18%, followed by pearly penile papule [figure 2] in 16% and fde in 12% cases in the present study . The study by acharya et al . Reported infections as commonest disorder contributing 40% cases . Genital vitiligo as most common disorder (16%) was observed in an another study, which is almost similar to our study . Khoo and cheong had 14.3% pearly penile papule as most common nonvenereal dermatoses, which are similar to this study (16%). Seventy - four patients (74%) were from the urban area while twenty - six patients (26%) belong to rural background . Fifty - two (52%) patients were married and the remaining forty - eight (48%) patients were unmarried . Scrotum was involved in 60% and penis in 30% while both scrotum and penis were affected in 10% cases in our study . Genital vitiligo could be an exclusive finding, or it can be associated with generalized vitiligo . Genital vitiligo [figure 1] accounted for 18% cases as commonest disorder in our study and is seen in all age group from young adult to older age group . This is in contrast with the study conducted by karthikeyan et al ., where the entire patients with vitiligo were in older age group . Ten patients in our study had associated vitiligo elsewhere while eight patients had only genital vitiligo . They were present in 16% cases in our study [figure 2], which is almost similar to the study conducted by khoo and cheong they are frequently mistaken as warts and misdiagnosed as tyson's gland or ectopic sebaceous gland of fordyce . All the patients with pearly penile papule came to visit opd in apprehension of some venereal disease . Fixed drug eruptions were observed in 12% of cases in our study as third most common disorder ., where only 3 cases had fde and all of them because of cotrimoxazole . In our study, various drugs were implicated such as, nonsteroidal antiinflammatory drugs, sulphonamides, ornidazole, fluconazole, ampicillin, etc ., half of our patients with fde had oral involvement also . Recorded scabies as most common nonvenereal dermatoses accounting for 30 cases (15%), while it was present in only 10% cases in our study [figure 3]. Lichen planus was present in 9% cases in our study that is in contrast [figure 5] with puri and puri where it was seen in only 6.6% (3) cases and karthikeyan et al . Where it was seen in only 1 case . Contributory factors include, tight clothing, friction, maceration, atopy, over - washing, use of various toiletries, topical medicaments and indigenous preparations . Scrotal dermatitis [figure 4] accounted for 9% cases in our study inclusive of allergic contact dermatitis, irritant contact dermatitis . Sebaceous cysts of scrotum [figure 6] were observed in 7% cases in our study, while it was second most common finding (14%) by karthikeyan et al . They were observed in only 3.7% cases by khoo and cheong all of our cases were asymptomatic and from younger age group . Multiple sebaceous cysts scrotum dermatophytic infection was present in 5% cases in our study as scaly pruritic plaques over scrotum . Lichen sclerosus (ls) is chronic inflammatory dermatoses which are associated with substantial discomfort and morbidity with an unknown etiology . Ls was observed in 3 cases in our study [figure 7], while it was seen in only 2 cases by karthikeyan et al . Clinical findings in cases of ls in this study were found to be in concordance with the literature review . Lichen sclerosus - sclerosus and de - pigmentation of prepuce and glans with phimosis around 2% of the world population have psoriasis, but it is possible that many more could have ano - genital psoriasis at some time . Genital appearance could be challenging to interpret, especially in uncircunscribed individuals because a mucosal site is affected rather than keratinized skin . Reported a solitary case of psoriasis of glans penis while acharya et al . Reported 5 cases of psoriasis over genitalia zoon's balanitis or plasma cell balanitis [figure 8] was observed in 2% cases in this study that had not been reported by acharya et al ., it is a disorder of middle and older age in uncircumcised male, the etiology remains unknown . Zoon's balanitis or plasma cell balanitis lymphangiectasia of scrotum [figure 9] was seen in 2 cases . One case each of lichen nitidus, granuloma annulare, papulo - necrotid tuberculid and squamous cell carcinoma [figure 10] were also observed in our study . It is very important to distinguish between venereal and nonvenereal genital dermatoses, as these nonvenereal disorders are a considerable concern to patients causing mental distress and feeling of guilt . Also, these nonvenereal disorders are quiet difficult in making a diagnosis by the treating physicians . A comprehensive understanding of the various presentations, their etiology is, therefore, essential . This study was quiet useful in understanding the epidemiological, clinical and etiological characteristics of various nonvenereal genital dermatoses.
Myxoma is a benign neoplasm of mesenchymal origin composed of undifferentiated stellate cells in a myxoid stroma, which were differentiated into altered fibroblasts and lost their ability to produce normal collagen18). Myxoma can arise in a variety of locations; heart, subcutaneous and aponeurotic tissues, bone, genitourinary tract and skin, etc . The most frequent site for myxoma is the heart, and bony myxoma is almost exclusively limited to the jaws and palate, representing 40 - 50% of all head and neck myxomas216). The soft tissue myxoma is rare, and occurs mostly in the large skeletal muscles . The myxomas that arise in the skeletal muscles are generally called intramuscular myxomas (ims). The most of ims are detected in the fourth to sixth decades of life with slight female predominance16). I m accounts for about 3 - 17% of the all myxomas, and the incidence is about 1 case per million population per a year51823). To our knowledge, we report the eighth case of a myxoma developed within the lumbosacral paraspinal muscle (including thoracolumbar paraspinal muscle), which had some different characteristics from the typical i m, and brief review of the literature will be followed . A 62-year - old female was referred to our hospital for mass - like lesion detected on magnetic resonance imaging (mri) of her spine . She presented with low - back pain just for 3 months without radiating leg pain, and the symptom was slowly progressive . But, interestingly, she had the previous history of bone harvest on the right posterior superior iliac spine (psis) for the cervical fusion operation 15 years ago in the other hospital . Physical examination revealed a large palpable mass on the right paraspinal area in the lumbosacral region, but there was no associated tenderness in this region . Her neurologic examination at the time of admission was fully normal, and there was no abnormal finding on the laboratory tests and the electrophysiological studies such as electromyography (emg) and somatosensory evoked potential (ssep). Mri demonstrated a multi - lobulated mass in the right lumbosacral paraspinal muscles in contact with the posterior elements of the l4 and l5 vertebra (fig . This well - circumscribed mass was measured approximately 4.03.56.5 cm in size, and the lesion was hypointense on t1-weighted image (t1wi) and hyperintense on t2-weighted image (t2wi). There were heterogenously enhancing foci inside the mass with peripheral and septal enhancement on the post - contrast t1wi . On computed tomography (ct) scan of the lumbosacral region, this mass had low density and some calcification particularly on the inferior portion of the mass (fig . There was no bony destruction or invasion around the mass . In the process of a full diagnostic workup, we found her previous abdominal ct images in picture archiving computer system (pacs), which had been performed for evaluation of her abdominal pain 4 months ago (fig . 2c, d). In the previous abdominal ct, although it was slightly smaller than the mass of recent lumbar ct, the hypodense lesion was also seen at the same area . It was notable that calcification inside the mass was not detected on the previous abdominal ct . We planned to conduct the complete excision, instead of simple biopsy (for example, ct - guided biopsy), because the tumor was not only symptomatic, but also the operation had fairly limited risk to the patient . The tumor was encapsulated and lobulated with grayish white surface, and there was no evidence of the infiltration or invasion into the surrounding bony structures . 3). According to the frozen biopsy during the operation, the mass was revealed to myxoid tumor, and the final histopathological findings were consistent with intramuscular myxoma . This mass was composed of prominent myxoid stroma with a distinct presence of spindle and stellate cells, and while pyknotic nuclei and karyorrhectic nuclei were also seen (fig . Numerous micro - calcifications were also found, especially in the peripheral capsular area of the tumor . The patient was neurologically free and experienced complete relief of her low - back pain postoperatively . Since stout et al18). Outlined the diagnostic criteria and defined myxoma at 1948, the intramuscular myxoma has been known as the most rare sub - type in these tumor category . Ims have been reported at the various locations such as thighs, buttocks, upper limbs, shoulders, and paravertebral area . Of those ims, paraspinal regions are the most uncommon site, and so far, only 14 cases of paraspinal ims have been reported . And to the best of our knowledge, this report is just the eighth case of paraspinal i m in the lumbosacral area including one case of thoracolumbar lesion, and the first case in asian population (table 1)891114151720). Ims usually appear as well - circumscribed homogeneous low - density lesions within the skeletal muscle on ct scan, and have low - signal intensity on t1wi and high - signal intensity on t2wi . Bancroft el al.1) described that the peri - tumoral fat rind on t1wi and increased signal in the adjacent muscle on t2wi are the most reliable radiologic features for distinguishing intramuscular myxoma from other myxoid tumor . But in this case, this finding was not definite on the mri . On gross inspection, the tumor has usually oval shape and shows well - circumscribed appearance, however its fibrous tissue often has incomplete pseudocapsule to result in infiltration of adjacent musculature510). And in general the tumor has a glistering gray - white appearance and contains gelatinous material and cyst filled with fluid inside the mass . Histologically, ims are composed of well - localized myxomatous connective tissue surrounded by a collagenous fibrous connective tissue22). Ims are often confused with other pathologic conditions such as ganglion or neurofibroma, and furthermore, with other mucoid sarcomas such as liposarcomas, chondrosarcomas, low - grade fibromyxoid sarcoma and malignant histiocytomas, because these sarcomas are also accompanied by the myxomatous degeneration . However, several critical clues had been presented, which help to distinguish ims from other myxoid lesions . First, ims have poor vascularization and a paucity of cells (lipoblasts or chondroblasts), in contrast to myxoid liposarcoma which has a rich, delicate, plexiform capillary vascular network . Secondly, ims lack nuclear atypia, necrosis, and prominent mitotic activity . In addition, on the enhanced mri, soft tissue sarcomas showed heterogeneity, intense enhancement, septation, ill - defined margins and invasion of adjacent structure against the ims . Moreover, ims often do not have a true capsule, and therefore could infiltrate to the adjacent striated muscle, resulting in muscle atrophy which appear as a peri - tumoral fat rind on t1wi . Additionally, doyle et al.4) presented the muc4 protein as a specific marker for distinguishing from low - grade fibromyxoid sarcroma . Ims usually present as a solitary slow growing painless mass, however multiple myxomas can occur on rare specific condition, for example, mazabraud's syndrome . The clinical manifestations of ims generally are non - specific, but rather presenting symptoms depend on the affected locations . In our case, the patient also presented with ambiguous low back pain and this symptom was resolved after total resection of the tumor . In general, ims are known as slowly growing and painless tumors, whereas there are some differences from typical natures of ims in this case . First, it had been just asymptomatic mass, but it developed to symptomatic lesion in a very short time (about 3 months). There was demonstrable difference in size of the tumor; widthlengthheight=3.03.05.4 cm to 4.03.56.5 cm . In addition, a new calcification inside the mass was accompanied with tumor growth, while there is no internal calcification in most cases of i m . Third, the location of i m was adjacent to the previous operation site (iliac bone harvest site). Although the pathogenesis of ims remains obscure, there are several theories for explaining the pathogenesis . According to one of the theories, stellate cells may be altered fibroblasts that fail to produce mature collagen5). By glazunov and puckov, " viral origin " wirth et al.24) proposed the fibrous dysplasia as another theory for the pathogenesis of ims, and fibrous dysplasia usually precedes the ims by several years . We assumed that interruption of paraspinal muscles by previous operation could induce fibrous dysplasia to arise i m . Though there was no report about the prevalence by race due to the rarity of the incidence of ims, previous many reports were almost confined to the western population . Our case is the first case of i m in asian people, which developed in the lumbosacral paraspinal musculature . The clinical course of ims is known to be nearly benign, and the treatment for ims is the total surgical resection . There is no report of metastasis or malignant change, but occasionally local recurrence resulting from incomplete resection has been reported6913). Therefore, if possible, wide excision with clean margins is recommended to avoid local recurrence, since ims have an infiltrative tendency to surrounding muscles . We report the first case of the paraspinal intramuscular myxoma developed in the lumbosacral area in asian population . Intramuscular myxomas are uncommon benign tumors, especially in the paraspinal area, and moreover it is difficult to make a diagnosis before operation because of the lack of characteristic clinical history and radiographic findings, however, neurosurgeons should be aware of this entity.
The preeruptive migration of a tooth across the midline is termed as transmigration . Initially, the term transmigration was used only if the entire length of the impacted canine had migrated and crossed the midline of the mandible . However, javid modified the definition by suggesting that one half or more of the length of the tooth was required to cross the midline in order to be considered as transmigration [2, 3]. Joshi suggested that rather than the distance of migration, the tendency of a canine to cross the barrier of the midline suture was more important . Though a number of factors have been proposed, the aetiology and exact process of transmigration are indeterminate . The prevalence of transmigration in different populations and ethnic groups was the subject of several studies and was reported to be between 0.1 and 0.34% [57]. In india only one study has been conducted on transmigrated mandibular canines . To the best of our knowledge, the present study is the first to report the prevalence of both transmigrated maxillary and mandibular canine in india . In the current study, we determine the prevalence of transmigrant maxillary and mandibular canines in a north indian population and describe the clinical and radiographic features of patients with transmigrant impacted canines . A prospective study of 3000 panoramic radiographs (1695 males and 1305 females) was conducted in two dental colleges in haryana for 8 months from september 2013 to april 2014 . The radiographs of completely edentulous patients and paediatric patients below the age of 10 years were excluded . The radiographs were screened for transmigrant canines by a single examiner experienced in dentomaxillofacial radiology . The examiner in the study was blinded to patient data during the radiographic examination procedure . An impacted canine was considered transmigrated when the tip of the crown of the canine regardless of its length had crossed the midline . The panoramic radiographs were screened for radiographically identified position of the transmigrated tooth, retained canine, associated pathologies, and other coexisting dental anomalies (other than third molar). Each image's contrast and brightness were optimized to produce the best image for viewing under standardized conditions . After diagnosing a transmigrant canine radiographically, clinical evaluation was done for the patient's age and sex, symptoms, the presence of associated pathology, and the treatment provided . As diagnosis of transmigrated canine is an objective assessment and these teeth are clearly visible there was no requirement for an interexaminer validity . Statistical analysis was done using spss version 11 and the chi - square test was done . Canine positioned mesioangularly across the midline, labial or lingual to the anterior teeth . Canine positioned mesioangularly across the midline, labial or lingual to the anterior teeth . Canine horizontally impacted near the inferior border of the mandible inferior to the apices of the incisor teeth . Canine horizontally impacted near the inferior border of the mandible inferior to the apices of the incisor teeth . Canine horizontally impacted near the inferior border of the mandible below the apices of posterior teeth . Canine horizontally impacted near the inferior border of the mandible below the apices of posterior teeth . Canine positioned vertically in the midline with the long axis of the tooth crossing the midline . Canine positioned vertically in the midline with the long axis of the tooth crossing the midline . The relationship (position / angulation) of maxillary transmigrant canine with median palatine suture and the distance travelled in contralateral arch past midline suture by transmigrated canine along its long axis was also evaluated . The maxillary transmigrated canine was approximated into three parts along its long axis as tip of crown till approximate halfway of crown, halfway of crown till cement - enamel junction (cej), and lastly beyond cej irrespective of root length of transmigrated canine (figure 7). There were 20 patients (12 males and 8 females) observed in our study with transmigrated canines . The mean age of patients with transmigrated canines was found to be 24.1 years (age range 1550 years). The prevalence of mandibular canine transmigration was 0.5% (15/3000) whereas the prevalence of 0.16% (5/3000) was recorded for the maxillary canine transmigration (p <0.05) (figures 1, 2, 3, 4, 5, and 6). There was no statistically significant difference for gender with 12 transmigrant mandibular canines in males (0.7%) as compared to 8 subjects with transmigrant canines (0.61%). Left - side predominance was observed for mandibular canine transmigration with 11 patients observing left mandibular canine migrating towards right side and four cases migrating from the right side towards left side (p <0.5). A similar finding was observed in maxillary canine transmigration with 3 cases depicting a left maxillary canine migration as compared to two cases depicting right side transmigration . There was presence of the retained primary canine with respect to transmigrated canine in seven cases (35.0%). The presence of other dental anomalies was present in 45% cases of transmigration (p> 0.05). Applying mupparapu's classification for mandibular canine transmigration (15 cases), type 1 transmigration was the commonest type found in our study (10 cases), followed by types 2 and 4 (2 cases each) and 1 case of type 5 transmigration . No type 3 transmigration case was found in the study . 2 cases (10.0%) of transmigrated canines only the tip of crown till approximate half of crown positioned in the median palatine suture was observed in 60% cases of transmigrant maxillary canines (figure 7). Four maxillary transmigrant canines were positioned in a horizontal or perpendicular direction (8090) to median palatine suture as compared to single case with mesioangular impaction (table 3). In the present study, the prevalence of maxillary and mandibular canine transmigrated canines was evaluated which is in contrast to the only previous indian study related to transmigrated teeth, that focussed only on mandibular canines . Transmigration, intraosseous migration of unerupted tooth, is a rare phenomenon [5, 6]. Early diagnosis with a timely orthodontic or surgical intervention can help dentists preserve the canines, which play an important role, in both aesthetics and function in the human dentition . The research on prevalence of transmigrated canines has been scarce with only a handful of studies conducted on this rare phenomenon (table 4) [516]. In accordance with the previous studies, a greater predilection of mandibular canine transmigration (0.5%) was observed in our study as compared to maxillary canine transmigration (0.16%). Aktan et al . Had observed a similar predilection of 0.14% for maxillary transmigrated canines and found a prevalence of 0.34% for transmigrated mandibular canines, while gunduz found a 0.1% prevalence of transmigrated canines in 12,000 patients [6, 7]. The prevalence of transmigrated maxillary canine (0.16%) found in our study was found to be similar to all the studies, though a greater prevalence of 0.44% was documented in one study . The prevalence of transmigrated mandibular canine was, however, found to be variable ranging from 0.004% by mupparapu to 0.46% by kumar et al . However, the overall prevalence of transmigration of 0.66% was found to be slightly higher than the other studies . Zvolanek's case series failed to find any transmigrant canines in 4000 individuals whereas javid had identified one transmigrant canine after examining 1,000 panoramic radiographs . The difference in prevalence can be attributed not only to ethnic differences but also to type of the study population whether it is orthodontic or general population . Similar to the findings reported by aydin et al ., we also found that transmigrant canines occur more frequently in male patients (60%) than in female patients (40%). Gonzlez - snchez et al . Who found a greater prevalence of transmigrated canines in females [15, 18]. In an extensive review by sumer et al . They had postulated a slight female predilection (1.6: 1). Aras et al . Had, however, observed no gender predilection in their study of 6000 patients . The reason for the gender predilection is not clear, though females typically tend to report frequently for aesthetics as compared to males . In our study there was a marginally increased number of males (1695 males; 1305 females) that possibly could have resulted in greater number of transmigrated canines being observed in males, though no statistically significant difference was found (p> 0.05). The reported patient age at presentation of the transmigrant canine varies from 8 years to 69 years . In our study the average age the mean age for maxillary transmigrant canines (32 years) was found to be much higher as compared to transmigrant mandibular canine (21.5 years). This increased mean age could be attributed to a greater path travelled as well as median palatine barrier that has to be crossed by maxillary canine to transmigrate as compared to relatively easier path for mandibular canine . Nevertheless, this finding could be coincidental and more studies regarding transmigrant maxillary canine can prove that whether transmigrant maxillary canine is likely to be encountered in a slightly higher age group as compared to mandibular canine . Moreover, the lack of previous radiographic records further makes this finding difficult to ascertain . Canine transmigration involved a left - side tooth more often (14/20 cases; 70%) than the right canine (6/20; 30%). This finding was in accordance with the findings observed by kumar from south india, celikoglu from turkey, and mazinis in greece [8, 13, 16]. However aydin et al . And aras et al . Had not found any side predilection in their study [10, 12]. In the 28 cases documented by joshi, though no prevalence was reported, 53.6% were left - side teeth, 32.1% were right - side ones, and 14.6% were bilateral cases . Bilateral canine transmigrations are rare and very few case reports have been documented in literature [2024]. Was the first to report the bilateral occurrence of transmigrated canines and later mupparapu et al . All documented bilateral transmigrant canine cases have involved mandibular canines except a single reported case of bilateral maxillary transmigrated canines in study conducted by aktan et al . The findings in the current study did not reveal any case of bilateral transmigration . Despite the first case of transmigration reported by caldwell in 1955, a possible etiological factor of transmigration postulated was retention or premature loss of deciduous teeth . 35% of the cases in our study had retained primary canine with respect to the transmigrated tooth . Other factors postulated were crowding, spacing, supernumerary teeth, unfavourable alveolar arch length, fractures with displacement of tooth bud, and an excessive crown length of the permanent canine [9, 18]. Crowding of the mandibular incisors was present in only one case (5.0%) in our study . Joshi had stated that the presence of cyst, tumor, or odontoma may facilitate transmigration of the canine . In the current study, . Evaluated that impacted transmigrant canines were not associated with any pathology in 6,000 patients . Bruzst suggested that the canine tooth bud is situated in front of the lower incisors and that facial growth pushes the canine towards the contralateral side . The future studies of transmigration should focus also on the patient's siblings and parents for the possible hereditary pattern . Pippi and kaitsas had postulated a pericoronal osteolytic area, because of an anomalous secretion of signal molecules either due to genetic origin or inflammatory stimuli . Therefore, a symmetrical pericoronal enlargement takes place that represents a locus minoris resistentiae, towards which the impacted canine moves . This movement stops when the tooth finds a mechanical obstacle (e.g., jaw cortical bone). Vichi and franchi had suggested that agenesis of the adjacent teeth, in particular, the lateral incisor, may favour retention of the primary canine, and the excess of space in the dental arch may account for the absence of a correct guide for eruption . Though there were 2 concurrent cases of agenesis of maxillary lateral incisors and one case of congenitally missing maxillary canine with mandibular canine transmigration, none of these cases were sufficient to warrant a statistical analysis . The authors correlated the association of transmigration with other dental anomalies (other than third molars) but could not find any significant finding . The rarity of transmigrant canines in our study and other published studies in literature makes the correlation studies difficult . The reason why an impacted canine transmigrates might be due to concoction of all the above factors that could possibly lead to varying presentations of particular transmigrant tooth . The likely reasons for a greater number of mandibular transmigrant cases are voluminous mandibular symphysis and an emphasised buccal inclination of the lower incisors, as well as the typical conic shape of the canine root and crown . Transmigration of an impacted maxillary canine is rare due to the negligible distance between the apexes of the maxillary incisors and the floor of the nasal fossae and to the presence of the midpalatal suture, which is a considerable barrier against maxillary canine migration . In three cases (60%), tip of crown till less than halfway of crown of transmigrant maxillary canines was positioned in the median palatine suture whereas in 40% of the cases more than half of crown (till cementoenamel junction) of transmigrant maxillary canine was observed to cross the median palatine barrier . The above result is in agreement with other studies where crown of transmigrant maxillary canines could traverse only till cement - enamel junction beyond median palatine suture and no case has yet been reported where a maxillary canine has completely crossed into the contralateral arch [6, 11, 16]. Currently it is not known how much eruptive force is required but the transmigrant maxillary canine might be having a greater amount of horizontal component of eruptive force to penetrate the resistance of dense median palatine suture . The type of the angulation formed between the midsagittal plane and unerupted canine decides the course of transmigration . It has been suggested that when the angle exceeds 50, mandibular canines are likely to migrate and cross the midline . However if the angulation is less than 30, transmigration is unlikely . Though radiographic technique dictates the appearance of radiographic position of transmigrated maxillary canine, a horizontal position (8090) of transmigrant maxillary canine was observed in four cases, whereas mesioangular position (45) was observed in one case . Shapira and kuftinec had suggested that the migrating maxillary canine probably tunnels through the bone as median palatine suture typically remains intact radiographically in all previously documented cases . Thus a higher angulation (approximately 80) as compared to mandibular canine might be required for an impacted maxillary canine to overcome strong anatomical barrier of median palatine suture and hence a horizontal position is more likely to transmigrate as compared to a mesioangular position . However more studies are required to corroborate the above finding of angulation and position making a difference in penetration of suture as there is still paucity of reported cases of maxillary transmigrant canines . According to mupparapu the most likely position of the transmigrant mandibular canine is mesioangular impaction (type 1) and the erupted transmigrant canines especially in vertical position in midline are the rarest (type 5). In our study, type 1 transmigration was the pattern most frequently encountered in accordance with the previous studies . These findings were similar to the studies conducted by kumar et al, mazinis et al . [6, 8, 16] the type 3 canine transmigration appears to be the rarest form of mandibular transmigration rather than type 5 after reviewing all the published studies on transmigration . The classification by mupparapu (2002) could not be used for maxillary transmigrated teeth, though there was mild variation in the patterns of transmigrated maxillary canines in position and angulation . The transmigrated canines are mostly asymptomatic, though, rarely complications like follicular space enlargement, chronic infection with fistula, pain due to impingement, resorption of adjacent teeth, and swelling as well as ectopic eruption could occur . In our study we observed two cases (10.5%) of transmigrant canine with follicular space enlargement . None of the patients in the present study were symptomatic and no root resorption was evident in adjoining teeth of the transmigrant canines . Mazinis et al . Had however observed a single case of root resorption in lateral incisor and had postulated usage of cone beam computed tomography (cbct) for accurate localisation and assessing the root resorption . Proposed treatment strategies for transmigrated mandibular canines include surgical removal, transplantation, and surgical exposure with orthodontic alignment . The most preferred treatment for migrated canines is surgical extraction [8, 18]. This is especially true when the mandibular arch is crowded and requires therapeutic extractions to correct the incisor crowding . It is however imperative to anesthetise the nerve on the originating side as transmigrated teeth maintain their nerve connection to the originating side where the tooth germ is formed . If the mandibular incisors are in normal position with sufficient space for transmigrated canine, then transplantation can be undertaken . Wertz stated that if the crown of a transmigrated tooth migrates past the opposite incisor area, or if the apex migrates past the apex of the adjacent lateral incisor, it might be mechanically impossible to bring it into place . Had suggested that when the transmigrated canine is accessible, and especially if it is symptomatic, removal of the impacted tooth is recommended . If asymptomatic, transmigrant canine should be periodically evaluated radiographically and kept under observation . Since none of our cases were symptomatic, removal of impacted transmigrated tooth was not considered . All the patients were kept on periodic follow - up and referred for orthodontic treatment with varying management modalities planned like orthodontic traction and surgical exposure with orthodontic treatment . According to the aktan et al ., except for the canine, no tooth type shows a tendency for transmigration in the dental arch . However, kara et al . Had observed two transmigrant cases of lateral incisor and three cases of premolar in their study . In our study most of the transmigrant cases are impacted and rarely erupt as was observed in studies conducted by aktan et al . And only one erupted transmigrated canine (5.0%) was observed in our study as compared to 2.2% of erupted transmigrated canines in a study by kara et al . . The prevalence of transmigrant maxillary canine is almost similar (0.13%0.20%) in all the studies conducted on transmigration [6, 10, 12]. The panoramic radiographs must be used for evaluation of impacted canines and the usage of cbct should be implemented in advanced treatment planning as it would provide more precise information about the localisation of transmigrated canine and its relationship to surrounding teeth and median palatine suture . Research studies on transmigration should focus not only on the possible complications like root resorption and follicular spaces but also on the distance beyond the midline migrated by the transmigrated teeth as no literature is yet available . Further research is required to validate that a horizontally impacted maxillary canine is more likely to transmigrate as compared to mesioangularly impacted maxillary canine . Canine transmigration is of vital significance in dentistry as it can create orthodontic, esthetic, surgical, and interceptive complications . Hereditary patterns of the transmigration should be further investigated as its etiology is not yet ascertained . Type 3 canine is the rarest form of transmigrant mandibular canine and a classification system for maxillary transmigrant canine to aid in the diagnosis and management is long overdue.
We conducted a retrospective cohort analysis of trends in dengue diagnoses among hospitalized patients using the national inpatient sample, the largest all - payer database of hospital discharges in the united states . The database is maintained as part of the healthcare cost and utilization project by the agency for healthcare research and quality and consists of a 20% stratified sample of discharges from nonfederal acute care hospitals (11). We first extracted all discharges from the national inpatient sample for hospital admission (i.e., the denominator of the incidence rate) from 2000 through 2007 . Among this population, we then identified cases of dengue fever coded as either a primary or secondary diagnosis (i.e., code 061, dengue fever), according to the international classification of diseases, ninth revision, clinical modification . For each yearly incidence rate, we calculated a 95% exact binomial confidence interval . To determine whether a significant trend in hospitalizations of patients with dengue fever occurred during the study period, we fit a logistic regression model using yearly incidence as the dependent variable and year as the independent variable . In addition, to accommodate the temporal association in the yearly incidence, we fit the model using generalized estimating equations, assuming an autoregressive correlation structure . We also calculated the ratio of the yearly incidence rates at the beginning and the end of the study period (i.e., rates in 2000 and 2007). We tested whether this incidence ratio is significantly different from one using the fisher exact test and computed a 95% exact confidence interval for the corresponding odds ratio using the hypergeometric distribution . (because the incidence rates are fairly low, the odds ratio closely approximates the incidence ratio .) Finally, we used the monte carlo variant of the fisher exact test to investigate possible geographic variation in the incidence rate among the 4 us census regions . All statistical analyses were performed by using r version 2.10.1 (r foundation for statistical computing; www.r-project.org) and sas version 9.2 (sas institute inc ., cary, sc, usa . ). The length of stay for these patients ranged from 0 to 35 days (median 3 days). For the monte carlo variant of the fisher exact test, we found the incidence rates for the 4 us census regions were homogenous for all years, except for 2004 and 2007 . In these 2 years, the northeast region had the highest incidence rate (p<0.0001 for each year). Over the study period, the estimated number of dengue cases more than tripled from 81 cases in 2000 to 299 cases in 2007 . The trend in the incidence of patients hospitalized with dengue during the study period was upward and significant (trend estimate 0.1313, model - based se 0.0258; p<0.0001) (figure). The increase from 2000 to 2007 was also significant (incidence ratio 3.5641, 95% confidence interval 2.02936.6232; p<0.0001). National estimates of dengue yearly incidence rates and 95% exact binomial confidence intervals (error bars), calculated by using data from the national inpatient sample, united states, 20002007 . The trend (dotted line) is based on a logistic regression model fit by using generalized estimating equations . Note that the trend is curvilinear in the incidence rate, yet linear in the log odds of the incidence . We found a dramatic increase in the number of hospitalizations for patients with dengue fever in the united states . This increase is not surprising considering that 1) the number of cases in disease - endemic regions has increased in recent years, and 2) a substantial number of travelers annually enter the united states from the tropics and subtropics (12). Although infrequent, severe consequences of dengue infection may occur in returning travelers . As individual travelers increasingly make multiple visits to dengue - endemic areas, the risk for severe dengue infections may similarly increase . A survey of 219 travelers who received treatment for dengue in europe showed that 23 (11%) had severe clinical manifestations, including internal hemorrhage, plasma leakage, shock, and marked thrombocytopenia (13). We were unable to ascertain whether mosquito - borne hemorrhagic fever (international classification of diseases, ninth revision, code 065.4) also increased because the code appears quite infrequently, making statistical inferences unreliable . We also attempted to use deaths as a marker for disease severity, but we could not detect an increase in disease severity in our analysis because number of deaths was insufficient to accurately estimate a mortality rate . Dengue and dengue hemorrhagic fever have been described as potential public health threats for residents of the us mainland (14). Despite the proximity of circulating dengue virus to the continental united states and the spread of the vector mosquitoes (aedes aegypti and ae . Albopictus) to at least 26 states (15), autochthonous cases in the continental united states have been relatively rare (5) until the recent florida outbreak . The increase in reported cases that we have documented highlights a potential risk for dengue spread within the united states . Although dengue fever was previously classified as reportable in some states, it did not become a reportable illness at the national level until 2010 . Thus, some time is required before cases reported to public health departments can be used to establish reliable statistical estimates of national trends . Furthermore, the number of cases may not be linked to other relevant clinical data . The major limitation to our study is that we used administrative data, and thus we did not have access to laboratory data or patients travel histories . Nevertheless, our results indicate that the decision to make dengue fever a reportable disease in the united states was warranted and that increased vigilance focused on these new surveillance data is needed . In addition, administrative data, as we describe here, can be used to estimate the effects and severity of illness attributable to dengue.
Analysis of flow over a vertical surface with simultaneous heat and mass transfer from different geometrics embedded in porous media has many engineering and geophysical applications such as geothermal reservoirs, drying of porous solids, thermal insulation, enhanced oil recovery, packed - bed catalytic reactors, and underground energy transport . The various aspects of the pioneering work of sakiadis on continuous surfaces have been investigated by many authors . Again, the studies of magnetohydrodynamics (mhd) flow for electrically conducting fluid past a porous vertical surface are important from technological point of view . Pioneering work of hartmann on the flow fluid under the influence of uniform transverse magnetic field has led to an extensive study on mhd flow problems by several researchers . These studies have a bearing on industrial applications such as mhd power generators, mhd pumps, accelerators, aerodynamic heating, electrostatic precipitation, polymer technology, petroleum industry, purification of crude oil and fluid droplets and sprays . Furthermore, a lot of research work concerning the mhd flow has been obtained under different physical effects by the investigators like nigam and singh, makinde, muthucumaraswamy et al ., geindreau and auriault and apelblat . In most cases, the hall and ion slip terms were ignored in applying ohm's law, as they have no marked effect for small and moderate values of the magnetic field . However, the current trend for the application of magnetohydrodynamics is towards a strong magnetic field, so that the influence of electromagnetic force is noticeable as studied by cramer and pai . Under these conditions, the hall current and ion slip are important and they have a marked effect on the magnitude and direction of the current density and consequently on the magnetic force term . The effects of hall current on mhd free convection flow along a vertical surface with or without mass transfer have been studied by number of authors: sato, tani, pop, raptis and ram, hossain et al . [1315], pop and watanabe, acharya et al ., aboeldahab and elbarbary, acharya et al ., and so forth . Hall current with ion slip has important engineering applications in the problem of magnetohydrodynamics generators and hall accelerators as well as in flight magnetoaerodynamics . Furthermore, attia has investigated the effects of heat and mass transfer in elasticoviscous fluid past an impulsively started infinite vertical plate by considering ion slip . In the present study, we propose to investigate the effects of simultaneous heat and mass transfer on the free convection mhd flow of an incompressible electrically conducting viscoelastic fluid characterized by walters liquid (model b) past an impulsively started infinite vertical plate embedded in a porous medium considering hall and ion slip effect into account with chemical reaction . Solutions are presented in graphical forms for various parametric values entering into the fluid flow region . The constitutive equation for walters liquid (model b) is (1)ik=pgik+ik,ik=20eik2k0eik, where ik is the stress tensor, p is isotropic pressure, gik is the metric tensor of a fixed coordinate system x, vi is the velocity vector, and the contravariant form of e is given by (2)eik=eikt+vme, mikv, mkeimv, miemk . It is the convected derivative of the deformation rate tensor e defined by (3)2eik = vi, k+vk, i . Here, 0 is the limiting viscosity at the small rate of shear which is given by (4)0=0n()d, k0=0n()d with n() being the relaxation spectrum . This idealized model is a valid approximation of walters liquid (model b) taking very short memories into account so that terms involving (5)0tnn()d, n2, have been neglected . We consider the unsteady flow of an incompressible electrically conducting viscoelastic fluid past an impulsively started infinite vertical porous plate with oscillating temperature and concentration embedded in a porous medium in the presence of a transversely imposed magnetic field with a coordinate system (x, y, z), where x-axis is oriented vertically upwards along the plate and y-axis is taken as normal to the plane of the plate while z-axis is taken along the width of the plate as shown in figure 1 . Since the plate is of infinite length, all the physical variables are function of y and t only . In order to make the flow model more ideal, the investigation is restricted to the following assumptions. (i)the plate is electrically nonconductive and is subjected to a constant suction velocity v0. (ii)the magnetic reynolds number is so small that the induced magnetic field can be neglected . Also, the electrical conductivity of the fluid is reasonably low and hence the ohmic dissipation may be neglected. (iii)the electron pressure is constant. (iv)the energy dissipated due to internal friction of the fluid particles is negligible. (v)in the absence of an externally applied electric field and with negligible effects of polarization of the ionized gas, the electric field vector is assumed to be zero.with the foregoing assumptions and under the usual boundary layer and boussinesq approximations, the governing equations of motion are as follows . The magnetic reynolds number is so small that the induced magnetic field can be neglected . Also, the electrical conductivity of the fluid is reasonably low and hence the ohmic dissipation may be neglected . The energy dissipated due to internal friction of the fluid particles is negligible . In the absence of an externally applied electric field and with negligible effects of polarization of the ionized gas, the electric field vector momentum equation is (7) ut+vuy=02uy2k0(3uty2+v3uy3) + g(tt)+g(cc) b02[(1+bebi)u+bew][(1+bebi)2+be2]uk,wt+vwy=02wy2k0(3wty2+v3wy3) b02[(1+bebi)wbeu][(1+bebi)2+be2]wk. Energy equation is (8)tt+vty=ktcp2ty2 . Concentration equation is (9)ct+vcy=d2cy2kl(cc), where (10)tt=, cc= subject to the boundary conditions which are (11)y=0: u=u0, w=0,=aeit, =beit,y: u0, w0,0, 0 . For the sake of normalization of the flow model, we introduce the following nondimensional quantities: (12)=v0y, t = v02t4, u = uv0,w = wv0, =a, =b, pr=cpkt, gr = g(tt)av03, m=b02v02, k = kv022,sc=d, =klv02k = k0v022, gm = g(cc)bv03 . We get the following governing equations which are dimensionless: (13)14utu=2u2k[143ut23u3] m{(1+bebi)u+bew}(1+bebi)2+be2 + gr+gmuk,(14)14wtw=2w2k[143wt23w3] m[(1+bebi)wbeu](1+bebi)2+be2wk,(15)14t=1pr22(16)14t=1sc22. The relevant boundary conditions are (17)=0: u=1, w=0,=eit, =eit,: u0, w0,0, 0 . Introducing the complex variable (18)=u(,t)+iw(,t), where i=-1, (13) and (14) transform to single partial differential equation (19)14t=22k[143t233] m[(1+bebi)ibe](1+bebi)2+be2k+gr+gm subject to boundary conditions (20)=0: =1, =eit, =eit,: 0, 0, 0 . In order to solve (15), (16), and (19) under the boundary conditions of (20), we assume that (21)(,t)=0()eit,(22)(,t)=0()eit,(23)(,t)=0()eit . Using (21) and (22) into (15) and (16), we get (24)0=e(/2)(pr+pr2+ipr),0=e(/2)(sc+sc2+(isc+4sc)). Using 0, 0 in (21) and (22), respectively, we get the solutions for the temperature and the concentration fields which are as follows: (25)=eit(/2)(pr+pr2+ipr)=eit(/2)(sc+sc2+(isc+4sc)). Substituting the values of (23) in (19), we get (26)k0+(1ik4)0+0(a1ia2)0 = gr0gm0, where primes denote differentiation with respect to . The corresponding transformed boundary conditions are (27)=0: 0=eit,: 00 . To solve (26), we use the multiparameter perturbation technique as follows: (28)0=00()+k01(), as k 1, for small shear rate . Using (28) in (26) and equating the coefficients of like powers of k, we get (29)00+00(a1ia2)00=gr0gm0,01+01(a1ia2)01=i40000, subject to modified boundary condition (30)=0, 00=eit, 01=0,, 000, 010 . Solving (29) under the boundary conditions (30), we get (31)0=(c1e6+c3e2+c5e4) + i(c2e6+c4e2+c6e4). From (23), we get (32)=0eit={(c1e6+c3e2+c5e4)hhl+i(c2e6+c4e2+c6e4)}eit . Now, equating the real and imaginary part, we get the axial and transverse components of velocity as follows: (33)u=(c1e6+c3e2+c5e4)cost (c2e6+c4e2+c6e4)sint, w=(c2e6+c4e2+c6e4)cost + (c1e6+c3e2+c5e4)sint . The axial component of the shearing stress at the plate for primary velocity is (34)1=(uk[142ut2u2])=0=(c16+c32+c54)cost + (c26+c42+c64)sint k[14{(c16+c32+c54)sinthhhhhhhlh+(c26+c42+c64)cost}hhhhhh{(c162+c322+c542)costhhhhhhhhh(c262+c422+c642)sint}14]. The transverse component of the shearing stress at the plate for secondary velocity is (35)2=(wk[142wt2w2])=0=(c26+c42+c64)cost (c16+c32+c54)sint k[14{(c26+c42+c64)sinthhhhhhhh(c16+c32+c54)cost}hhhlhh(c262+c422+c642)costhhlhhh+14(c162+c322+c542)sint]. The rate of heat transfer in terms of the nusselt number is given by (36)nu=()=0=12(pr+pr2+ipr)eit . The rate of mass transfer coefficient in terms of the sherwood number is given by (37)sh=()=0=12(sc+sc2+(isc+4sc))eit . The objective of the present paper is to investigate the effects of ion slip on viscoelastic fluid flow past an impulsively started infinite vertical plate embedded in a porous medium with chemical reaction . In order to get a physical insight of the effects of flow parameters on the flow problem under consideration, we make graphical illustration for velocity field in figures 211 and shearing stress in figures 1221 . The parameters k = 1, t = /2 are kept fixed throughout the discussion . The nonzero values of the parameter k characterize the viscoelastic fluid and k = 0 represents the newtonian fluid flow phenomenon . Figures 2 and 3 reveal the effects of both the magnetic parameter m and the viscoelastic parameter k on the fluid velocity components u and w. in general, application of transverse magnetic field has the tendency to decrease the velocity due to resistive lorentz force . In both of the figures, the enhancement of viscoelasticity modifies the primary fluid velocity u and secondary fluid velocity w in comparison with newtonian fluid flow phenomenon . Also, the growth of magnetic parameter m shifts the fluid velocity in downward direction for both of the velocity components . Grashof number studies the behavior of free convection and it is defined as the ratio of buoyancy force to viscous force . It plays an important role in heat and mass transfer technology . In this study, the results are discussed for the flow past an externally cooled plate (gr> 0) and flow past an externally heated plate (gr <0). Figures 4 and 5 are depicted for positive values of the buoyancy parameter gr which corresponds to the cooling problem . It is experienced from figures 4 and 5 that the rise of grashof number (gr) with the increasing values of viscoelastic parameter magnifies fluid flow velocity in both types of velocity components . Figures 6 and 7 exhibit the effects of viscoelastic parameter on fluid velocity for the flow past a heated plate (gr <0) and for the flow past a cooled plate (gr> 0). It is seen from the figures that, for the flow past a heated plate, the flow nature of newtonian fluid diminishes as compared to the flow past a cooled plate . Furthermore, with the amplification of viscoelastic parameter, the velocity pattern for heated plate gradually decreases in comparison with newtonian fluid but a reverse trend is observed in case of cooled plate for both primary and secondary velocity components . Figures 8 and 9 represent the graphs of primary and secondary velocity components for different values of viscoelastic parameter (k) when the effect of hall current is accounted . From these figures, we note that the enhancement of hall current declines the trend of both primary and secondary velocity profiles along with the increment of viscoelastic parameter . Figures 10 and 11 depict the influence of ion slip parameter bi on fluid velocity with viscoelastic parameter . Analyses of the graphs show that the rising effect of ion slip parameter is to increase the velocity profiles for both newtonian and non - newtonian cases . After knowing the velocity field, it is very important from a physical point of view to know the effect of viscoelastic parameter on resistive force or viscous drag . The resistive force or viscous drag on the surface of the body due to the motion of the fluid is known as the shearing stress . To get the physical behavior of the axial shearing stress and transverse shearing stress, figures 1221 have been plotted to notify the nature of shearing stress formed at the externally cooled surface (gr> 0) with the variation of viscoelastic parameter (k) for different values of flow parameters: prandtl number (pr), hall parameter (be), ion slip parameter (bi), radiation (r), grashof number for mass transfer (gm), and chemical reaction parameter (). Prandtl number plays a significant role in heat transfer flow problems as it helps to study the simultaneous effects of momentum and thermal diffusion in fluid flow . Figures 12 and 13 represent the nature of axial and transverse components of shearing stress experienced by newtonian as well as non - newtonian fluid against prandtl number . Analysis of the graph shows that the magnitude of the axial shearing stress rises with the variation of viscoelastic parameter, but, on the other hand, a reverse trend is observed in case of transverse shearing stress . Figures 14 and 15 have been plotted to discuss the axial and transverse shearing stress against hall parameter (be). It is seen that near the surface of the plate, the magnitude of axial shearing stress for non - newtonian fluid gradually increases as compared to newtonian fluid, but a descending pattern is encountered in case of transverse shearing stress . The effects of ion slip parameter (bi) on the shearing stress for newtonian as well as non - newtonian fluids at the plates are revealed in figures 16 and 17 . From these graphs, we observe that the rising trend of ion slip parameter shows a growth in axial shearing stress along with the increasing values of k in comparison with newtonian fluid, but a reverse physical nature is observed for transverse shearing stress . The consequences of free convection parameter for mass transfer (gm) are noticed in figures 18 and 19 . Gm> 0 indicates that the free stream concentration is less than the concentration at the boundary surface . These figures mainly discuss the nature of resistive force of fluid flow during the various positive values of grashof number for mass transfer (gm). The figures reveal that the increasing values of gm lead to subdue the magnitude of axial shearing stress for newtonian fluid in comparison with viscoelastic fluid . Furthermore, the transverse shearing stress for newtonian fluid accelerates whereas resistivity diminishes away from the plate for viscoelastic fluid . Figures 20 and 21 display the variation of shearing stresses against chemical reaction parameter with viscoelastic parameter . The graphs enable the fact that as the chemical reaction parameter increases, the resistivity of the non - newtonian fluids is enriched for axial shearing stress and there would be a significant decrease of magnitude in case of transverse shearing stress away from the surface as compared to simple fluid . The effects of ion slip on unsteady mhd flow of an incompressible electrically conducting viscoelastic fluid past an impulsively started vertical plate embedded in a porous medium with chemical reaction in presence of heat and mass transfer are studied in this paper . Some of the important conclusions of this paper are as follows.the flow field is significantly affected with the variation of viscoelastic parameter.the effect of ion slip parameter on velocity is prominent throughout the boundary layer in presence of other flow parameters.the rates of heat transfer, that is, nusselt number, and rate of mass transfer, that is, sherwood number, are not significantly affected during the variations of viscoelastic parameter throughout the fluid flow phenomenon.the axial and transverse components of shearing stress are prominently affected by the viscoelastic parameter along with other flow parameters . The effect of ion slip parameter on velocity is prominent throughout the boundary layer in presence of other flow parameters . The rates of heat transfer, that is, nusselt number, and rate of mass transfer, that is, sherwood number, are not significantly affected during the variations of viscoelastic parameter throughout the fluid flow phenomenon . The axial and transverse components of shearing stress are prominently affected by the viscoelastic parameter along with other flow parameters.
The development of simple creatinine - based equations to estimate kidney function has contributed to raise the epidemics of chronic kidney disease (ckd) to the category of major public health problem . Ckd is an important contributor to the burden of non - communicable diseases, leading to poor quality of life and severely reduced life expectancy . Ckd also has a substantial impact on societal costs, by increasing per se the need for healthcare services and contributing to work disability . Further, the consequences of ckd are of importance for many other aspects of healthcare, including the incidence of other comorbidities [1, 5] or impairment on the diagnostic accuracy of laboratorial analyses and medical devices [68] dependent on protein or fluid retention by the failing kidneys . Notwithstanding that creatinine is a very common and low - cost assessment in healthcare [9, 10], systematic screening programs are lacking and, in most societies, the awareness of ckd among both physicians and patients remains very low [11, 12]. Many drugs have nephrotoxic effects per se or by forcing hydrophilic drug hyperfiltration in damaged kidneys . Further, significant morbidity is experienced due to medication accumulation and exposure in ckd, commonly leading to adverse events, prolonged length of hospital stay, increased healthcare cost and even death [14, 15], even when drug clearance is mediated by non - renal mechanisms . Drug - induced nephrotoxicity indeed accounts for 25% of episodes of acute kidney injury, a figure that can be as high as 70% among the elderly . Due to low ckd awareness, the risk for inappropriate drug utilization (idu) in the community is high [13, 15, 1921], representing a likely important risk factor for ckd progression and complications [22, 23]. Sweden is a northern european country with universal healthcare access and a population of 9.8 million inhabitants as of 2015, with nearly 25% living in the county of stockholm . The burden of ckd in sweden, especially of earlier ckd stages, and its impact on society and healthcare utilization is currently unknown . Awareness of the extent of the problem is needed to estimate and plan for allocation of resources on disease prevention strategies as well as diagnosis and early management . Assessment of plasma creatinine is one of the most common routine laboratory analyses performed on wide indications in clinical practice, both in the emergency setting and in regular check - ups of patients with various disorders . The extent of this occasional creatinine testing is unknown, but can provide important information on kidney function in order to inform patients and guide clinical practice . The stockholm creatinine measurement (scream) project is a collaborative project between karolinska institutet and the stockholm county council (sll by swedish acronym), to ascertain the burden of ckd and the extent of idu in the region . This article provides an overview of the scream objectives and goals, details of the laboratory data extraction and linked data sources . Scream objectives are focussed around key research methodologies: clinical epidemiology, health services research, pharmacoepidemiology and health economics . The following four specific aims were initially identified: to determine the prevalence of ckd in the region of stockholm, sweden, and ascertain its clinical consequences in terms of comorbid complications and healthcare resource utilization.to determine healthcare- and socioeconomic - related risk factors for progression of ckd.to identify idu in the context of kidney dysfunction (incorrectly dosed or nephrotoxic drug prescription) and to document its adverse clinical consequences.to establish the safety and effectiveness of common drugs in individuals with ckd . To determine the prevalence of ckd in the region of stockholm, sweden, and ascertain its clinical consequences in terms of comorbid complications and healthcare resource utilization . To determine healthcare- and socioeconomic - related risk factors for progression of ckd . To identify idu in the context of kidney dysfunction (incorrectly dosed or nephrotoxic drug prescription) and to document its adverse clinical consequences . To establish the safety and effectiveness of common drugs in individuals with ckd . Three laboratory companies (aleris, unilabs and karolinska) performed the vast majority of all clinical chemistry laboratory tests of the region . Each laboratory provider extracted the requested laboratory tests for all patients who had these measurements undertaken throughout the region . The inclusion criteria considered to enter scream was to be a stockholm resident with a valid personal identifying number undertaking at least one measurement of creatinine in primary, secondary or tertiary care during 200611 . All creatinine tests analysed during the period were included, as well as other laboratory measurements routinely linked to ckd and used to guide ongoing patient assessment, monitor disease progression and identify relevant outcomes (table 1). Table 1.routine laboratory tests collected in screamserum tests creatinine cystatin - c albumin haemoglobin haemoglobin a1c glucose c - reactive protein parathyroid hormone blood lipids: cholesterol, low density lipoprotein- and high density lipoprotein - cholesterol, triglycerides electrolytes: k, hco, na, ca, po, co2 thyroid hormones: thyroid stimulating hormone, triiodothyronine and thyroxine, both total and free prothrombin time (international normalized ratio)urine tests dipstick albuminuria albumin - to - creatinine ratio routine laboratory tests collected in scream each laboratory test included, in addition to the details of the test, a unique patient identifier (swedish personal identification number), the date on which the test was performed, method used and units of measurement . Concerning serum creatinine, all laboratory providers had since 2005 implemented either enzymatic or corrected jaffe method (alkaline picrate reaction), both methods being traceable to isotope dilution mass spectroscopy standards . Creatinines analysed locally (on site) were largely included, as most analysing machines were connected to the laboratory data system . Inter- as well as intra - laboratory variation was considered minimal, with the three laboratories being frequently audited for quality and harmonization by the national organization equalis (www.equalis.se). However, in the absence of patient data such as height and weight, estimation of kidney function by established equations becomes less accurate for individuals below 18 years of age, and we did not consider them in our analyses . Patient - identified laboratory data were sent from each laboratory provider to sll's subcontracted company tieto sweden ab (http://www.tieto.se/). The personal identifier was used to link the laboratory data to the administrative health data register of this region (vrdanalysdatabasen, val; stockholm regional healthcare data warehouse) (figure 1). Val is established by sll and contains information on all consultations in primary and secondary care (defined as specialist outpatient care), as well as hospitalizations . Both public and private healthcare providers within the region report each patient visit to val . Data for primary care was available since 2003, and for secondary care and hospitalization since 1993 . Each healthcare visit was accompanied by the date (and when applicable discharge date), the centre accessed and medical department, therapeutic procedures undertaken and established diagnoses according to the international classification of diseases version 10 (icd-10) coding system . Identification of the type of healthcare visit in which the laboratory test was performed is important to differentiate test results obtained during a hospitalization (potentially influenced by acute illness) from those performed in the out - patient setting, which may better reflect stable medical conditions . Comorbid history will be primarily defined based on the disease domains of the charlson comorbidity score [26, 27]. The presence of one or more diagnostic codes in any position up to 3 years prior to measurement entry will be considered to define comorbidity history . Patient demographics included sex and date of birth (month and year), migration procedures (when applicable date of registration as county citizen and date of departure / emigration), and ascribed municipality of residency according to the national classification of statistics sweden . Information on race is not available in sweden by law, and in accordance with the general population characteristics, it was assumed that all patients were of caucasian origin . Icd-10 codes and therapeutic procedures were retrieved for each individual since 1997 and up to 31 december 2012 . The scream dataset was thereafter sent to the national board of health and welfare, to be linked to the following national quality registers: the swedish prescribed drug registry, a nationwide registry instituted in 2005 and collecting information on all prescription drugs dispensed at swedish pharmacies . It also contains information on the practice (primary health care centre or clinic) and profession / medical specialty of the prescriber . Drugs were reported by their generic names as well as active principle atc codes, together with the number of defined daily doses dispensed and the costs (both reimbursed expenditure and patient co - payment). This register did not contain information on over - the - counter pharmaceuticals, nor ambulatory or in - hospital care drugs given at hospitals . Drug dispensing data were requested for each individual since 2006 and up to 31 december 2012 . The swedish population registry recorded, on a monthly basis, information on vital status for each swedish citizen with virtually no loss to follow - up . In case of death, vital status information was requested for each individual from inclusion in scream up to 31 december 2012 . The longitudinal integration database for health insurance and labour market studies (lisa by swedish acronym) (http://www.scb.se/en_/services/guidance-for-researchers-and-universities/scb-data/longitudinal-integration-database-for-health-insurance-and-labour-market-studies-lisa-by-swedish-acronym/.eng) combined information from several sociodemographic population registers . All swedish citizens older than 16 years residing in sweden as of 31 december were registered and the information was updated on a yearly basis . The swedish renal registry included swedish patients referred to a nephrologist and diagnosed with ckd . Inclusion was encouraged to all hospitals from ckd stage 3b and considered mandatory from the first diagnosis of ckd stage 4 . Information from patient visits during the non - dialysis phase and until renal replacement therapy start (first chronic dialysis or renal transplantation) was collected, with additional data collection on primary kidney disease, laboratory tests (such as uric acid) and in - hospital provided drugs to treat kidney diseases . Upon start of dialysis, a patient visit was recorded annually detailing in addition dialysis modality and specifications (dialysis dose, fistula / graft, etc . ). Information from this registry was requested since its establishment in 1999 up to 31 december 2012 . After all requested data were linked, patient identification was substituted by a random identifier, safeguarding patient privacy and confidentiality . The key to decipher the identifiers was kept at the national board of health and welfare for amendments, follow - up expansions and/or new linkages to the extraction . Only then, the complete scream project, including more than 200 datasets, was sent to the responsible investigators . Data were analysed according to our university rules and ethical standards and the project was reviewed and approved by the ethical committee of stockholm . In parallel with building the scream dataset sll was developing a clinical decision support system for the prescription of pharmaceutical drugs to patients with reduced kidney function (njuren). Njuren was an electronic application integrated to the health - records that assists prescribers by automatically estimating kidney function using the latest available creatinine assessment, gender and age . By combining this information with the information on prescribed drugs the prescriber was informed, when applicable, of kidney drug contraindications, nephrotoxicity, and the need for dose adjustment and/or safer drug alternatives for the patient . This current scream extraction will serve to document idu rates and its clinical consequences in the region of stockholm . Scream will identify which drugs, medical specialties and population segments are susceptible to idu, and this knowledge will be used to develop educational / corrective programs . During 2017, a second laboratory extraction will take place (scream-2), adding laboratory assessments and clinical practices for the period 201216 . This will serve to assess the effectiveness of njuren implementation by comparing the expected change in inappropriate drug utilization rates among njuren participating centres . This analysis of program effectiveness will motivate the expansion of njuren to other regions in sweden . Additionally, the second data extraction will allow capturing recent drugs and clinical practice changes . Three laboratory companies (aleris, unilabs and karolinska) performed the vast majority of all clinical chemistry laboratory tests of the region . Each laboratory provider extracted the requested laboratory tests for all patients who had these measurements undertaken throughout the region . The inclusion criteria considered to enter scream was to be a stockholm resident with a valid personal identifying number undertaking at least one measurement of creatinine in primary, secondary or tertiary care during 200611 . All creatinine tests analysed during the period were included, as well as other laboratory measurements routinely linked to ckd and used to guide ongoing patient assessment, monitor disease progression and identify relevant outcomes (table 1). Table 1.routine laboratory tests collected in screamserum tests creatinine cystatin - c albumin haemoglobin haemoglobin a1c glucose c - reactive protein parathyroid hormone blood lipids: cholesterol, low density lipoprotein- and high density lipoprotein - cholesterol, triglycerides electrolytes: k, hco, na, ca, po, co2 thyroid hormones: thyroid stimulating hormone, triiodothyronine and thyroxine, both total and free prothrombin time (international normalized ratio)urine tests dipstick albuminuria albumin - to - creatinine ratio routine laboratory tests collected in scream each laboratory test included, in addition to the details of the test, a unique patient identifier (swedish personal identification number), the date on which the test was performed, method used and units of measurement . Concerning serum creatinine, all laboratory providers had since 2005 implemented either enzymatic or corrected jaffe method (alkaline picrate reaction), both methods being traceable to isotope dilution mass spectroscopy standards . Creatinines analysed locally (on site) were largely included, as most analysing machines were connected to the laboratory data system . Inter- as well as intra - laboratory variation was considered minimal, with the three laboratories being frequently audited for quality and harmonization by the national organization equalis (www.equalis.se). However, in the absence of patient data such as height and weight, estimation of kidney function by established equations becomes less accurate for individuals below 18 years of age, and we did not consider them in our analyses . Patient - identified laboratory data were sent from each laboratory provider to sll's subcontracted company tieto sweden ab (http://www.tieto.se/). The personal identifier was used to link the laboratory data to the administrative health data register of this region (vrdanalysdatabasen, val; stockholm regional healthcare data warehouse) (figure 1). Val is established by sll and contains information on all consultations in primary and secondary care (defined as specialist outpatient care), as well as hospitalizations . Both public and private healthcare providers within the region data for primary care was available since 2003, and for secondary care and hospitalization since 1993 . Each healthcare visit was accompanied by the date (and when applicable discharge date), the centre accessed and medical department, therapeutic procedures undertaken and established diagnoses according to the international classification of diseases version 10 (icd-10) coding system . Identification of the type of healthcare visit in which the laboratory test was performed is important to differentiate test results obtained during a hospitalization (potentially influenced by acute illness) from those performed in the out - patient setting, which may better reflect stable medical conditions . Comorbid history will be primarily defined based on the disease domains of the charlson comorbidity score [26, 27]. The presence of one or more diagnostic codes in any position up to 3 years prior to measurement entry will be considered to define comorbidity history . Patient demographics included sex and date of birth (month and year), migration procedures (when applicable date of registration as county citizen and date of departure / emigration), and ascribed municipality of residency according to the national classification of statistics sweden . Information on race is not available in sweden by law, and in accordance with the general population characteristics, it was assumed that all patients were of caucasian origin . Icd-10 codes and therapeutic procedures were retrieved for each individual since 1997 and up to 31 december 2012 . The scream dataset was thereafter sent to the national board of health and welfare, to be linked to the following national quality registers: the swedish prescribed drug registry, a nationwide registry instituted in 2005 and collecting information on all prescription drugs dispensed at swedish pharmacies . It also contains information on the practice (primary health care centre or clinic) and profession / medical specialty of the prescriber . Drugs were reported by their generic names as well as active principle atc codes, together with the number of defined daily doses dispensed and the costs (both reimbursed expenditure and patient co - payment). This register did not contain information on over - the - counter pharmaceuticals, nor ambulatory or in - hospital care drugs given at hospitals . Drug dispensing data were requested for each individual since 2006 and up to 31 december 2012 . The swedish population registry recorded, on a monthly basis, information on vital status for each swedish citizen with virtually no loss to follow - up . In case of death, vital status information was requested for each individual from inclusion in scream up to 31 december 2012 . The longitudinal integration database for health insurance and labour market studies (lisa by swedish acronym) (http://www.scb.se/en_/services/guidance-for-researchers-and-universities/scb-data/longitudinal-integration-database-for-health-insurance-and-labour-market-studies-lisa-by-swedish-acronym/.eng) combined information from several sociodemographic population registers . All swedish citizens older than 16 years residing in sweden as of 31 december were registered and the information was updated on a yearly basis . The swedish renal registry included swedish patients referred to a nephrologist and diagnosed with ckd . Inclusion was encouraged to all hospitals from ckd stage 3b and considered mandatory from the first diagnosis of ckd stage 4 . Information from patient visits during the non - dialysis phase and until renal replacement therapy start (first chronic dialysis or renal transplantation) was collected, with additional data collection on primary kidney disease, laboratory tests (such as uric acid) and in - hospital provided drugs to treat kidney diseases . Upon start of dialysis, a patient visit was recorded annually detailing in addition dialysis modality and specifications (dialysis dose, fistula / graft, etc . ). Information from this registry was requested since its establishment in 1999 up to 31 december 2012 . After all requested data were linked, patient identification was substituted by a random identifier, safeguarding patient privacy and confidentiality . The key to decipher the identifiers was kept at the national board of health and welfare for amendments, follow - up expansions and/or new linkages to the extraction . Only then, the complete scream project, including more than 200 datasets, was sent to the responsible investigators . Data were analysed according to our university rules and ethical standards and the project was reviewed and approved by the ethical committee of stockholm . In parallel with building the scream dataset sll was developing a clinical decision support system for the prescription of pharmaceutical drugs to patients with reduced kidney function (njuren). Njuren was an electronic application integrated to the health - records that assists prescribers by automatically estimating kidney function using the latest available creatinine assessment, gender and age . By combining this information with the information on prescribed drugs the prescriber was informed, when applicable, of kidney drug contraindications, nephrotoxicity, and the need for dose adjustment and/or safer drug alternatives for the patient . This current scream extraction will serve to document idu rates and its clinical consequences in the region of stockholm . Scream will identify which drugs, medical specialties and population segments are susceptible to idu, and this knowledge will be used to develop educational / corrective programs . During 2017, a second laboratory extraction will take place (scream-2), adding laboratory assessments and clinical practices for the period 201216 . This will serve to assess the effectiveness of njuren implementation by comparing the expected change in inappropriate drug utilization rates among njuren participating centres . This analysis of program effectiveness will motivate the expansion of njuren to other regions in sweden . Additionally, the second data extraction will allow capturing recent drugs and clinical practice changes . The coverage of scream was assessed by comparing the number of scream individuals with the complete stockholm population . For that purpose, this included adult (18 years old) citizens of the stockholm region during 200611 (n = 1 706 259 individuals). Citizenship was ascertained by being censored in any of the 26 municipalities of the region at least once during the study period . For comparative purposes, summary statistics for age and sex strata as well as number of citizens per municipality were provided and compared with scream . Since assessment of creatinine was particularly indicated in specific comorbidities, we ascertained the coverage of scream in cardiovascular disease and diabetes mellitus patients . For this, we classified individuals according to the presence of at least one relevant icd-10 code during the period coming from a secondary or tertiary healthcare consultation . Cardiovascular disease was defined with the following four charlson domains [26, 27]: acute myocardial infarction, congestive heart failure, peripheral vascular disease and cerebrovascular disease; diabetes mellitus was defined with the two charlson domains of diabetes . Finally, being this an extraction of individuals seeking healthcare, we expected to have a sicker population with an over - representation of occurring deaths . To that end, we compared the number of deaths per calendar year in scream with those recorded for the general population . The coverage of scream was assessed by comparing the number of scream individuals with the complete stockholm population . For that purpose, this included adult (18 years old) citizens of the stockholm region during 200611 (n = 1 706 259 individuals). Citizenship was ascertained by being censored in any of the 26 municipalities of the region at least once during the study period . For comparative purposes, summary statistics for age and sex strata as well as number of citizens per municipality were provided and compared with scream . Since assessment of creatinine was particularly indicated in specific comorbidities, we ascertained the coverage of scream in cardiovascular disease and diabetes mellitus patients . For this, we classified individuals according to the presence of at least one relevant icd-10 code during the period coming from a secondary or tertiary healthcare consultation . Cardiovascular disease was defined with the following four charlson domains [26, 27]: acute myocardial infarction, congestive heart failure, peripheral vascular disease and cerebrovascular disease; diabetes mellitus was defined with the two charlson domains of diabetes . Finally, being this an extraction of individuals seeking healthcare, we expected to have a sicker population with an over - representation of occurring deaths . To that end, we compared the number of deaths per calendar year in scream with those recorded for the general population . Scream identified a total of 1 344 197 individuals with at least one serum creatinine assessed during the study period . For this analysis, we excluded individuals not residing in the stockholm county (n = 85 667) and those younger than 18 years old (n = 140 023). Therefore, the analysis of scream coverage and representativeness was based on 1 118 507 stockholm citizens in the adult age range . Roughly, one - third of the stockholm population underwent at least one creatinine assessment annually (25, 34, 35, 36, 34 and 33% across the years 200611, respectively). Since different individuals were assessed every year, the number of unique individuals with creatinine assessed during the whole period 200611 corresponded to 66% of the stockholm population . The coverage was equally distributed throughout the 26 municipalities of the region, ranging from 62% coverage in stockholm city, to 72% in norrtlje (figure 2). The coverage was slightly higher for women than for men in the age range of 1864 years, thereafter becoming equal between the sexes . Because creatinine assessment is more frequently assessed in older ages, scream's coverage increased with age; scream captured 50% of all individuals in the range of 1844 years old,> 75% of individuals aged 4564 years and> 90% of all individuals aged 65 years old . The distribution of age and sex in scream was very similar to that of the stockholm population (figure 3b). 3.proportion of the stockholm population included in scream, stratified by age and sex (a); age and sex distribution in scream and in the complete stockholm population (b). Proportion of the stockholm population included in scream, stratified by age and sex (a); age and sex distribution in scream and in the complete stockholm population (b). As many as 98% (121 785 versus 124 092) of all individuals with a cardiovascular disease (cvd)-related diagnosis are captured by scream . Likewise, 97% (78 125 versus 80 188) of individuals with a diabetes code were included . The proportion of cvd and diabetes cases in scream and in the general population was rather similar (figure 4), with an expected slight overrepresentation of both diseases in scream . During 200611, 91 353 deaths occurred in the general population . According to our 66% population coverage, 61 000 deaths were to be anticipated in scream . In all, 89% (n = 81 270) of deaths were captured in scream, being the expected difference attributed to the selection criterion of individuals in need of healthcare . 4.proportion of individuals with cvd (a) or diabetes mellitus (b) ascertained by icd-10 codes from secondary or tertiary healthcare consultations during 200611 in the stockholm population and in scream . Proportion of individuals with cvd (a) or diabetes mellitus (b) ascertained by icd-10 codes from secondary or tertiary healthcare consultations during 200611 in the stockholm population and in scream . The scream cohort represents to date the largest patient material to estimate the burden and implications of ckd in sweden, and probably one of the most comprehensive healthcare extractions to assess the implications of kidney function in healthcare management . The coverage and representativeness of the region of stockholm is high and in accordance with both the commonness of creatinine assessment in healthcare, and medical indications for creatinine tests . The richness of information retrieved, including both laboratory values and complete healthcare utilization data is unique and can be used for the study of other chronic medical conditions beyond ckd . Strengths of scream are the personal identity number unique to all swedish citizens and the possibility to continuously enrich data, increasing completeness and minimizing loss to follow - up . Since healthcare in sweden is tax - financed (to a large extent including also the cost of medications), we believe that confounding related to socioeconomic status, affecting the probability of adhering to the treatment, is lower than in other countries . However, there are important limitations as well; first, we use the existence of creatinine assessment to define our study cohort, which although reflects standard clinical practices, limits our study to subjects who have sought medical care and had a creatinine test undertaken (selection bias). The indications for creatinine measurement in scream are unknown . However, judging by the high representativeness of the stockholm population, especially among the older age stratum, this is unlikely to invalidate our findings . Finally, we lack certain clinical variables of importance such as body mass index, blood pressure and lifestyle factors (smoking habits, exercise), which may confound our conclusions . Nevertheless, some of them can be indirectly captured by specific ascertainment of comorbidities and medications . Other healthcare extractions have been performed in the past, but representativeness has seldom been estimated in those . Scream is a cohort by indication of assessment, and from this analysis, it can be concluded that scream findings can be well generalized to the elderly individuals (65 years old) of our region (> 90% coverage). Likely, scream can be fairly generalized to individuals in the age range 4564 years (> 75% coverage). The lowest representativeness is found for younger ages (<45 years), which may not have the same indications for creatinine testing . This prompts the need to stratify future study results across these age ranges and ponder both patient selection and conclusions having coverage and testing by indication in mind . The coverage of diseases with indications for creatinine testing is also virtually complete (> 97%), that is, all patients with diabetes or cvd during the observation period have had at least one creatinine test . Thus, scream has an excellent quality to study cardio - renal and diabeto - renal complications, treatments and management . In an ulterior sphere, this analysis shows that creatinine is already measured in a larger proportion of the community . Although screening programs are necessary, improved patient care can be achieved by interpreting the information that we already have and integrated in the medical decision process . Community approaches to pharmacy - idu are scarce, with previous evidence focussing on restricted population segments such as the elderly or restricted to in - hospital admissions and single - centre experiences [13, 15, 1921]. It has been demonstrated that clinical decision support systems to guide medication dosing improved the appropriateness of dosage by 13% and appropriateness of frequency by 24% in the usa . In a cross - sectional trial from belgium, the implementation of a computer - assisted medical support system in the intensive care unit - setting, reduced by 3-fold the rate of medication prescription errors in patients with renal insufficiency . We are positive that our efforts to identify idu coupled with the implementation of njuren in our region may improve this . To conclude, scream is a region - representative repository of laboratory data linked to regional and national registers that can be used for health services and health policy research in which kidney function has or may have an impact . Research collaborations are certainly welcome, including as well the possibility to link scream data to other swedish registers via each citizen's personal identification number . The scream project has obtained financial support from sll, the regional agreement on medical training and clinical research (alf) between sll and karolinska institutet, the swedish medical research council (vetenskapsrdet), martin rind s and westman s foundations.
Aside from non - melanoma skin cancer, prostate cancer (pc) is the most common cancer in men in the united states and europe, with a rapid increasing incidence in the last two decades . Radiotherapy is one of the most important curative options in treating localized pc . For high - risk pc, standard dose (70 gy) plus long - term anti - androgen therapy became the gold standard after the publication of eortc 22863 and rtog 92 - 02 trial results . Furthermore, pollack et al . Shown that dose escalation (78 gy) was a key point for biochemical control . Many studies have confirmed positive effect of dose escalation on disease local control using radiation therapy [58]. However, dose escalation delivered through 3d external beam radiation therapy (ebrt) was associated with a higher rate of gastro - intestinal (gi) and genito - urinary (gu) toxicities . Using intensity modulated radiation therapy (imrt), zelefsky et al . Shown that a total dose of 81 gy could be delivered to the prostate increasing the rate of biochemical control without increasing the rate of gu and gi toxicities . Intensity - modulated radiation therapy for pc is generally performed through 8 to 9 consecutive weeks; nevertheless, due to the low / ratio of the prostatic tissue, clinical research was directed toward hypofractionation concept . Randomized trials comparing standard radiation dose (2 gy / f, 1.8 gy / f) versus soft hypofractionated regimen (2.5 gy / f to 3 gy / f) using ebrt confirmed that the two regimens were equivalent in terms of acute and late toxicity [11, 12]. High - dose rate (hdr) brachytherapy boost following ebrt delivering a total dose of 46 gy to the pelvis or prostatic fossa, represents a smart technique to not only increase the dose to the prostate, but also significantly reduce the number of round trips to the radiation facility . In february 2009, we integrated the hdr brachytherapy boost program in pc treatment . We report the results in terms of acute gu and gi toxicity observed between these three different regimens . Between february 2009 and july 2013, 216 consecutive patients underwent a hdr brachytherapy boost for pc . Over the study period, two different treatment planning systems (tps) were used: plato (nucletron bv, veenendaal, the netherlands) from february 2009 to may 2012, and oncentra brachy (nucletron, an elekta company, elekta ab, stockholm, sweden) from june 2012 until now . In order to avoid any comparative bias, we analysed the first 124 patients treated with the same tps (plato). From 02/2009 to 01/2010, 30 patients (group 1 = g1; 24%) were treated with 3 6 gy over 2 days . In order to make this procedure more comfortable for the patient and less time - consuming for the medical staff, from 01/2010 to 01/2011, 52 patients (group 2 = g2; 42%) received 2 fractions of 9 gy over 2 days, while from 01/2011 to 05/2012, 42 patients (group 3 = g3; 34%) were treated with a single fraction of 14 gy . This observational study retrospectively analysed the acute gu and gi toxicities occurred during the first 6 months after the implant in the 3 treatment groups . All the patients underwent imaging studies, including magnetic resonance, computed tomography, and bone scan . Appropriate patient selection was performed according to the american brachytherapy society (abs) consensus guidelines for hdr prostate brachytherapy . Patients were treated with a first course of ebrt, delivering 46 gy in 23 fractions . The dose was delivered to the icru point using high - energy x - photons (> 10 mv). According to the calculated risk of lymph node involvement, the clinical target volume (ctv) was the whole pelvis or the prostatic fossa (prostate and seminal vesicles) for high- (15%) and low - risk (<15%) of lymph node involvement, respectively . The planning target volume (ptv) was defined as a 1 cm margin around the ctv in all directions, and reduced to 5 mm at the prostate rectal interface . Short and long - term androgen deprivation therapy (adt) was proposed to intermediate and high - risk patients, respectively . This procedure was performed under general anaesthesia . Using transrectal ultrasound (trus) guidance, 15 to 17 catheters (sharp needles; nucletron, an elekta company, elekta ab, stockholm, sweden) were implanted transperineally and periurethrally through a dedicated template . After recovery, post - implant planning ct - scan was directly performed in the radiation oncology department for treatment planning purposes . Ctv (prostate) and organs at risk (oar urethra and rectum) were outlined . The prostatic portion of the urethra was outlined plus a 10 mm additional margin above the base and below the apex, while the rectum was outlined from the anorectal junction to the rectosigmoid junction . Brachytherapy boost delivered 3 6 gy, 2 9 gy, and 1 14 gy for g1, g2, and g3, respectively . Considering / 1.5 gy for prostatic tissue, eqd2 (equivalent dose at 2 gy / fraction) were 39 gy, 54 gy, and 62 gy for g1, g2, and g3, respectively . Dose constraints for prostate were v100 (ctv receiving 100% of the prescribed dose)> 95%, v150 <35%, and v200 <15%, d90 (dose delivered to 90% of ctv)> 105%, and d100%> 80% . Dose constraints used for oars relied on the brachytherapy regimen groups . Because of the slight dose escalation between g1/g2 (eqd21.5 39 gy, 54 gy) and g3 (eqd21.5 62 gy), more restrictive dose constraints were considered for g3 patients . Consequently, for g1/g2, vu125 (percentage of the urethra volume receiving 125% of the prescribed dose) and vr100 (percentage of the rectum volume receiving 100% of the prescribed dose) should be less than 1%, while for g3 patients, vu115 (percentage of the urethra volume receiving 115% of the prescribed dose) and vr90 (percentage of the rectum volume receiving 90% of the prescribed dose) should be less than 1% (fig . Dose - volume adaptation was manually achieved using graphical optimization (plato, nucletron bv, veenendaal, the netherlands) by dwell location and time variation . Patients treated with multiple fractions after a single insertion were re - scanned before each fraction, followed by re - optimization with regard to the risk of catheter migration . The first fraction was given post - operatively (at day 0), while the following fractions were given at day 1 (with at least 6 hours interval between the 2 and 3 fraction for g1). Dose distribution obtained after planning using plato (nucletron bv, veenendaal, the netherlands) on sagittal (a) and transversal (b) view patients were followed at 1 month after the implant, then every 6 months with an assessment of serum prostate specific antigen level (psa), physical examination, patient symptom assessment, and digital rectal examination in case of rising psa . Acute gu and gi toxicities were scored according to the common terminology criteria for adverse event (ctcae v3.0). For gu complications, we analysed stricture / stenosis, frequency / urgency, retention, incontinence, cystitis and bladder spasms while for gi complications we analysed haemorrhoids, anal incontinence, and proctitis . Because a longer follow - up is needed to assess sexual toxicity, and due to the high number of patients who received adt, this complication was not assessed . Biochemical failure was defined as psa nadir plus 2 ng / ml . Data were entered and stored on an access file, and then transferred into r.2.14.1 software for statistical analysis . Test or fisher's exact test was used for category comparison . Student test or anova or non - parametric kruskal - wallis was performed to test variables expressed as categories versus continuous variables . If this test was significant, we used the test of tukey or nemenyi test to compare these categories . We also showed the median survival and confidence intervals at 95% of the studied population (kaplan - meier method). Tests of significance were two - sided, and considered significant when the p value was 0.05 or less . Between february 2009 and july 2013, 216 consecutive patients underwent a hdr brachytherapy boost for pc . Over the study period, two different treatment planning systems (tps) were used: plato (nucletron bv, veenendaal, the netherlands) from february 2009 to may 2012, and oncentra brachy (nucletron, an elekta company, elekta ab, stockholm, sweden) from june 2012 until now . In order to avoid any comparative bias, we analysed the first 124 patients treated with the same tps (plato). From 02/2009 to 01/2010, 30 patients (group 1 = g1; 24%) were treated with 3 6 gy over 2 days . In order to make this procedure more comfortable for the patient and less time - consuming for the medical staff, from 01/2010 to 01/2011, 52 patients (group 2 = g2; 42%) received 2 fractions of 9 gy over 2 days, while from 01/2011 to 05/2012, 42 patients (group 3 = g3; 34%) were treated with a single fraction of 14 gy . This observational study retrospectively analysed the acute gu and gi toxicities occurred during the first 6 months after the implant in the 3 treatment groups . All the patients underwent imaging studies, including magnetic resonance, computed tomography, and bone scan . Appropriate patient selection was performed according to the american brachytherapy society (abs) consensus guidelines for hdr prostate brachytherapy . Patients were treated with a first course of ebrt, delivering 46 gy in 23 fractions . The dose was delivered to the icru point using high - energy x - photons (> 10 mv). According to the calculated risk of lymph node involvement, the clinical target volume (ctv) was the whole pelvis or the prostatic fossa (prostate and seminal vesicles) for high- (15%) and low - risk (<15%) of lymph node involvement, respectively . The planning target volume (ptv) was defined as a 1 cm margin around the ctv in all directions, and reduced to 5 mm at the prostate rectal interface . Short and long - term androgen deprivation therapy (adt) was proposed to intermediate and high - risk patients, respectively . This procedure was performed under general anaesthesia . Using transrectal ultrasound (trus) guidance, 15 to 17 catheters (sharp needles; nucletron, an elekta company, elekta ab, stockholm, sweden) were implanted transperineally and periurethrally through a dedicated template . After recovery, post - implant planning ct - scan was directly performed in the radiation oncology department for treatment planning purposes . Ctv (prostate) and organs at risk (oar urethra and rectum) were outlined . The prostatic portion of the urethra was outlined plus a 10 mm additional margin above the base and below the apex, while the rectum was outlined from the anorectal junction to the rectosigmoid junction . Brachytherapy boost delivered 3 6 gy, 2 9 gy, and 1 14 gy for g1, g2, and g3, respectively . Considering / 1.5 gy for prostatic tissue, eqd2 (equivalent dose at 2 gy / fraction) were 39 gy, 54 gy, and 62 gy for g1, g2, and g3, respectively . Dose constraints for prostate were v100 (ctv receiving 100% of the prescribed dose)> 95%, v150 <35%, and v200 <15%, d90 (dose delivered to 90% of ctv)> 105%, and d100%> 80% . Dose constraints used for oars relied on the brachytherapy regimen groups . Because of the slight dose escalation between g1/g2 (eqd21.5 39 gy, 54 gy) and g3 (eqd21.5 62 gy), more restrictive dose constraints were considered for g3 patients . Consequently, for g1/g2, vu125 (percentage of the urethra volume receiving 125% of the prescribed dose) and vr100 (percentage of the rectum volume receiving 100% of the prescribed dose) should be less than 1%, while for g3 patients, vu115 (percentage of the urethra volume receiving 115% of the prescribed dose) and vr90 (percentage of the rectum volume receiving 90% of the prescribed dose) should be less than 1% (fig . Dose - volume adaptation was manually achieved using graphical optimization (plato, nucletron bv, veenendaal, the netherlands) by dwell location and time variation . Patients treated with multiple fractions after a single insertion were re - scanned before each fraction, followed by re - optimization with regard to the risk of catheter migration . The first fraction was given post - operatively (at day 0), while the following fractions were given at day 1 (with at least 6 hours interval between the 2 and 3 fraction for g1). Dose distribution obtained after planning using plato (nucletron bv, veenendaal, the netherlands) on sagittal (a) and transversal (b) view patients were followed at 1 month after the implant, then every 6 months with an assessment of serum prostate specific antigen level (psa), physical examination, patient symptom assessment, and digital rectal examination in case of rising psa . Acute gu and gi toxicities were scored according to the common terminology criteria for adverse event (ctcae v3.0). For gu complications, we analysed stricture / stenosis, frequency / urgency, retention, incontinence, cystitis and bladder spasms while for gi complications we analysed haemorrhoids, anal incontinence, and proctitis . Because a longer follow - up is needed to assess sexual toxicity, and due to the high number of patients who received adt, this complication was not assessed . Data were entered and stored on an access file, and then transferred into r.2.14.1 software for statistical analysis . Test or fisher's exact test was used for category comparison . Student test or anova or non - parametric kruskal - wallis was performed to test variables expressed as categories versus continuous variables . If this test was significant, we used the test of tukey or nemenyi test to compare these categories . We also showed the median survival and confidence intervals at 95% of the studied population (kaplan - meier method). Tests of significance were two - sided, and considered significant when the p value was 0.05 or less . Patient and treatment characteristics are reported in table 1 . Regarding patient (age, risk group factors) and treatment (ebrt, adt) data, the majority of the patients were classified as high (73.4%) or intermediate (19.3%) risk regarding to d'amico classification . Sixty - five percent of the patients (81 patients) received whole pelvic ebrt while 80% (99 cases) underwent adt . The median time interval between ebrt and hdr brachytherapy boost was 14 days (ranged 0 - 43). Patient characteristics according to the treatment group n number of patients, ebrt external beam radiation therapy, adt androgen deprivation therapy, int rt / brachy time interval between the end of ebrt and high - dose rate brachytherapy expressed in days dosimetric data are reported in table 2 . Because no significant dosimetric differences were noticed between g1/g2 patients regarding 1, 2 and 3 post - implant ct - scan (data not shown), the 1 ct - scan was used for dosimetric comparisons between the 3 treatment groups . Median ctv were equivalent in 3 groups (36, 33, and 32 cc for g1, g2, and g3, respectively, p = 0.088). D90 for g3 (105%) was significantly decreased compared to the d90 calculated for g1 (114%), and g2 (111%) (p <0.001) as well as v100 (97, 96, and 95% for g1, g2, and g3, respectively; p = 0.013), while v150 and v200 significantly decreased from g1 to g3 (v150: 45, 36, and 27% for g1, g2, and g3, respectively; p <0.001 and v200: 16, 12, and 9 for g1, g2, and g3, respectively p <0.001). Dose homogeneity index (dhi) for g3 (69%) was significantly better compared to dhi noticed for g1 (54%) and g2 (61%) (p <0.001). For oars (urethra and rectum), dosimetric results were significantly improved in g3 compare to g1 and g2 (p <0.001) (table 2). Assuming 3 gy for urethra and rectum, eqd2 of the dose delivered by the boost to oars significantly decreased from g1 to g3 for both urethra (d10u: 52, 47, and 46 gy for g1, g2, and g3, respectively; p <0.001) and rectum (d2r: 28, 27, and 26 gy for g1, g2, and g3, respectively; p <0.001) (table 2). Dosimetric data and equivalent doses at 2 gy for ctv (eqd2 / 1.5 gy) and organs at risk (eqd2 / 3 gy) according to the treatment group ctv clinical target volume (prostate), d90 dose delivered to 90% of ctv expressed in percentage and in eqd2 with /1.5 gy, d100 dose delivered to 100% of ctv expressed in percentage and in equivalence of the dose at 2 gy per fraction (eqd2) with / 1.5 gy, v100 ctv receiving 100% of the prescribed dose expressed in percentage and in cubic centimetres, v150 ctv receiving 150% of the prescribed dose expressed in percentage and in cubic centimetres, v200 ctv receiving 200% of the prescribed dose expressed in percentage and in cubic centimetres, dhi dose homogeneity index, d0.1u dose delivered to 0.1 cc of the urethral volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d1u dose delivered to 1 cc of the urethral volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d10u dose delivered to 10% of urethral volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d30u dose delivered to 30% of urethral volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d01r dose delivered to 0.1 cc of the rectal volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d1r dose delivered to 1 cc of the rectal volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d2r dose delivered to 2 cc of the rectal volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy the median follow - up was 25 months (ranged 8 - 46.9). Genito - urinary and gi complication grades for each treatment group were summarized in table 3 . The rates of acute gi and gu toxicities observed at 1 and 6 months after hdr brachytherapy boost were mainly grade 1 with few grade 2 (gu: 5% at 1 month; gi: 1% at 6 months). No grade 3 toxicity was observed in the whole population, while one patient in group 1 developed grade 4 gu toxicity consisting in a post - operative septic syndrome treated in intensive - care unit during 14 days . Analysis of genito - urinary (gu) and gastro - intestinal (gi) complications observed at 1 and 6 months after hdrb boost regarding to the complication grade (ctcv3.0) and the different fractionation schemes percentages of complication among patients who developed gu and/or gi complications gu genito - urinary complications, gi gastro - intestinal complications, fract . Schemes fractionation schemes regarding the acute gu and gi toxicities occurred in the 3 treatment groups, no significant differences were observed at 1 and 6 months after the hdr brachytherapy boost between g1, g2, and g3 treatment groups (table 3). Patient and treatment characteristics are reported in table 1 . Regarding patient (age, risk group factors) and treatment (ebrt, adt) data, the majority of the patients were classified as high (73.4%) or intermediate (19.3%) risk regarding to d'amico classification . Sixty - five percent of the patients (81 patients) received whole pelvic ebrt while 80% (99 cases) underwent adt . The median time interval between ebrt and hdr brachytherapy boost was 14 days (ranged 0 - 43). Patient characteristics according to the treatment group n number of patients, ebrt external beam radiation therapy, adt androgen deprivation therapy, int rt / brachy time interval between the end of ebrt and high - dose rate brachytherapy expressed in days dosimetric data are reported in table 2 . Because no significant dosimetric differences were noticed between g1/g2 patients regarding 1, 2 and 3 post - implant ct - scan (data not shown), the 1 ct - scan was used for dosimetric comparisons between the 3 treatment groups . Median ctv were equivalent in 3 groups (36, 33, and 32 cc for g1, g2, and g3, respectively, p = 0.088). D90 for g3 (105%) was significantly decreased compared to the d90 calculated for g1 (114%), and g2 (111%) (p <0.001) as well as v100 (97, 96, and 95% for g1, g2, and g3, respectively; p = 0.013), while v150 and v200 significantly decreased from g1 to g3 (v150: 45, 36, and 27% for g1, g2, and g3, respectively; p <0.001 and v200: 16, 12, and 9 for g1, g2, and g3, respectively p <0.001). Dose homogeneity index (dhi) for g3 (69%) was significantly better compared to dhi noticed for g1 (54%) and g2 (61%) (p <0.001). For oars (urethra and rectum), dosimetric results were significantly improved in g3 compare to g1 and g2 (p <0.001) (table 2). Assuming 3 gy for urethra and rectum, eqd2 of the dose delivered by the boost to oars significantly decreased from g1 to g3 for both urethra (d10u: 52, 47, and 46 gy for g1, g2, and g3, respectively; p <0.001) and rectum (d2r: 28, 27, and 26 gy for g1, g2, and g3, respectively; p <0.001) (table 2). Dosimetric data and equivalent doses at 2 gy for ctv (eqd2 / 1.5 gy) and organs at risk (eqd2 / 3 gy) according to the treatment group ctv clinical target volume (prostate), d90 dose delivered to 90% of ctv expressed in percentage and in eqd2 with /1.5 gy, d100 dose delivered to 100% of ctv expressed in percentage and in equivalence of the dose at 2 gy per fraction (eqd2) with / 1.5 gy, v100 ctv receiving 100% of the prescribed dose expressed in percentage and in cubic centimetres, v150 ctv receiving 150% of the prescribed dose expressed in percentage and in cubic centimetres, v200 ctv receiving 200% of the prescribed dose expressed in percentage and in cubic centimetres, dhi dose homogeneity index, d0.1u dose delivered to 0.1 cc of the urethral volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d1u dose delivered to 1 cc of the urethral volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d10u dose delivered to 10% of urethral volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d30u dose delivered to 30% of urethral volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d01r dose delivered to 0.1 cc of the rectal volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d1r dose delivered to 1 cc of the rectal volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy, d2r dose delivered to 2 cc of the rectal volume expressed in percentage of the prescribed dose and in eqd2 with / 3 gy genito - urinary and gi complication grades for each treatment group were summarized in table 3 . The rates of acute gi and gu toxicities observed at 1 and 6 months after hdr brachytherapy boost were mainly grade 1 with few grade 2 (gu: 5% at 1 month; gi: 1% at 6 months). No grade 3 toxicity was observed in the whole population, while one patient in group 1 developed grade 4 gu toxicity consisting in a post - operative septic syndrome treated in intensive - care unit during 14 days . Analysis of genito - urinary (gu) and gastro - intestinal (gi) complications observed at 1 and 6 months after hdrb boost regarding to the complication grade (ctcv3.0) and the different fractionation schemes percentages of complication among patients who developed gu and/or gi complications gu genito - urinary complications, gi gastro - intestinal complications, fract . The acute gu and gi toxicities occurred in the 3 treatment groups, no significant differences were observed at 1 and 6 months after the hdr brachytherapy boost between g1, g2, and g3 treatment groups (table 3). Dose escalation for prostate cancer radiation treatment represents a key point in order to improve biochemical control . According to the meta - analysis published by viani et al ., high - dose radiotherapy was superior to conventional dose radiotherapy in preventing biochemical failure in all risk groups, suggesting that high - dose radiotherapy should be offered to all patients regardless of their risk status . The low / ratio indicates that prostate tumours are more sensitive to high radiation doses per fraction than most other malignancies . Therefore, many investigators have directed their efforts to deliver hypofractionated ebrt . In this field, the use of brachytherapy boost in addition to pelvic ebrt up to 46 gy represents a smart and attractive option [22, 23]. Indeed, brachytherapy allows an intrinsic dose escalation due to the overdose volumes generated by the interstitial position of the radioactive source within the prostatic tissues, and lead to a biological advantage over external beam radiation boost technique . Brachytherapy boost has not only a potential advantage on clinical outcome, but also allows to make the treatment more acceptable for the patient due to less transportations to the medical insurance for reimbursement, and for the medical staff dealing with a less time consuming procedure . In this field, hdrb appears to give at least equivalent clinical outcomes compared to low - dose rate brachytherapy . A matched - pair analysis comparing prostate cancer ebrt + hdr vs. ebrt alone demonstrated a significant advantage in 5-year biochemical control in favour to the combination modality . Two randomized trials have confirmed the superiority of the combination therapy using either low or high - dose rate iridium sources in terms of biochemical control with at least similar late gu and gi toxicity with a potential advantage for acute rectal complications . However, these studies used as conventional ebrt arm a total dose around 70 gy which is now considered as a low dose regarding biochemical control probability . Nevertheless, even in case of very high - dose delivered to the prostate (86.4 gy) using a imrt technique, deutsch et al . Showed in a retrospective comparative analysis between ebrt vs. brachytherapy boost that the biochemical control remained significantly better in the brachytherapy group (3 7 gy). Whatever, it remains difficult to compare ebrt with brachytherapy boost techniques, because the dose delivered by brachytherapy will be always higher compare to the same prescribed dose delivered by ebrt (overdose volumes). This fact introduces an initial bias, which complicates the comparison between the two boost techniques . Taking all together, currently, there is no standard fractionation scheme, and numerous protocols have been tested with various numbers of fractions (ranged from 1 to 5) and dose per fraction (ranged from 4.75 to 15 gy). It appears more comfortable for both patient and medical staff to reduce as far as possible the number of brachytherapy fractions respecting a similar biological effect, and clinical outcome in terms of toxicity and efficacy . In this filed, we started our brachytherapy boost program using 3 fractions in 2 days (3 6 gy), while one fraction of 14 gy became our standard protocol allowing to implant the needles, treat the patient and remove all the materials in the same day (one day procedure). According to acute gi and gu toxicities, hoskin et al . Reported in a phase iii randomised trial that hdrb boost technique could significantly reduce gi toxicity with equivalent gu complication rates compared to those observed with ebrt . Regarding acute gi and gu toxicities, we reported mainly grade 1 complications and especially no significant differences between the 3 fractionations schemes . Furthermore, the dose distribution improved according to the learning curve, although the dose constraints for oars were more restrictive . Our results in terms of eqd2 for urethra and rectum (boost eqd2 + ebrt 46 gy) respect the recently published gec - estro recommendations, which propose for urethra d0.1cc and d10 120 gy eqd2 with d30 105 gy eqd2, and for rectum d2cc 75 gy eqd2 . Currently, the right fractionation remains under debate, but following strict implantation rules, there is a strong rational to propose a prostate cancer hdrb boost using a single fraction, providing similar results in terms of acute toxicity, and more comfortable procedure for patient and less time consuming for medical staff . However, longer follow - up is needed to confirm that single fraction of hdrb used as boost will give consistent results in terms of clinical outcome . The right fractionation remains under discussion, but prostate cancer hdrb boost using a single fraction (providing similar results in terms of acute toxicity) is more comfortable for the patient, less time consuming for the medical staff.
Common failure of the root canal obturation process is because of the presence of gaps and porosities at the sealer / dentin interface . Several studies have shown that filled root canals can allow the re - colonization of micro - organisms . The presence of micro - organisms and their products in the treated root canal can led to failure of the root canal treatment and the necessity of retreatment . The analysis of the dentin / sealer interface allows the determination of which materials and filling techniques could obturate the root canals with less gaps and voids . Several microscopy techniques are currently used to evaluate the sealer / dentin interface, including stereomicroscopy, scanning electron microscopy (sem), transmission electron microscopy, and confocal laser scanning microscopy (clsm). In comparison to conventional sem, clsm has the advantage of providing detailed information about the presence and distribution of sealers or dental adhesives inside dentinal tubules in the total circumference of the root canal walls at relative low magnification as 100x through the use of fluorescent rhodamine marked sealers . Placement and activation of a sealer into the root canal system should be done in a manner which is predictable and completely covers the dentin walls . Accepted means of sealer placement include the use of endodontic files or reamers, lentulo spirals, gutta - percha cones, ultrasonic files, and counter - clockwise motion of rotary systems . Very few studies have been done to evaluate the efficacy of different methods of sealer placement and activation effectively . Mta fillapex is a sealer that is composed of mta and has excellent flow, radiopacity, easy handling, working time, and low solubility . Because of its suitable physicochemical properties and excellent biocompatibility and bioactivity, hence, this study was planned to evaluate percentage and the depth of dentinal tubule sealer penetration of mta fillapex when agitated with three different techniques and to compare it with ah plus using clsm as the evaluative tool . Sixty teeth with single canal and free of cracks, caries, resorption, calcification, previous endodontic treatment, and less than 10- root curvature were selected and stored in distilled water until use . For standardization of the root length (10 mm), the crowns were resected with a diamond disc in a slow speed straight handpiece under constant water cooling . The working length was determined by inserting #15 k - file (dentsply maillefer) into the canal until it was just seen at the apical foramen and then 0.5 mm was subtracted from this length . The roots were instrumented by using the protaper universal root canal files (dentsply maillefer) in a sequential manner from s1 till f5 . Canals were irrigated between files with 2 ml of 3% naocl (sree rayalaseema alkalies and allies chemicals limited, india). To eliminate the smear layer 2 ml of 17% ethylenediaminetetraacetic acid (edta) (ph 7.7) for 3 min was used, followed by a final rinse of 2 ml distilled water . The roots were randomly divided into three groups on the basis of sealer activation technique utilized: g1: ultrasonic (woodpecker dte - d5 ultrasonic scaler, china); g2: lentulo spiral (dentsply, maillefer); g3: counter - clockwise motion (x smart, densply, maillefer, ballaigues, switzerland). Each group was further divided into two subgroups on the basis of type of sealer . Subgroup a utilized ah plus sealer while subgroup m utilized mta fillapex sealer . Ah plus sealer and mta fillapex sealer were mixed according to the manufacturer instructions and to allow analysis under the clsm, each sealer was labelled with rhodamine b (mayor diagnostics, mumbai, india) to an approximate concentration of 0.1%(by weight). Group 1 (g1): the ultrasonic unit (woodpecker dte - d5 ultrasonic scaler, china) was used in endo mode with a satelec ultrasonic endodontic tip k15 sonofile (dentsply tulsa) for activation . The file was inserted 2 mm short of the working length inside the canal and was ultrasonically activated for 20 seconds . Gutta - percha was compacted 1 mm below the canal orifice and the teeth were sealed with cavit (3 m, espe). In g1a ah plus sealer and in g1 m, mta fillapex sealer was used . Group 2 (g2): a size 30 lentulo spiral (dentsply, maillefer) was selected that would not bind in the prepared canal and that would reach the prepared working length . The lentulo spiral was rotated at speed of 300 rpm keeping the instrument 2 mm from apex for 20 seconds . Thereafter, the obturation of canal was conducted as described for g1 . In g2a ah plus sealer and in g2 m, mta fillapex sealer was used . Group 3 (g3): f1 protaper file attached in handpiece of endodontic micromotor (x - smart, dentsply tulsa) was kept 2 mm short of apex and rotated at 300 rpm in reverse mode for 20 seconds . Thereafter, the obturation of canal was conducted as described for g1 . In g3a ah plus sealer and in g3 m, mta fillapex sealer was used . The specimens were kept in an incubator at 37c and 100% humidity for 2 days . Each root was sectioned at 90- to the long axis by using diamond disc removing two, 1 mm sections at 3 mm and 6 mm from the apex . Coronal surface of each section was polished with sand paper (politriz, arotec, cotia, sp, brazil). The dentin segments were examined on a confocal microscope (olympus fluoview fv 1000). The respective absorption and emission wave lengths for the rhodomine b were 540 nm and 590 nm . Dentin samples were analyzed using the 10x lens . To calculate the percentage of sealer penetration around the root canal, first each image was imported into the iob software and the circumference of root canal measured using its ruler tool . Next, areas along the canal walls in which the sealer penetrated into dentinal tubules were outlined and measured using the same method [figure 1]. Subsequently, the percentage of root canal sealer penetration in that section was established . To determine the maximum depth of penetration, the point of deepest penetration was measured from the canal wall to the maximum depth of penetration [figure 2]. The effects of sealer agitation techniques and type of sealer on depth and percentage of sealer penetration were analyzed, by performing nonparametric kruskal - wallis for overall analysis, and a series of mann - whitney u tests for pairwise comparison using statistical package of social sciences (spss) statistics 20 software . Confocal laser scanning microscopic photograph showing calculation of percentage of penetration of sealer in the dentinal tubules (original magnification 10) confocal laser scanning microscopic photograph showing calculation of depth of penetration of sealer in the dentinal tubules (original magnification 10) sixty teeth with single canal and free of cracks, caries, resorption, calcification, previous endodontic treatment, and less than 10- root curvature were selected and stored in distilled water until use . For standardization of the root length (10 mm), the crowns were resected with a diamond disc in a slow speed straight handpiece under constant water cooling . The working length was determined by inserting #15 k - file (dentsply maillefer) into the canal until it was just seen at the apical foramen and then 0.5 mm was subtracted from this length . The roots were instrumented by using the protaper universal root canal files (dentsply maillefer) in a sequential manner from s1 till f5 . Canals were irrigated between files with 2 ml of 3% naocl (sree rayalaseema alkalies and allies chemicals limited, india). To eliminate the smear layer 2 ml of 17% ethylenediaminetetraacetic acid (edta) (ph 7.7) for 3 min was used, followed by a final rinse of 2 ml distilled water . The roots were randomly divided into three groups on the basis of sealer activation technique utilized: g1: ultrasonic (woodpecker dte - d5 ultrasonic scaler, china); g2: lentulo spiral (dentsply, maillefer); g3: counter - clockwise motion (x smart, densply, maillefer, ballaigues, switzerland). Each group was further divided into two subgroups on the basis of type of sealer . Ah plus sealer and mta fillapex sealer were mixed according to the manufacturer instructions and to allow analysis under the clsm, each sealer was labelled with rhodamine b (mayor diagnostics, mumbai, india) to an approximate concentration of 0.1%(by weight). Group 1 (g1): the ultrasonic unit (woodpecker dte - d5 ultrasonic scaler, china) was used in endo mode with a satelec ultrasonic endodontic tip k15 sonofile (dentsply tulsa) for activation . The file was inserted 2 mm short of the working length inside the canal and was ultrasonically activated for 20 seconds . Canal was obturated with protaper f5 gutta - percha cone . Gutta - percha was compacted 1 mm below the canal orifice and the teeth were sealed with cavit (3 m, espe). In g1a ah plus sealer and in g1 m, mta fillapex sealer was used . Group 2 (g2): a size 30 lentulo spiral (dentsply, maillefer) was selected that would not bind in the prepared canal and that would reach the prepared working length . The lentulo spiral was rotated at speed of 300 rpm keeping the instrument 2 mm from apex for 20 seconds . Thereafter, the obturation of canal was conducted as described for g1 . In g2a ah plus sealer and in g2 m, mta fillapex sealer was used . Group 3 (g3): f1 protaper file attached in handpiece of endodontic micromotor (x - smart, dentsply tulsa) was kept 2 mm short of apex and rotated at 300 rpm in reverse mode for 20 seconds . Thereafter, the obturation of canal was conducted as described for g1 . In g3a ah plus sealer and in g3 m, mta fillapex sealer was used . The specimens were kept in an incubator at 37c and 100% humidity for 2 days . Each root was sectioned at 90- to the long axis by using diamond disc removing two, 1 mm sections at 3 mm and 6 mm from the apex . Coronal surface of each section was polished with sand paper (politriz, arotec, cotia, sp, brazil). The dentin segments were examined on a confocal microscope (olympus fluoview fv 1000). The respective absorption and emission wave lengths for the rhodomine b were 540 nm and 590 nm . Dentin samples were analyzed using the 10x lens . To calculate the percentage of sealer penetration around the root canal, first each image was imported into the iob software and the circumference of root canal measured using its ruler tool . Next, areas along the canal walls in which the sealer penetrated into dentinal tubules were outlined and measured using the same method [figure 1]. Subsequently, the percentage of root canal sealer penetration in that section was established . To determine the maximum depth of penetration, the point of deepest penetration was measured from the canal wall to the maximum depth of penetration [figure 2]. The effects of sealer agitation techniques and type of sealer on depth and percentage of sealer penetration were analyzed, by performing nonparametric kruskal - wallis for overall analysis, and a series of mann - whitney u tests for pairwise comparison using statistical package of social sciences (spss) statistics 20 software . Confocal laser scanning microscopic photograph showing calculation of percentage of penetration of sealer in the dentinal tubules (original magnification 10) confocal laser scanning microscopic photograph showing calculation of depth of penetration of sealer in the dentinal tubules (original magnification 10) the percentage and depth of sealer penetration of different groups and intergroup comparison are summarized in tables 1 and 2, respectively . Shows mean and standard deviation of depth and percentage of sealer penetration of various groups at 3 and 6 mm levels intergroup comparison of depth and percentage of sealer penetration at 6 and 3 mm levels mean percentage and depth of sealer penetration of mta fillapex and ah plus sealer was greatest when ultrasonics was used for agitation and statistically significant (p <0.001) than lentulo spiral and counter - clockwise agitation techniques . Irrespective of method used for agitation, the percentage and depth of sealer penetration for mta fillapex was significantly greater (p <0.001) than ah plus . The percentage and depth of sealer penetration for mta fillapex and ah plus were greater and highly significant (p <0.001) at 6 mm than 3 mm . Removal of smear layer and use of sealer to attain and impervious seal between the core material and root canal walls is considered an essential step of root canal treatment . Sealers which can penetrate into the dentinal tubules exert bactericidal effect by having a closer contact with the residual bacteria within the tubules . In addition, sealer plugs inside the dentinal tubules provide a mechanical interlocking, thereby improving the retention of the filling material and reducing the microleakage along the root canal walls . Thus, both percentage and depth of sealer penetration might influence the outcome or success rate of endodontic therapy . Factors influencing sealer depth penetration in dentinal tubules are presence / absence of dentinal permeability (the number and the diameter of tubules), root canal dimension, presence of water, and physical and chemical properties of the sealer . The compound effect of physical properties viz flow, surface tension, solubility, viscosity, chemical composition, and working and setting time may influence penetration . Flow which is defined as the ability of a sealer to penetrate in irregularities, lateral canals, or dentinal tubules of the root canal system is one of the main physical factors to influence the tubular penetration . As most endodontic sealers are pseudoplastic, their flow increases with increase of shear rate . On comparison with conventional sealers patel dv et al ., and gharib sr et al ., found better depth of penetration of resin - based sealer in dentinal tubules . Mta fillapex has excellent biocompatibility and bioactivity and displayed significantly higher ph up to 7 days period than ah plus . Studies based on the physical properties reported significantly greater flow of mta fillapex than ah plus . Therefore, this comparative study was conducted to evaluate and compare the quality of sealing of ah plus and mta fillapex . Clinically, rapid insertion or activation of a pseudoplastic sealer into the canal would decrease viscosity and increase the flow of sealer . As very few studies have been conducted on the effect of sealer activation / placement and activation on sealing ability of root canal sealers therefore in the present study three activation techniques (ultrasonics, lentulo spiral, and rotary counter - clockwise motion) were chosen and the sealer distribution was analyzed . Amount of sealer, extent of activating instrument, and time for activation were standardized to minimize the errors . All the analyzed activation techniques failed to show a consistent adaptation of both the sealers to the total circumference of the root canal walls . However, mta fillapex showed significantly better (p <0.001) percentage and depth of sealer penetration than ah plus with all the techniques of sealer activation . Kuc, ia et al ., also reported greater mta fillapex sealer penetration than ah plus . Zhou h et al ., and silva ej et al ., found mta fillapex significantly more flowable than ah plus and attributed difference in composition and smaller particle size of the sealer for this property . Application of ultrasonics for activation led to significantly more (p <0.001) percentage and depth of both the sealers and is accordance to guimaraes bm et al ., the explanation for this is that the oscillating files in ultrasonics transmits the acoustic microstreaming energy and cause a greater depth of dentinal sealer penetration and coverage of root canal walls in the same manner as it promotes the penetration of irrigants in an area of anatomic complexities and the dentinal tubules . Although activation of both the sealers with lentulo spiral resulted in more percentage and depth of penetration as compare to counter - clockwise rotation however, the difference was statistically insignificant (p> 0.05). This is one of the pioneer studies evaluating effect of methods of activation on mta fillapex so it was difficult to compare the results obtained with previous studies . Lentulo spiral rotates and the spring portion pushes the sealer centrifugally while in counter - clockwise rotation with a file, the sealer tends to pool toward the tip of file but is not forced toward the walls . Irrespective of technique of activation and type of sealer, the depth, and percentage of penetration of sealer was significantly better at the 6 mm level than 3 mm level . These findings are similar to other studies and could be because the number and diameter of dentinal tubules decreases on descending apically in the root canal and superior removal of smear layer . In addition, the apical dentin is irregular in direction and density; even some areas are devoid of dentinal tubules . Under the parameters of present study, it can be concluded that mta fillapex sealer exhibited better percentage and depth of penetration in the radicular dentinal tubules than ah plus . Ultrasonic activation of pseudoplastic sealers can significantly increase its percentage and depth of penetration and level of root canal can affect both, percentage, and depth of penetration of sealers in the dentinal tubules.
A systematic review of literature was performed to identify the prevalence of adhd in iranian children and adolescents (up to 18 years old) from 1977 . Inclusion criteria were: 1) conducting a study by generalizability of the produced data to the population of the same age; 2) using valid instruments to assess adhd diagnosis or symptoms; and 3) presenting a prevalence of adhd or attention deficit and / or hyperactivity symptoms . Pubmed, isi web of science, psychinfo, iranpsych, iranmedex, irandoc were searched . Irandoc, iranmedex and iranpsych are iranian databases of which the last one is especially for psychiatry and psychology literature . Key words for search were: prevalence, rate, attention deficit, hyperactivity, adhd, behavioral problems, impulsivity, iran, tehran (and names of other cities which have universities), conners rating scale, rutter rating scale, wender utah rating scale . All iranian psychiatry and psychology journals were reviewed by hand searching to identify published but not indexed articles . All the booklets of iranian psychiatry, psychology, and epidemiology congress were reviewed in hand searching . Gray literature review was performed to identify final reports of research projects, desertions, and theses not published . Irandoc and iranmedex were searched for final reports up to 2003 after that searched by hand searching . 80 (60 in farsi and 20 in english) papers were screened in electronic and hand search by title and abstract; of which 64 (45 in farsi and 19 in english) were rejected as they were either unrelated or repeated in some sources . In the gray literature search, 57 final reports were screened by abstract; of which 50 were rejected or deleted . It should be noted that we deleted some of the reports as some universities refused to give us the full text of the final reports . Quality assessment was performed on full texts of 13 papers and 7 final reports which were approved in the last stage . For quality assessment quality assessment checklist for prevalence studies which assesses sampling, measurement and analysis method of studies: does the study design yield a sample of respondents representative of defined target group, do the survey instruments yield reliable and valid measures of adhd and other key concepts (also for translated version), and were special features of the sampling design accounted for in the analysis or subgroups prevalence rates reported? They reviewed each article and report, and if they both approved an article or a report, we accepted the material; but if one of them rejected the material, a third person reviewed it again . In quality assessment, 10 papers and 5 final reports were accepted . Some papers were excluded due to not reporting the prevalence or lack of randomization in sampling . Due to so many heterogeneities in instruments and in the rate of reported prevalence, we did not conduct analysis, and described the results in tables and graphs . Pubmed, isi web of science, psychinfo, iranpsych, iranmedex, irandoc were searched . Irandoc, iranmedex and iranpsych are iranian databases of which the last one is especially for psychiatry and psychology literature . Key words for search were: prevalence, rate, attention deficit, hyperactivity, adhd, behavioral problems, impulsivity, iran, tehran (and names of other cities which have universities), conners rating scale, rutter rating scale, wender utah rating scale . All iranian psychiatry and psychology journals were reviewed by hand searching to identify published but not indexed articles . All the booklets of iranian psychiatry, psychology, and epidemiology congress were reviewed in hand searching . Gray literature review was performed to identify final reports of research projects, desertions, and theses not published . Irandoc and iranmedex were searched for final reports up to 2003 after that searched by hand searching . 80 (60 in farsi and 20 in english) papers were screened in electronic and hand search by title and abstract; of which 64 (45 in farsi and 19 in english) were rejected as they were either unrelated or repeated in some sources . In the gray literature search, 57 final reports were screened by abstract; of which 50 were rejected or deleted . It should be noted that we deleted some of the reports as some universities refused to give us the full text of the final reports . Quality assessment was performed on full texts of 13 papers and 7 final reports which were approved in the last stage . For quality assessment, we used quality assessment checklist for prevalence studies which assesses sampling, measurement and analysis method of studies: does the study design yield a sample of respondents representative of defined target group, do the survey instruments yield reliable and valid measures of adhd and other key concepts (also for translated version), and were special features of the sampling design accounted for in the analysis or subgroups prevalence rates reported? They reviewed each article and report, and if they both approved an article or a report, we accepted the material; but if one of them rejected the material, a third person reviewed it again . In quality assessment, 10 papers and 5 final reports were accepted . Some papers were excluded due to not reporting the prevalence or lack of randomization in sampling . Due to so many heterogeneities in instruments and in the rate of reported prevalence, we did not conduct analysis, and described the results in tables and graphs . However, 3 studies have been conducted on preschool - aged children and only one study addressed university students . The instruments of studies were conners (teacher and parents), rutter (teacher and parents), and csi-4 (teacher and parents) questionnaire . Conners for parents has 48 questions and its cut off point is 15(chronbach's alpha:%91). Conner for teachers has 28 questions and its cut off point is 15(chronbach's alpha:%96). Characteristics of studies included in the analyses reliability of rutter test by test - retest is% 90 and its validity is% 76.7(p=0.001). Reliability of csi-4 for parents is% 87 and for teachers is% 89 and its cut off is 6 . It has facial validity and its sensitivity is% 62 with the specificity of%83.it should be noted that conners, rutter and csi-4 calculate adhd according to their scoring system . What is obtained from these scales tehran has the most prevalence rate of adhd, while sanandaj has the least prevalence . It can be related to more social stigma and less access to psychiatric consultation in cities like sanandaj . Figure 2 shows the prevalence of hyperactive type of adhd in girls and boys . The prevalence of hyperactive type of adhd in girls and boys the prevalence of inattentive type of adhd in girls and boys . We have conducted a systematic review of studies addressing prevalence rates of adhd in iran . Our results should be interpreted with caution because of the large variability found in all the conducted analyses . Probably the different prevalence rates across studies could be accounted for by different methodologies (8, 13, 14). Methodological features were coded for each study, including sample size, response rate, information source (e.g., parents, teachers, children), and whether diagnosis followed the dsm or not . According to dsm, it is possible to diagnose a child who shows symptoms in only one dimension . Some impairing symptoms, but not all, must be shown at home and at school . Unfortunately, many of iranian studies have only used rating scales to diagnose adhd in the children . Moreover, different rating scales have been used by such as conners, rutter and csi-4 . There was not any representative community sample in our studies, and this affects the reported prevalence rate . An analysis of epidemiological studies in the usa found that community samples gave higher prevalence rates than school samples (mean prevalence: 10.3% for community samples vs. 6.9% for school samples) (15, 16). It is generally agreed that the prevalence of adhd is significantly higher in boys than in girls, especially in children . Thus, the ratio of males to females in the sample population can affect the apparent prevalence and may need to be taken into account . For example, three studies have shown decreases in the prevalence with increasing age over the age range 10 - 20 years (17), 8 - 15 years (18), and 6 - 14 years (19). Thus, even within studies conducted on children, the age range of the sample is likely to affect the apparent prevalence . Most of the studies in our country have been conducted in school - aged children . Although analysis were done in similar age groups, but the heterogeneity did not decrease . Supporting this conclusion, a multisite trial study reported that using a uniform diagnostic protocol yields adhd patients who are highly similar across clinics in africa, australia, europe, and north america (20). It is difficult to make exact comparisons between studies because the estimated prevalence is highly influenced by the means of assessment and the type of sample recruited . It is necessary to take these factors into account when comparing data from different studies . One limitation of the literature we reviewed is the reliance of many studies on rating scale measures rather than interviews with patients and parents . Unlike rating scale methods, interview - based procedures come close to reproduce the results one might expect from a clinical evaluation and are better able to incorporate the impairment and pervasiveness criteria of the dsm diagnoses of adhd . As discussed by hartman et al (21), it is possible that improved operational symptoms could lead to increased measurement precision and a better assessment of the validity of diagnostic categories . The other limitation was that some studies had reported only the mean score of the indexes . Theses figures could not be accounted because it was not clear how many subjects were above the cutoff point.
Exposure to ionizing radiation generates several reactive oxygen species (ros), like hydroxyl radical, hydrogen peroxide, aqueous electron, and superoxide in the aqueous medium . Such ros have harmful effects on macromolecules such as lipids, proteins, and dna and damages cellular function, leading to multiple disorders and dysfunctions in the human body (1, 2). As several studies in the literature have indicated, radiation response is a multi - factorial scenario that depends on several parameters, such as the nuclear material content, cell reproduction, tissue and organ revival, and biological repair . From a biological viewpoint, it is important to consider how radiation response may moderate the responses of tissues and organs to gamma irradiation . From this aspect, genetic properties are highly crucial in most diseases and disorders, such as ataxia - telangiectasia (at) (3), xeroderma pigmentosum (4), and breast cancer (5), and an important characteristic in the radiosensitivity of cells, tissues and organs (5). Approximately 10% of normal individuals in similar physical and environmental conditions exhibit incremented response to ionizing radiations (6). Increased radiosensitivity of breast cancer patients has been reported in several different studies (79). Hence, identifying a prognostic marker for radiotherapy is one of the main goals of the radiation biology and radiation protection field . If the personal complication risks of ionizing radiation are recognized, the risk could be reduced in the small ratio of highly sensitive people through dose reduction in patients and the defining of more limited rules in radiation protection regulations (10). Some origins of radiosensitivity, including physiological, genetic, and epigenetic sources, have been proposed in cancer patients and normal individuals (11, 12). Several studies have marked that abo blood groups are associated with the risk of ischemic heart disease and developing severe manifestation of atherosclerosis (13). Results from the framingham study and other reports showed the occurrence of ischemic heart disease might be higher in subjects with blood group a (13). In addition, patients with blood groups a or o are more often afflicted with pernicious anemia (14). The incidences of gastric carcinoma (15), peptic ulcer (16), and ovarian cancer (5) are much more extensive in persons with blood types a, o, and b, respectively . Nonetheless, it was explained that the abo blood group is an inherent characteristic (17). Thereafter, the alleles for the blood groups were recognized in the same place on chromosome 9 at 9q34.1q34.2 (18). These findings demonstrated that one s blood group is a genetic trait . Despite such investigations, a preliminary clinical study showed that the radiation response of type o patients suffering from carcinoma of the cervix is better than those of other blood groups (19). Meanwhile, an elevated frequency of micronuclei (mn) in the a blood group was reported in peripheral blood lymphocytes of normal individuals (20). Regarding these findings - and given the variation in radiosensitivity in the normal population - this study aimed to evaluate chromosomal radiosensitivity of peripheral blood lymphocytes in normal individuals with rh and rh factors . In this experimental study, informed consent was acquired from 50 healthy human volunteers (25 rh+ and 25 rh) in the a blood group who ranged in age from 22 to 23 years old . All responders completed a questionnaire to provide information on their lifestyle and exposure to chemical and physical agents . Having a history of known irradiation, smoking, drug treatment, and alcohol consumption were exclusion criteria . Finally, samples were divided into two separate groups (control and exposed blood samples). The aliquots of blood samples were exposed to gamma radiation of a low let co source (theratone780 manufactured by canada) at dose rate of 70 cgy / min, with source - to - sample distance (ssd) = 80 cm, a field size of 10 10 cm, and at room temperature . Blood samples were irradiated with a total dose of 2 gy, and a vial of unexposed blood samples was kept as the control group . The cbmn assay was carried out using the standard technique proposed by earlier investigations (21), with minor modifications (22). Hence, 0.5ml of peripheral blood samples were added to 4.5ml of a cell culture medium (rpmi-1640) containing sodium bicarbonate supplemented with 10% fetal calf serum (fcs), antibiotics, and 1% l - glutamine . One hundred microliters of phytohaemagglutinin (pha, sigma) was added to stimulate lymphocytes . Forty - four hours after pha stimulation, cytochalasin - b (cyt - b, sigma) at a final concentration of 6g / ml was added, and binucleated lymphocytes were harvested after 72 h. the cells were then collected by centrifugation at 2,000 rpm for 10 min (boecho u-320 r), and supernatant was decanted . Two to three microliters of fresh hypotonic solution 0.075 m kcl was added to the solution remaining at the bottom of tubes and centrifuged at 1,200 rpm for 7 min . Again, the supernatant was poured off, and 5ml of fixature including methanol: glacial acetic acid (6/1) were quickly mixed with solution at the bottom of the tubes . The induction of mn was evaluated using a double - blind score of 1000 binucleated cells (bnc) with a light microscope set to 40x magnification . Independent - sample t - test was used to determine the statistical significance of the difference between rh and rh samples . In addition, a paired - samples t - test was used to analyze the difference between the control and exposed samples . The study was approved by the ethical committee of the faculty of medical sciences at babol university of medical sciences (babol, iran). In this experimental study, informed consent was acquired from 50 healthy human volunteers (25 rh+ and 25 rh) in the a blood group who ranged in age from 22 to 23 years old . All responders completed a questionnaire to provide information on their lifestyle and exposure to chemical and physical agents . Having a history of known irradiation, smoking, drug treatment, and alcohol consumption were exclusion criteria . Finally, samples were divided into two separate groups (control and exposed blood samples). The aliquots of blood samples were exposed to gamma radiation of a low let co source (theratone780 manufactured by canada) at dose rate of 70 cgy / min, with source - to - sample distance (ssd) = 80 cm, a field size of 10 10 cm, and at room temperature . Blood samples were irradiated with a total dose of 2 gy, and a vial of unexposed blood samples was kept as the control group . The cbmn assay was carried out using the standard technique proposed by earlier investigations (21), with minor modifications (22). Hence, 0.5ml of peripheral blood samples were added to 4.5ml of a cell culture medium (rpmi-1640) containing sodium bicarbonate supplemented with 10% fetal calf serum (fcs), antibiotics, and 1% l - glutamine . One hundred microliters of phytohaemagglutinin (pha, sigma) was added to stimulate lymphocytes . Forty - four hours after pha stimulation, cytochalasin - b (cyt - b, sigma) at a final concentration of 6g / ml was added, and binucleated lymphocytes were harvested after 72 h. the cells were then collected by centrifugation at 2,000 rpm for 10 min (boecho u-320 r), and supernatant was decanted . Two to three microliters of fresh hypotonic solution 0.075 m kcl was added to the solution remaining at the bottom of tubes and centrifuged at 1,200 rpm for 7 min . Again, the supernatant was poured off, and 5ml of fixature including methanol: glacial acetic acid (6/1) were quickly mixed with solution at the bottom of the tubes . The induction of mn was evaluated using a double - blind score of 1000 binucleated cells (bnc) with a light microscope set to 40x magnification . Independent - sample t - test was used to determine the statistical significance of the difference between rh and rh samples . In addition, a paired - samples t - test was used to analyze the difference between the control and exposed samples . The study was approved by the ethical committee of the faculty of medical sciences at babol university of medical sciences (babol, iran). The results of this study showed an increased mean frequency of mn in binucleated cells after irradiation (figure 1). As table 1 shows, the mean values of mn in binucleated cells were 196 18.23 and 169 17.11 for rh and rh samples, respectively (table 1). The results indicated that the average mn is significantly higher in rh than in rh in the irradiated group (p<0.001) (table 1). Table 2 shows that the frequency of mn cells significantly increased in both rh and rh irradiated (2gy) samples whereas the percentage of binucleated, trinucleated, and tetranucleated cells decreased after irradiation . The ndi value for the rh group was 1.81 and 1.39 before and after irradiation, respectively . For the rh type, the parameter declined from 1.85 to 1.40 following gamma irradiation . Regarding the ndi in table 2, approximately 20% of the radiotherapy patients showed a wide range of normal tissue reactions (23). Recognizing patients with high, moderate, or low radiosensitivity would help develop a good plan for the radiotherapy of patients with the highest cure rate in terms of the patient s individual adaptation (10). The explanation of criteria that modify the total effects of ionizing radiation is one of the main subjects of research efforts in radiobiology . Some studies have explained that a correlation exists between tumor response and the level of 8-oxo - dg in the urine of lung and breast cancer patients (23). Hence, several studies have been initiated to investigate whether radiosensitivity is an individualized subject in patients radiation response . The main purpose of this study was to investigate the role of rh in radiation response of healthy people . Hoeller et al . Showed that individual radiosensitivity measured by lymphocytes might be a good way to predict fibrosis in breast cancer patients after radiotherapy (24). In the field of radiobiology, the cbmn cyt assay for pbl is a suitable bio - dosimetry device to measure in - vivo and in - vitro radiosensitivity and cancer reproducibility (25). Lymphocytes are chosen because of their easy availability, simple method of culture, and ease of sample collection . Therefore, in our study, cbmn cyt assay as one of the standard techniques for genetic toxicology evaluation in human cells was selected to measure chromosomal aberrations in rh and rh healthy people . As expected, the average number of mn increased significantly after the 2gy gamma irradiation of lymphocyte cells . This result concurs with the earlier findings indicating a higher level of mn after gamma irradiation (21, 26). The current study showed that the mean frequency of mn in the rh group was roughly 16% higher than in the rh group . This result might be due to the association of rh alleles on chromosome 9 and particular alleles of genes responsible for dna double strand break repair . However, more molecular investigations need to evaluate more precisely the main reason for this event . Indicated that the a blood group has more mn frequency compared to other blood types (20). They showed that group a has a higher frequency of mn than did groups ab, b, and o (20). In another study, they showed that left - handed breast cancer women are more radiosensitive than right - handed women (18). The background mn frequency in the present study was 3.75 0.53 and 5.35 0.90 for the rh and rh groups, respectively . This value is in fair agreement with the cited value of iaea epr bio - dosimetry manual by international atomic energy agency (27). Regarding the iaea report, the background mn frequencies range from 0 to 40 per 1000 bn cells (27). For the ndi counted prior to and after irradiation, the value decreased in both rh and rh groups after 2gy gamma irradiation, indicating a reduction in cell proliferation . However, no significant difference occurred between these two blood types after irradiation . As the present study indicated, a decrease yield of binucleated and polynucleated cells was observed after gamma irradiation . In contrast, the frequency of mononucleated cells increased after gamma irradiation . Because carrying out standard cytokinesis blocked micro - nuclei protocol was time consuming, choosing a larger study population the present study suggested that chromosomal radiosensitivity of lymphocytes in normal individuals with rh factor is higher than their rh counterparts . This finding revealed that the rh factor can be considered one of the hereditary traits affecting individuals radiosensitivity and can be a valuable item in radiation protection regulations . However, further in - vivo and in - vitro studies are needed for validation.
Cancer is a bitter truth that affects the life of millions of people and has turned into one of the greatest tensions in their life . It is also one of the main causes of mortality in iran . Among all types of cancer, breast cancer is one of the main causes of death and disease all over the world whose incidence is increasing day by day, especially in developing countries . According to statistics, in the usa, the new cases of breast cancer number 235,030 among which 4430 cases end in patients death . Breast cancer is considered as the most common type of cancer and the fifth major reason for death among iranians . It is also worth noting that the mean age of contracting cancer by iranian women is 47.148.8 years . Although iranian women contract cancer 10 years earlier than women from developed countries, progress in treatment and care services has led to iranian patients improved survival and more successful outpatient treatments . Thus, due to increased use of outpatient services for cancer patients and due to the decrease in referral to hospitals and patients improved survival and caregivers willingness for taking care of patients at home, the first step of care giving to cancer patients has moved from hospitals to houses . Family members and close friends have taken up the responsibility for taking care of these patients . Informal caregivers are family members, spouses, or friends who provide support to the cancer patient . According to statistics, two - thirds of cancer patients never use the facilities and services provided by the community for fulfilling their daily needs . Caregivers are, in fact, mediums for transferring information from the patients to the medical team and vice versa to make sure patients treatment plans are performed perfectly . They are also considered as vital members of the support team for coordinating appointments with the specialists and labs . As cancer is considered as a family issue and not only an individual and personal condition, it changes patients family identity, as well as family members roles and daily performance . Taking care of cancer patients is usually coupled with many challenges, a wide range of problems, and issues for the caregivers . So, maintaining the relation with the medical team is necessary for providing care to patients . By providing training and knowledge on caregiving skills, and interacting with family members, the medical team plays a significant role in enabling the family members to maintain and improve the care provided to patients . However, working with caregivers is a new and difficult role for the medical team as the physicians may know the significance of training the caregivers but they may overlook the significance of caregivers need for the training . Poor interaction of treatment personnel with patients families and inadequate support to the patients lead to informational contrast in families and patients as well as incorrect application of resources that may ultimately increase the tension and pressure in these conditions . Although caregivers are part of the patient support network, they are, as a frail population, deprived of the attention they need and are overlooked by the healthcare system . Thus, thorough identification and assessment of their needs and experiences is of great importance . Interventions made for helping caregivers are even considered as the standard component of patients treatment . Caregivers problems have been studied in some researches, but inadequate attention has been given to caregivers of cancer patients . On the other hand, most of the studies conducted on caregivers are related to western countries that are much different from eastern, especially muslim, countries . Thus, the findings of western countries would prove inapplicable and inefficient in eastern countries . Caregiving is a multifaceted experience that is connected to race, nationality, cultural values, beliefs, and family systems . Considering the cultural and religious values as well as the strong familial relations, iranian families provide much care to the cancer patients and are considered as the strongest support system to patients; they actually are willing to take care of the patients . Considering the emphasis placed by international cancer organizations on the significance of the spouses and family members experiences and a necessity for studies on familial caregivers and the network of friends in different cultural groups, the authors decided to study caregivers understanding of their own needs with regard to the treatment team in a varied range of caregivers selected from family members of breast cancer patients . The main focus of the study is on caregivers of breast cancer patients, as breast cancer is the most common type of cancer in iran and patients have a greater chance of survival in the initial stages of diagnosis . On the other hand, iranian women's contraction age of breast cancer these two factors, i.e. Higher survival rate and contraction by younger women, have made the caregivers and families to be more involved and make a greater and longer contribution . Despite the widespread incidence of this type of cancer and the important role of iranian families as the best source of care for patients, insufficient information is available on this subject in iran . This research has been motivated by researcher's experience with caregivers issues, as well as the significance of the subject and was conducted due to the lack of domestic studies on the subject . A qualitative design, based on a conventional content analysis approach, was adopted to attain the objective of the study in 2013 . The participants were selected through purposeful sampling from family members taking care of cancer patients who had referred to specialized cancer centers and support societies . The participants were over 18 and were able to read, write, and state their experiences . The researcher introduced herself and stated the main objectives of the study to gain participants trust in the confidentiality of the interviews . The authors followed the ethical observations, and ensured the participants their freedom to discontinue their cooperation with the study at any moment they desired . Interviews contained open - ended unstructured question and started with the general request, please speak about your experiences about your interactions with the treatment team; this was followed by semi - structured explorative questions . Further interviews were conducted based on the analysis of previous interviews and the obtained data . The interviews took 30 to 60 minutes and were conducted with prior arrangement with participants and in their favourite venues . The criterion for stopping sampling was repetition of data and data saturation that yielded no new data . The conventional content analysis method with an inductive approach was employed for data analysis . To this end, after transcription of interviews and several iterations of review, meaning units were identified in the form of sentences or paragraphs in transcribed dialogs, and primary codes or open codes were extracted . Then, based on centrality and conceptual similarity, the main categories and subcategories were extracted . Next, the first transcriptions as well as primary and final categories were reread several times . Changes in the number, content, and name of categories were made in each review iteration up to the point in which primary categories, main categories, and subcategories were finalized . Guba and lincoln criteria including credibility, conformability, dependability, and transferability were used to ensure data accuracy . Enough time was allocated to data gathering; in - depth interviews were conducted in multiple sessions and at different times and places; the findings were reviewed by participants and supervisors; the data were analyzed by colleagues; and a variety of participants with regard to their gender, age, marital status, degree of closeness to the patient, educational level, and socio - economic condition were selected . The age of participants ranged from 20 to 69 with a mean age of 37.5 years . Most of the participants were women (69.6%), married (65.2%), and patients children (34.8%) with academic education (56.5%). Most of them (43.5%) earned between 5,000,000 and 7,000,000 rials of iran as their monthly salary . The mean of care time was 7.1 h / day (with a range of 1.512 h) [table 1]. Four categories were extracted from the analysis results of data gathered from the participants: a) caregivers contribution and acknowledging them in the treatment system, b) training in efficient caregiving, c) efficient interaction of the medical team with caregivers, and d) easy and dynamic access to the medical services [table 2]. Characteristics of family caregivers of breast cancer patients results obtained from data analysis most of the participants were willing to contribute to be considered and acknowledged in the treatment system . However, in most of the cases, the treatment team did not allow them to contribute and help, and thus, the participants were marginalized . Consultation with patient and caregiver and explaining to them about the treatment plan and the prescribed medicine were among the points the caregivers mentioned for several times as contributions they liked to have with regard to patients issues . Nevertheless, they did not receive any information on these issues and were not consulted . They did not provide any information to us either on the prescribed drugs or the things they were doing . They never mention these to the patient and caregiver and never ask for their opinion . (a 24-year - old caregiver, a patient's daughter) the caregivers emphasized that they liked to feel they were valuable and wanted to be seen by the treatment team . The caregiver feels obligated to be with the patient during the treatment process to decrease his / her stress . The treatment team is expected to pay attention to the caregiver and recognize his / her role . The fact we (the caregivers) are not seen at all is much annoying . (a 20-year - old caregiver, a patient's daughter) the findings of the study confirm that caregiving by family caregivers is on an uncertain and misty path . They felt like travelers who were not prepared and trained adequately, even though they were with the patient on this uncertain path . In my opinion, it is very important that one is prepared and knows what to do and how to care . I think caring is a skill and one needs to be trained so one can support the patient and be of any benefit to the patient . (a 38-year - old patient a patient's husband) unpreparedness and lack of training and information were mentioned as one of the stressors of caregiving . (a 38-year - old caregiver, a patient's husband) many of them were confused about their role and had to refer to external resources for information and guidance . (a 32-year - old caregiver, a patient's son) another concerning issue for caregivers is the patients spiritual and mental mood . A need for guidance and training on how to treat patients and support them mentally and spiritually was emphasized repeatedly by participants . They do nt explain clearly how one should treat cancer patients . Even on their mental and spiritual mood . The patients have the same stress and fear of death and that is enough for them . (a 24-year - old caregiver, a patient's daughter) caregivers are mediums between the treatment team and the patient during the treatment process to make sure the treatment procedure is conducted fully and patients receive adequate and efficient support . For the realization of this, it is necessary for the treatment team and caregivers to have a mutual and efficient interaction and relationship with one another . It was mentioned by the participants that their role and status was not respected by the treatment team and they did not have a good inter - personal relationship with each other . (a 38-year - old caregiver, a patient's husband) some participants mentioned short visits made by the doctor and the presence of other people or patients at the doctor's room during their visit . I had made a list of questions to ask when we wanted to go to doctor's office . But when we went in, there were other patients and their companions and i did nt find the time to ask any questions at all . (a 38-year - old caregiver, a patient's husband) receiving information in a simple and comprehensible language and without any medical jargons was important . When they explain something to me, i like it to be comprehensible to me . (a 20-year - old caregiver, a patient's daughter) perceiving cancer as an untreatable disease in many cultures and as a taboo in iran causes much stress in patients and their families . Many caregivers considered the diagnosis and disclosure moment as a critical and extremely difficult moment . The moment the doctor said it was cancer, i said, oh, cancer! The name of cancer kills one . (a 38-year - old caregiver, a patient's husband) another significant and noticeable issue in the caregivers experience was how the news of cancer diagnosis was disclosed by the treatment team . To understand the caregivers condition and sympathize with them at the moment of diagnosis the doctor told us quite frankly and without standing on any circumstances that the cancer had spread to her lungs and bones . I did nt expect the doctor to tell us this way, without any introduction and preparation . (a 28-year - old caregiver, a patient's sister) easy and dynamic interaction with the treatment team, and access to the team members even through phone and at any time of the night or day to obtain their advice on the correct and necessary measures at emergency conditions were participants other concerns . It would have been really great if they gave us a nurse's contact number so we could contact her in emergencies or to ask our questions . (a 24-year - old caregiver, a patient's daughter) some participants mentioned long waiting time for visits to doctors in government hospitals and access to other health services such as the appointments diagnostic, therapeutic and drug approval . Our waiting time for mri is 3 months . (a 28-year - old caregiver, a patient's sister) others known cancer as a particular disease and they were willing to attention to these patients and putting them in priority to access services . Cancer is as a particular disease and it is different from other diseases . So, patients should take priority for treatment, medication, tests and other things . (a 54-year - old caregiver, a patient's mother) another issue of concern for the caregivers is the presence of equipped medical centers, with other services . Most of the participants were willing to contribute to be considered and acknowledged in the treatment system . However, in most of the cases, the treatment team did not allow them to contribute and help, and thus, the participants were marginalized . Consultation with patient and caregiver and explaining to them about the treatment plan and the prescribed medicine were among the points the caregivers mentioned for several times as contributions they liked to have with regard to patients issues . Nevertheless, they did not receive any information on these issues and were not consulted . They did not provide any information to us either on the prescribed drugs or the things they were doing . They never mention these to the patient and caregiver and never ask for their opinion . (a 24-year - old caregiver, a patient's daughter) the caregivers emphasized that they liked to feel they were valuable and wanted to be seen by the treatment team . The caregiver feels obligated to be with the patient during the treatment process to decrease his / her stress . The treatment team is expected to pay attention to the caregiver and recognize his / her role . The fact we (the caregivers) are not seen at all is much annoying . The findings of the study confirm that caregiving by family caregivers is on an uncertain and misty path . They felt like travelers who were not prepared and trained adequately, even though they were with the patient on this uncertain path . In my opinion, it is very important that one is prepared and knows what to do and how to care . I think caring is a skill and one needs to be trained so one can support the patient and be of any benefit to the patient . (a 38-year - old patient a patient's husband) unpreparedness and lack of training and information were mentioned as one of the stressors of caregiving . (a 38-year - old caregiver, a patient's husband) many of them were confused about their role and had to refer to external resources for information and guidance . (a 32-year - old caregiver, a patient's son) another concerning issue for caregivers is the patients spiritual and mental mood . A need for guidance and training on how to treat patients and support them mentally and spiritually was emphasized repeatedly by participants . They do nt explain clearly how one should treat cancer patients . Even on their mental and spiritual mood . The patients have the same stress and fear of death and that is enough for them . Caregivers are mediums between the treatment team and the patient during the treatment process to make sure the treatment procedure is conducted fully and patients receive adequate and efficient support . For the realization of this, it is necessary for the treatment team and caregivers to have a mutual and efficient interaction and relationship with one another . It was mentioned by the participants that their role and status was not respected by the treatment team and they did not have a good inter - personal relationship with each other . (a 38-year - old caregiver, a patient's husband) some participants mentioned short visits made by the doctor and the presence of other people or patients at the doctor's room during their visit . I had made a list of questions to ask when we wanted to go to doctor's office . But when we went in, there were other patients and their companions and i did nt find the time to ask any questions at all . (a 38-year - old caregiver, a patient's husband) receiving information in a simple and comprehensible language and without any medical jargons was important . When they explain something to me, i like it to be comprehensible to me . (a 20-year - old caregiver, a patient's daughter) perceiving cancer as an untreatable disease in many cultures and as a taboo in iran causes much stress in patients and their families . Many caregivers considered the diagnosis and disclosure moment as a critical and extremely difficult moment . The moment the doctor said it was cancer, i said, oh, cancer! The name of cancer kills one . (a 38-year - old caregiver, a patient's husband) another significant and noticeable issue in the caregivers experience was how the news of cancer diagnosis was disclosed by the treatment team . To understand the caregivers condition and the doctor told us quite frankly and without standing on any circumstances that the cancer had spread to her lungs and bones . I did nt expect the doctor to tell us this way, without any introduction and preparation . The doctor shattered us that way . (a 28-year - old caregiver, a patient's sister) easy and dynamic interaction with the treatment team, and access to the team members even through phone and at any time of the night or day to obtain their advice on the correct and necessary measures at emergency conditions were participants other concerns . It would have been really great if they gave us a nurse's contact number so we could contact her in emergencies or to ask our questions . (a 24-year - old caregiver, a patient's daughter) some participants mentioned long waiting time for visits to doctors in government hospitals and access to other health services such as the appointments diagnostic, therapeutic and drug approval . Our waiting time for mri is 3 months . (a 28-year - old caregiver, a patient's sister) others known cancer as a particular disease and they were willing to attention to these patients and putting them in priority to access services . Cancer is as a particular disease and it is different from other diseases . So, patients should take priority for treatment, medication, tests and other things . (a 54-year - old caregiver, a patient's mother) another issue of concern for the caregivers is the presence of equipped medical centers, with other services . The results of the study showed that the most significant needs of caregivers are contribution, being acknowledged in the treatment system, training on efficient caregiving, medical teams interaction with them, and easy and dynamic access to the medical services . At the beginning of the treatment, decisions need to be made with regard to treatment options . Thus, providing information to and efficient contribution from the patients and caregivers are vital in making decisions . Our results confirm the results of other iranian studies indicating lack of awareness of the disease, diagnosis, treatment process, and plan of patients and their families . Despite iranian doctors belief that family members need to be aware of treatment decisions and contribute to the treatment from the initial stages of diagnosis, this has been overlooked in iran's treatment and healthcare system . Other western studies also show that caregivers are marginalized in the decisions made by the treatment team and patients, and they are not informed on the nature of the disease, treatment program, and patient's condition, while they need to contribute actively and the treatment team should view them as part of the caring team and share their knowledge and information with the caregivers . Training for caregivers was their other need . Despite the major role of caregivers in caring, this has not been addressed in iran's treatment and healthcare system adequately and there is no comprehensive treatment program in this regard . Our results confirm the results of other studies indicating non - provision of training and information by oncologist personnel to caregivers with regard to caring for cancer patients, disease development symptoms, mental spiritual support to patients, and interaction with them . Access to services and interaction with healthcare professionals was also mentioned as another important issue . Other studies also have emphasized caregivers familiarity with medical literature and having a honest, clear conversation with them . Our results confirm the results of other studies indicating the importance of support to caregiver by treatment teams and access and relationship with the treatment personnel . Another aspect deemed as important by the participants was treatment teams skill for disclosing the cancer diagnosis news . However, studies have emphasized caregivers emotional feedback to the diagnosis and prognosis of cancer and the necessity for providing emotional support to caregivers individually . Unfortunately, iran lacks supportive caring, such as consultation, even for patients themselves . Due to specialists poor skills of disclosing bad news and their poor management of the emotions of patients who are aware of the diagnosis as caregivers are valuable members of treatment team and assist the healthcare system to achieve its treatment goals for patients, they need to interact with the treatment team so that they receive necessary services, equipment, and information . On the other hand, it has been proved that recognizing caregivers as separate people with their own special needs will have beneficial effects on both the patients and caregivers . Thus, understanding their experiences, preferences, and needs in their interaction with the treatment team is of prime importance in the care system for cancer patients . This will help to make effective interventions to this end by correct use of limited resources . The findings of this study, which were obtained from caregivers viewpoints, provide a better understanding of their needs in their interaction with the treatment team . These findings can also assist with providing a framework for effective, culture - sensitive, and appropriate medical interventions in cancer healthcare system planning . Thus, caregivers need to be viewed as a standard part of cancer care programs, and a comprehensive family - centered care program needs to be designed so that the needs of familial caregivers, as the first responders to cancer patients problems, are addressed in healthcare policies.
A 33-year - old female patient presented at the emergency room with epigastric pain, nausea, and vomiting that had persisted for a day . The patient had a history of child birth eight months prior, and had proximal deep vein thrombosis (dvt) on the left common femoral vein during the 36th week of pregnancy . An inferior vena cava (ivc) filter had been inserted instead of anticoagulation due to the high risk of bleeding during pregnancy . Removal of the ivc filter was recommended one month after insertion, but the patient refused for personal reasons . On arriving at the emergency room, her vital signs were stable and the laboratory examination did not show abnormalities other than mild leukocytosis . The patient's prothrombin time international normalized ratio value at the emergency room was 1.31 due to irregular intake of warfarin . A computed tomography (ct) scan revealed that one of the ivc filter legs had penetrated the ivc wall and caused a duodenal perforation (fig . A protruding ivc filter leg was observed in the lumen of the third portion of the duodenum (fig . 2). In addition, the duodenum mucous membrane on the opposite side showed erythema, erosion, and nodular changes, resembling chronically progressing penetration . An emergency laparotomy was performed in order to remove the ivc filter and to repair the duodenum . Because there were concerns regarding the possible ivc rupture during surgery, a cannula was placed in the superior vena cava to provide extracorporeal circulation when needed . Also, the femoral artery and femoral vein were isolated for cannulation . The portions of the ivc and the duodenum, including the penetrations, were isolated behind the colon . When the duodenum was lifted up, we found the ivc filter leg between the ivc and the duodenum . We then cut the ivc filter leg and removed the ivc filter leg remnant from the duodenum portion . The ivc was found to be densely adhered to the surrounding tissue including the aorta, and fixed to the adjacent structures . We concluded that isolation and removal of the ivc filter leg remnant in the ivc were more dangerous due to the risk of rupture . The remaining leg protruding from the ivc side to the duodenum was cutoff and removed (fig . 3). After the surgery, ileus occurred and the patient started to eat by mouth after 14 days . The ivc filter is known as an effective and safe method for preventing fatal pulmonary thromboembolism in patients with deep vein thrombosis . The ivc filter is indicated in dvt patients when any of the following symptoms are present: contraindication of anticoagulation, major bleeding, need for surgery within 2 weeks, severe and prolonged thrombocytopenia, and recurrent dvt disease despite anticoagulation . Complications related with the ivc filter can be categorized as insertion - related complications, device failure, and late complications . Insertion - related complications include pneumothorax, hemorrhage, filter misplacement, excessive tilt, and vascular injury . Late complications include recurrent pulmonary embolus, caval occlusion, filter migration, and filter leg perforation . The titanium greenfield filter showed a 3.5% perforation rate, the stainless steel greenfield filter showed a 4.4% rate, the bird's nest filter showed a 38% rate, and the simon nitinol filter showed a 37% rate . Most patients with perforations do not show any symptoms and the perforation rarely causes clinical problems . However, there are cases where the filter can damage surrounding organs and cause severe complications in the aorta, duodenum, ureter, or retroperitoneal space [4 - 6]. Pulsation of the aorta and respiratory motion are thought to be the main cause of caval penetration of the filter leg . In our case, the cause of the ivc wall penetration seemed to be chronic progression of the ivc filter leg, nine months after the ivc filter insertion; subsequently, it finally penetrated into the duodenum . Based on the preoperative ct venography, the ivc flow was maintained and there was no dvt below the ivc level . Also, based on the observations in the operation room, although cardiopulmonary bypass was in standby before surgery, removal of the ivc filter was thought to be more risky . Thus, a partial resection of the ivc filter leg that was penetrating the duodenum was performed, and the ivc was reinforced . Currently, the ivc filter is widely used and only 50% of them are removed from patients . Most of the remaining ivc filters are asymptomatic and do not cause clinical problems . However, in order to prevent potential sequelae caused by ivc filter leg penetration, like that reported in this case, the removal of the ivc filter, when possible, is preferred.
The primary objective of root canal treatment is debridement to achieve thorough disinfection and obturation of the canal space . Development of a periapical lesion signifies the presence of bacteria in the root canal system, in particular in its apical portion where they can exist in a biofilm and pose a challenge to treatment.1,2 an important role of root canal irrigation is to remove the residual bacterial biofilm from the inaccessible, un - instrumented surfaces.3 biofilms can be defined as polysaccharide matrix enclosed bacterial populations adherent to each other and/or to surfaces or interfaces.4 irrigants acts as a flush to remove organic and inorganic debris that would otherwise be left in the canal after mechanical instrumentation.5 root canal irrigants should have a broad antimicrobial spectrum and high efficacy against anaerobic and facultative microganisms organized in biofilms . The exact mechanism by which a biofilm is removed from the root canal surface is only a speculation and the exact means is not known . It was further speculated that the removal of a bacterial biofilm would be influenced by a number of factors and their interactions, including canal dimensions, properties of an irrigant, irrigant regimen and the properties of the biofilm.6 irrigation devices play an important role in the delivery of the irrigant solution to the end point of canal preparation in removing intra - canal debris . The modes of irrigation include static irrigation as well as dynamic irrigation (active and passive ultrasonic irrigation [pui]). Two types of ultrasonic irrigation has been described in the literature: one where irrigation is combined with simultaneous ultrasonic instrumentation (ultrasonic instrumentation) and another related to the non cutting action of the ultrasonically activated file called as passive ultrasonic instrumentation.7 the aim of the present study was to evaluate the efficiency of two irrigating techniques - static and dynamic (passive ultrasonic instrumentation) irrigation in the elimination of bacterial biofilm . The objectives of this study were (1) to use an ex vivo split tooth model to simulate the bacterial biofilm (2) to use the above biofilm model to assess the influence of various factors like canal taper, volume of the irrigant, mode of irrigation, open port of the needle upon the removal of bio - molecular film at various levels of root canal . Forty extracted human permanent maxillary central incisors teeth with straight roots and single canals, were collected and stored in 4% formal saline . The forty teeth were divided into two groups each with twenty samples for static irrigation and ultrasonic irrigation . The root canals were prepared to tip sizes (20, 40) and tapers (0.04, 0.08). After access preparation was done, the tooth length was determined by placing a size 10 stainless steel k file (mani inc ., all the teeth were decoronated to give a standardized working length of 17.5 mm; the working length was set at 0.5 mm short of the apical foramen . The canals were prepared to tip size 20, 40 with tapers 0.04 and 0.08 using system gt instruments (dentsply malliefer, switerzland) in a 70:1 controlled - torque, low speed rotary handpiece at 300 rpm according to subgroup designation (table 1) following manufacturers protocol . During instrumentation, each tooth was irrigated with 50 ml of 2.5% sodium hypochlorite (ammdent, india), delivered with a 3 ml monoject luer lock syringe with a 25-guage needle (dentsply rinn, usa). Each tooth was partly embedded in silicone putty (aquasil putty impression material, dentsply usa) to create a set matrix which allowed reassembly of the tooth for irrigation tests after splitting . The tooth was then grooved on the buccal and palatal aspects along its entire length using a diamond disc (axis dental corporation, usa) and placed longitudinally on another silicone matrix, to allow cushioning when split into two halves using an osteome and mallet . The two halves of the canal were randomly assigned as side a or b. four layers of organic collagen solution (in 0.6% acetic acid; type i rat tail collagen, first link ltd ., birmingham, uk) mixed with chinese calligraphic ink (pebeo fine arts, france), in a ratio of 5:1, were applied to the canal surfaces using a small brush and allowed to gel into a three - dimensional matrix by evaporation of the acetic acid solvent at room temperature for 48 h. each split half of the tooth was equally divided into coronal, middle and apical sections and marked with a sharp pencil . Digital images were taken before and after irrigation with 9, 18, 27 and 36 ml of sodium hypochlorite using a digital camera (sony dsc, sony corporation, usa). Irrigation experiments were done for static irrigation and ultrasonic irrigation using sodium hypochlorite . From a sample of 40 teeth, 20 teeth (a1-a4) were irrigated with 2.5% sodium hypochlorite, delivered with a monoject endodontic 3 ml syringe through a luer lock 25 guage max - i - prob needle (dentsply rinn) at a rate of 6 ml / min . The irrigating needle was inserted 2 mm short of the working length and a total of 36 ml of solution was delivered in twelve 3 ml boluses (each 3 ml bolus delivered over 30 s with sufficient duration to allow syringe recharging and canal surface evaluation after 9, 18, 27 and 36 ml). The open side port of the needle faced the canal side designated a, in a fixed orientation . The protocol for irrigation was the same as for static irrigation with the addition of an ultrasonically activated file tip of size 15 k - file performed after introducing the first 3 ml of the irrigant, followed by delivery of the next 6 ml in two 3 ml boli . The digital images were analyzed using imagej software (national institute of health, usa) to quantify residual canal coverage by the stained collagen . In total, the means and standard deviation of the percentage of canal surface coverage with residual collagen after irrigation were calculated for each corono - apical section on each side of the canal . The generalized estimating equation (gee) approach (stata 9; stata corporation: college station, tx, usa, 2005) was used to investigate the influence of the potential factors (corono - apical level of canal, canal dimension, volume of irrigant, mode of irrigation and orientation of irrigation needle port) on the efficacy of irrigation using percentage of canal coverage with residual collagen as the independent variable . The clustering effect of the four repeated measurements taken on the same canal surface was accounted for in the gee linear regression model . For static irrigation of 2.5% sodium hypochlorite one for facing the needle and another opposite to the side of needle (graphs 1 and 2). One for facing the needle and another opposite to the side of needle (graphs 3 and 4). Mean percentage of canal surface coverage with residual collagen on the surface facing the side port of needle after static irrigation (a: samples from static irrigation group, 1 - 4 denote different canal dimensions [1= size 20/0.04; 2 = size 20/0.08; 3 = size 40/0.04 and 4 = size 40/0.08], c: coronal third of canal, m: middle third of canal and a: apical third of canal). Mean percentage of canal surface coverage with residual collagen on the surface opposite to the side port of needle after static irrigation (a: samples from static irrigation group, 1 - 4 denote different canal dimensions [1 = size 20/0.04; 2 = size 20/0.08; 3 = size 40/0.04 and 4 = size 40/0.08], c: coronal third of canal, m: middle third of canal and a: apical third of canal). Mean percentage of canal surface coverage with residual collagen on the surface facing the side port of needle after passive ultrasonic irrigation (b: samples from the pui group, 1 - 4 denote different canal dimensions [1 = size 20/0.04; 2 = size 20/0.08; 3 = size 40/0.04 and 4 = size 40/0.08], c: coronal third of canal, m: middle third of canal and a: apical third of canal). Mean percentage of canal surface coverage with residual collagen on the surface opposite to the side port of needle after passive ultrasonic irrigation (b: samples from the pui group, 1 - 4 denote different canal dimensions [1 = size 20/0.04; 2 = size 20/0.08; 3 = size 40/0.04 and 4 = size 40/0.08), c: coronal third of canal, m: middle third of canal and a: apical third of canal). Forty extracted human permanent maxillary central incisors teeth with straight roots and single canals, were collected and stored in 4% formal saline . The forty teeth were divided into two groups each with twenty samples for static irrigation and ultrasonic irrigation . The root canals were prepared to tip sizes (20, 40) and tapers (0.04, 0.08). After access preparation was done, the tooth length was determined by placing a size 10 stainless steel k file (mani inc ., all the teeth were decoronated to give a standardized working length of 17.5 mm; the working length was set at 0.5 mm short of the apical foramen . The canals were prepared to tip size 20, 40 with tapers 0.04 and 0.08 using system gt instruments (dentsply malliefer, switerzland) in a 70:1 controlled - torque, low speed rotary handpiece at 300 rpm according to subgroup designation (table 1) following manufacturers protocol . During instrumentation, each tooth was irrigated with 50 ml of 2.5% sodium hypochlorite (ammdent, india), delivered with a 3 ml monoject luer lock syringe with a 25-guage needle (dentsply rinn, usa). Each tooth was partly embedded in silicone putty (aquasil putty impression material, dentsply usa) to create a set matrix which allowed reassembly of the tooth for irrigation tests after splitting . The tooth was then grooved on the buccal and palatal aspects along its entire length using a diamond disc (axis dental corporation, usa) and placed longitudinally on another silicone matrix, to allow cushioning when split into two halves using an osteome and mallet . The two halves of the canal were randomly assigned as side a or b. four layers of organic collagen solution (in 0.6% acetic acid; type i rat tail collagen, first link ltd ., birmingham, uk) mixed with chinese calligraphic ink (pebeo fine arts, france), in a ratio of 5:1, were applied to the canal surfaces using a small brush and allowed to gel into a three - dimensional matrix by evaporation of the acetic acid solvent at room temperature for 48 h. each split half of the tooth was equally divided into coronal, middle and apical sections and marked with a sharp pencil . Digital images were taken before and after irrigation with 9, 18, 27 and 36 ml of sodium hypochlorite using a digital camera (sony dsc, sony corporation, usa). Irrigation experiments were done for static irrigation and ultrasonic irrigation using sodium hypochlorite . From a sample of 40 teeth, 20 teeth (a1-a4) were irrigated with 2.5% sodium hypochlorite, delivered with a monoject endodontic 3 ml syringe through a luer lock 25 guage max - i - prob needle (dentsply rinn) at a rate of 6 ml / min . The irrigating needle was inserted 2 mm short of the working length and a total of 36 ml of solution was delivered in twelve 3 ml boluses (each 3 ml bolus delivered over 30 s with sufficient duration to allow syringe recharging and canal surface evaluation after 9, 18, 27 and 36 ml). The open side port of the needle faced the canal side designated a, in a fixed orientation . For the rest 20 teeth (b1-b4), pui was done . The protocol for irrigation was the same as for static irrigation with the addition of an ultrasonically activated file tip of size 15 k - file performed after introducing the first 3 ml of the irrigant, followed by delivery of the next 6 ml in two 3 ml boli . The digital images were analyzed using imagej software (national institute of health, usa) to quantify residual canal coverage by the stained collagen . In total, the means and standard deviation of the percentage of canal surface coverage with residual collagen after irrigation were calculated for each corono - apical section on each side of the canal . The generalized estimating equation (gee) approach (stata 9; stata corporation: college station, tx, usa, 2005) was used to investigate the influence of the potential factors (corono - apical level of canal, canal dimension, volume of irrigant, mode of irrigation and orientation of irrigation needle port) on the efficacy of irrigation using percentage of canal coverage with residual collagen as the independent variable . The clustering effect of the four repeated measurements taken on the same canal surface was accounted for in the gee linear regression model . For static irrigation of 2.5% sodium hypochlorite one for facing the needle and another opposite to the side of needle (graphs 1 and 2). One for facing the needle and another opposite to the side of needle (graphs 3 and 4). Mean percentage of canal surface coverage with residual collagen on the surface facing the side port of needle after static irrigation (a: samples from static irrigation group, 1 - 4 denote different canal dimensions [1= size 20/0.04; 2 = size 20/0.08; 3 = size 40/0.04 and 4 = size 40/0.08], c: coronal third of canal, m: middle third of canal and a: apical third of canal). Mean percentage of canal surface coverage with residual collagen on the surface opposite to the side port of needle after static irrigation (a: samples from static irrigation group, 1 - 4 denote different canal dimensions [1 = size 20/0.04; 2 = size 20/0.08; 3 = size 40/0.04 and 4 = size 40/0.08], c: coronal third of canal, m: middle third of canal and a: apical third of canal). Mean percentage of canal surface coverage with residual collagen on the surface facing the side port of needle after passive ultrasonic irrigation (b: samples from the pui group, 1 - 4 denote different canal dimensions [1 = size 20/0.04; 2 = size 20/0.08; 3 = size 40/0.04 and 4 = size 40/0.08], c: coronal third of canal, m: middle third of canal and a: apical third of canal). Mean percentage of canal surface coverage with residual collagen on the surface opposite to the side port of needle after passive ultrasonic irrigation (b: samples from the pui group, 1 - 4 denote different canal dimensions [1 = size 20/0.04; 2 = size 20/0.08; 3 = size 40/0.04 and 4 = size 40/0.08), c: coronal third of canal, m: middle third of canal and a: apical third of canal). The mean percentage of canal surface coverage with residual collagen after static and pui are summarized graphically . The mean percentage of canal surface with residual collagen increased with: (a) coronal level of canal . (b) decrease in apical size and taper of canal preparation (c) decrease in the volume of the irrigant . The canal surface facing the open side port of the needle had less residual collagen after irrigation than the opposing surface . The efficacy of irrigation measured as percentage of canal coverage with residual collagen was significantly influenced by all the five explanatory variables . In descending order of effect, the relative ranking was: (i) corono - apical level of canal, (ii) volume of irrigation, (iii) canal dimension,(iv) mode of irrigation and (v) orientation of the needle port . The efficacy of irrigation measured as percentage of canal coverage with residual collagen was significantly influenced by all the five explanatory variables . In descending order of effect, the relative ranking was: (i) corono - apical level of canal, (ii) volume of irrigation, (iii) canal dimension,(iv) mode of irrigation and (v) orientation of the needle port . It is generally accepted that one of the main causes of periapical disease is the bacterial infection of the root canal system . In cases of asymptomatic, persistent post treatment periapical radiolucencies, clinicians should consider the necessity of removing the extra - radicular factors like a biofilm . Few bacteria can attach to type i collagen in dentine through expression of surface adhesions and biofilms . In the clinical setting, biofilm removal from the instrumented part of the canal may be facilitated by the mechanical contact of the instruments . In the non - instrumented part of the canal, the biofilm is likely to be removed as a function of progressive chemical dissolution, along with a limited amount of fluid agitation as the fluid environment is likely to be viscous dominant . In view of such difficulties, this study used a split tooth model simulating a bacterial biofilm along the canal walls and comparing the efficacy of two irrigating techniques - static and dynamic (pui) irrigation in the elimination of bacterial biofilm . This model was in accordance with the similar studies done by huang et al.6 it was further speculated that the removal of the bacterial biofilm would be influenced by a number of factors and their interactions, including canal dimensions, properties of irrigant, irrigation regimen and the properties of the biofilm . The aim of the present study was to evaluate the efficiency of two irrigating techniques - static and dynamic (passive ultrasonic instrumentation) irrigation in the elimination of a bacterial biofilm by the physical and chemical action of irrigation . The test model used in the present study was designed to quantify the relative importance of factors like canal taper, volume of the irrigant, mode of irrigation, open port of the needle upon the removal of bio - molecular film at various levels of root canal . In the present study to prepare the split tooth study model, human maxillary central incisors with single root and straight canals were chosen . Further in this study design, type 1 rat tail collagen was used as a surface adherent test substrate, exhibiting the hydrodynamic properties of a biofilm because: they are visible when applied in root surface . Had good adhesion to the root dentin and can be removed by physical abrasion or fluid turbulence (flushing action of irrigants). The film gradually dissolved and disintegrated from the surface by the irrigation and the removal helped by the physical abrasion or fluid turbulence iv among the available materials, it was the most cost economic . The selected collagen, which was neither dried nor frozen, retained its native long molecule state, without denaturation in 0.6% acetic acid solution . On application the collagen then gels into a three - dimensional matrix after evaporation of the acidic solution, a state that is reversible on addition of acetic acid (type i rat tail collagen). It should aggregate with the collagen so as not to be decolourized by either sodium hypochlorite . The chinese calligraphic ink (peebo, france) met all the requirements and retained the black colour after exposure to an equal volume of 2.5% naocl for 1 day . The removal characteristics of the stained rat tail collagen layer on the canal surfaces were tested using sodium hypochlorite, and water . It was found that water did not remove the layer but the sodium hypochlorite did, leaving a clean surface without evidence of ink spread into dentinal tubules at or beyond the site of initial application . Four layers of the stained collagen gave good contrast and a predictable thickness of coating (5 - 15 um), a dimension within the range of bacterial film thickness.8 a max - i - probe 25-guage needle (dentsply, usa) was used in this study . The max - i - probe has a closed - ended, side vented channel, which delivers the irrigant laterally . Salzgeber et al.9 showed that increasing the volume of an irrigant significantly reduced the bacterial load, regardless of the irrigation needle depth . Huang et al.6 have used 4 different volumes of sodium hypochlorite and found that the larger volume of 36 ml significantly removed more collagen material from the tooth surface when compared with smaller volumes . Thus in the present study different volume regimens were chosen (9, 18, 27 and 36 ml) in accordance with these studies . Narrow canals compromise the efficacy of irrigation and may need to be enlarged and the taper increased to allow for effective irrigation . Salzgeber et al.9 advocated the need for the canals to be enlarged to a 40-file, so that the maximum irrigants is in direct contact with the apical debris . In the present study, the teeth samples were enlarged to apical preparation sizes 20 and 40 in accordance with the above literature . In the present study, canals were prepared to 20 and 40 tip size and a taper of 0.04 and 0.08 . Review of the literature have shown that the tip of the needle have been placed from 1 mm to 10 mm short of the apical end of the canal . In this present study, to standardize the depth of the needle penetration for all the samples, the tip was maintained at 2 mm short of the working length . Moreover this was the deepest penetration without the needle tip binding against the canals prepared to apical size 20 with a 0.04 taper . It is desirable to control the irrigant delivery by rate and pressure throughout a study.12,13 the average rate of irrigant delivery in this study was set at 6 ml / min with a gauge 25 needle as it seemed clinically optimal and allowed the operator to maintain a constant force during extrusion.11 this study design employed a special imaging method (imagej, nih, usa) to quantify residual canal coverage by the stained collagen which renders three - dimensional irregularities on the root canal surface into two - dimensional surfaces on the images . The gee approach (stata 9, usa) was used to investigate the influence of potential factors (coronal - apical level, canal dimension, volume of irrigant, mode of irrigation and orientation of irrigation needle port) on the efficacy of irrigation using percentage of canal coverage with residual collagen as the independent variable . The data was analyses using linear regression models . This ex vivo model was used to test the influence of several factors like (a) volume of irrigation, (b) canal dimension and taper, (c) mode of irrigation, (d) corono - apical level of canal and (e) canal surface, using 2.5% solution of sodium hypochlorite in the removal of bacterial biofilm . In static irrigation, the volumes of irrigation in the 9, 18, 27 and 36 ml did remove significant amount of collagen . This can be probably due to the higher velocity and volume of irrigant flow created in the canal . Also the mechanical flushing action of the irrigant (naocl) would have been facilitated by the pui . In this study, teeth samples are prepared to apical preparation sizes 20 and 40 with 0.04 and 0.08 taper . For static irrigation, the canal dimensions at 0.04 taper removed collagen less in the apical region, when compared with the 0.08 taper . For pui the collagen was removed more with 0.08 taper, when compared with 0.04 taper . Overall, collagen was removed more with pui than with static irrigation, especially in the apical region and with 0.08 taper . More the taper and larger the canal dimension, more will be the removal of the collagen and this can be attributed to the better apical flushing by the irrigant . In this study the collagen removal was more in the apical section when compared with the middle and coronal sections . The efficacy of irrigation was thus marked apically because of the greater fluid exchange / interaction that could have occurred in the apical area . The root canal surface facing the open side port of the needle appeared more cleaner than the opposite side, reinforcing the notion of the flushing effect of the irrigant in addition to the chemical effect . The collagen was removed more for the pui technique in the facing side of the needle than with the static irrigation . The ability of irrigant to penetrate into areas not instrumented by mechanical instrumentation is critical for debridement and disinfection of the root canal system . Previous studies have shown that pui resulted in significantly cleaner canals than hand filing alone . Flushing action of naocl facilitated by the pui.much higher velocity and the volume of the irrigant volume.oscillations of the ultrasonic activated file.14pui combines acoustic waves with the chemical action of the irrigant and generates microstreaming . This microstreaming moves the solution against the root canal surfaces, enhancing mechanical cleansing of the canal walls and bacterial destruction . Flushing action of naocl facilitated by the pui . Much higher velocity and the volume of the irrigant volume . Oscillations of the ultrasonic activated file.14 pui combines acoustic waves with the chemical action of the irrigant and generates microstreaming . This microstreaming moves the solution against the root canal surfaces, enhancing mechanical cleansing of the canal walls and bacterial destruction . In static irrigation, the volumes of irrigation in the 9, 18, 27 and 36 ml did remove significant amount of collagen . This can be probably due to the higher velocity and volume of irrigant flow created in the canal . Also the mechanical flushing action of the irrigant (naocl) would have been facilitated by the pui . In this study, teeth samples are prepared to apical preparation sizes 20 and 40 with 0.04 and 0.08 taper . For static irrigation, the canal dimensions at 0.04 taper removed collagen less in the apical region, when compared with the 0.08 taper . For pui the collagen was removed more with 0.08 taper, when compared with 0.04 taper . Overall, collagen was removed more with pui than with static irrigation, especially in the apical region and with 0.08 taper . More the taper and larger the canal dimension, more will be the removal of the collagen and this can be attributed to the better apical flushing by the irrigant . In this study the collagen removal was more in the apical section when compared with the middle and coronal sections . The efficacy of irrigation was thus marked apically because of the greater fluid exchange / interaction that could have occurred in the apical area . The root canal surface facing the open side port of the needle appeared more cleaner than the opposite side, reinforcing the notion of the flushing effect of the irrigant in addition to the chemical effect . The collagen was removed more for the pui technique in the facing side of the needle than with the static irrigation . The ability of irrigant to penetrate into areas not instrumented by mechanical instrumentation is critical for debridement and disinfection of the root canal system . Previous studies have shown that pui resulted in significantly cleaner canals than hand filing alone . Hence in this study, several reasons can be attributed to this factor . Flushing action of naocl facilitated by the pui.much higher velocity and the volume of the irrigant volume.oscillations of the ultrasonic activated file.14pui combines acoustic waves with the chemical action of the irrigant and generates microstreaming . This microstreaming moves the solution against the root canal surfaces, enhancing mechanical cleansing of the canal walls and bacterial destruction . Flushing action of naocl facilitated by the pui . Much higher velocity and the volume of the irrigant volume . Oscillations of the ultrasonic activated file.14 pui combines acoustic waves with the chemical action of the irrigant and generates microstreaming . This microstreaming moves the solution against the root canal surfaces, enhancing mechanical cleansing of the canal walls and bacterial destruction . In static irrigation, the volumes of irrigation in the 9, 18, 27 and 36 ml did remove significant amount of collagen . This can be probably due to the higher velocity and volume of irrigant flow created in the canal . Also the mechanical flushing action of the irrigant (naocl) would have been facilitated by the pui . In this study, teeth samples are prepared to apical preparation sizes 20 and 40 with 0.04 and 0.08 taper . For static irrigation, the canal dimensions at 0.04 taper removed collagen less in the apical region, when compared with the 0.08 taper . For pui the collagen was removed more with 0.08 taper, when compared with 0.04 taper . Overall, collagen was removed more with pui than with static irrigation, especially in the apical region and with 0.08 taper . More the taper and larger the canal dimension, more will be the removal of the collagen and this can be attributed to the better apical flushing by the irrigant . In this study all tooth samples were divided into coronal, middle and apical sections . The collagen removal was more in the apical section when compared with the middle and coronal sections . The efficacy of irrigation was thus marked apically because of the greater fluid exchange / interaction that could have occurred in the apical area . The root canal surface facing the open side port of the needle appeared more cleaner than the opposite side, reinforcing the notion of the flushing effect of the irrigant in addition to the chemical effect . The collagen was removed more for the pui technique in the facing side of the needle than with the static irrigation . The ability of irrigant to penetrate into areas not instrumented by mechanical instrumentation is critical for debridement and disinfection of the root canal system . Previous studies have shown that pui resulted in significantly cleaner canals than hand filing alone . Hence in this study, several reasons can be attributed to this factor . Flushing action of naocl facilitated by the pui.much higher velocity and the volume of the irrigant volume.oscillations of the ultrasonic activated file.14pui combines acoustic waves with the chemical action of the irrigant and generates microstreaming . This microstreaming moves the solution against the root canal surfaces, enhancing mechanical cleansing of the canal walls and bacterial destruction . Oscillations of the ultrasonic activated file.14 pui combines acoustic waves with the chemical action of the irrigant and generates microstreaming . This microstreaming moves the solution against the root canal surfaces, enhancing mechanical cleansing of the canal walls and bacterial destruction . Within the limitations of this study, it can be concluded that a bacterial biofilm, adherent to the root canal surface at the apical third of the root canal may be efficiently removed by: sufficient enlargement of the canal by increasing the canal taper.agitation of an irrigant, with an passive ultrasonically activated file.irrigation of the canal with a side vent needle like max-i-probe.large volumes of irrigant imparting lavaging effect . Irrigation of the canal with a side vent needle like max - i - probe.
Over the last few years, the number of people infected with multidrug - resistant bacteria has been rising in a number of countries, among them germany . As a characteristic feature, this inhibits the efficacy of antibiotic treatment, with serious consequences for the health of infected individuals, the utilization of resources in the health care sector and the availability of resources in the economy in general . Following an infection with multidrug - resistant bacteria, a number of effects may arise, each representing an economic cost: first, an individual will suffer from the risk of severe impairment of her health, including premature death . Next, appropriate treatment will take more time to produce the desired health outcome or will require more resources which must be diverted from other uses in the health care sector . In addition, the economy will incur losses in productivity whenever infected individuals belong to the workforce . Finally, aggravating these already serious consequences, infected individuals may spread multidrug - resistant bacteria, thus inflicting harm on other individuals and increasing the economic cost to society . A first objective of the present contribution is to work out in more detail the economic effects which an infection or a colonization with multidrug - resistant bacteria may bring about . Building on this account, it will be argued that the status quo is inefficient . Next, an approach will be outlined which can be used to determine whether strategies to prevent or to reduce infections with multidrug - resistant bacteria provide a net benefit to society . In order to be able to implement a strategy that has been shown to be advantageous in this sense, in essence, both objectives require a regional management of multidrug - resistant bacteria . By taking into account the entire health care sector within a region in order to carry out the tasks outlined above, it is important to be clear about how to assess the costs and benefits associated with strategies to prevent or reduce infections with multiresistant bacteria . Furthermore, the range of effects which need to be taken into account will depend on the perspective of the analysis . More specifically, while the (in-)efficiency of the status quo or of an intervention can only be evaluated from the perspective of society, the issue of (in-)appropriate incentives must be judged from the individual perspective . First, it is helpful to distinguish three types of costs . Direct costs represent the monetary value of resource utilization that takes place due to the production and consumption of health care . Apart from the cost of health care itself, this also includes any cost incurred by the patient in order to receive care, e.g., travel costs . The next type is given by indirect costs which cover the productivity losses, also valued in monetary units, due to illness or premature death . Finally, intangible costs capture the loss in utility or welfare for the patient which results from a reduction in her health status (including death). In comparison with the other two types, intangible costs are more difficult to measure because it is necessary to develop a valuation from scratch . Intangible costs can be measured either in monetary units (i.e., as willingness - to - pay) or in other units such as quality - adjusted life years (qalys). Next, strategies which aim to prevent infections with multidrug - resistant bacteria or to control their impact will usually involve a reduction in the associated costs . Relying on a similar categorization as on the cost side, these benefits may turn up either as direct benefits, or as indirect benefits, or finally as intangible benefits . Whereas direct benefits (or indirect benefits) accrue as reductions in direct costs (or indirect costs), intangible benefits arise from improvements in health status or an extension of length of life . From the individual perspective of an economic unit such as, e.g., a physician, an insurer, or a patient, it makes sense to take into account only those costs and benefits which accrue to the unit itself . Thus, a decision is individually rational if it maximizes benefits over costs for the unit, thereby reaping the greatest net benefit . In contrast, the societal perspective takes into account the costs and benefits of all decision units . Hence, a decision is rational from the perspective of society, or efficient for short, if it maximizes the net benefit that accrues to society . Otherwise, it is inefficient and it is at least possible to make everyone better off by moving to an efficient decision . It is important to realize that both the perspectives are necessary to explain why inefficient decisions involving a waste of resources are taken and how they can be improved upon . According to a basic assumption that will also be imposed below, economic units always act individually rational, i.e., they always maximize their own net benefit . Simple though it is, this assumption nevertheless can be quite helpful in explaining inefficient decisions . More precisely, such decisions are likely to occur whenever the associated costs and benefits do not fall exclusively on the unit who takes them . E.g., patients infected with multidrug - resistant bacteria are likely to receive inefficient health care if the providers have no incentive to take into account all the costs and benefits associated with the treatment they provide . Applying the societal perspective provides the only way to identify those strategies to prevent or control infections that are efficient . In order to promote their implementation, it is essential to modify the incentives which the providers of health care face such that applying an efficient strategy is in their own interest, i.e., becomes individually rational . As claimed above, this demonstrates that both the societal perspective and the individual perspectives of the relevant players must be taken into account in order to arrive at an efficient provision of health care to patients infected with multidrug - resistant bacteria . Individuals who are colonized with multidrug - resistant bacteria but not yet infected usually do not suffer from any immediate consequences . However, there are substantial risks involving serious medical problems and economic costs, both for the individuals themselves as well as for other individuals who get in touch with them . First, there is the risk of developing an infection, imposing direct and intangible costs and possibly also indirect costs as outlined above . The available studies, usually focusing on one type of bacteria and on a single disease, clearly demonstrate a considerable increase in length - of - stay and in direct hospital costs,,,, . Even though the reductions in health status due to an infection with multidrug - resistant bacteria are less well documented, the associated rise in mortality indicates that intangible costs may well turn out to be very high,, . At the same time, individuals colonized with multidrug - resistant bacteria put other individuals at risk because their presence increases the probability that further colonization will take place . An analysis of the costs of multidrug - resistant bacteria from the societal perspective must take these costs into account as well . Economically speaking, they represent external costs because the colonization of one individual may exert a negative impact upon the health status of other individuals, . In fact, the external effects may reach very far, both with respect to the number of individual s concerned and over time . Consider the use of antibiotics: even with proper use in treatment, eventually resistant bacteria will turn up . However, antibiotic misuse on the part of either health care providers or patients substantially accelerates the evolution of resistant strains . Thus, a flawed treatment of patients today imposes a cost on future patients . In economic terms, efficacy of antibiotic treatment can be viewed as a common social good which, due to wrong incentives for the major players in the health care sector, suffers from the tragedy of the commons . In order to move towards a solution of the current problems with multidrug - resistant bacteria, it is necessary to identify strategies for improving the provision of health care . In a further step, care must be taken to make sure that promising strategies will also be implemented by the relevant actors . Applying both steps in a systematic manner regularly and based on appropriate empirical data to check available strategies to prevent or control infections, one obtains a management of multidrug - resistant bacteria . Given the problems of the status quo as outlined above, a change from simply dealing with multidrug - resistant bacteria ad hoc to a proper management can be expected to result in a substantial reduction in the associated costs for society . As a first step, it is necessary to evaluate the costs and benefits to society arising from strategies to prevent infections with multidrug - resistant bacteria . Strategies that confer a net benefit to society improve on the current provision of health care and contribute to attaining the goal of an efficient management . Observe that the term strategy is defined in a narrow sense: in particular, it refers to a specific group of patients receiving care from a specific provider at a specific place . Thus, if the benefits of, e.g., a screening strategy exceed the associated costs, it is nevertheless possible that a further modification, for example by focusing on a different group of patients, may provide an ever higher net benefit to society . There are good reasons to tackle these tasks within a regional management of multidrug - resistant bacteria . As its name suggests, a regional management has a clear focus on a specific region but implies no further restrictions otherwise . Thus, it represents a comprehensive approach which is not confined to a single branch of the health care system . Furthermore, in view of the problems outlined above it seems necessary to adopt a regional management because such an approach offers a unique opportunity to apply the perspective of society to strategies to prevent or control infections with multidrug - resistant bacteria . This includes the setup and the continuous maintenance of a database relating to multidrug - resistant bacteria in order to record the prevalences of colonized individuals or infected patients as well as other relevant data . A major objective would be to take into account the entire process of the development and also the spread of colonization and infections with multidrug - resistant bacteria . In some contrast to the prevailing approach with its focus on hospitals and on the control of infections, a regional management offers the prospect to recognize both the contribution of other health care providers and of strategies to prevent infections in order to deal with infections with multidrug - resistant bacteria in a manner that provides a maximum net benefit to society . At present, a major problem is that appropriate data on the costs of colonization with multidrug - resistant bacteria are not available . To be sure, nevertheless, there are still no or at least no systematic data on some of the cost effects explained above . Referring to infections with multidrug - resistant bacteria, this is true for the direct costs outside of hospitals as well as for the intangible costs due to a reduction in length of life or a deterioration of health status . In addition, there is no evidence on the probabilities with which colonized individuals develop an infection or cause other individuals either to be colonized or infected with multidrug - resistant bacteria . These data would need to be collected with respect to the type of bacteria (pathogen) and possibly also with respect to individual attributes such as, e.g., age or prevailing chronic diseases . Note that the lack of data on some important cost effects also has further implications . Since the benefits of strategies to prevent or control infections arise primarily as reductions in the direct, indirect or intangible costs of multidrug - resistant bacteria, the full benefits to society are not known either . More specifically, there is a lack of data on the impact of efforts to prevent infections, and also on the valuation of the corresponding health effects by the affected individuals . In order to explain the present scale of infections with multidrug - resistant bacteria in germany, it is necessary to investigate the incentives of health care providers to employ strategies to prevent or control infections . Consider a health care provider who applies a screening test to patients deemed at risk such that patients will be isolated until the result becomes available and will be sanified if the test yields a positive result . In many instances, for the provider this means incurring a certain cost with the prospect of only a small expected benefit: first, the remuneration may be insufficient (perhaps even nil) and/or the strategy may not generate enough cost savings for the provider . The latter reason applies, e.g., to ambulatory physicians because the higher cost of treatment due to an infection will occur primarily in hospitals . Furthermore, the additional benefits due to averted infections of other individuals will have no direct effect on the provider under consideration either . A similar statement applies to the careful and cautious therapy with antibiotics which may also contain the development and the spread of multidrug - resistant bacteria: again, the cost falls primarily upon the prescribing physician whereas the benefit turns up elsewhere in society . Even though it is not possible currently to describe the economic impact of colonization with multidrug - resistant bacteria in full, the lack of sufficiently strong incentives for health care providers can safely be taken to imply that too little effort is devoted to the prevention or the control of infections . As a gauge, consider the costs and benefits to society of strategies to prevent or control infections with multidrug - resistant bacteria: as argued above, health care providers implementing such strategies will usually reap only part of the associated benefit while bearing the full cost . Therefore, the incentives facing health care providers are too weak to induce them to provide efficient care with respect to the protection from infections with multidrug - resistant bacteria . In turn, this implies that, from the perspective of society, there will be too many infections imposing an overall cost that is unnecessarily high . If a strategy to prevent infections with multidrug - resistant bacteria conferring a net benefit to society has been identified, the next task is to make sure that it will be implemented . In essence, this means setting appropriate incentives: the net benefit to society needs to be distributed among the relevant actors in a manner such that it is individually rational for everybody to apply the strategy . More specifically, this implies an appropriate remuneration of health care providers, covering at least their own cost due to the effort to prevent or control infections with multidrug - resistant bacteria . Moreover, it may make sense to promote the cooperation among different sectors of the health care system, given that multidrug - resistant bacteria turn up not only in hospitals but also in ambulatory care or in nursing homes . Applying a regional management in a suitable manner also offers the prospect of overcoming the tragedy of the commons problem that besets the current approach to multidrug - resistant bacteria . By implementing the perspective of society in an entire region, it will be possible to take into account most of the substantial cost effects that are now external . Improving surveillance as well as providing reliable information on the appropriate use of antibiotics may help to improve the efficacy of treatment for future patients . In this sense, a successful regional management may also contribute to save costs in the health care sector and to improve the health of patients in the future . The current provision of health care to individuals who are colonized or infected with multidrug - resistant bacteria suffers from a number of shortcomings due to, e.g., a lack of data and inappropriate incentives for providers . Implementing suitable strategies to control or prevent infections offers the prospect of reducing the costs associated with multidrug - resistant bacteria substantially, either by improving the health status of patients or by easing the financial burden on the health care system . For several reasons, a regional management represents an approach that can be trusted to improve on the status quo and to reap substantial net benefits for society by better dealing with the problems caused by multidrug - resistant bacteria . It is true that building up and maintaining a regional management of multidrug - resistant bacteria will involve a considerable cost . However, given the high costs of the status quo which will continue to rise unless precautionary measures are taken, this cost will be more than offset by the benefits that can be reaped by implementing efficient strategies to prevent or control infections . A regional management of multidrug - resistant bacteria represents the first step towards a better integration of the health care system . If this step is taken successfully, it may also act as a role model for dealing with other problems in the provision of health care . At any rate, the potential benefits appear to be sufficiently high to more than justify embarking on this first step.
Craniosynostosis is a wonderfully descriptive term, meaning " the skull has fused bone " . However, it is also a fairly nonspecific clinical designation, encompassing multiple presentations ranging from isolated single suture involvement to multi - sutural fusions5). Multi - sutural fusion can occur as isolated fusions or with associated anomalies that occur outside the skull . Epidemiologic studies have suggested that the incidence of craniosynostosis may be as high as 1 in 1700 live births612). For example, early theories regarding the pathogenesis of this condition, based on clinical observations and an experimental animal model, included intrauterine constraint811). Over the last quarter century, there have been many advances in the understanding of craniosynostosis, resulting in more rational management of the problems associated with this condition, both in its simple form and its syndromic manifestations315). These advances have included reduced emphasis on the technical aspects of this disease and greater emphasis on its pathology and natural history, along with more accurate analysis of the morphology of the craniofacial skeleton1310). More recently, genetic studies have identified the genetic loci of many craniosynostosis syndromes, as well has determining the downstream pathways associated with disease development . Recent clinical and genetic studies have identified multiple forms of human craniosynostosis, each associated with mutations within various growth factor signaling pathways . Knowledge gathered from these investigations may result in the future development of alternative strategies to enhance or perhaps even replace current approaches for the treatment of craniosynostosis . Craniosynostosis was first treated surgically in the late 1800s, using techniques such as fragmentation of the cranial vault and linear craniectomies . These early procedures were associated with high rates of reossification and poor esthetic outcomes, mandating many subsequent procedures7). At present, however, simple craniectomy is used only in patients with transient cranial decompression . These early procedures have been supplanted by surgical remodeling of the affected area of the cranial vault and orbits . Surgery is generally performed at age 69 months to take full advantage of the regenerative capacity of the skull at this age . Three stages of growth have determined the approach to the management of craniosynostosis3). During this time, cerebral growth is the greatest, and craniosynostosis may have detrimental effects on the developing brain . The original surgical approach consisted of excision of the premature suture or sutures by linear craniectomy, allowing the growing brain to expand . If urgent decompression was required, fronto - orbital advancement and multiple suture excisions were performed, resulting in lateral and posterior expansion . After the second year, cerebral growth slows; nevertheless, severe craniostenosis may still lead to papilledema and potential visual failure . The cranial capacity must therefore be expanded by large bilateral decompressive craniotomies, generous fronto - orbital advancement, or a combination of these procedures . Definitive facial surgery can be performed, beginning at age 10 years, although waiting until maturity may achieve better esthetic outcomes . Patients with crouzon syndrome who have proptosis and/or maxillary hypoplasia are especially likely to need surgical treatment1214). Early surgical correction has been limited by a late diagnosis and the risks associated with intraoperative blood loss, which was less effectively managed in the past than currently13). Since the beginning of the 21st century, many centers have tended to determine whether a less invasive procedure could be performed at an earlier stage with acceptable risk . Endoscopic linear craniectomy, with postoperative application of helmets and cranial remodeling through small skin incisions, has led to cosmetic results comparable to those of more invasive procedures if performed during the first 46 months of life, with acceptable blood loss and operative risks13). More recently, the combination of frontal advancement and posterior cranial enlargement during the first months of life has been found to protect both the ocular and posterior cranial structures . Currently, a free bone flap (floating technique) or springs are used to allow for cerebral growth until a rigid fronto - orbito - maxillary advancement can be performed13). The advent of osteodistraction has lowered the age for facio - maxillary advancement (35 years), and may avoid the necessity of repeating fronto - orbital procedures . This has reduced the risks of dural tears and postoperative cerebrospinal fluid fistulas in a significant proportion of patients . As the procedures used to remodel the calvarial vault are extensive although the mortality rate has been reported to be as high as 2.3%, most international studies had mortality rates of 1.5% to 2%7). Most deaths were attributed to hemorrhagic complications, but various other causes have been reported, including air emboli, cerebral edema, and respiratory infections4). Attention to intraoperative hemodynamics and careful postoperative intensive care unit monitoring are critical in minimizing overall morbidity and mortality rates . During this time, cerebral growth is the greatest, and craniosynostosis may have detrimental effects on the developing brain . The original surgical approach consisted of excision of the premature suture or sutures by linear craniectomy, allowing the growing brain to expand . If urgent decompression was required, fronto - orbital advancement and multiple suture excisions were performed, resulting in lateral and posterior expansion . After the second year, cerebral growth slows; nevertheless, severe craniostenosis may still lead to papilledema and potential visual failure . The cranial capacity must therefore be expanded by large bilateral decompressive craniotomies, generous fronto - orbital advancement, or a combination of these procedures . Definitive facial surgery can be performed, beginning at age 10 years, although waiting until maturity may achieve better esthetic outcomes . Patients with crouzon syndrome who have proptosis and/or maxillary hypoplasia are especially likely to need surgical treatment1214). Early surgical correction has been limited by a late diagnosis and the risks associated with intraoperative blood loss, which was less effectively managed in the past than currently13). Since the beginning of the 21st century, many centers have tended to determine whether a less invasive procedure could be performed at an earlier stage with acceptable risk . Endoscopic linear craniectomy, with postoperative application of helmets and cranial remodeling through small skin incisions, has led to cosmetic results comparable to those of more invasive procedures if performed during the first 46 months of life, with acceptable blood loss and operative risks13). More recently, the combination of frontal advancement and posterior cranial enlargement during the first months of life has been found to protect both the ocular and posterior cranial structures . Currently, a free bone flap (floating technique) or springs are used to allow for cerebral growth until a rigid fronto - orbito - maxillary advancement can be performed13). The advent of osteodistraction has lowered the age for facio - maxillary advancement (35 years), and may avoid the necessity of repeating fronto - orbital procedures . This has reduced the risks of dural tears and postoperative cerebrospinal fluid fistulas in a significant proportion of patients . As the procedures used to remodel the calvarial vault are extensive, complications can occur following surgery for craniosynostosis . Although the mortality rate has been reported to be as high as 2.3%, most international studies had mortality rates of 1.5% to 2%7). Most deaths were attributed to hemorrhagic complications, but various other causes have been reported, including air emboli, cerebral edema, and respiratory infections4). Attention to intraoperative hemodynamics and careful postoperative intensive care unit monitoring are critical in minimizing overall morbidity and mortality rates . The combination of early technical success with recent advances in treatment14) has indicated the necessity of multidisciplinary management . The overall approach can be distilled into six principles9): 1) care should be provided by multi - disciplinary teams; 2) care should be a protocol - driven process, with all forms of care defined and delivered optimally; 3) care should be longitudinal, as age, healing and growth processes; 4) secure financial support is needed to implement such longitudinal care11); 5) competent professionals should be involved in ongoing education and training in teaching and research; and 6) research should explore causes, treatment strategies, and treatment outcomes . Centers with the appropriate vision and infrastructure are necessary to optimize the care of patients with craniosynostosis, as well as to enhance scientific knowledge and education about this disease . Several investigations have evaluated the roles of various growth factors and cytokines in determining the fate of sutures . Fibroblast growth factors (fgfs) are particularly important, as mutations in their receptors have been implicated in many craniosynostosis syndromes . Mutations in three of the four known fgf receptors have been associated with premature pathologic suture fusions1617). Recent advances in developmental biology and genetics have identified some of the events governing suture fate, highlighting multiple axes of cellular signaling with the potential for clinical manipulation . Such knowledge and comprehension may facilitate therapeutic translations, ultimately enhancing or perhaps even replacing contemporary modalities for treating craniosynostosis.
The health of the elderly has elicited considerable social attention . With the social and economic development, health care conditions and living standards improve continuously, thus improving the physical health of the elderly and prolonging their life span . According to statistics from the chinese ministry of health, in 60 years of development, the average life span has increased by 39.5 years, reaching 74.8 years old in 2010 (1). However, mental health problems of the elderly are increasingly becoming prominent . Serious psychological problems not only hurt physical and mental health and family happiness of the elderly, but also cause various social problems . Therefore, studying the mental health of the chinese elderly is important . Since the reform and opening - up policy, china has sacrificed high environmental cost for rapid economic development, consequently bringing serious threats to public health . Approximately 300,000 to 500,000 people are killed early by indoor air pollution every year in china (3). However, controversy has arisen in the academe concerning the effect of air pollution on mental health . The tradeoff between rapid economic development and environmental degradation has complicated this controversy (4). Economic development improves the quality of living and social welfare, and thereby, improves the mental health of the public . However, with the further worsening of environmental problems, its negative effects on people s mental health have gradually surfaced . Such a complicated relationship introduces difficulties in comprehending the influence of air pollution and other environmental problems on mental health . Meanwhile, china is a country with an outstanding urban - rural dual structure . Significant differences occur between urban and rural regions in terms of medical insurance, living standards, social culture, and lifestyle . Whether those differences will be reflected on the mental health level of the elderly in urban and rural areas is an important question to be considered when studying the mental health of chinese elderly . Thus, this paper addresses three problems by using data from china, namely, 1) will air pollution influence the mental health of the elderly? 2) is mental health different between the elderly from urban and rural regions? In what ways? Medical and sociological research reported that the so2 concentration in air would threaten physical health and even cause respiratory system diseases (5). Being healthy is not only being free from diseases or weakness, but also the perfect combination of body, mental, and social adaptation (6). Hence, research on health also focuses on mental health . Clark et al . Studied the 9 to 10-year - old british children and noted that air pollution (no2) in schools will not influence their cognition and health . After the no2 level was controlled, they found that aircraft noise and road traffic noise have significant different effects on cognition and health of children (7). A study on 1,546 samples collected from salt lake county, utah, usa found that higher so2 and no2 particles would increase risks of suicide (8). From a medical perspective, tiny particles in air pollution will hurt the nervus centralis in humans after entering the brain and may cause depression and apoplexy (9). Considering the physiological and psychological differences of the elderly with children and youth, the relationship between the mental health of the elderly and air pollution has elicited considerable attention in medical research attention . Studied the 699 elderly males and found the internal relation between pm2.5 and people s mood (optimistic or pessimistic) (10). However, yi et al . Stated in a study of 732 old people (65 years old) in boston that no correlation had been found between air pollution and depressive symptoms (11). These two studies report different results primarily because most medical researchers are based on individuals or non - random samples, which may incur selection bias . Therefore, we shall maintain a cautious attitude toward the corresponding research conclusions and policy implications . Scholars have also focused on the relationship between various social demographic characteristics and mental health . Small changes in smoking, drinking, sports, and diet would significantly affect both physical and mental health (12). Reducing smoking and increasing recreational sports some scholars explored mental health from the perspective of social relations, and pointed out that social communication positively affected the health of the aged (14, 15). Some scholars studied the mental health of the elderly from the perspective of urban - rural difference . A study on 404 old people (60 years old) done in chinese rural and urban regions, found a significant difference in mental health between these regions (16). The urban old people have better mental health than do rural old people (17). However, mikael et al . Offered the opposite conclusion, and they believed that rural old people had better mental health than urban old people had (18). Studied 64,000 old people (65 years old) in hong kong and revealed a significant correlation between depression and chronic diseases, such as cardiovascular disease and coronary artery disease (19). Moreover, norstrand and xu analyzed the health conditions of chinese old people (65 years old) from the perspective of social relations, and found a significant relationship between mental health of urban old people and social relations (20). Based on demography, mental health deteriorated greatly with aging, while physical health and income could improve mental health (21). To sum up, although many research results concerning the mental health of the elderly have been achieved, mental health is characterized by complicated and diverse influencing factors . These factors lead different research perspectives and thereby inconsistent conclusions, which may be caused by the lack of comprehensive and representative data . Moreover, only few studies on the relationship between mental health of chinese elderly and air pollution have been reported . Based on previous studies, this paper establishes a model to analyze comprehensively the influencing factors of the mental health of the elderly in china from the perspective of urban - rural difference and by combining local air quality, individual characteristics, and lifestyle . This study also applies the representative data from china health and retirement longitudinal survey (charls) (22) and regional air pollution to conduct verify the established analysis model . Medical and sociological research reported that the so2 concentration in air would threaten physical health and even cause respiratory system diseases (5). Being healthy is not only being free from diseases or weakness, but also the perfect combination of body, mental, and social adaptation (6). Hence, research on health also focuses on mental health . Clark et al . Studied the 9 to 10-year - old british children and noted that air pollution (no2) in schools will not influence their cognition and health . After the no2 level was controlled, they found that aircraft noise and road traffic noise have significant different effects on cognition and health of children (7). A study on 1,546 samples collected from salt lake county, utah, usa found that higher so2 and no2 particles would increase risks of suicide (8). From a medical perspective, tiny particles in air pollution will hurt the nervus centralis in humans after entering the brain and may cause depression and apoplexy (9). Considering the physiological and psychological differences of the elderly with children and youth, the relationship between the mental health of the elderly and air pollution has elicited considerable attention in medical research attention . Studied the 699 elderly males and found the internal relation between pm2.5 and people s mood (optimistic or pessimistic) (10). However, yi et al . Stated in a study of 732 old people (65 years old) in boston that no correlation had been found between air pollution and depressive symptoms (11). These two studies report different results primarily because most medical researchers are based on individuals or non - random samples, which may incur selection bias . Therefore, we shall maintain a cautious attitude toward the corresponding research conclusions and policy implications . Scholars have also focused on the relationship between various social demographic characteristics and mental health . Small changes in smoking, drinking, sports, and diet would significantly affect both physical and mental health (12). Reducing smoking and increasing recreational sports could improve physical and mental health (13). Some scholars explored mental health from the perspective of social relations, and pointed out that social communication positively affected the health of the aged (14, 15). Some scholars studied the mental health of the elderly from the perspective of urban - rural difference . A study on 404 old people (60 years old) done in chinese rural and urban regions, found a significant difference in mental health between these regions (16). The urban old people have better mental health than do rural old people (17). However, mikael et al . Offered the opposite conclusion, and they believed that rural old people had better mental health than urban old people had (18). Studied 64,000 old people (65 years old) in hong kong and revealed a significant correlation between depression and chronic diseases, such as cardiovascular disease and coronary artery disease (19). Moreover, norstrand and xu analyzed the health conditions of chinese old people (65 years old) from the perspective of social relations, and found a significant relationship between mental health of urban old people and social relations (20). Based on demography, mental health deteriorated greatly with aging, while physical health and income could improve mental health (21). To sum up, although many research results concerning the mental health of the elderly have been achieved, mental health is characterized by complicated and diverse influencing factors . These factors lead different research perspectives and thereby inconsistent conclusions, which may be caused by the lack of comprehensive and representative data . Moreover, only few studies on the relationship between mental health of chinese elderly and air pollution have been reported . Based on previous studies, this paper establishes a model to analyze comprehensively the influencing factors of the mental health of the elderly in china from the perspective of urban - rural difference and by combining local air quality, individual characteristics, and lifestyle . This study also applies the representative data from china health and retirement longitudinal survey (charls) (22) and regional air pollution to conduct verify the established analysis model . The data were from the 2013 charls published by the national school of development of peking university . The charls project covered 28 provinces, cities, and municipalities in china and investigated the health, economy, and family of the population (45 years old). After missing and inappropriate data were deleted, 6,630 effective samples for final statistics were chosen, including 3,930 rural samples and 2,700 urban samples . Air pollution data of different regions were provided by china city statistical yearbook (23). Chinese ministry of health announced that mental diseases, like depression and senile dementia, were the main threats to the health of the chinese elderly (24). Surveys reported that the population aged 65 or more years has a 4.4% chance of having depression . As a result, this paper evaluated the mental health degree of the elderly using cognition and depression data in the charls questionnaire . Using the method of the center for epidemiological studies depression scale (ces - d) as reference (25), charls designed a simple questionnaire of 10 problems, including feeling annoyed by trifles, difficulties in concentration, feeling down in the dumps, encountering difficulties in doing anything, being hopeful for the future, feeling frightened, experiencing poor sleep, feeling happy, feeling lonely, and encountering difficulties in continuing life . Each problem has four answers based on time span, namely, 0 for <1d; 1 for 1d <answer<2d; 2 for 3d <answer<4d; 3 for 5d <answer<7d . Among these 10 questions, finally, scores of these 10 problems are added to identify cognition and depression degrees of respondents . The higher the ces - d score is, the more depressed the elderly will be . Based on the design principle of ces - d, therefore, the influencing factors of the psychological disorder of the elderly were further observed . Based on the processing method of yi et al . And zhao yaohui et al . (2,11), respondents with more than 10 ces - d scores were determined to have depression symptoms, that is, having a psychological disorder . (26), was measured by so2 discharge in cities and counties in which the respondents live . The study area covered 123 cities and counties in 28 chinese provinces . To further analyze the effect of respondent background on mental health, this paper controlled demographic variables, including age, gender, marriage, education, and physical health . Among the variables, physical health was measured with chronic disease and physical disability . Meanwhile, the lifestyle of the elderly was introduced as a variable, including smoking, drinking, social activity participation, and employment . The definition of variables was shown in table 1 . Definition of variables included in the study note: 1) chronic disease data in charls included 12 common diseases of the elderly, namely, hypertension, hyperlipidemia, diabetes mellitus, cancer, chronic lung diseases, liver disease, heart disease, apoplexy, kidney disease, gastric disease or digestive system disease, arthritis or rheumatism, and asthma . 2) education background was divided into four groups, namely, illiteracy, primary school, middle school, as well as vocational education and junior college or above . 3) disability included five options, namely, physical disability, brain damage, blindness or half - blindness, deafness or half - deafness, and dumb or serious stammer . In this paper, a virtual variable total ces - d score is the indicator of mental health of the elderly, which ranges between 0 and 30 . Using the least square method the basic model is: yi=+1rurali+2soi+3soi2+zixi+i yi={yi,0yi300,yi>30 or yi<0 where yi is a potential variable . Yi is the total ces - d score of respondent i, which is used to assess their mental health . So2 drainage has two effects on mental health of the elderly . On the one hand, so2 drainage amount reflects the industrial and economic development of cities, thereby affecting mental health of local residents through income level . On the other hand therefore, the square of so2 drainage was added into the model analysis to determine the influence factor of air quality on mental health . Rurali is the urban - rural variable, 1 for urban regions and 0 for rural regions . Xi is the control variable, involving age, gender, marriage, education, physical health, smoking, drinking, social activity, and employment . Respondents will be evaluated depressed when yi (ces - d score) in equation increases to a certain extent (10 score). A virtual variable di is defined, which represents if respondent i has psychological disorder (yes:1; no: 0), which is identified by the presence of depressive symptoms . Di and yi are then correlated: di{1,11yi300,0yi<11 considering that di is a 0, 1 binary discrete variable, we chose the binary selection probit model to analyze the influencing factors of psychological disorder of the elderly . The data were from the 2013 charls published by the national school of development of peking university . The charls project covered 28 provinces, cities, and municipalities in china and investigated the health, economy, and family of the population (45 years old). After missing and inappropriate data were deleted, 6,630 effective samples for final statistics were chosen, including 3,930 rural samples and 2,700 urban samples . Air pollution data of different regions were provided by china city statistical yearbook (23). Chinese ministry of health announced that mental diseases, like depression and senile dementia, were the main threats to the health of the chinese elderly (24). Surveys reported that the population aged 65 or more years has a 4.4% chance of having depression . As a result, this paper evaluated the mental health degree of the elderly using cognition and depression data in the charls questionnaire . Using the method of the center for epidemiological studies depression scale (ces - d) as reference (25), charls designed a simple questionnaire of 10 problems, including feeling annoyed by trifles, difficulties in concentration, feeling down in the dumps, encountering difficulties in doing anything, being hopeful for the future, feeling frightened, experiencing poor sleep, feeling happy, feeling lonely, and encountering difficulties in continuing life . Each problem has four answers based on time span, namely, 0 for <1d; 1 for 1d <answer<2d; 2 for 3d <answer<4d; 3 for 5d <finally, scores of these 10 problems are added to identify cognition and depression degrees of respondents . The higher the ces - d score is, the more depressed the elderly will be . Based on the design principle of ces - d, therefore, the influencing factors of the psychological disorder of the elderly were further observed . Based on the processing method of yi et al . And zhao yaohui et al . (2,11), respondents with more than 10 ces - d scores were determined to have depression symptoms, that is, having a psychological disorder . (26), was measured by so2 discharge in cities and counties in which the respondents live . The study area covered 123 cities and counties in 28 chinese provinces . To further analyze the effect of respondent background on mental health, this paper controlled demographic variables, including age, gender, marriage, education, and physical health . Among the variables, physical health was measured with chronic disease and physical disability . Meanwhile, the lifestyle of the elderly was introduced as a variable, including smoking, drinking, social activity participation, and employment . The definition of variables was shown in table 1 . Definition of variables included in the study note: 1) chronic disease data in charls included 12 common diseases of the elderly, namely, hypertension, hyperlipidemia, diabetes mellitus, cancer, chronic lung diseases, liver disease, heart disease, apoplexy, kidney disease, gastric disease or digestive system disease, arthritis or rheumatism, and asthma . 2) education background was divided into four groups, namely, illiteracy, primary school, middle school, as well as vocational education and junior college or above . 3) disability included five options, namely, physical disability, brain damage, blindness or half - blindness, deafness or half - deafness, and dumb or serious stammer . In this paper, a virtual variable disability was designed . Total ces - d score is the indicator of mental health of the elderly, which ranges between 0 and 30 . Using the least square method the basic model is: yi=+1rurali+2soi+3soi2+zixi+i yi={yi,0yi300,yi>30 or yi<0 where yi is a potential variable . Yi is the total ces - d score of respondent i, which is used to assess their mental health . So2 drainage has two effects on mental health of the elderly . On the one hand, so2 drainage amount reflects the industrial and economic development of cities, thereby affecting mental health of local residents through income level . On the other hand therefore, the square of so2 drainage was added into the model analysis to determine the influence factor of air quality on mental health . Rurali is the urban - rural variable, 1 for urban regions and 0 for rural regions . Xi is the control variable, involving age, gender, marriage, education, physical health, smoking, drinking, social activity, and employment . Respondents will be evaluated depressed when yi (ces - d score) in equation increases to a certain extent (10 score). A virtual variable di is defined, which represents if respondent i has psychological disorder (yes:1; no: 0), which is identified by the presence of depressive symptoms . Di and yi are then correlated: di{1,11yi300,0yi<11 considering that di is a 0, 1 binary discrete variable, we chose the binary selection probit model to analyze the influencing factors of psychological disorder of the elderly . Total ces - d score is the indicator of mental health of the elderly, which ranges between 0 and 30 . Using the least square method the basic model is: yi=+1rurali+2soi+3soi2+zixi+i yi={yi,0yi300,yi>30 or yi<0 where yi is a potential variable . Yi is the total ces - d score of respondent i, which is used to assess their mental health . So2 drainage has two effects on mental health of the elderly . On the one hand, so2 drainage amount reflects the industrial and economic development of cities, thereby affecting mental health of local residents through income level . On the other hand therefore, the square of so2 drainage was added into the model analysis to determine the influence factor of air quality on mental health . Rurali is the urban - rural variable, 1 for urban regions and 0 for rural regions . Xi is the control variable, involving age, gender, marriage, education, physical health, smoking, drinking, social activity, and employment . Respondents will be evaluated depressed when yi (ces - d score) in equation increases to a certain extent (10 score). A virtual variable di is defined, which represents if respondent i has psychological disorder (yes:1; no: 0), which is identified by the presence of depressive symptoms . Di and yi are then correlated: di{1,11yi300,0yi<11 considering that di is a 0, 1 binary discrete variable, we chose the binary selection probit model to analyze the influencing factors of psychological disorder of the elderly . The descriptive statistics of all samples, urban samples, and rural samples are listed in table 2 . The average ces - d score of all samples is 8.09, which is close to the critical value of depression in the simple ces - d questionnaire (10). The proportion of respondents with higher than 10 ces - d scores and evident depressive symptoms reaches as high as 27.8% . However, no significant difference was observed between urban and rural samples in terms of age, gender, marriage, physical health indicators, and social activity . The average age of the elderly was 67.66 years, and 67% old people had at least one chronic disease . With respect to lifestyle, smoking and drinking respondents accounted for 46% and 41% of the total samples, respectively . In terms of educational background, urban samples present nearly 10 times higher proportions of vocational education or above than rural samples . Furthermore, deposit per capita of the urban samples was more than four times higher than that of the rural samples, but employment showed the opposite . A total of 63% rural samples had jobs, but only 28% of urban samples were employed . Urban and rural samples had both similarities and differences in terms of mental health and various influencing factors, which deserves further analysis . 1) reflects obvious differences among all samples, urban samples, and rural samples . The ces - d distribution of rural samples inclines to the right end of all samples and urban samples because the average ces - d score of rural samples is 8.79, which is higher than that of all samples (8.09) and urban samples (7.08). Moreover, the ces - d score of female samples is at the right end of the male samples . Density distribution of ces - d this paper conducted a tobit regression on all samples, urban samples, and rural samples using the total ces - d score as dependent variable . Explanatory variables include air quality, urban - rural variable, demographic variables, and lifestyle variable . Tobit regression results on the influencing factors of the mental health of the chinese elderly 1).t statistics in parentheses . 2). p<0.05, * * p<0.01, * * * p<0.001 . Two - tailed test . Table 3 shows that the regression result of urban - rural variable is negative (the coefficient is 1.43, p<0.001), indicating that the ces - d score of urban old people is significantly lower than that of rural old people . This result reflects that urban old people have better mental health than rural old people do . So2 drainage has a significant negative effect on the mental health of all samples, urban samples, and rural samples (p<0.001). This result reflects that so2 drainage lowers the ces - d score significantly, that is, reducing the depression degree of the elderly . The square of so2 drainage is positive, which means that increasing so2 drainage will intensify the depression degree of the elderly (p<0.001). The effect of so2 drainage on the mental health of the elderly presents a positive u - shaped the regression results of age, gender, and marriage are the same . With the increase in age, the mental health of both urban and rural old people improves significantly (the regression coefficients of all samples, urban samples, and rural samples are 0.062, 0.07, and 0.057, respectively, (p<0.001). The regression results of gender show that in both urban and rural areas, females have far better mental health than males (p<0.001). The regression coefficients of all samples, urban samples, and rural samples are 1.77, 1.38, and 2.07, respectively different educational backgrounds affect urban and rural old people in similar ways but to various extents . Compared with the control group, primary school, middle school, and vocational education significantly however, only vocational education or higher educational background greatly improve the mental health of rural old people (p<0.01). The regression coefficients of all samples, urban samples, and rural samples are 2.02, 1.78, and 2.13, respectively . Viewed from all samples, smoking samples show poorer mental health than non - smoking samples . Regression coefficients of urban and rural samples have the same direction, but they have no statistical significance perhaps because old people struggling with mental problems often ease their mental problems by smoking . However, rural samples prefer drinking to smoking to alleviate mood . Therefore, drinking could significantly improve the mental health of rural samples (p<0.01). Deposit and employment are vital to the mental health of rural old people and are the main way for them to maintain life, which is why rural old people with more deposit and employment have better mental health (p<0.001). By contrast, these two factors are insignificant to urban old people . The probit model estimation results of psychological disorder of all samples, urban samples, and rural samples are shown in table 4 . To better analyze the effect of explanatory variables on psychological disorder of the aged, table 4 presents the marginal effect of explanatory variables . According to the estimation result of all samples, rural old people are more likely to suffer psychological disorder than urban old people are (the marginal effect is 0.074, (p<0.001). So2 drainage imposes a great effect, presenting a positive u - shaped variation (p<0.001), which reflects that air pollution affects the psychological disorder and mental health of the elderly in the same way . Probit model estimation results of psychological disorder of the chinese elderly 1). Z statistics in parentheses . 2). Two - tailed test aging could greatly reduce the probability of psychological disorder (p<0.001). On the average marginal effect, the probability of psychological disorder of females is 11% higher than that of males, which is significant at 1% level . The probability of psychological disorder of married samples is 4.9% lower than that of single samples (p<0.001). Viewed from all samples and urban samples, middle school and higher educational background could significantly reduce risks of psychological disorder (p<0.001). However, only vocational education, junior college, and higher educational background could greatly reduce risks of psychological disorder of rural samples (p<0.01). Compared with physically healthy samples, disabled samples have 12.5% higher risks of having mental diseases (p<0.001). However, social activity could reduce the risk of mental diseases by 6.1% (p<0.001). Drinking could significantly lower the probability of psychological disorder of rural samples (p<0.01), possibly because rural old people in china prefer to drink at parties . Compared with samples without chronic diseases, samples with one or more chronic diseases have 4.7% higher probability of psychological disorder and those with three or more chronic diseases have 10.6% higher probability of psychological disorder . For rural samples, deposit amount and employment are inversely proportional to the probability of psychological disorder (the marginal effects are 0.002 and 0.043, respectively) the descriptive statistics of all samples, urban samples, and rural samples are listed in table 2 . The average ces - d score of all samples is 8.09, which is close to the critical value of depression in the simple ces - d questionnaire (10). The proportion of respondents with higher than 10 ces - d scores and evident depressive symptoms reaches as high as 27.8% . However, no significant difference was observed between urban and rural samples in terms of age, gender, marriage, physical health indicators, and social activity . The average age of the elderly was 67.66 years, and 67% old people had at least one chronic disease . With respect to lifestyle, smoking and drinking respondents accounted for 46% and 41% of the total samples, respectively . In terms of educational background, urban samples present nearly 10 times higher proportions of vocational education or above than rural samples . Furthermore, deposit per capita of the urban samples was more than four times higher than that of the rural samples, but employment showed the opposite . A total of 63% rural samples had jobs, but only 28% of urban samples were employed . Urban and rural samples had both similarities and differences in terms of mental health and various influencing factors, which deserves further analysis . 1) reflects obvious differences among all samples, urban samples, and rural samples . The ces - d distribution of rural samples inclines to the right end of all samples and urban samples because the average ces - d score of rural samples is 8.79, which is higher than that of all samples (8.09) and urban samples (7.08). Moreover, the ces - d score of female samples is at the right end of the male samples . This paper conducted a tobit regression on all samples, urban samples, and rural samples using the total ces - d score as dependent variable . Explanatory variables include air quality, urban - rural variable, demographic variables, and lifestyle variable . Tobit regression results on the influencing factors of the mental health of the chinese elderly 1).t statistics in parentheses . 2). * p<0.05, * * p<0.01, * * * p<0.001 . Two - tailed test . Table 3 shows that the regression result of urban - rural variable is negative (the coefficient is 1.43, p<0.001), indicating that the ces - d score of urban old people is significantly lower than that of rural old people . This result reflects that urban old people have better mental health than rural old people do . So2 drainage has a significant negative effect on the mental health of all samples, urban samples, and rural samples (p<0.001). This result reflects that so2 drainage lowers the ces - d score significantly, that is, reducing the depression degree of the elderly . The square of so2 drainage is positive, which means that increasing so2 drainage will intensify the depression degree of the elderly (p<0.001). The effect of so2 drainage on the mental health of the elderly presents a positive u - shaped curve . The regression results of age, gender, and marriage are the same . With the increase in age, the mental health of both urban and rural old people improves significantly (the regression coefficients of all samples, urban samples, and rural samples are 0.062, 0.07, and 0.057, respectively, (p<0.001). The regression results of gender show that in both urban and rural areas, females have far better mental health than males (p<0.001). The regression coefficients of all samples, urban samples, and rural samples are 1.77, 1.38, and 2.07, respectively different educational backgrounds affect urban and rural old people in similar ways but to various extents . Compared with the control group, primary school, middle school, and vocational education however, only vocational education or higher educational background greatly improve the mental health of rural old people (p<0.01). The regression coefficients of all samples, urban samples, and rural samples are 2.02, 1.78, and 2.13, respectively . Viewed from all samples, smoking samples show poorer mental health than non - smoking samples . Regression coefficients of urban and rural samples have the same direction, but they have no statistical significance perhaps because old people struggling with mental problems often ease their mental problems by smoking . Therefore, drinking could significantly improve the mental health of rural samples (p<0.01). Deposit and employment are vital to the mental health of rural old people and are the main way for them to maintain life, which is why rural old people with more deposit and employment have better mental health (p<0.001). By contrast, these two factors are insignificant to urban old people . The probit model estimation results of psychological disorder of all samples, urban samples, and rural samples are shown in table 4 . To better analyze the effect of explanatory variables on psychological disorder of the aged, table 4 presents the marginal effect of explanatory variables . According to the estimation result of all samples, rural old people are more likely to suffer psychological disorder than urban old people are (the marginal effect is 0.074, (p<0.001). So2 drainage imposes a great effect, presenting a positive u - shaped variation (p<0.001), which reflects that air pollution affects the psychological disorder and mental health of the elderly in the same way . Probit model estimation results of psychological disorder of the chinese elderly 1). Z statistics in parentheses . 2). Two - tailed test aging could greatly reduce the probability of psychological disorder (p<0.001). On the average marginal effect, the probability of psychological disorder of females is 11% higher than that of males, which is significant at 1% level . The probability of psychological disorder of married samples is 4.9% lower than that of single samples (p<0.001). Viewed from all samples and urban samples, middle school and higher educational background could significantly reduce risks of psychological disorder (p<0.001). However, only vocational education, junior college, and higher educational background could greatly reduce risks of psychological disorder of rural samples (p<0.01). Compared with physically healthy samples, disabled samples have 12.5% higher risks of having mental diseases (p<0.001). However, social activity could reduce the risk of mental diseases by 6.1% (p<0.001). Drinking could significantly lower the probability of psychological disorder of rural samples (p<0.01), possibly because rural old people in china prefer to drink at parties . Compared with samples without chronic diseases, samples with one or more chronic diseases have 4.7% higher probability of psychological disorder and those with three or more chronic diseases have 10.6% higher probability of psychological disorder . For rural samples, deposit amount and employment are inversely proportional to the probability of psychological disorder (the marginal effects are 0.002 and 0.043, respectively). This paper conducted a tobit regression on all samples, urban samples, and rural samples using the total ces - d score as dependent variable . Explanatory variables include air quality, urban - rural variable, demographic variables, and lifestyle variable . Tobit regression results on the influencing factors of the mental health of the chinese elderly 1).t statistics in parentheses . 2). * p<0.05, * * p<0.01, * * * p<0.001 . Two - tailed test . Table 3 shows that the regression result of urban - rural variable is negative (the coefficient is 1.43, p<0.001), indicating that the ces - d score of urban old people is significantly lower than that of rural old people . This result reflects that urban old people have better mental health than rural old people do . So2 drainage has a significant negative effect on the mental health of all samples, urban samples, and rural samples (p<0.001). This result reflects that so2 drainage lowers the ces - d score significantly, that is, reducing the depression degree of the elderly . The square of so2 drainage is positive, which means that increasing so2 drainage will intensify the depression degree of the elderly (p<0.001). The effect of so2 drainage on the mental health of the elderly presents a positive u - shaped curve . The regression results of age, gender, and marriage are the same . With the increase in age, the mental health of both urban and rural old people improves significantly (the regression coefficients of all samples, urban samples, and rural samples are 0.062, 0.07, and 0.057, respectively, (p<0.001). The regression results of gender show that in both urban and rural areas, females have far better mental health than males (p<0.001). The regression coefficients of all samples, urban samples, and rural samples are 1.77, 1.38, and 2.07, respectively different educational backgrounds affect urban and rural old people in similar ways but to various extents . Compared with the control group, primary school, middle school, and vocational education however, only vocational education or higher educational background greatly improve the mental health of rural old people (p<0.01). The regression coefficients of all samples, urban samples, and rural samples are 2.02, 1.78, and 2.13, respectively . Viewed from all samples, smoking samples show poorer mental health than non - smoking samples . Regression coefficients of urban and rural samples have the same direction, but they have no statistical significance perhaps because old people struggling with mental problems often ease their mental problems by smoking . Therefore, drinking could significantly improve the mental health of rural samples (p<0.01). Deposit and employment are vital to the mental health of rural old people and are the main way for them to maintain life, which is why rural old people with more deposit and employment have better mental health (p<0.001). The probit model estimation results of psychological disorder of all samples, urban samples, and rural samples are shown in table 4 . To better analyze the effect of explanatory variables on psychological disorder of the aged, table 4 presents the marginal effect of explanatory variables . According to the estimation result of all samples, rural old people are more likely to suffer psychological disorder than urban old people are (the marginal effect is 0.074, (p<0.001). So2 drainage imposes a great effect, presenting a positive u - shaped variation (p<0.001), which reflects that air pollution affects the psychological disorder and mental health of the elderly in the same way . Probit model estimation results of psychological disorder of the chinese elderly 1). Z statistics in parentheses . 2). Two - tailed test aging could greatly reduce the probability of psychological disorder (p<0.001). On the average marginal effect, the probability of psychological disorder of females is 11% higher than that of males, which is significant at 1% level . The probability of psychological disorder of married samples is 4.9% lower than that of single samples (p<0.001). Viewed from all samples and urban samples, middle school and higher educational background could significantly reduce risks of psychological disorder (p<0.001). However, only vocational education, junior college, and higher educational background could greatly reduce risks of psychological disorder of rural samples (p<0.01). Compared with physically healthy samples, disabled samples have 12.5% higher risks of having mental diseases (p<0.001). However, social activity could reduce the risk of mental diseases by 6.1% (p<0.001). Drinking could significantly lower the probability of psychological disorder of rural samples (p<0.01), possibly because rural old people in china prefer to drink at parties . Compared with samples without chronic diseases, samples with one or more chronic diseases have 4.7% higher probability of psychological disorder and those with three or more chronic diseases have 10.6% higher probability of psychological disorder . For rural samples, deposit amount and employment are inversely proportional to the probability of psychological disorder (the marginal effects are 0.002 and 0.043, respectively). Based on the effect of urban - rural difference on mental health and psychological disorder of the elderly, rural old people are facing more serious mental health problems due to poor living environment, entertainment culture conditions, and health care facilities . For example, deposit amount and employment could significantly improve the mental health of rural samples, but only slightly influence the mental health of urban samples . This result indicated that the aged in rural areas needed more assets to buy basic insurances, like medical service . Drinking could improve the mental health of rural samples, but did not influence urban samples . The aged in urban areas paid more attention on their health and drank less, while the aged in rural areas enjoyed limited social activities and entertainment activities . The effects of air pollution on mental health and disorder of the elderly confirm the opinion proposed early in this paper, i.e., during the industrialization in china, so2 drainage facilitates urban economic development, improves people s living standard, enriches life of the aged, and thus improves the mental health and reduces risks of psychological disorder of the elderly . However, with the further increase of so2 drainage, the negative effects of air pollution on the mental health of the elderly become more prominent . The conclusion that the mental health of the elderly is improved with the increase of age agrees with the opinions of luan wenjing and li jianxin s studies (17, 21) possibly because old people become introversive and easy - going with age, thus having better mental health conditions (28). The female elderly have far poorer mental health and more serious psychological disorders than do males . This result revealed that in both urban and rural areas, chinese males have higher position in the family and additional social relationships, thus having better mental health . Higher educational background could improve mental health and greatly reduce the probability of psychological disorder (the coefficient is increasing continuously). This result implied that the elderly with higher educational background have richer and more diversified spiritual and cultural life, as well as pay more attention to knowledge on mental health . In terms of chronic diseases, this result showed the close relationship between mental health and physical health of the elderly . Hence, more attention must be paid on psychological changes of the elderly with poor physical health . Owing to the data limitations, these results, based on the cross - sectional micro - data, are more likely to be established within a specific period . Further research is necessary to analyze the change trend of the mental health of the elderly in china from the perspective of urban - rural difference when the data are available . Results show that air quality significantly influences mental health and psychological disorder of the aged, showing a positive u - shaped curve ., local governments should consider the influence of air quality on the mental health of the aged . Specifically, regions with developed industries should adopt strict environmental protection measures to reduce effectively the emission of air pollutants . Moreover, local governments should be aware of the differences in mental health of urban and rural old people when making associated policies . Governments should reasonably allocate public resources, remove city - countryside dualization barrier, and accelerate urban - rural integration . Special attention should be paid to the psychological conditions of the female elderly and the aged with chronic diseases and physical disability . Great efforts should be made to enrich social activity of and increase employment opportunities for the elderly . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, and redundancy) have been completely observed by the authors.
The discovery and analysis of ncrnas has become an important step in the understanding of the genomics behind human disease . Genomics in humans has in the past tended to concentrate on the small percentage (12%) of genomic space coding for proteins . Since the vast majority of human ncrnas lie in non - coding regions including introns and intergenic spaces, ncrna classes in general, and in particular micrornas (mirnas) and short interfering rnas (sirnas) are of great interest in disease studies . In some cases mirnas have been implicated in various cancers with altered expression levels appearing to be associated with the genetic alterations seen in malignancies (ryther et al ., pathogen interaction networks between humans and their viruses (aurrecoechea et al ., 2009), rna interference (rnai) has also been raised as an option for medical treatment of human diseases including cancer (garlapati et al ., 2011), viruses (khaliq et al ., 2010; haasnoot and berkhout, 2011), and transplantation (zhang et al ., 2011b). Rna interference in general, is a process where small rnas (e.g., mirnas and sirnas) are used by a protein macromolecule, to target and then to cleave transcribed mrnas, hence interfering with the expression of a targeted gene . There are a number of different pathways for this interference (collins and penny, 2009; batista and marques, 2011; ketting, 2011), with three main proteins or their like, being typically required . These proteins are dicer, argonaute, and rna - dependent rna polymerase (rdrp). Finding homologs to these proteins in protist species is usually the first step in determining that rnai exists in that species . However, as can be seen in species such as giardia lamblia and entamoeba histolytica, some of these proteins may not contain all the domains we expect to find (macrae et al ., 2006; carlton et al ., 2007; batista and marques, 2011). It is thus, very likely that protist rnai pathways will differ from their well - studied multicellular counterparts, and that understanding these differences will enable a far more efficient use of rna as a molecular tool . Rna interference has been used to understand gene expression levels and the changes that occur at different stages of disease, different stages of life cycle or development, and differences in environmental conditions (typically with mirna studies). Genes can also be specifically knocked - out using gene silencing studies to investigate the effects of particular parts of metabolic pathways . This has been done in some protists, in particular kinetoplasts where rnai has been used as a tool in genomic studies for trypanosoma brucei (reviewed in kolev et al ., 2011), and to a lesser extent in leishmania braziliensis subgenus viannia (lye et al ., 2011). The difficulty in protist rnai research is that the small rnas that are used in rnai (i.e., mirnas and sirnas), are not easily isolated and characterized . Genomic - wide sequencing is also furthering studies on how a host species reacts to pathogens in immune and preventative responses . One non - protist example is where high - throughput sequencing was used to characterize mirna levels and identify novel mirnas involved in avian influenza virus (aiv) infection of chicken (aurrecoechea et al ., 2009). In this study, sequences were matched not only to genomic sequences but to mature mirna sequences previously lodged in mirbase (finn et al ., 2006), allowing for insertions and deletions of 14 nt . Profiling analysis compared infected and non - infected tissue to identify mirnas that changed expression upon infection . Mapping of the sequences also revealed that many mirnas are grouped in clusters on the chicken chromosomes and up- or down - regulated together . Results from this study suggest that different mirna regulation mechanisms may exist on host response to virus infection with some genes up regulated to aid host immune response and down regulation of targets to aid inhibition of virus replication . For example in the wang et al . Study 377 mirnas were identified from chicken lung tissue but only 149 mirnas were identified from tracheae . The techniques for analysis will be similar but will require a greater understanding of the typical features of the different mirna classes in the protist of study . There are many different classes of ncrnas found in protists (table 1), and only some of these such as mirnas and sirnas and sometimes small nucleolar rnas (snornas) are involved in rnai . Other ncrnas such as trnas and rrnas are relatively easy to characterize because they look familiar to those already studied, but there are classes such as snornas, that are harder to find and classify because either their sequence or their action, is novel . Previously, there were two main approaches to ncrna identification (figure 1). Approach, involved the isolation of expressed rna in a designated size range, cloning, sequencing then finally, northern blotting to confirm size and conformational isotopes from rna modifications . This approach was costly both in laboratory expenditure and time, and was not very practical on a genomic scale . The other approach took a sequenced genome and computationally screened it for candidate ncrnas, using mathematical models based on the sequence and structural characteristics of a class of ncrna . This second approach, although it could be applied on a genomic scale, often produced masses of candidates that would then have to be experimentally tested by the first approach . Another issue with the computational approach is that only a single class could be searched at a time, and one had to know what that class looked like both in sequence and secondary structure in order to find it . Permitting more flexibility however, results in more false positives, a circumstance that can quickly overload the computer and its user . High - throughput sequencing permits the genome - wide sequencing of ncrnas from expressed rna (the power of the first approach), and for rapid comparison to known classes (the power of the second approach), as well as the characterization of novel ncrnas (figure 1). The disadvantage of this type of sequencing is that it demands a different type of computational analysis than previously used with ncrnas (see later). Both the traditional laboratory approach and the more recent high - throughput sequencing approach begin with the isolation of total rna from culture, followed by size selection of the rna by excising a given band from a polyacrylamide gel . Under the traditional approach the excised rna is cloned then sequenced by sanger sequencing to obtain candidate mirna sequences . With high - throughput sequencing the rna is sequenced directly without cloning and bioinformatics is used to select the best candidates . Computational approaches do not begin with biological samples but instead use mathematical models based on known ncrnas to search an already sequenced genome . Both the traditional and computational approaches require that candidate gene expression be confirmed by additional laboratory work . Key: molecular biology stages are represented by the flask icon . All other stages use rna genomic and bioinformatics procedures . + these protists all contain rnase p rna, rnase mrp rna, trnas, rrnas, snrnas, and snornas; * atypical protein structure . Short interfering or silencing rnas (sirnas) are similar to mirnas but are produced from double stranded precursors instead of folded single - stranded precursors . What makes this mechanism of great interest is that the gene silencing is highly specific, and also highly potent in where only a few copies of an small rna (2225 nt long) can demonstrate wide ranging affects . In plants, they have been highly investigated for their role in virus response (ridanpaa et al ., 2003; pantaleo, 2011), but in humans and mice they are under intense study for therapeutic medicine (recently reviewed in burnette et al ., 2005; khaliq et al ., a combination of host and viral genes has been used as a sirna - based treatment for hepatitis c virus (hcv; ashfaq et al ., 2011). Developing rnai based treatments for hcv are an important are of research, since there is currently no vaccine available for hcv due to a high degree of strain variation . Another factor is that the current drug treatment (with a pegylated interferon /ribavirin combination) is costly, has significant side effects and is not always effective (ashfaq et al ., 2011). Rnai offers new and less harsher types of treatment especially for viral diseases, but there are still challenges in this area in systematic sirna delivery and distribution to appropriate tissue (vaishnaw et al ., 2011). Small nucleolar rnas typically have roles in the modification of other ncrnas such as rrna, small nuclear rnas (snrnas), and possibly even mrnas (ridanpaa et al ., 2003; bompfunewerer et al ., 2005; gardner et al ., 2010; khanna and stamm, 2010). Snornas are between 60 and 150 nt long and fall into two main classes based on conserved sequence motifs, h / aca (sometimes called snoras), and c / d (sometimes called snords). Snornas may have a high potential for use as markers for diseases either by having mutated sequences or differential expression . In one example some snornas were discovered to have a higher expression in some lung cancer cells than in non - cancerous cells, and thus have a potential as markers for the early detection of this cancer (liao et al ., 2010). The examples above focus on how ncrnas from the host can be used to study gene expression from healthy, diseased, and sometimes treated tissue . A second type of study looks at the ncrnas from the pathogen itself to understand where the pathogen could be vulnerable and hence open to new treatment options . This is where there is less work published since until now the discovery and characterization of ncrnas in most pathogens was slow and laborious . Until very recently ncrnas in prokaryotes (often called small rnas) were not commonly thought of as being important in pathogenic studies . The characterization of the crispr system and small rna pathways (e.g., hfq - binding srnas) has made us more aware that an rna - based backbone exists just as much in prokaryotes as in eukaryotes (for review see biggs and collins, 2011; collins and biggs, 2011). Eukaryotic pathogens (e.g., nematodes, yeast, and protists) have received a little more attention but lag behind our understanding of host (typically human and mice) ncrnas (review by batista and marques, 2011). Rna interference in the protist t. brucei (causative agent of sleeping sickness) was characterized early on as functional (ngo et al ., 1998), only months after it was demonstrated in the nematode caenorhabditis elegans (fire et al . However, rnai mechanisms can be lost from a lineage, as demonstrated in some yeast (drinnenberg et al ., 2009, 2011) and some trypanosomatids (lye et al ., 2011). Thus, even though rnai is considered to be an ancient system that was likely to be present in the last common ancestor of eukaryotes (collins and chen, 2009), it does not follow that it will be still be present . 2011) have revealed that rnai in trypanosomatids loss in multiple lineages are in most cases correlated with the lack of mobile elements (kolev et al ., 2011; the lack of rnai but presence of mobile element - like sequences in one lineage of trypanosomatids t. cruzi falls against that trend (kolev et al ., 2011) indicating that there is much more to be learned about the evolution of rnai mechanisms . One of the issues contributing to the slow progress in understanding protist rnai is that many of these pathogens (and especially the protists) do not yet have well annotated genomes . Others may have genomes sequenced (some fungi and nematodes) but genes are annotated based on sequence similarity to the few very well known genomes . Very small genes such as those for mirnas and sirnas can be extremely hard to characterize based on sequence similarity . Hence, the arrival of high - throughput sequencing has enabled these ncrnas to be tackled in a slightly easier manner, but it has meant the incorporation of more bioinformatics into these projects . High - throughput sequencing of pathogens and especially protists in practice uses the power of computational biology combined with the expression from real rna . With this technology we can look at mirnas, sirnas, snorna, and even longer ncrnas from a pathogenic protist and however, first we have to characterize the ncrna classes from our species of choice . Here we will use the two pathogenic protists, g. lamblia and trichomonas vaginalis as examples to highlight the issues and possible solutions with high - throughput small rna sequencing . It should be noted however, that these issues and solutions are not confined to protist genomics, but are also generally applicable to other species including prokaryotes . Pathogenic protists are responsible for a host of human diseases that affect millions worldwide, but not surprisingly ncrna research in these pathogens has lagged behind protein - based research . Over the last decade, there are two protist species, g. lamblia and t. vaginalis that are of interest in the characterization of their rnas because of their evolutionary distance from other eukaryotes (collins and penny, 2005, 2009; collins and chen, 2009; chen et al ., 2011). G. lamblia is a diplomonad anaerobic protist that infects humans and other mammals . When ingested, the cysts hatch into trophozoites in the small intestine causing diarrhea and growth hindrance in children . It is a significant pathogen for the immune - compromised and those in developing countries affected by malnutrition . The most common treatment for giardiasis is metronidazole (flagyl), which unfortunately has unpleasant side effects including nausea and dizziness, but more importantly has potential carcinogenic properties . Metronidazole is not approved by the fda in the usa for treatment in human medicine for this reason . Trichomonas vaginalis is an anaerobic parabasalid protist that causes the sexually transmitted disease trichomoniasis in humans . Despite its name, trichomoniasis is symptomatic as an std, but it can also lead to adverse pregnancy outcomes and be associated with an increased risk of human immunodeficiency virus (hiv) transmission (cudmore et al ., trichomonas differs from giardia in that it does not have a cyst stage, so infection is directly by the trophozoites being transferred from patient to patient . Treatment of trichomoniasis also includes metronidazole, but studies have shown at least 5% of cases are resistant to this drug (cudmore et al ., 2004). Other pathogenic protists include plasmodium falciparum (malaria), e. histolytica (amebic dysentery) and t. brucei (sleeping sickness) and t. cruzi (chagas disease). Although drug treatments for all these diseases are available there is still a need for further development, especially for giardia and trichomonas where problems with treatment persist . Thus, the use of ncrnas and rnai is especially applicable to eukaryotic pathogens such as giardia and trichomonas as both a tool and a potential treatment option . Giardia and trichomonas were completed before this technology appeared meaning that their assembly was slow and most is still in the form of large pieces (supercontigs). This should not put any researcher off using such genomes since genomes like this are very usable for high - throughput small rna studies . From a number of studies, all using these annotated genomes, we can see that giardia and trichomonas like their distant multicellular human host contain a rich collection of ncrnas (chen et al ., 2007, 2008, 2009). These include rnase p (piccinelli et al ., 2005), rnase mrp (chen et al ., 2011) snornas (yang et al ., 2005; chen et al ., 2007, 2011), spliceosomal snrnas (chen et al ., 2008), mirnas (saraiya and wang, 2008; chen et al ., 2009; zhang et al ., 2009), and antisense transcripts (teodorovic et al ., 2007) studies on trichomonas ncrnas show that the currently known ncrnas also exhibit typical features of eukaryotes (piccinelli et al ., 2005; simoes - barbosa et al ., 2008; smith and johnson, 2011) including rnase p (piccinelli et al ., 2005), rnase mrp (piccinelli et al ., 2005), snrnas (simoes - barbosa et al ., 2008), and some snornas (chen et al ., 2007, 2009, 2011). However, some classes of ncrnas (especially the snornas) contain some features that are not typical . It is high - throughput sequencing that offers the opportunity to investigate these novel classes of ncrna . High - throughput sequencing of small rnas requires an rna sample of high quality and reasonably high concentration . An issue with many protists is that they are not culturable so rna is sometimes extracted from patient samples . Therefore, obtaining enough clean and uncontaminated rna for genomic sequencing is sometimes not easy or possible unless from a culturable strain . Additionally, lab strains carry the risk that they may contain genetic differences from their clinical relatives . Advances in sequencing protocols have meant that the amount of rna required for sequencing (genomic, transcriptomic, and to some extent small rna) is reduced, and it is hoped that with further improvements in protocols, more protists will be sequenced . Once a sample of rna is obtained, then the next stage is to decide which ncrnas are going to be sequenced . A sample of total rna contains some rnas that can drown out any underrepresented ncrnas in samples so mrnas, rrnas, and trnas must be removed as much as possible . A typical procedure is to run the sample on a polyacrylamide gel, then excise a band corresponding to the size for sequencing (e.g., mirnas, sirnas:1930 nt, snornas:50200 nt; chen et al ., 2009). Gel isolation, although contributing to the reduction in the amount of rna available for sequencing, offers flexibility and selectivity in the class of ncrna being examined . Overall, small rna sequencing experiments are individualistic with all the advantages and disadvantages that come with the use of developing technology . The actual sequencing of small rna samples is typically performed by the operator of the technology, whether at a service provider or in - house facility . There are currently choices of sequencing platforms for high - throughput sequencing, and highly likely that there will be even more choice in the near future . For small rna sequencing the platform selected will depend on the length of the ncrna to be sequenced . Some platforms such as the roche 454 produce long sequences (4001000 nt) which is really only suitable if trying to sequence very long ncrnas . Small rnas can be done on this platform but it is not optimized for this type of work . Presently available platforms that generate short sequences are the illumina systems (hiseq, miseq, and genome analyzer) and the solid system.because they enable sequences of short length to be obtained (3650 nt) these systems are ideal for sequencing small ncrnas such as mirnas and sirnas and as you will see later, can also be used to sequence ncrnas of longer lengths . For a review of these platforms and their use in small rna studies in general there are also different rna - based protocols available, including those that combine mrna and ncrna sequencing (i.e., without need for the mrna to have polya tails; yang et al ., 2011). This is very useful for prokaryotic sequencing and also for combining mrna and ncrna data in a single run . However, small rna sequencing is the typical protocol used for human ncrna analysis . The field of high - throughput sequencing is very dynamic with platforms and protocols constantly being added and upgraded . It is therefore best to consult with the platform operator on what is available, prior to submitting samples . If the idea is to compare mirnas and their expression levels across different conditions (i.e., ncrna gene expression) then there is one further consideration . There can be affects in running samples in different partitions of the sequencing apparatus (e.g., in different lanes or partitions on a flowcell). To avoid an excess of statistical analysis to take lane - effect into account, it is suggested that both samples are run in the same partition or partitions and barcoded to aid sorting after the sequencing . The analysis of data from high - throughput sequencing is quite unlike other genomic analysis and can be daunting to the newcomer . Unfortunately the analysis of ncrna data is one of the lesser - described protocols in high - throughput sequencing meaning that there are fewer automated pipelines, and the software is primarily at a command - line level . Only now are guidelines to small rna analysis being published (e.g., mccormick et al ., 2011) and this is primarily for mammalian and plant research . What follows in this section is a general approach to the analysis of short (<25 nt) and medium / long (> 50 nt) ncrna sequencing data from high - throughput sequencing (figure 2). A general approach for using high - throughput sequencing data to search for small and larger ncrnas . Small ncrnas such as mirnas and sirnas will produce data containing the ncrna sequence plus adaptor sequence . Once the adaptor is trimmed off, the sequence is ready for mapping and further genomics . When longer ncrnas are sequenced, they are fragmented to a predetermined size by the sequencing process . After sequencing this approach was successfully used for the analysis of ncrnas from giardia lamblia (see text for details). The output from high - throughput sequencing is typically a file of short sequences (often termed short - reads or reads) accompanied by a quality score for each nucleotide in each sequence . This is termed fastq format with four lines for each sequence (cock et al ., 2010). However, because of the high sensitivity of this type of sequencing, the raw data will also contain sequences, including sequencing primers and contaminants, which occur in all high - sequencing datasets . Data filtering is therefore the first step to analysis and for small rna sequencing it can permit the separation of reads into those likely to be from small ncrnas (mirnas and sirnas) and those from medium ncrnas . Adaptors are the short sequences that are added to the ends of the rna fragment during the sequencing process . Additionally, during rna work (due to the fragments being very short) we can also get adaptor dimers forming and these will show up in the results . Adaptor sequence information is available from the sequencing vendors and can be removed from the data using text - mining or sequence manipulation software . One example is the fastx - toolkit that contains scripts not only to remove adaptor sequences, but can also remove sequences containing too many n s and those sequences of lower quality . Other quality assessment tools include many commercial software packages, and freeware such as fastqc, which can help guide data - filtering requirements . Biologically ncrnas may be of different lengths in the cell, and this can cause numerous issues for downstream analysis where computer programs prefer the data length to be consistent . It is therefore particularly recommended that sequences are trimmed to a common nucleotide length before mapping . For work with protist small rna data such as that from giardia and trichomonas (used in studies by chen et al ., 2007, 2008, 2009, 2011), this basic approach of filtering sequences prior to further analysis was used . It was found that by filtering those sequences with adaptors, and those without, into different datasets, ncrnas of different length ranges (small and medium) could be characterized (figure 3). Ncrnas processes characterized in giardia lamblia and their likely cellular locations in the trophozoite stage, and in relation to the major rna processes of transcription and translation . Giardia has two nuclei which appear identical and replicate at the same time and all of the processes within transcription, rnp biogenesis, transcriptional regulation, and core rna processing could be expected to be found in both nuclei . Rnai may also be important in viral defense since some sirnas found in high - throughput sequencing do map to the stable giardiavirus (unpublished results). It is not known yet whether giardia has rna storage granules but their presence in trypanosoma does not preclude them here . A similar diagram can be visualized for trichomonas except that it only has one nucleus . Mapping (or matching) of the short - read sequences to a reference genome is the easiest approach for finding ncrnas from protists . Since there are still only a relatively few classes of ncrnas characterized for many protists this approach can yield limited results but is worth doing to characterize medium length ncrnas such as snornas, snrnas, rnase p, and rnase mrp . Smaller ncrna classes such as mirnas and sirnas may have too many sequence differences over their short length to permit accurate mapping directly to known sequences, but mapping of mirnas is possible to their longer pre - mirna precursors, and sirnas to potential target sequences . Mapping tools such as bwa (li and durbin, 2009), bowtie (langmead et al ., 2009), and soap2 (aurrecoechea et al ., 2009), as well as however, all of this software was designed for dealing with sequences longer than 22 nt, so software parameters must be changed for mapping short ncrnas such as mirnas and sirnas . One of the key parameters to adjust is the seed length for the alignments, which is where the initial contact between the read and the reference genome is made . Allowing more mismatches, e.g., up to n = 3 mismatches per seed (the standard is n = 2) may only be beneficial if the species being sequenced is different from the reference genome . Often when a short - read maps to multiple places, only one place is reported and this is typically assigned by random . Since we commonly have mirnas and sirnas match both at their place in the genome and at their target position in the genome, multiple matches are normal, thus reporting settings should be adjusted for the reporting of all hits . In practice, mirnas have been characterized from protists using a mixture of traditional, computational, and high - throughput sequencing approaches . Some mirnas from giardia have been shown to be derived from snornas (saraiya and wang, 2008; kolev and ullu, 2009), with two of these, mir2 and mir4 consequently shown to regulate the expression of variant surface proteins (vsps), involved in resistance to host intestinal proteases (prucca et al ., 2008; garlapati et al ., 2011). A computational study of mirnas from giardia (zhang et al ., 2009) identified 50 mature mirna candidates, some of which also targeted vsp genes . Using high - throughput sequencing these vsp targeting mirna candidates were also found (chen et al ., 2007, 2009) as well as many other mirnas conforming to the expected size range of 2527 nt (macrae et al ., 2006;, mirnas have been characterized using traditional (lin et al ., 2009), and high - throughput sequencing approaches (chen et al ., 2009, the use of mirna - based software such as miranda, aided in the selection of strong mirna candidates . In practice, although high - throughput sequencing is sequencing biologically expressed rnas (unlike computational analysis), there may also be some mrna degradation products and contaminants that may slip through filtering . Studies in giardia and trichomonas have highlighted that confirmation procedures from another source (including mirna - based software), is still required . Expression levels of some trichomonas mirnas have also been examined showing that one mirna tva - mir-001 has a significantly lower expression level in the ameboid stage (lin et al . Most of the small rna work to date in giardia and trichomonas has focused on mirnas but sirnas are also present (chen et al ., some sirnas appear to map in giardia to a long - tandem repeat rnas (girep-15) which show a high degree of sequence similarity with a number of vsp genes (chen et al ., in addition, both giardia and trichomonas contain stable rna viruses and high - throughput sequencing has resulted in some reads mapping to these viruses (unpublished results). Small rnas from other protists have also been characterized using traditional or computational methods . In t. cruzi, where there standard rnai proteins have not been found, small rnas derived from trnas have been characterized which are usually recruited to specific cytoplasmic granules (garcia - silva et al ., 2010). T. brucei does contain standard rnai pathways and computational studies have uncovered mirnas that target another type of antigenic variation variant surface glycoproteins (vsgs; rudra et al ., 2007). Some mirna candidates have also been characterized in e. histolytica using a computational study (de et al ., 2006), but research here has focused on using dsrna for gene silencing (reviewed by kolev et al ., 2011; zhang et al . Studies of protist mirnas to date are showing that rnai has an important role in antigenic variation and hence the parasite s survival in its host . Learning more about this system could enable more effective strategies to prevent and treat a range of protist diseases . To characterize medium length ncrnas, such as snornas, rnase p, and rnase mrp rnas (overlapping consensus fragments) from the mapping data . De novo assembly tools such as velvet (zerbino and birney, 2008) and abyss (simpson et al ., 2009) which assemble fragments without any prior alignment to a reference genome, are again primarily designed for working with longer sequences, and in practice did not perform well with data from giardia and trichomonas data (chen et al . However, with careful parameter choice and a bit of experimentation, it is not inconceivable that these tools could be useful in the assembly of long (> 200 nt long) ncrnas, such as those discovered to be crucial for human and mouse epigenetics . An easier way to generate consensus contigs can be to use the results from mapping and convert them to contig sequences using software such as the mpileup function of samtools (li et al ., 2009), or the now depreciated tools in maq (li et al ., 2008) the use of a reference genome to guide the assembly of contigs means that areas separated by low coverage of reads can be joined for further analysis . This technique was used to find medium length ncrnas such as rnase p and rnase mrp rnas from giardia (chen et al ., 2011). Although the rnase p rna has been previously identified from giardia, the closely related rnase mrp rna was found by forming larger contigs from short - reads then using the infernal rna comparison software (nawrocki et al ., 2009) to compare these contigs to ncrnas from rfam . Using this method rnase p and mrp rnas were either characterized, confirmed for had ambiguous genomic positions clarified (chen et al ., 2011). Comparative genomes has been used to characterize snornas from trypanosomatids including leishmania (liang et al ., 2007) and t. brucei (uliel et al ., 2004; 2005) can be used to characterize snornas from both classes c / d box and h / aca . Often it can be necessary to change parameters within this software to permit changes in expected secondary structure . C / d box snornas direct 2-o methylation, and are relatively easy to identify based on conserved sequence elements (stem - loop features) and complementary binding to the target rnas . H / aca box snornas direct pseudouridylation, and often exhibit more variable features due to their shorter length of conserved elements and discontinuous complementary target binding regions . A combined experimental and computation approach using high - throughput sequencing enabled both of these snornas classes to be characterized in g. lamblia, snoscan was modified to search for snornas in the giardia wb genome but these adjustments caused a loosening of parameters, and hence, an increase in false positives being reported . Positive hits were compared to high - throughput sequencing results to filter through the large number of false positives . When the sequencing results were combined with information from potential ribosomal pseudouridylation sites, other snornas were characterized from both giardia and trichomonas (chen et al ., 2011). There are many snornas even from well characterized species (i.e., humans) that do not have identified targets . These are termed orphan snornas (bazeley et al ., 2008) and it is likely that such snornas will be found in protists, but perhaps not as closely associated with splicing (giardia and trichomonas have few introns). However, without potential binding sites to check results against, much more laboratory work will be required to characterize these orphan snornas, even those potential candidates found by high - throughput sequencing . High - throughput sequencing has opened the door on more research into the use of ncrnas either as tools for investigating protist biology, markers for disease detection or progression, or as potential avenues for treatment . Although there is a long way to go to catch up with ncrna analysis from host species (e.g., human and mouse), the genomic sequencing of many pathogenic protists is already permitting genome - wide screens of ncrnas such as mirnas and sirnas . Studies of protist mirnas to date are showing that rnai has an important role in antigenic variation and hence the parasite s survival in its host . Learning more about these systems other areas of active research are now looking at the integration of regulatory rna (mirna and sirna) data with protein gene expression sequencing data (i.e., rna - seq, or mrnaseq), to characterize how mirnas control their targets, and are themselves controlled, in different environments . In effect, this is a combination of mirna expression and target expression, all coming from the same sample . The use of high - throughput sequencing to uncover and characterize ncrnas has both the biological relevance of traditional laboratory approaches and the genome - wide scale of the computational approaches . However, it does require the understanding of both the biological and computational aspects of rna analysis . Although software both for mapping, assembly and sequence manipulation was written for longer mrnas and genomic sequencing, it can be applied to short ncrnas such as mirnas and sirnas with careful parameter adjustment . It is likely that in the next few years we will see further development of the small rna sequencing protocols that are available especially as they rise in importance in the medical research world . What is needed to meet this rise is a general upskilling of molecular researchers to deal with the increased bioinformatics that this new technology brings, and further development of software pipelines to make it easier to adapt rna software to non - mammalian and non - plant species . Protist biology is very different and their ncrna systems are delivering us many surprises (collins and penny, 2009). It is clear that genome biology of host and pathogens can no longer exclude the analysis of non - coding sequences . The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Intraorally, it presents as a soft - tissue swelling of the maxillary anterior mucolabial fold, lateral to midline leading to obliteration of the nasolabial fold . It is submucosal and extraosseous, spreads in the labial sulcus and expands all the soft tissues outwards . Extraorally, it appears as a swelling between the upper lip and nasal aperture causing elevation of nasal ala . The terms nasolabial and nasoalveolar cysts have been used synonymously, as these are similar cysts with the only difference that the former is a lesion located entirely within the soft tissues and the latter causes maxillary alveolar bone erosion . A 69-year - old female reported to the dental college with a chief complaint of an extraoral swelling on the right side of her upper lip and elevation of the alar base [figure 1a] since 7 - 8 months . History of present illness revealed that the swelling had increased in size since its initiation but was not associated with any pain, drainage through oral / nasal cavity or difficulty in breathing through nasal obstruction . Furthermore, there was no history of dacryocystitis / watery eyes but there was a swelling intraorally corresponding to this extraoral swelling which was obliterating the labial vestibule on that side . Intraoral examination revealed bilateral smooth, soft, fluctuant masses lateral to the central incisor teeth in the maxillary labial vestibule . The right - sided mass was well - defined [figure 1b] and measured approximately 2 3 cm and left - sided mass measured 1.5 1.5 cm when palpated bimanually . Aspiration biopsy from right - sided mass revealed straw - colored fluid [figure 1c]. (a) clinical picture showing extraoral swelling and elevation of alar base on the right side . (b) clinical picture showing intraoral swelling in the labial vestibule on the right side . (c) straw - colored fluid on aspiration from the right - sided cyst occlusal radiograph [figure 2a] showed a large scooped - out radiolucent area with cupping surrounding upper left central incisor (ul1) in the middle and coronal thirds of its root, but not in the periapical area . The cysts were enucleated [figure 2b and c] using intraoral approach under local anesthesia and adrenaline (1:80,000). A full - thickness incision was reflected along the crest of maxillary alveolar ridge in the canine to canine region and blunt dissection by periosteal elevator exposed two smooth, well - circumscribed cystic swellings just below the nasal floor . There had been scooping of the anterior maxilla on the left side due to expansion of the cyst . In contrast, morphology of alveolar bone on right side was not affected . In the same appointment, extractions of hopeless teeth were also done and the wound was closed with loop sutures using 3 - 0 non - absorbable black mersilk after debridement and irrigation with normal saline . (c) enucleation of left cyst histopathology of the right - sided cyst revealed a cystic cavity lined by variable thickness of epithelium predominantly made up of many layered stratified squamous cells along with a few mucosal cells [figure 3]. At few places, stratified squamous and pseudostratified columnar epithelia with many cilia and goblet cells were also evident [figure 4a and b]. Left - sided cyst specimen showed two or more layered stratified squamous lining epithelium with thin capsule [figure 5]. Photomicrograph of a section of right cyst showing epithelial lining predominantly made of many layered stratified squamous cells with few mucosal cells (h&e stain, 100) (a) photomicrograph of right cyst: stratified squamous epithelium with mucous cells and cilia (h&e stain, 400). (b) photomicrograph of right cyst: pseudostratified ciliated columnar epithelium with goblet cells (h&e stain, 400) photomicrograph of left cyst showing cystic cavity lined by stratified squamous non - keratinized epithelium with thin capsule (h&e stain, 40) patients seek medical assistance when the cyst becomes infected or cause a cosmetic defect . In the present case, intraoral swellings were present in the labial vestibule on both sides, lateral to the maxillary central incisors, which is the most common symptom associated with this cyst . As the nasolabial cysts arise in soft tissues, in most cases there are no radiographic changes . Occasionally, pressure resorption of the underlying bone may occur, in that case, it is known as nasoalveolar cyst, where the cyst being external to the alveolar bone, produces cupping of the underlying cortical plate . In the present report, erosion of bone in a cupping fashion around ul1 was observed as a sequel of pressure from the cyst . Routine intraoral radiographs are not diagnostic for nasoalveolar cysts but assist in excluding other odontogenic or non - odontogenic cysts . Periapical inflammatory lesions can be excluded by vitality testing of the associated teeth and the absence of radiolucency in the periapical region on intraoral radiographs . Globulomaxillary and nasopalatine duct cysts can easily be mistaken as nasolabial cysts; however, both of these are intraosseous lesions . In the present case,, the rarefaction was observed in the middle and coronal third of the root of ul1 . However, cohen and hertzanu have reported a case of nasolabial cyst with a high growth potential that resulted in the erosion of maxillary alveolus, invaded supporting structures in the region of incisors and caused displacement of these teeth . In the present case also, there were signs of displacement of adjacent teeth . Distolabial migration of the crowns of upper central incisors was primarily due to loss of periodontal support caused by periodontitis in addition to pressure from the cyst . 10.6% of nasolabial cysts are bilateral, this finding suggests that these cysts are developmental in origin . Etiopathogenesis of this cyst involves two theories suggesting that it arises: as a fissural cyst which develops from embryonic nasal epithelium entrapped between the merging maxillary processes and the medial and lateral nasal processesas a developmental cyst arising from the remnants of the embryonic nasolacrimal ducts, due to histological similarities to the latter . Furthermore, this cyst is in the area of the naso - optic furrow and proliferation of the nasolacrimal duct has been seen at this site . Although this theory may need some revision, due to the finding of apocrine secretion along the surface of the nasolabial cyst, it seems to be the most accepted one at this stage . The fact that high percentage of linings demonstrate pseudostratified columnar epithelium, often with cilia or numerous goblet cells, is compatible with origin from primitive ductal or glandular structures . So, the most likely origin is from the nasolacrimal duct . In case presented here, the cystic lining was predominantly stratified squamous type, although few areas showed pseudostratified ciliated columnar epithelium . Su et al ., in their scanning electron microscopic examination revealed that cilia observed in the light microscope were in fact numerous short globular or irregular microvilli which probably resulted from lack of stimulation by air . As a fissural cyst which develops from embryonic nasal epithelium entrapped between the merging maxillary processes and the medial and lateral nasal processes as a developmental cyst arising from the remnants of the embryonic nasolacrimal ducts, due to histological similarities to the latter . Furthermore, this cyst is in the area of the naso - optic furrow and proliferation of the nasolacrimal duct has been seen at this site . Although this theory may need some revision, due to the finding of apocrine secretion along the surface of the nasolabial cyst, it seems to be the most accepted one at this stage . The fact that high percentage of linings demonstrate pseudostratified columnar epithelium, often with cilia or numerous goblet cells, is compatible with origin from primitive ductal or glandular structures . So, the most likely origin is from the nasolacrimal duct . In case presented here, the cystic lining was predominantly stratified squamous type, although few areas showed pseudostratified ciliated columnar epithelium ., in their scanning electron microscopic examination revealed that cilia observed in the light microscope were in fact numerous short globular or irregular microvilli which probably resulted from lack of stimulation by air . In the present case, bimanual palpation (best method of diagnosing this cyst by placing one finger in the labial sulcus and the other on the nasal floor) revealed soft, fluctuant masses bilaterally . Dimension of the cyst in the soft tissues of the labial sulcus was more (2 3 cm) on the right side which also produced an extraoral swelling because this cyst was exclusively an extraosseous one . In contrast, the left - sided cyst measured relatively less (1.5 1.5 cm) in the soft tissues with no corresponding extraoral swelling because the cyst was occupying space between the vestibular sulcus and alveolar bone as was evident on surgical exploration . The preferred method of treatment of this cyst is surgical excision by intraoral sub - labial approach, although care must be exercised to prevent perforation and collapse of the lesion . The surgical outcome of this method is generally successful; however, complications associated with the procedure include facial swelling, gingival numbness, decreased sensation of the teeth, and wound infection . An alternative transnasal approach has been suggested that allows endoscopic marsupialization of the cystic cavity . In the present case, full thickness incision was given on the crest of the ridge instead of sub - labial incision (and a mucoperiosteal flap was reflected with a periosteal elevator to expose the cysts) because the surgical procedure was inclusive of extractions of adjacent mutilated teeth . So, in the presented case; clinical, radiographic and histological features of the lesions were suggestive of nasolabial cyst on the right side and nasoalveolar cyst (residing on the alveolar surface) on the left side, which were successfully enucleated by a modification of intraoral sublingual surgical approach . In 1-year
The treatment options for graves' disease consist of antithyroid drugs, radioactive iodine (rai) therapy, and surgery . Rai therapy has been used for the treatment of graves' disease for more than seven decades, and in most studies, it has not been associated with an increased risk of cancer . On the other hand, several studies have proposed the potential increase in thyroid malignancies with rai treatment [2 - 4]. Here, we describe a very rare case of anaplastic thyroid carcinoma (atc) associated with graves' disease, following rai treatment . A 42-year - old woman was referred to our clinic in 1998, because of palpitations, hand tremor, and laboratory findings of hyperthyroidism (serum thyrotropin [thyroid - stimulating hormone, tsh] 0.05 iu / ml [reference range, 0.17 to 4.05], free thyroxine 4.01 ng / dl [reference range, 0.79 to 1.86], and positive tsh receptor antibody at 201.7% [percent binding inhibition index, radioreceptor assay, reference range; 15.0%]), and had been diagnosed as graves' disease . A technetium-99 m thyroid scan showed enlarged and homogeneously increased uptake, and ultrasonography revealed a diffuse goiter without nodules . The patient received 200 mg / day of propylthiouracil and -adrenergic blocking agents for 1 year . However, due to recurrent hyperthyroidism, she received rai therapy twice with an administered dose 370 mbq (10 mci), 296 mbq (8 mci), each . Two years later, she received thyroid hormone replacement due to subsequent hypothyroidism at a local clinic . In september 2010, over 10 years later, she revisited us with complaints of hoarseness, dysphagia, and dyspnea, which had been presented for 2 months . An anterior neck mass, which was hard and fixed, and some lymph nodes (lns) around the mass were palpable . Her chest radiography showed a tracheal narrowing and deviation to the right without active lung lesion . On computed tomography (ct) of the neck, positron emission tomography - ct, an intense hypermetabolic lesion was noted in the thyroid gland and lns on her left neck without distant metastasis . A total thyroidectomy with selective neck dissection, tracheal sleeve resection, and end - to - end anastomosis were performed . Subsequently, she had concurrent cisplatin - based chemoradiotherapy for her neck and superior mediastinum (total 6,600 cgy/33 fractions). After 4 months of follow - up, despite the aggressive treatment, the disease progressed . On neck ct, a new focal necrotic mass on the left side of her neck was noted, suggesting local recurrence . The mass extended up to the tracheal lumen and the patient died of sudden uncontrolled bleeding in the airway . The patient in this case received rai therapy twice for recurrent hyperthyroidism . After more than 10 years, she developed respiratory symptoms and was finally diagnosed with atc . This case raises questions about the causal relationship between rai therapy for graves' disease and the subsequent occurrence of thyroid cancer . Although it has been widely accepted that rai is safe, some studies have suggested that rai therapy is possibly associated with a small increased risk of thyroid cancer [2 - 4]. Degroot reported that the rai treatment can induce an increase of thyroid antibodies, such as tsh receptor antibody, and it was suggested that thyroid stimulating antibodies play an important role in thyroid carcinogenesis . These antibodies also can stimulate angiogenesis, which contributes to thyroid tumor cell growth, by activating vascular endothelial growth factor . Sturgis reported cytomorphologic alterations in thyroid follicular epithelium, which is associated with rai therapy . Before treatment, graves' disease typically lack significant atypia, while after rai therapy, nuclear atypia, and progressive cellular metaplasia could be seen microscopically . Furthermore, it was prominent in those who remain in a hyperthyroid state despite rai treatment . In addition to our case, two previously reported cases of atc after rai therapy for documented graves' disease are summarized in table 1 . Baker reported a 64-year - old woman with atc 12 years after rai therapy . Because thyroid toxicity persisted after one course of rai therapy, the patient received a second therapy in 4 months . Gossage et al . Reported a 46-year - old woman who had her first rai therapy, when aged only 23 years . Although she underwent partial thyroidectomy prior to the rai therapy, hyperthyroidism recurred and she received several subsequent doses of radioiodine . An analysis of these cases is noteworthy, because since the interval to cancer diagnosis was over 10 years in all cases, it supports the importance of long - term vigilance in patients receiving rai therapy . In addition, these cases had something in common in terms of the occurrence of atc after repetitive, small doses of rai therapy . The cooperative thyrotoxicosis therapy follow - up study showed that thyroid neoplasms developed in children treated with lower, rather than higher, doses of rai therapy, which is compatible with our finding . Therefore, it can be hypothesized that multiple courses of rai therapy, with small doses, may play a role in carcinogenesis, especially in the poorly differentiated type . . Demonstrated that i treatment inactivates the tumor suppressor gene that is frequently found in atc rather than in the differentiated type . However, to discuss the causal relationship between rai therapy and the occurrence of cancer, the number of reports is too small, therefore a population - based study is required and this is a limitation of our report . In addition to the rai therapy, we cannot exclude that graves' disease itself might also increase the risk of cancer . Although our patient had no thyroid nodule by ultrasonography, the palpable thyroid nodules found in graves' patients have a 16.9% malignancy rate, which is higher than that of the general population . Moreover, the finding that thyroid autoantibody stimulates the malignant transformation of the thyroid gland supports this suggestion . It is not clear that the occurrence of atc reported here was influenced by rai therapy or, alternatively, it may only represent the delayed recognition of a rare change in the natural history of graves' disease . Nevertheless, this report is worthwhile since it presents a very rare case of atc that occurred 11 years after rai therapy for graves' disease.
It is very important to distinguish m. abscessus from other nontubercular mycobacteria (ntm) as antimycobacterial therapy is significantly more complex . A 53-year - old male patient from gujarat (india), with a history of hypertension, diabetes mellitus, hypothyroidism, and a stable dilated cardiomyopathy was shifted to our hospital with a prolonged pyrexia, which had remained undiagnosed and unresponsive to standard antimicrobial treatment . A coronary angiography had been performed 8 weeks earlier, which showed normal coronary anatomy . Three weeks following the angiography, the patient developed moderate- to high - grade fever for which standard investigations, routine blood cultures, and imaging were performed and were inconclusive . He was transferred to our care 6 weeks into his febrile illness with no clear diagnosis . On examination, the patient was febrile, toxic, and had an early diastolic murmur in the aortic area . The investigations revealed hemoglobin 8.3 g / dl, white blood cells 9200 cells / mm, platelets 2,23,000/mm, serum creatinine level 2.2 mg / dl, and erythrocyte sedimentation rate 88 mm / h . Urine analysis showed 3 - 5 red blood cells (rbcs)/high power field (hpf) and a raised 24-h urine protein of 1527 mg/24 h was also noted . On ultrasonography, there was mild hepatosplenomegaly, but there was no evidence of focal lesions in the spleen or liver . Chest roentgenogram revealed mildly prominent pulmonary vasculature with cardiomegaly but no significant lung parenchymal pathology . The two - dimensional echocardiography (2d echo) done at previous hospital showed left ventricular ejection fraction (lvef) of 30%, normal heart valves, and global hypokinesia . A repeat 2d echo at our center showed an evidence of mild aortic regurgitation with three discrete small mobile vegetations on the left and right coronary cusps of the aortic valve . Choroidal infiltrates as a stigmata of infective emboli were noted, but there was no evidence of embolization to skin or any other organ . Double stranded antibody (anti - dsdna), and anti nucleophilic cytoplasmic antibodies (anti - anca) were negative and complement levels (c3 and c4) were within the normal range . The patient was treated for heart failure and was started on ceftriaxone at a dose of 1 g twice daily intravenously as empirical treatment for infective endocarditis . Bactec myco / f lytic medium was then used, which demonstrated the rapidly growing organisms as ntm . Linezolid and clarithromycin were empirically added while awaiting the sensitivity and speciation of the ntm . Meanwhile, species identification using the line probe assay technique revealed the organism to be m. abscessus and drug sensitivities were subsequently obtained . The organism was found to be sensitive to amikacin, clarithromycin, linezolid, and tobramycin, with intermediate sensitivity to cefoxitin but was found to be resistant to doxycycline, imipenem, cefepime, ceftriaxone, minocycline, and amox clavulanic acid . The two antibiotics (linezolid and clarithromycin) were continued and the other sensitive drugs isoniazid, ethambutol, and cefoxitin were added to the treatment regimen . The patient defervesced for the first time after 7 weeks of continuous fever 1 week into the above treatment . Linezolid was withheld for 10 days in view of significant bone marrow suppression but was later reinstituted in lower doses . After 3 weeks of therapy with a combination of five antibiotics, the creatinine stabilized at 2.6 mg / dl having previously reached a peak of 3.6 mg / dl . The valvular vegetations, however, were present and blood cultures still grew the same mycobacteria despite the patient having defervesced . Aortic valve replacement surgery was considered but was withheld in view of multiple comorbidities and patient's reluctance to undergo any procedure . A repeat 2d echo at 4 weeks post treatment showed no change in the vegetation size and the patient was discharged on request on all five antimycobacterial antibiotics . Subsequently, the patient developed worsening cardiac failure and died of pulmonary edema and renal dysfunction despite aggressive medical management in a local intensive care unit . Postprocedure mycobacterial infections have been often reported, especially in india where glutaraldehyde is used as a sterilizing solution for surgical and other equipments . Risk factors for bacteremia following catheterization include obesity, duration of the procedure, the number of balloons used, and the number of skin punctures performed . Infections complicating coronary angiography include sepsis, endocarditis, suppurative pancarditis, stent infection, septic arthritis, epidural abscess, necrotizing fasciitis, and groin wound infection . The most common causative organism of bacteremia following cardiac catheterization is staphylococcus aureus, particularly in the obese . Native valve endocarditis with ntm such as m. abscessus is rare and often missed without appropriate blood cultures . In cases of postprocedure endocarditis, early diagnosis and prolonged combination therapy are obligatory to minimize mortality, as m. abscessus is inherently resistant to multiple antibiotics . Although there are no specific drug protocols for treatment of pulmonary m. abscessus disease, a macrolide - based regimen is often used combined with surgical debridement for successful therapy . Evidence - based management for other nonpulmonary diseases and specifically for m. abscessus endocarditis is lacking due to sheer rarity of the condition and treatment is based on in vitro drug susceptibility . Typically, antibiotics with maximum in vitro activity against m. abscessus isolates include amikacin, clarithromycin, tigecycline, and cefoxitin . To a lesser extent, combination therapy is mandatory and should be administered for at least 2 - 4 months . Tigecycline and linezolid are newer antibiotics with efficacy against m. abscessus and the recommended treatment length with these drugs is 6 - 12 months . Among the carbapenems, clarithromycin and linezolid were used in combination as aminoglycosides were contraindicated due to renal failure . The cause of the raised creatinine was thought to be multifactorial, secondary to diabetic nephropathy, hypertension, and congestive cardiac failure . The possibility of septic emboli causing nephritis seems low owing to only 3 - 5 rbcs / hpf in urine . Isoniazid and ethambutol were added as standard antituberculous therapy with the hope of some benefit in such a desperate situation . M. abscessus infection is not always considered a medically curable condition, but prolonged antibiotic therapy may result in chronic suppression of the disease . In vitro drug susceptibility patterns do not always correlate with clinical responses and measurements of minimum inhibitory concentrations of antibiotics do not always predict their therapeutic effects . Endocarditis caused by m. abscessus generally has a very poor prognosis despite combination antimicrobial therapy, as only two of the 10 documented cases described in the literature have survived after treatment . Surgical intervention to replace the infected valve would have been worth attempting if the patient had been willing . However, in view of the dismal treatment outcomes shown by previous studies, it remains uncertain if early institution of appropriate therapy or valve replacement would have altered the ultimately fatal outcome with this pathogen . Nosocomial infection control needs to be strengthened particularly at smaller centres where complex interventions are increasingly being conducted.
Rheumatoid arthritis (ra) is an autoimmune disease of unknown etiology, characterized by chronic polyarthritis that induces joint damage and functional disability . Tumor necrosis factor (tnf) plays a key role in the ra pathogenesis, and anti - tnf agents have shown good efficacy in treating refractory ra, especially when methotrexate (mtx) and disease - modifying anti - rheumatic drugs (dmards) proved ineffective . As the first clinically approved tnf blocker, infliximab has been widely used in ra treatment . However, approximately 30% of ra patients achieved inadequate response . Clinical parameters related with ra diagnosis or prognosis, such as c - reactive protein (crp) and disease activity score 28 (das28), failed to predict clinical response to infliximab . Thus, considering the high cost, adverse effects, and prolonged disease severity in non - responders, a search for predictive biomarkers was needed . In recent years, gene expression profiling has been successfully used on tissue samples or blood to identify biomarkers in various disorders . Compared to synovial biopsy in ra, peripheral blood samples were easy to obtain and facilitated dynamic monitoring, which could also reflect the inflammatory condition . Thus, several studies tested the gene expression pattern of peripheral blood mononuclear cells (pbmcs) in ra patients to identify genetic markers for predicting subsequent infliximab response . Unfortunately, huge discrepancies between studies restrained the clinical application of those reported biomarkers, and no systematic reviews have concentrated on this . Therefore, we conducted a systematic bioinformatics analysis of microarray data in public databases to identify the most recognized differential genes associated with ra infliximab response, and then test the viability of these genes as predictive biomarkers . Gene expression omnibus (geo) (https://www.ncbi.nlm.nih.gov/geo/) and arrayexpress (http://www.ebi.ac.uk/arrayexpress/) are international public repositories that archive and freely distribute microarray data submitted by the research community . We searched related microarray data in the databases using key words including infliximab, anti - tnf or anti - tumor necrosis factor in combination with rheumatoid arthritis or all clinical information and published articles of the microarray datasets were reviewed independently by 2 researchers . Microarray datasets were included if they satisfied the following criteria: (i) diagnosis of ra was based on the 1987 revised criteria of the american college of rheumatology (acr) for the classification of ra or on the 2010 acr / european league against rheumatism (eular) classification criteria; (ii) patients responded inadequately to mtx; (iii) nave to anti - tnf treatment or had an interval of at least 3 months after the last treatment; (iv) peripheral blood samples were collected before first administration of infliximab, and total rna was separated from pbmcs to analyze the global expression pattern of numerous well - annotated genes by microarray; and (v) response to infliximab was assessed at least 6 weeks later according to certain criteria . For those eligible datasets, the normalized data of gene expression profiling were downloaded from the databases of geo or arrayexpress . For each microarray dataset, all samples were divided into different groups according to the therapeutic outcomes . Differential genes associated with infliximab response were analyzed using the gcrma package of r language (version 3.2.3, https://www.r-project.org/). The genes recognized by half of the datasets were regarded as candidate biomarkers for predicting infliximab response . P<0.05 was considered significant, and p values from these analyses were not adjusted for multiple testing . Acr and eular response criteria are commonly used to measure individual response in clinical trials, dependent on the extent of change and the level of disease activity reached . In the included datasets, gse58795 and gse42296 were randomly or prospectively designed datasets using the acr and eular response criteria, respectively . Thus, we chose these datasets as validation sets to determine the accuracy of the biomarkers in predicting infliximab response . Roc (receiver operating characteristic) analysis was conducted to assess the predictive power of each biomarker . If no single biomarker showed good predictive power, a logistics regression model was established to evaluate the predictive accuracy of multiple gene models . In gse42296, blood samples were also collected for microarray testing at week 2 after first infliximab treatment, and the data were used to assess the predictive power of biomarkers at the early phase of treatment . In gse58795, another 29 patients were randomly divided into control group and received placebo treatment, and 13 patients were categorized as moderate response at week 14 . Through comparing the biomarkers predictive values between infliximab and placebo groups, the specificity and accuracy of these biomarkers could be assessed indirectly . To overview biological aspects of the biomarkers, the online tool toppgene (https://toppgene.cchmc.org/) was used for a gene ontology analysis of the molecular function, biological processes, and cellular components in the biomarkers . Gene expression omnibus (geo) (https://www.ncbi.nlm.nih.gov/geo/) and arrayexpress (http://www.ebi.ac.uk/arrayexpress/) are international public repositories that archive and freely distribute microarray data submitted by the research community . We searched related microarray data in the databases using key words including infliximab, anti - tnf or anti - tumor necrosis factor in combination with rheumatoid arthritis or all clinical information and published articles of the microarray datasets were reviewed independently by 2 researchers . Microarray datasets were included if they satisfied the following criteria: (i) diagnosis of ra was based on the 1987 revised criteria of the american college of rheumatology (acr) for the classification of ra or on the 2010 acr / european league against rheumatism (eular) classification criteria; (ii) patients responded inadequately to mtx; (iii) nave to anti - tnf treatment or had an interval of at least 3 months after the last treatment; (iv) peripheral blood samples were collected before first administration of infliximab, and total rna was separated from pbmcs to analyze the global expression pattern of numerous well - annotated genes by microarray; and (v) response to infliximab was assessed at least 6 weeks later according to certain criteria . For those eligible datasets, the normalized data of gene expression profiling were downloaded from the databases of geo or arrayexpress . For each microarray dataset, all samples were divided into different groups according to the therapeutic outcomes . Differential genes associated with infliximab response were analyzed using the gcrma package of r language (version 3.2.3, https://www.r-project.org/). The genes recognized by half of the datasets were regarded as candidate biomarkers for predicting infliximab response . P<0.05 was considered significant, and p values from these analyses were not adjusted for multiple testing . Acr and eular response criteria are commonly used to measure individual response in clinical trials, dependent on the extent of change and the level of disease activity reached . In the included datasets, gse58795 and gse42296 were randomly or prospectively designed datasets using the acr and eular response criteria, respectively . Thus, we chose these datasets as validation sets to determine the accuracy of the biomarkers in predicting infliximab response . Roc (receiver operating characteristic) analysis was conducted to assess the predictive power of each biomarker . If no single biomarker showed good predictive power, a logistics regression model was established to evaluate the predictive accuracy of multiple gene models . In gse42296, blood samples were also collected for microarray testing at week 2 after first infliximab treatment, and the data were used to assess the predictive power of biomarkers at the early phase of treatment . In gse58795, another 29 patients were randomly divided into control group and received placebo treatment, and 13 patients were categorized as moderate response at week 14 . Through comparing the biomarkers predictive values between infliximab and placebo groups, the specificity and accuracy of these biomarkers could be assessed indirectly . To overview biological aspects of the biomarkers, the online tool toppgene (https://toppgene.cchmc.org/) was used for a gene ontology analysis of the molecular function, biological processes, and cellular components in the biomarkers . Eight microarray datasets [1219] were identified with 183 pre - treated blood samples of infliximab responders and 191 non - responders (geo accession numbers: gse3592, gse8350, gse12051, gse42296, gse58795, gse20690, gse33377, and gse78068). Eight different platforms were involved (gpl3064, gpl5460, gpl2507, gpl6244, gpl10379, gpl4133, gpl5175, and gpl6480). The dataset of gse19821 was excluded for absence of response categories . Among the eligible datasets, most ra patients were diagnosed according to the acr (1987) criteria . Before infliximab treatment, all patients suffered from moderate to severe disease activity, and responded inadequately to disease - modifying anti - rheumatic drugs (dmard) and/or mtx . Most datasets used the acr or eular response criteria, and categorized the therapeutic outcomes into response and non - response, while the criteria based on clinical disease activity index (dai) and serum crp level were used in gse78068 and gse20690, respectively . The differential genes in gse33377 were obtained from the supplementary material of published studies due to the poor annotated files in the microarray dataset . Only 5 genes were recognized by at least half of the datasets: fkbp1a (fk506 binding protein 1a), fgf12 (fibroblast growth factor 12), ano1 (anoctamin 1, calcium activated chloride channel), lrrc31 (leucine rich repeat containing 31), and akr1d1 (aldo - keto reductase family 1, member d1) (figure 1). Gene ontology analysis indicated that these 5 genes were significantly associated with the regulation of transmembrane transport (table 2). The 5 most recognized genes were regarded as candidate biomarkers, while gse58795 and gse42296 were chose as validation sets . No single gene showed good power in predicting infliximab response (auc <0.85). However, the logistics regression model of these 5 genes showed good accuracy in predicting infliximab response (auc=0.963 in gse58795 and 1.000 in gse42296) (figure 2a, 2b). At week 2 after the first infliximab infusion, the 5-gene model in peripheral blood samples also showed a good accuracy in predicting the infliximab response at week 14 (auc=1.000 in gse42296) (figure 2d). In the placebo treatment group, the 5-gene model failed to predict the response to placebo (auc=0.697) (figure 2c), indirectly indicating the specificity of the 5-gene model in predicting infliximab response . Eight microarray datasets [1219] were identified with 183 pre - treated blood samples of infliximab responders and 191 non - responders (geo accession numbers: gse3592, gse8350, gse12051, gse42296, gse58795, gse20690, gse33377, and gse78068). Eight different platforms were involved (gpl3064, gpl5460, gpl2507, gpl6244, gpl10379, gpl4133, gpl5175, and gpl6480). The dataset of gse19821 was excluded for absence of response categories . Among the eligible datasets, most ra patients were diagnosed according to the acr (1987) criteria . Before infliximab treatment, all patients suffered from moderate to severe disease activity, and responded inadequately to disease - modifying anti - rheumatic drugs (dmard) and/or mtx . Most datasets used the acr or eular response criteria, and categorized the therapeutic outcomes into response and non - response, while the criteria based on clinical disease activity index (dai) and serum crp level were used in gse78068 and gse20690, respectively . The differential genes in gse33377 were obtained from the supplementary material of published studies due to the poor annotated files in the microarray dataset . Only 5 genes were recognized by at least half of the datasets: fkbp1a (fk506 binding protein 1a), fgf12 (fibroblast growth factor 12), ano1 (anoctamin 1, calcium activated chloride channel), lrrc31 (leucine rich repeat containing 31), and akr1d1 (aldo - keto reductase family 1, member d1) (figure 1). Gene ontology analysis indicated that these 5 genes were significantly associated with the regulation of transmembrane transport (table 2). The 5 most recognized genes were regarded as candidate biomarkers, while gse58795 and gse42296 were chose as validation sets . No single gene showed good power in predicting infliximab response (auc <0.85). However, the logistics regression model of these 5 genes showed good accuracy in predicting infliximab response (auc=0.963 in gse58795 and 1.000 in gse42296) (figure 2a, 2b). At week 2 after the first infliximab infusion, the 5-gene model in peripheral blood samples also showed a good accuracy in predicting the infliximab response at week 14 (auc=1.000 in gse42296) (figure 2d). In the placebo treatment group, the 5-gene model failed to predict the response to placebo (auc=0.697) (figure 2c), indirectly indicating the specificity of the 5-gene model in predicting infliximab response . Response prediction has significant health and economic benefits, but the prediction based on clinical parameters and disease activity scores could not satisfy the need for accuracy . With the development of high - throughput gene detection technology, gene expression profiling has been used in identifying genes associated with disease diagnosis, prognosis, and treatment . Several studies tested the mrna expression profiles in peripheral blood samples from ra patients before infliximab treatment, and detected gene panels discriminating between future responders and non - responders . Although synovial biopsy samples could reflect the degree of inflammation at a specific joint and the response to infliximab more directly, it was difficult to obtain such samples, particularly from multiple sites and at different time points . The most common approach to searching for biomarkers is to identify individual genes showing statistically significant differences between responders and non - responders . After systematic analyses, we found that differential genes identified in these studies were not consistent with each other, and different gene sets were reported to distinguish between responders and non - responders . First, most studies were limited in sample size, and the response criteria varied from studies (acr, eular, serum crp, or dai). Different platforms contained different gene - oriented probes, and not all platforms were well - annotated . Sampling, rna amplification and labeling, and the hybridization conditions might also affect the results . Thus, we analyzed the differential genes associated with the subsequent infliximab efficacy according to the criteria of each study . Considering that there was no overlap of differential genes across all 8 studies, the genes recognized by at least half of the studies were chosen as candidate biomarkers . Then, 2 random - prospective - designed datasets were used to test the predictive power of candidate biomarkers . Five genes were identified for the validation (fkbp1a, fgf12, ano1, lrrc31, and akr1d1). Gene ontology analysis indicated that the 5 genes were significantly associated with the regulation of transmembrane transport . However, no single gene showed good predictive power, but the 5-gene model of logistics regression had an accurate prediction at baseline, as well as at the early phrase of treatment . Currently, most, if not all, therapeutic effect prediction studies focused on the predictive values of single genes rather than multiple genes . Our study indicated an excellent performance of joint prediction by multiple genes, which could also illuminate the low predictive values of differential genes in eligible studies . First, we believe this is the first systematic study to identify the circulating biomarkers for predicting ra infliximab response through bioinformatics analysis of the microarray data from public databases . Second, a 5-gene model was established and showed good predictive power, and it was also applicable at the early phrase of treatment . The models of fkbp1a, fgf12, ano1, lrrc31, and akr1d1 in peripheral blood is useful for efficiently predicting the response to infliximab treatment in rheumatoid arthritis patients.
The concept of this treatment started in the united states in the 1980s in randomized controlled studies in different cancers, resulting in reported significant increases in survival and disease - free period.13 its efficacy is enhanced by 20%30% when cell - based immunotherapy is combined with other conventional treatment methods . Interferon (ifn) was one of the first cytokines found to have anticancer effects, and it was introduced into the combined modality regimens used to treat small cell lung cancer (sclc) in the early 1980s in an attempt to overcome the problem of early relapse . Ifns are a group of proteins and glycoproteins produced by cells in response to viral stimulation.4 ifns have immunoregulatory effects on antibody production, natural killer (nk) and t cell activation, macrophage function, delayed - type hypersensitivity, and major histocompatibility complex molecule expression.57 they have also been shown to have antiproliferative effects and antiangiogenic properties.8 type 1 ifns (the ifn- family and ifn-) are known to inhibit tumor cell growth and stimulate the immune system.9 it has also been demonstrated that recombinant ifn- induces immunomodulation and has antiproliferative activity . Combinations of ifn- and ifn- demonstrate synergistic antiviral and antiproliferative activity.1012 in addition, synergistic or additive interactions between ifn- and cytotoxic drugs, particularly cisplatin, have been demonstrated in the experimental setting1315 and in clinical phase ii studies.16 however, only a few randomized clinical trials have been conducted to evaluate the possible advantage of adding immunomodifiers to conventional induction therapy.17 in the past, our group has reported significant antitumor activity of ifn- and improved survival in patients with sclc.18 the aim of this study was to determine whether in patients with sclc the addition of either ifn- or ifn- or their combination with standard induction chemotherapy would improve response rates and survival at acceptable toxicity . Furthermore, we evaluated the immunological responses in the peripheral blood by measuring the levels of nks and some subsets of t lymphocytes (cd4 + t cells, cd8 + t cells, and nk t cells [ctls]) and determining whether these immunoregulatory observations correlated with the progress of the disease . Chemo - nave patients with histology - confirmed sclc (limited or extensive disease), according to the veterans administration lung study group definition modified by the international association for the study of lung cancer, were eligible . Exclusion criteria included age> 72 years, karnofsky index <70, central nervous system metastasis at the time of diagnosis, creatinine clearance lower than 60 ml/ min, and serious concomitant disease . From our department, 170 patients were initially recruited, from whom 164 patients were finally evaluable for assessment of response and toxicity . Pretreatment investigations included: general medical examination; chest radiography and computed tomography of the chest, upper abdomen, and brain; tissue sampling by bronchoscopy or mediastinoscopy; and a bone scan . All patients signed an informed consent form . A randomized, nonblinded trial was conducted . Randomization was stratified for stage, and patients were divided into four groups as follows: group a, the control group, received conventional chemotherapy; group b received chemotherapy and ifn- (3 million iu) subcutaneously three times a week; group c received ifn- (3 million iu) subcutaneously three times a week; and group d received ifn- and ifn- (1.5 million iu of each) 3 times a week subcutaneously . Chemotherapy was the same for all groups and consisted of eight cycles with carboplatin 5.5 mg / m intravenously on day 1, ifosfamide 3.5 g / m intravenously (with mesna) on day 1, and etoposide 200 mg / m total dose taken orally on days 1 through 3, every 28 days, which has been shown to improve overall survival compared with standard chemotherapy in the lu21 trial.1921 after the completion of eight cycles of chemotherapy, patients were restaged by computed tomography scans of the brain, thorax, and upper abdomen . Those found to have limited disease received primary site irradiation (4550 gy in 25 fractions) and prophylactic cranial irradiation (25 gy in 12 fractions).22 immunotherapy was continued throughout these treatments and during the follow - up period as maintenance therapy . Antiemetic treatment consisted of 5-hydroxytryptamine receptor antagonists, and acetaminophen 500 mg was given to treat ifn - related influenza - like symptoms . Blood was taken before each course of chemotherapy and during follow - up to measure the total amount of t lymphocytes and their subgroups cd4 + t cells, cd8 + t cells, nkt (natural killer t) cells, and nk cells . Patients who showed disease progression at any time were taken off study medication but were followed up for survival analysis . The study was initiated in may 2004, and patient enrollment ended on february 2007 . The primary end - point of the study was survival; the secondary end points were response after induction therapy and the fluctuation of the immunologic indices during the course of the disease (figure 1). Statistical analysis was performed using the statistical package for the social sciences (spss; ibm corporation, armonk, n y, usa). Differences in the four groups for clinical characteristics, response, and toxicity were assessed by chi - square statistics . Survival was compared using kaplan meier statistics and log - rank test . A p - value of, 0.05 was considered significant . Chemo - nave patients with histology - confirmed sclc (limited or extensive disease), according to the veterans administration lung study group definition modified by the international association for the study of lung cancer, were eligible . Exclusion criteria included age> 72 years, karnofsky index <70, central nervous system metastasis at the time of diagnosis, creatinine clearance lower than 60 ml/ min, and serious concomitant disease . From our department, 170 patients were initially recruited, from whom 164 patients were finally evaluable for assessment of response and toxicity . Pretreatment investigations included: general medical examination; chest radiography and computed tomography of the chest, upper abdomen, and brain; tissue sampling by bronchoscopy or mediastinoscopy; and a bone scan . Randomization was stratified for stage, and patients were divided into four groups as follows: group a, the control group, received conventional chemotherapy; group b received chemotherapy and ifn- (3 million iu) subcutaneously three times a week; group c received ifn- (3 million iu) subcutaneously three times a week; and group d received ifn- and ifn- (1.5 million iu of each) 3 times a week subcutaneously . Chemotherapy was the same for all groups and consisted of eight cycles with carboplatin 5.5 mg / m intravenously on day 1, ifosfamide 3.5 g / m intravenously (with mesna) on day 1, and etoposide 200 mg / m total dose taken orally on days 1 through 3, every 28 days, which has been shown to improve overall survival compared with standard chemotherapy in the lu21 trial.1921 after the completion of eight cycles of chemotherapy, patients were restaged by computed tomography scans of the brain, thorax, and upper abdomen . Those found to have limited disease received primary site irradiation (4550 gy in 25 fractions) and prophylactic cranial irradiation (25 gy in 12 fractions).22 immunotherapy was continued throughout these treatments and during the follow - up period as maintenance therapy . Antiemetic treatment consisted of 5-hydroxytryptamine receptor antagonists, and acetaminophen 500 mg was given to treat ifn - related influenza - like symptoms . Blood was taken before each course of chemotherapy and during follow - up to measure the total amount of t lymphocytes and their subgroups cd4 + t cells, cd8 + t cells, nkt (natural killer t) cells, and nk cells . Patients who showed disease progression at any time were taken off study medication but were followed up for survival analysis . The study was initiated in may 2004, and patient enrollment ended on february 2007 . The primary end - point of the study was survival; the secondary end points were response after induction therapy and the fluctuation of the immunologic indices during the course of the disease (figure 1). Statistical analysis was performed using the statistical package for the social sciences (spss; ibm corporation, armonk, n y, usa). Differences in the four groups for clinical characteristics, response, and toxicity were assessed by chi - square statistics . Survival was compared using kaplan meier statistics and log - rank test . A p - value of, 0.05 was considered significant . From 170 patients initially recruited, 164 were finally eligible for assessment . From six patients who were not evaluated, four were excluded because of violation of protocol, and two patients in group c were lost because they did not attend . Median survival was 10 months for group a (95% confidence interval [ci], 9.310.6 months), 10.3 months for group b (95% ci, 7.1313.5 months), 8.3 months for group c (95% ci, 6.89.8 months), and 11 months (95% ci, 9.212.8 months) for group d. if we compare all groups together, there is no significant difference in survival, even with pairwise comparison among four groups (p . Interestingly, when patients with only limited disease were considered, a significant survival benefit in group b was revealed compared with control group a (p = 0.039), group c (p, 0.005), and group d (p = 0.038). Median survival for each group in all stages and for limited disease separately is shown in table 3 . Ten patients in the ifn- group (25%) and two in group d (ifn- and ifn-), but none in the other groups, survived for 3 years or longer . Assessment of fluctuations was based on three different time measurements: before treatment initiation; before the fourth cycle of treatment; and at the time of disease progression . The analysis of immunological measurements revealed improvement of immune markers accompanied by clinical improvement, whereas deterioration of all markers was accompanied, although not significantly, by disease progression (time series analysis p <0.05; figures 2 and 3). Significantly more patients in the immunotherapy groups than in the chemotherapy group experienced toxicity, but in groups b, c, and d, symptoms were mild and well manageable . Assessment of fluctuations was based on three different time measurements: before treatment initiation; before the fourth cycle of treatment; and at the time of disease progression . The analysis of immunological measurements revealed improvement of immune markers accompanied by clinical improvement, whereas deterioration of all markers was accompanied, although not significantly, by disease progression (time series analysis p <0.05; figures 2 and 3). Significantly more patients in the immunotherapy groups than in the chemotherapy group experienced toxicity, but in groups b, c, and d, symptoms were mild and well manageable . The aim of this study was to investigate, in a randomized trial, the efficacy of adjunctive immunotherapy in sclc therapy . Our previous pilot study17 has shown that combination therapy with ifn- is effective for sclc, particularly in those patients with limited disease . The present trial has confirmed that the addition of ifn- provides a good survival benefit in limited sclc, whereas administration of ifn- has no place as monotherapy in sclc treatment.23,24 immunomodulatory activity of ifn- and ifn- concerned in intensification of major histocompatibility complex i and ii molecule expression in cancer cells surface, thereby increasing the recognition and killing of these cells . Ifns also directly activate other immune cells, such as macrophages and nk cells, with the difference being that ifn- protects cancer cells from the lytic activity of nk cells,25 which partly explains the absence of therapeutic effect in patients in group c, who received ifn- only . Ifn- seems to have lower direct antitumor activity compared with either ifn- alone or ifn- and ifn- in combination, as shown in cancer cell lines.26 clinically, ifn- proved to be effective in the treatment of hairy cell leukemia and chronic myelogenous leukemia.27 in contrast, many other tumors, among them sclc, seem to resist cytokines, but they respond to ifns in their early stage.28 one hypothesis is that the reset of the immune system in limited disease is faster than in extensive disease; this is also an explanation for the better response of limited disease in general . Another possible explanation could be that despite chemotherapy affecting the immune system of the host negatively, ifns exert a positive influence on the immune system, and patients with limited disease have more reserve to respond to ifn stimuli . Fischer et al29 found significant reduction of interleukin 2, ifn-, ifn-, and tumor necrosis factor in patients with sclc, which improved after tumor control with chemotherapy . This immunomodulatory activity of chemotherapy seems to partly explain both the improvement of immune marker levels in patients who responded in chemotherapy and also the deterioration of them when patients experienced progressive disease in the control group . However, the lack of clinical effect is an indication of insufficient activation of cells produced or weakness of migration at the tumor site, evidenced by their appearance in large numbers in the peripheral blood . First, the total number of patients investigated was small; this is the main limitation of the study . Second, relatively low doses of cytotoxic agents were chosen to avoid a potentiation of the myelosuppressive effect by ifns.14 toxicity was characterized by fever and a higher grade of myelosuppression in the ifn groups, as could have been expected . Increased neutropenia in group d shows increased toxicity when ifns are combined rather than received alone, even if their total dose remains the same . That is why sclc is uncommon in women and why the vast majority of patients enrolled in the study are men.30 in summary, among the cytokines used in this study, only ifn- appears to confer a survival benefit to patients with sclc who have limited disease . Because of the low number of randomized patients, the clinical relevance of these findings remains to be defined . Further clinical studies on the efficacy of ifn- as an adjunctive and/or maintenance treatment are warranted, as immunotherapy remains a challenge for the treatment of lung neoplasms.
Lennox gastaut syndrome (lgs) is an uncommon but refractory epileptic encephalopathy of childhood, with a peak onset at 35 years of age.13 it is classified by the international league against epilepsy as a symptomatic generalized syndrome and requires a triad of clinical characteristics for diagnosis.4 first, patients exhibit an interictal slow spike - and wave pattern (less than 2.5 hz) on the electroencephalogram with paroxysmal fast rhythms (10 hz) during sleep . Tonic seizures (often during sleep), atonic or drop attack seizures, and atypical absence seizures are the most common types; however, patients may also exhibit myoclonic generalized tonic - clonic, partial absence, and unclassified seizures . The third characteristic is cognitive impairment, with severe mental retardation found in more than half of patients.1,46 lgs accounts for only 1%4% of all childhood epilepsies, but is viewed as one of the most difficult epilepsies to control.4 in many cases the etiology is unknown, and the presentation of multiple seizure types often leads to antiepileptic drug (aed) polytherapy.7 the multidrug and high - dose regimens required increase the risk for drug interactions, thereby affecting adjunctive aed serum concentrations and increasing the risk of medication overdose or ineffective treatment . Moreover, most of the aeds have intolerable adverse effects or can exacerbate different seizures which occur within lgs . Only a few aeds have been shown to be effective for lgs.8 even with aed polytherapy, the long - term prognosis is poor, with a persistence of epilepsy in more than 75% of patients.9 this incomplete seizure control often results in significant lifetime health care costs for the patient.10 a patient s quality of life is very poor, with drop attacks being the most troubling presentation of lgs . These are the most physically damaging seizures, often causing head injuries due to falls.11 therefore, many patients require close supervision, protective headgear, or confinement . A reduction in drop attacks is one of the most clinically significant outcomes for patients with lgs.12 the treatment goals for lgs are to provide the best seizure control possible using the fewest medications, while limiting the risk of adverse events.13,14 currently, valproic acid is considered first - line therapy, and is considered effective for multiple seizure types in lgs; however, its use is not supported by controlled trials . Furthermore, the risk of life - threatening hepatotoxicity appears to be greater in children under two years of age, and valproate consequently tends to be avoided in this subgroup if other options are available.15 topiramate, lamotrigine, and felbamate have demonstrated efficacy as adjunctive therapies in randomized, placebo- controlled trials.1618 topiramate and lamotrigine are currently recommended as second - line therapy and have comparatively more favorable tolerability.15,19 felbamate lacks sedative effects, but it also carries serious risks of aplastic anemia and hepatotoxicity which limit its use.19 clobazam, a benzodiazepine with less sedative potential than other members of its class, is also a potential adjunctive treatment.20 other treatment options include zonisamide, a ketogenic diet, vagus nerve stimulation, or corpus callostomy.15,19 due to the limited success of treatments for lgs, there is still a great demand for novel medications to manage this syndrome . An ideal medication would be effective in reducing the multiple seizure types associated with lgs, have a tolerable side effect profile, and have limited drug drug interactions with other aeds . The antiepileptic efficacy and tolerability of rufinamide was established in a clinical trial which led the united states food and drug administration (fda) to grant it orphan drug status in 2004 . Rufinamide then received fda approval in early 2009 for adjunctive treatment of seizures associated with lgs in patients four years and older, as well as for adjunctive treatment of partial seizures in adults and adolescents . The purpose of this review is to analyze the current literature describing rufinamide use for the treatment of lgs . A pubmed search was conducted by the authors for available studies limited to the terms rufinamide and lennox - gastaut syndrome . Study population were evaluated for pediatrics and review article references were compared to search results . Rufinamide [1-(2,6-difluoro - phenyl)methyl-1h-1,2, 3-triazole- 4-carboxamide], banzel (eisai co., woodcliff lake, nj) is a third - generation aed with a triazole structure that has some similarity to lamotrigine.21 it mainly acts by prolonging the inactive state of sodium channels, inhibiting the firing of sodium - dependent action potentials.22 rufinamide is a lipophilic compound manufactured as 200 mg and 400 mg coated tablets.23 a suspension is not currently available; however, a study of extemporaneously compounded suspensions of rufinamide (40 mg / ml) in a 1:1 mixture of ora - plus and ora - sweet or ora - sweet sf were found to be stable for at least 90 days.24 rufinamide is not soluble in water and dissolves poorly in gastric contents . In a study of three healthy adult volunteers, a 600 mg oral dose was extensively absorbed (85%).25 absorption is believed to be dissolution rate - limited and takes place in the small bowel.26 the maximum plasma concentration (mean) was reached within 6.6 hours after a single 400 mg dose.27 in three studies analyzing the influence of food on absorption in healthy adult subjects, taking rufinamide within one hour from mealtimes did not significantly affect plasma concentrations . However, bioavailability is significantly increased with administration after a high - fat meal versus administration after prolonged fasting . Prolonged fasting may decrease plasma levels and decrease seizure protection.26 administration with food is recommended.23 rufinamide is approximately 34% bound to plasma proteins.26 it is extensively metabolized to a carboxylic acid derivative (cgp 47292) and to glucuronide conjugates of cgp 47292 . Very small amounts of the parent drug are found in the urine and feces . About 85% of the drug rufinamide is not believed to be metabolized via the cytochrome p450 system.27,28 it displays monoexponential elimination, with a mean half - life of about nine hours in adults.27 a population analysis in 117 children (411 years) and 99 adolescents (1217 years) determined that the pharmacokinetics of rufinamide were similar to those seen in adult studies . 23 in a pharmacokinetic study of 129 adults and children with lgs, a decrease in bioavailability was observed with higher doses in the children . Clearance is also proportional to body surface area; therefore, children display higher steady - state concentrations and lower clearance compared with adolescents or adults.29 rufinamide has a low potential for drug drug interactions due to its lack of protein binding and metabolism independent of the cytochrome p450 system . This lack of significant drug interactions has been demonstrated clinically through a pharmacokinetic analysis using data from five double - blind studies in adults and children.30 rufinamide was coadministered with carbamazepine (n = 903), valproic acid (n = 588), lamotrigine (n = 200), phenytoin (n = 299), phenobarbital (n = 149), and topiramate (n = 69). Rufinamide did not affect the clearance of topiramate or valproate, but increased the clearance of carbamazepine and lamotrigine, and decreased clearance of phenobarbital and phenytoin . However, these interactions were not considered clinically significant.30 another study demonstrated that rufinamide concentrations increased with concomitant valproate by 40% and 11% in children and adults, respectively . Therefore, dose reduction may be required with the initiation or withdrawal of concomitant valproate.29 rufinamide also resulted in a small increase in the clearance of the oral contraceptive ortho - novum (ethinyl estradiol and norethindrone); however, the clinical significance of this interaction has not been elucidated.31 in a randomized, double - blind, placebo - controlled trial of rufinamide in 138 children and adults with lgs, the majority of reported adverse effects were similar between placebo and rufinamide at a dose of 45 mg / kg . There were significant differences in somnolence (24.3% with rufinamide versus 12.5% with placebo) and vomiting (21.6% with rufinamide versus 6.3% with placebo).14 cognitive or psychiatric adverse events occurred in a lower percentage of patients taking rufinamide (17.6%) versus placebo (23.4%).14 polytherapy increased the risk of adverse effects . During the extension phase of this trial, a total of 113 (91.1%) patients experienced an adverse effect . The most commonly reported adverse effects were vomiting (30.6%), pyrexia (25.8%), upper respiratory tract infection (21.8%), and somnolence (21%). A total of 82 patients discontinued the medication prematurely, 9.7% due to adverse effects.32 the relationship between serum drug concentrations and adverse effects has also been analyzed in 1398 patients from both controlled and clinical studies . The most common adverse effects reported in controlled, double- blind pediatric studies were dizziness (13%), fatigue (17%), nausea (9%), vomiting (7%), diplopia (6%), and somnolence (7%).26,33 adverse effects were reported in a slightly higher percentage of adults (versus children), those with increased body weight, and in females . Increasing rufinamide plasma concentrations was also associated with an increase in adverse effects.33 a pooled analysis of seven clinical studies examined the incidence of adverse events specifically in the pediatric population (212 rufinamide - treated pediatric patients aged 316 years, 197 placebo - treated patients aged 417 years, and 391 patients in both double - blind or open - label extensions).34 overall, for all studies, 391 patients received an average dose of 41.67 mg / kg / day for 1224 months . The most commonly reported adverse events were vomiting (26.3%), headache (22.5%), and pyrexia (18.7%).34 the most common serious adverse effects that occurred in more than one patient were aggravated seizures (2.8%), status epilepticus (2%), pneumonia (2%), and vomiting (1.5%). Five possible cases of aed hypersensitivity syndrome was discovered retrospectively in children younger than 12 years and within the first four weeks of treatment . In the double - blind trials alone, the median dose of rufinamide was 41.96 mg / kg / day for an average duration of three months . The most common adverse events reported (rufinamide versus placebo) were somnolence (17% versus 8.1%), vomiting (16.5% versus 7.1%), and headache (16% versus 8.1%). Clinically relevant decreases in weight (7%) were seen only in the rufinamide group (11/188, 5.9%) versus an increased incidence of weight gain observed in the placebo group (15/178, 8.4%). The rates of psychiatric adverse events that occurred between rufinamide and placebo were comparable, at 10.4% versus 14.2%, respectively . Only one patient exhibited qt prolongation and another exhibited electrocardiographic changes (exact change not specified). In 18 healthy volunteers treated for 18 days with rufinamide, dose - dependent qt interval decreases were reported.35 rufinamide is therefore contraindicated in patients with a short qt interval due to risk of short qt syndrome (syncope, ventricular arrhythmia, possible sudden death).35 obtaining an electrocardiogram prior to initiating rufinamide would be prudent . Rufinamide is a lipophilic compound manufactured as 200 mg and 400 mg coated tablets.23 a suspension is not currently available; however, a study of extemporaneously compounded suspensions of rufinamide (40 mg / ml) in a 1:1 mixture of ora - plus and ora - sweet or ora - sweet sf were found to be stable for at least 90 days.24 rufinamide is not soluble in water and dissolves poorly in gastric contents . In a study of three healthy adult volunteers, a 600 mg oral dose was extensively absorbed (85%).25 absorption is believed to be dissolution rate - limited and takes place in the small bowel.26 the maximum plasma concentration (mean) was reached within 6.6 hours after a single 400 mg dose.27 in three studies analyzing the influence of food on absorption in healthy adult subjects, taking rufinamide within one hour from mealtimes did not significantly affect plasma concentrations . However, bioavailability is significantly increased with administration after a high - fat meal versus administration after prolonged fasting . Prolonged fasting may decrease plasma levels and decrease seizure protection.26 administration with food is recommended.23 rufinamide is approximately 34% bound to plasma proteins.26 it is extensively metabolized to a carboxylic acid derivative (cgp 47292) and to glucuronide conjugates of cgp 47292 . Very small amounts of the parent drug are found in the urine and feces . About 85% of the drug rufinamide is not believed to be metabolized via the cytochrome p450 system.27,28 it displays monoexponential elimination, with a mean half - life of about nine hours in adults.27 a population analysis in 117 children (411 years) and 99 adolescents (1217 years) determined that the pharmacokinetics of rufinamide were similar to those seen in adult studies . 23 in a pharmacokinetic study of 129 adults and children with lgs, a decrease in bioavailability was observed with higher doses in the children . Clearance is also proportional to body surface area; therefore, children display higher steady - state concentrations and lower clearance compared with adolescents or adults.29 rufinamide has a low potential for drug drug interactions due to its lack of protein binding and metabolism independent of the cytochrome p450 system . This lack of significant drug interactions has been demonstrated clinically through a pharmacokinetic analysis using data from five double - blind studies in adults and children.30 rufinamide was coadministered with carbamazepine (n = 903), valproic acid (n = 588), lamotrigine (n = 200), phenytoin (n = 299), phenobarbital (n = 149), and topiramate (n = 69). Rufinamide did not affect the clearance of topiramate or valproate, but increased the clearance of carbamazepine and lamotrigine, and decreased clearance of phenobarbital and phenytoin . However, these interactions were not considered clinically significant.30 another study demonstrated that rufinamide concentrations increased with concomitant valproate by 40% and 11% in children and adults, respectively . Therefore, dose reduction may be required with the initiation or withdrawal of concomitant valproate.29 rufinamide also resulted in a small increase in the clearance of the oral contraceptive ortho - novum (ethinyl estradiol and norethindrone); however, the clinical significance of this interaction has not been elucidated.31 in a randomized, double - blind, placebo - controlled trial of rufinamide in 138 children and adults with lgs, the majority of reported adverse effects were similar between placebo and rufinamide at a dose of 45 mg / kg . There were significant differences in somnolence (24.3% with rufinamide versus 12.5% with placebo) and vomiting (21.6% with rufinamide versus 6.3% with placebo).14 cognitive or psychiatric adverse events occurred in a lower percentage of patients taking rufinamide (17.6%) versus placebo (23.4%).14 polytherapy increased the risk of adverse effects . During the extension phase of this trial, a total of 113 (91.1%) patients experienced an adverse effect . The most commonly reported adverse effects were vomiting (30.6%), pyrexia (25.8%), upper respiratory tract infection (21.8%), and somnolence (21%). A total of 82 patients discontinued the medication prematurely, 9.7% due to adverse effects.32 the relationship between serum drug concentrations and adverse effects has also been analyzed in 1398 patients from both controlled and clinical studies . The most common adverse effects reported in controlled, double- blind pediatric studies were dizziness (13%), fatigue (17%), nausea (9%), vomiting (7%), diplopia (6%), and somnolence (7%).26,33 adverse effects were reported in a slightly higher percentage of adults (versus children), those with increased body weight, and in females . Increasing rufinamide plasma concentrations was also associated with an increase in adverse effects.33 a pooled analysis of seven clinical studies examined the incidence of adverse events specifically in the pediatric population (212 rufinamide - treated pediatric patients aged 316 years, 197 placebo - treated patients aged 417 years, and 391 patients in both double - blind or open - label extensions).34 overall, for all studies, 391 patients received an average dose of 41.67 mg / kg / day for 1224 months . The most commonly reported adverse events were vomiting (26.3%), headache (22.5%), and pyrexia (18.7%).34 the most common serious adverse effects that occurred in more than one patient were aggravated seizures (2.8%), status epilepticus (2%), pneumonia (2%), and vomiting (1.5%). Five possible cases of aed hypersensitivity syndrome was discovered retrospectively in children younger than 12 years and within the first four weeks of treatment . In the double - blind trials alone, the median dose of rufinamide was 41.96 mg / kg / day for an average duration of three months . The most common adverse events reported (rufinamide versus placebo) were somnolence (17% versus 8.1%), vomiting (16.5% versus 7.1%), and headache (16% versus 8.1%). Clinically relevant decreases in weight (7%) were seen only in the rufinamide group (11/188, 5.9%) versus an increased incidence of weight gain observed in the placebo group (15/178, 8.4%). The rates of psychiatric adverse events that occurred between rufinamide and placebo were comparable, at 10.4% versus 14.2%, respectively . Only one patient exhibited qt prolongation and another exhibited electrocardiographic changes (exact change not specified). In 18 healthy volunteers treated for 18 days with rufinamide, dose - dependent qt interval decreases were reported.35 rufinamide is therefore contraindicated in patients with a short qt interval due to risk of short qt syndrome (syncope, ventricular arrhythmia, possible sudden death).35 obtaining an electrocardiogram prior to initiating rufinamide would be prudent . Several trials have evaluated the effectiveness of rufinamide as an adjunctive treatment in lgs (see table 1). The efficacy of rufinamide as an adjunctive therapy for lgs was evaluated in a multicenter, randomized, placebo - controlled, parallel - group study in 138 patients aged 437 (mean 14) years of age.14 this study included a 12-week, double - blind phase followed by an open - label extension phase . Patients were randomized to placebo or twice - daily oral rufinamide titrated over 7 to 14 days to a daily target dose of 45 mg / kg in addition to their maintenance regimen . The two groups, placebo (n = 64) and rufinamide (n = 74), were equally distributed demographically except for a slightly higher age and weight in the rufinamide group . Rufinamide serum concentrations ranged from 4.95 to 48.15 g / ml . The median reduction from baseline in the frequency of drop attacks per 28 days was significantly greater for rufinamide versus placebo (42.5% versus + 1.4%, p <0.0001). The median reduction from baseline in frequency of all seizures was also significantly greater for study medication versus placebo (32.7% versus 11.7%, p = 0.0015). These results were associated with a greater proportion of responders (patients achieving 50% reduction in seizures per 28-day period) for rufinamide versus placebo . The responder rate for total seizures was 31.1% versus 10.9% and for drop attacks was 42.5% versus 16.7% for rufinamide versus placebo, respectively . A significant difference related to the parent / guardian global evaluation of the patient s condition at the end of the study was not demonstrated either . There were no significant differences compared with placebo in the incidence of adverse events, apart from somnolence and vomiting, which was higher in the rufinamide group . Following completion of this study, 123 patients (including 74 males) entered the open - label, treatment - extension phase with a median dose of 1800 mg / day for a median duration of 432 days.32 sixty percent of patients were treated for more than 18 months, 40% for more than two years, and 12% for three years or longer . Approximately half of the patients decreased their total daily dose of concomitant aeds during 30 months of adjunctive therapy with rufinamide . The reduction in median total seizure frequency was maintained, with some improvement noted in patients who continued treatment for up to three years . The responder rate for all seizures was also maintained, with 36.9% of patients having a 50% decrease in seizure activity during the open - label phase . In addition, 21.3% of patients achieved a 75% reduction in overall total seizures, with 29.1% of patients achieving a 75% reduction in tonic the long - term results of rufinamide in an open - label extension trial (trial presented above) were also published by the same research group.36 kluger et al observed patients from 10 to 1149 days, with a median of 432 days . After study termination had occurred (44 months), 33.9% of patients were still receiving rufinamide, whereas 66.1% had discontinued due to various reasons, including unsatisfactory therapeutic response (n = 51), adverse events (n = 12), or other unspecified reasons (n = 19). Notably, patients who had been receiving placebo (n = 59) during the double - blind treatment phase went from a 1.5% decrease in total seizure frequency to a 22% median reduction in total seizure frequency after two weeks on rufinamide . During the last 12 months of rufinamide treatment, response rates (50%) for total seizures and for tonic eight patients (6.8%) achieved seizure freedom within the last 12 months of rufinamide treatment . More serious adverse events were reported during the extension study (13.7%) compared with the double - blind study (2.7%). Serious adverse events reported and suspected to be treatment - related included rash, constipation, esophagitis, decreased weight, gastritis, anorexia, vomiting, lethargy, and status epilepticus . This trial demonstrated that rufinamide was able to maintain seizure control over the long term.36 a 12-week, retrospective, observational study conducted in europe by kluger et al included 45 children and 15 adults (mean age 14.5 years) with various refractory epilepsy syndromes . Researchers determined response rates by comparing frequency of seizures during the first four - week period of the trial before drug initiation versus seizure frequency with rufinamide during the last four weeks of observation . Seventeen patients with lgs responded to rufinamide (54.8%), with eight patients exhibiting a 50%75% seizure reduction, five a 75%99% reduction, and four achieving complete freedom from seizures during the last four weeks of observation.37 a three - year cost - efficacy analysis of rufinamide versus lamotrigine and topiramate for children with lgs was conducted in the united kingdom based on the current published literature.38 this study looked at the relationship between costs and quality of life years . Quality of life was based on response rates to each medication, as well as tolerability . However, the authors concluded that the extra expense for rufinamide was warranted if a patient s quality of life was improved, because lgs can be such a devastating condition . Currently, there are no direct comparisons of trial data for rufinamide versus other adjunctive therapies for lgs . In addition, a cochrane review of lgs treatment was unable to compare rufinamide effectively as an adjunctive therapy to other aeds due to a lack of direct comparison trials, as well as differences in trial designs and population.39 this review did suggest, however, that one specific medication has not been shown to be more effective than another and, thus, rufinamide, lamotrigine, topiramate, and felbamate should all be considered as an adjunctive treatment for lgs . Rufinamide may be a more efficacious option compared with current alternatives due to the fact that the patients included in the relevant clinical studies may have had more refractory forms of lgs and were already taking multiple aeds . The available literature to date examining rufinamide, a third - generation aed, for the treatment of lgs suggests that it is both a safe and effective adjunctive treatment option for patients who are refractory to therapy with multiple aeds . The fda - approved pediatric dosage is up to 45 mg / kg or 3200 mg / day divided into two doses, which is consistent with effective dosages from the trials . Tablets may be crushed for pediatric administration and the dose should be administered with a meal to increase absorption . 23 cost may be a challenge due to the availability of only brand - name dosage forms on the market . However, a pharmacoeconomic analysis38 suggests that increased quality of life with rufinamide outweighs its increased cost compared with alternatives . The most common adverse effects for rufinamide include somnolence, vomiting, dizziness, and fatigue, but these are relatively mild and typically do not warrant drug discontinuation . 33 rufinamide is a worthwhile adjunctive treatment for lgs due to its relative lack of clinically significant drug interactions and its acceptable adverse effect profile compared with older anticonvulsants . Additional studies are needed to further assess its short- and long - term efficacy and safety, as well as drug interactions . In addition, a direct comparison between rufinamide and alternative adjunctive treatments is warranted to assess its place in therapy . Rufinamide is a promising adjunctive agent for the treatment of pediatric lgs and may significantly decrease seizure frequency in patients who are refractory to other therapies.
A detailed description of patients with history of recurrent respiratory infections and progressive renal failure was given 1979 . The case under discussion has various rare features like young age of the patient, associated glomerulopathy causing nephrotic syndrome and normal renal function, which is showing gradual deterioration . Normal renal function at the time of diagnosis is known and in one large series published by bhandari et al . An 8-year - old male child presented with a history of facial puffiness and edema of one year duration . He was diagnosed to have nephrotic syndrome and was treated by a local physician with steroids . He did not improve, however, and was referred to this hospital for further management . There was no history of drug ingestion including exposure to mycotoxins or other herbal medicines . Patient had stunted growth with moon facies, secondary to steroids and had clinical features of rickets . His investigations revealed hemoglobin of 10 g / dl, blood urea 33 mg / dl and serum creatinine 0.7 mg / dl . He was human immunodeficiency virus non - reactive and hepatitis b surface antigen and hepatitis c virus and 24 h urine protein was 3565 mg / dl . Complete urine examination showed 2 + albumin, 4 - 6 red blood cells and granular casts . He was started on cyclophosphamide . However, there was no response even after 6 months . Biopsy showed 23 glomeruli, four of whom showed segmental sclerosis [figure 1]. Segmental glomerulosclerosis (thin arrow) and karyomegaly in tubular cells (thick arrow) (h and e, 400) karyomegaly in tubular epithelial cells (h and e, 400) (a) segmental sclerosis and tubular atrophy (periodic acid schiff, 200). (b) tubular atrophy and interstitial inflammation (h and e, 200). (c) interstitial fibrosis (masson's trichrome, 200) immunohistochemical stains for bk virus and cytomegalovirus were negative . He is under regular follow - up and his last creatinine has increased to 1.2 mg / dl . Kin is a rare disorder characterized by enlarged tubular epithelial cell nuclei and chronic interstitial nephritis . Histologically, presence of interstitial nephritis along with karyomegaly in the tubular epithelial cells is characteristic of this disorder . Karyomegalic cells have been identified in various tissues like astrocytes, schwann cells, intestinal smooth muscle and bile duct epithelium . Mcculloch et al ., reported kin in three adolescent patients treated with ifosfamide for ewings sarcoma . A familial clustering is known and frequency of human leukocyte antigen (hla)-a9 and hla - b35 haplotypes suggest the possibility of genetic susceptibility . Another genetic defect on chromosome 6, linked to major histocompatibility complex locus is also suspected . The morphological alterations in renal epithelial cells are thought to be the initial damage caused by either chemicals or viral agents, which in susceptible individuals lead to disruption of cells . Immunofluorescence and histological findings are negative in a large series presented by bhandari et al . In contrast our case clinically presented with nephrotic syndrome and also had histological evidence of focal segmental glomerulosclerosis (fsgs). As there is no positive family history or drug history and exposure to toxins, it is important to diagnose this entity as it is a progressive disorder leading to irreversible renal damage . Common etiological factors for both these lesions are toxins and drugs, patient on repeated questioning denied any history of drug or herbal medicine intake . There is no history of renal disease in the family and it unlikely to be familial because the chromosomal abnormalities described are different for both . Spoendlin et al ., reported four patients who were asymptomatic initially, but later experienced progressive renal failure . This particular case under discussion also initially presented with normal renal function and on follow - up was showing gradual deterioration . We present a rare case of kin who initially presented with normal renal parameters; interestingly, this patient's clinical presentation was of nephrotic syndrome whose biopsy also revealed fsgs . Hence, we think that the patient has idiopathic fsgs with an incidental kin, which is known to occur sporadically.
In a recent study we provided experimental evidence that sorla indeed is the molecular link between app and the retromer sorting complex . The cargo binding activity of the retromer is historically assigned to the trimer of vps26-vps29-vps35, whereas the snx subunit is responsible for membrane association (reviewed recently in refs). Numerous studies have previously identified vps35 as the receptor binding component . However, in our study we have demonstrated that vps26 binds directly to a cargo receptor, i.e., the cytoplasmic tail of sorla . This is in line with vps26 containing an arrestin fold, which is a conformation found to be responsible for cargo - binding activity in other proteins . Also, we found that the receptor interaction with vps26 was dependent on a phenylalanine residue located in a six amino acid fanshy - sequence in the tail of sorla . This agrees well with retromer subunits previously shown to prefer binding to aromatic sorting motifs . To study the functional relevance of the interaction between retromer and sorla, we generated a sorla mutant carrying six alanine residues instead of the fanshy motif (i.e., sorla fanshy6a). Using this sorla mutant we observed a mis - targeting to the endosomal compartments of the mutant receptor in line with retromer s role in retrograde transport . Subsequently, we focused on the localization of the sorla - fanshy6a by performing co - localization studies between non - mutated sorla (sorla - wt) and sorla - fanshy6awithin the same cell . These results confirmed that the mutated and the non - mutated sorla receptors localize differently, with sorla - wt found in the perinulear region corresponding to the tgn compartment and sorla - fanshy6a found in a more distal vesicular compartment representing late / recycling endosomes and/or tubular endosomal network (ten). This clearly shows that a lack of retromer binding alters receptor localization by impairing the retrograde trafficking from peripheral compartments (i.e., the ten) back to the golgi . Based on these observations, we speculated that this mislocalization might influence the function of sorla keeping app from processing . This was relevant based on previous findings where we showed that sorla exert part of its function by slowing app exit from the golgi into the secretory pathway whereby app avoids cleavage by the secretases . In order to analyze the exact role of sorla in retromer - dependent app sorting, we have now determined whether retromer is able to retrieve app from the endosomal compartments independent of its interaction with sorla . Accordingly, by triple immunohistochemical staining of sh - sy5y we found a strong co - localization between app and retromer (i.e., vps35) in the presence of sorla - wt (fig . 1). In contrast, there was very little co - localization between app and vps35 when cells express the sorla - fanshy6a mutant that is unable to associate with retromer (fig . 1). This set of data clearly demonstrates that sorla activity is needed to bridge retromer and app . Sorla - wt expression, but not sorla - fanshy6a, mediates co - localization between retromer and app . Sh - sy5y cells transfected with either sorla - wt (left panel) or sorla - fanshy6a (right panel) were stained with an antibody against the extracellular domain of sorla (in red), or the endogenously expressed proteins app (in green) and vps35 (in blue). Co - localization of the trimeric complex between app, vps35 and sorla - wt is indicated by white arrow heads . Having identified sorla as a protein binding both app and retromer, we next asked whether sorla also is involved in the mechanism where retromer is reported to decrease amyloid production . It was therefore of interest to determine how the sorla mutant, not retrieving back to the perinuclear region, behaved in terms of app trafficking and subsequent processing . First, to study app transport we used live cell imaging confirming previous data, which showed that sorla - wt significantly decreased the velocity and distance traveled by app . However, upon mutation of the fanshy motif this effect is completely abolished although the receptor mutant is still able to associate with app in the ten . Second, we also found that the app: sorla - fanshy6a complex formed in the peripheral ten did not lead to any decrease of a production compared with sorla - wt binding app early in the secretory pathway . These findings lead to a model where retromer and sorla co - operate in the retention of app in the perinuclear region, where sorla is the direct binding partner of app and retromer functions in the retrieval of sorla from the endosomal compartment back to early compartments of the secretory pathway (fig . 2). After mutation of the fanshy sequence, sorla is no longer able to interact with retromer . By disrupting this interaction, sorla app will then localize into other cellular compartments where app is more prone to secretase cleavage . Since endosomal processing leads to production of a, increasing endosomal delivery of app increases the amyloidogenic processing . This change in localization of app is important because sorla can only protect against app processing when located in the tgn . This is supported by the fact that sorla - fanshy6a cannot protect against app cleavage . When the retromer binding site is deleted sorla is not recycled back to tgn, but rather stays in the late endosomal compartment, where sorla has no impact on app . This model explains how genomic deficiency or misfunction of either sorla or retromer can influence app processing . These results not only shed new light on the mechanism of how retromer deficiency may lead to ad, but also provided new insight of how app sorting receptors are key players in a metabolism . Based on our studies, we believe that the retromer complex is indeed the adaptor protein necessary for the retrieval of sorla from the endosomes to the tgn, and the localization of sorla in the tgn together with app is of crucial importance for regulating the processing of app . Once disrupting the retromer binding site, the cellular distribution of sorla is changed, and sorla is no longer able to protect against the production of a.
It is predicted that by 2025, the prevalence of hypertension (htn) will increase by 60% to a total of 1.56 billion worldwide suggesting that htn remains a major public health problem . Htn in sub - saharan africa (ssa) has also been on the rise with reports indicating higher values in urban settings compared to rural settings [24]. The prevalence of htn in ssa ranges between 14.5% in rural eritrea, 32.9% in semiurban ghana, and 40.1% in urban south africa . Likewise, adequate blood pressure (bp) control has been on the decline, ranging between 1.7% in rural ghana, 4% in urban slum dwellers in nigeria, and 21.5% in urban kenya . In cameroon, the prevalence of htn spans from 5.7% in rural settings through 21.9% in semiurban to 47.5% in urban milieu, with a national average survey of 31.0% . Despite the relatively better bp control in urban over rural settings, levels of adequate bp control as low as 2% to 27.5% (in men) and 38.7% (in women) even though much has been done so far to reduce the incidence of hypertension in urban areas, this has not been the same in rural areas, with public health policies aimed at controlling hypertension mainly directed towards the large cities . Most of the studies done so far in these urban areas cannot therefore give a true estimate of the extent to which the rural population is affected by this major cardiovascular disease risk factor . An analysis of hypertension and its risk factors in rural cameroon will therefore help guide health policy decisions and provide baseline data for future studies aimed at addressing this problem . We therefore had the following as objectives: determining the prevalence of hypertension in the adult population in the moliwe health area in rural cameroon; identifying the risk factors associated with htn in this rural setting; determining the extent of bp control in known hypertensive patients in this locality . A community - based cross - sectional descriptive and analytical study was conducted over a 6-month period (june 20 to december 28, 2013) in the moliwe health area (mha). This principally rural health area is found in the limbe health district of the south west region of cameroon . A multistage sampling method was used to select participants from the 5 villages / settlements of the health area: bonadikombo, wotutu, ewongo, moliwe, and tomatal . The sample selected was self - weighted through the probability proportional to size method used . The data of 2005 cameroon census was used as a guide to the sampling frame with households considered as the primary sampling units . Using a random start, a systematic sample of households was then selected from a list of the approximate cumulative number of households of all the villages . This gave an estimate of the number of households to be selected from each village . As the secondary sampling units, two participants were then recruited from each selected household by simple random sampling from a list of eligible households occupants produced by the respective household heads . Three hundred and twenty - nine participants were thus recruited from bonadikombo, 113 from wotutu, 112 from moliwe, 96 from ewongo, and 83 from tomatal . A combined estimate of the prevalence of htn in rural rwanda, tanzania, and malawi of 22% was used . We recruited 733 participants by systematic and simple random sampling methods . At each selected household, a questionnaire was administered to each recruited participant to collect information on age, sex, level of education, marital status, status of htn, treatment of htn, smoking, quantity of alcohol intake, physical activity, diabetes status, and family history of htn . Blood pressure measurements were also done at the home of the participants by a casually dressed clinician not in white coat . Two measurements (in mmhg) were taken on the right arm using an automated electronic bp machine (omron m3 hem-7200-e omron matsusaka co. ltd ., kyoto, japan) through the standard procedure (jnc 7 recommendations). Heights in meters (m) and weights in kilograms (kg) were measured using standard procedures and were used to determine the body mass index (bmi) as follows: bmi = weight (kg)/[height (m) height (m)]. Who steps surveillance manual the data was entered into epi info statistical software, version 7.0 (cdc / who, atlanta, usa). Frequencies and means were obtained for appropriate variables; chi - square and fischer's exact tests were used as appropriate to test for associations between binary and categorical variables . A logistic regression model was built to assess the effect of factors found to be significantly associated with hypertension while controlling for potential confounders . An ethical clearance was obtained from the institutional review board of the faculty of health sciences, university of buea . Administrative approvals were obtained from the regional delegate of public health as well as from the local chiefs, quarter heads, and camp presidents concerned . All patients found to have elevated blood pressure values were advised and referred to the nearest health facility for proper management and follow - up . Of the 733 participants, 479 (39.5%) were between 20 and 29 years . The male to female ratio was 1: 1.2, 35.9% had primary education, and 50.5% were married . Less than half of the participants (47.3%) had normal bmi and 15% had sedentary life style . A small proportion, (2.9%) were known diabetics and 32.7% reported a family history of hypertension (table 1). Of the 733 participants recruited, 228 were hypertensive, giving a prevalence of 31.1% (95% ci: 27.834.6). Sixty - six of the 228 hypertensive participants (28.9%, 95% ci: 23.235.3) knew their status prior to our study . Of these 66 known hypertensive participants already aware of their status, 14 of them (21.2%, 95% ci: 12.133.3) had their blood pressures well controlled even though as high as 47 of them (71%, 95% ci: 58.781.7) were on antihypertensive treatment at that moment (figure 1). Age 40 and above, obesity, smoking, alcohol consumption, diabetes, low educational level, and marriage were found to be associated with hypertension (table 2). After adjusting for all significant factors using logistic regression, only age 40 and above, obesity, low educational level, and marriage there was a progressive increase in the prevalence of htn with age when age below 40 was used as the reference: from 3 folds in the 4059 years, through 8.3 folds in the 6079 years, to 11.6 folds in 80 years age groups . People who were obese had 2.8 times the odds of being hypertensive compared to the nonobese . Those who never went to school had 6.7 times the odds of having htn compared to those who had at least high school education (table 3). Since the new recommendations for htn diagnosis and management by jnc 7 in 2003, no study had been done in rural cameroon . The last published rural study on htn was as far back as 1998 . The 31.1% prevalence of htn observed in the mha is similar to the 31.0% reported in cameroon by kingue et al . And the 32.9% reported by amoah in semiurban ghana but higher than that reported elsewhere [3, 7, 8, 12, 16, 17]. However, our value was lower than 44.7% reported by williams et al . In rural ghana and 40% reported by chow et al . In rural and urban high, middle, and low income countries . Our study revealed low hypertension awareness similar to the 32.3% observed in rural ghana by addo et al . And the 32.6% observed by dzudie et al . In urban cameroon . However, this value is higher than the 11% reported in rural cameroon by mbanya et al . This variation between rural and urban settings could be explained by poor knowledge of the disease, lesser access to health facilities, and poverty, in rural compared to urban communities . Age was identified to be strongly associated with htn, supporting the findings of other studies [9, 12, 2123]. This is because as people get older, their blood vessels become harder and they are likely to have decreased baroreceptor sensitivity, increased responsiveness to sympathetic nervous system stimuli, altered renal and sodium metabolism, and an altered renin - aldosterone relationship thereby predisposing them to high blood pressure . Obesity, which is one of the common pathways between diabetes and htn, was also found to be strongly associated with htn in our study, as earlier documented [8, 26]. Low educational status and being married were strongly linked with htn in our study as reported in previous studies [21, 27, 28]. A poor understanding of the disease and its risk factors and the stress experienced my married couples, respectively, could put these groups at a higher risk . On the other hand, gender, family history of htn, overweight, and physical inactivity smoking, alcohol consumption, and diabetes mellitus which were associated with htn on bivariate analysis were not significantly associated with hypertension on multivariate analysis by logistic regression . The proportion of known hypertensives who were currently on antihypertensive treatment in our study was similar to the 64.9% reported in ghana but much higher than the 12.5% reported in cameroon . The high proportion on antihypertensives may be due to the increase in awareness of htn and the availability and access to antihypertensive medications in cameroon lately compared to 16 years ago . Despite this considerable proportion of hypertensive patients being on treatment, reported as low as 3% in rural nigeria and yuvaraj et al . Reported 12.5% in rural india however, higher proportions have been reported (33.1% and 63.2%) among hypertensives in urban and semiurban settings in cameroon, respectively, 56.8% in mexico, and 75.9% in urban india . In this context noncompliance to treatment is multifactorial in aetiology with reasons such as inadequate knowledge of htn, intolerance of side effects of medications, and low purchasing power . This study was cross - sectional which means participants declared hypertensive may not necessarily be hypertensive clinically . Also, our study was done in a small rural area whose findings may not necessarily apply to all rural cameroonian or sub - saharan african communities . Moreover, some known risk factors such as dyslipidaemia, salt intake, and psychosocial and socioeconomic status were not assessed in our study . Nevertheless, the large sample size, varied sampling method, and execution of standard measurement procedures make the findings of this study robust and accurate . Also, the white coat effect which could potentially produce an overestimation of the actual prevalence of hypertension in this setting was minimized since the casually dressed clinician without a white coat did the measurements from the participants' homes . Our study revealed that about one out of three adults in the moliwe health area could be hypertensive with only a quarter of them being aware of their status and a fifth of those on treatment having their blood pressure well controlled . This reveals how very much a cardiovascular risk the rural population may be exposed to . There is, therefore, need for massive improvement in awareness through education and repeated patient follow - up in these rural settings alongside the urban ones . Also, the importance of further research in other rural communities to assess trends and risk factors of hypertension and the extent of blood pressure control cannot be overemphasized.
This article describes the origins and rationale for the mcandrews leadership lecture and explains why the american chiropractic association honors george and jerome mcandrews . George mcandrews, a lawyer, administered a permanent injunction forbidding the american medical association s restraint of trade toward the chiropractic profession . The american chiropractic association has established the mcandrews leadership lecture to honor their contributions to the chiropractic profession . The american chiropractic association (aca) is a national professional organization established in 1962 that represents doctors of chiropractic in the united states . Its mission is to preserve, protect, improve, and promote the chiropractic profession and the services they provide for the benefit of the patients they serve . The aca vision includes the following:collaboration with other health care disciplines and integration into all health care delivery models that enhance individual health, public health, wellness, and safety.change in public policy, legislative, and regulatory arenas, resulting in a more effective us health care system.improved health care access and freedom of choice of health care providers for the american people, without discriminatory obstacles.increased value of health care for patients, policymakers, and the public through the high professional and educational standards of the chiropractic profession.healthier and more productive lives for the american people . Collaboration with other health care disciplines and integration into all health care delivery models that enhance individual health, public health, wellness, and safety . Change in public policy, legislative, and regulatory arenas, resulting in a more effective us health care system . Improved health care access and freedom of choice of health care providers for the american people, without discriminatory obstacles . Increased value of health care for patients, policymakers, and the public through the high professional and educational standards of the chiropractic profession . The aca convenes annually for its house of delegates (hod) meeting, typically in conjunction with its national chiropractic leadership conference . The annual hod meeting encompasses the governance and business of the aca and development of policy . The national chiropractic leadership conference is held annually for lobbying the us congress in matters of chiropractic legislation and is attended by doctors of chiropractic from various states and students from the chiropractic colleges and universities in the united states . The mcandrews leadership lecture was created by the aca to honor the mcandrews family, especially the substantial contributions by jerry mcandrews, dc, and george mcandrews, jd, to the chiropractic profession over their lifetimes (fig 1). In february 2015, the aca hod ratified a policy on the establishment of the annual mcandrews leadership lecture whereby it agreed to host an annual keynote address to its hod on health care policy . The speaker who is invited to deliver the mcandrews lecture is a living individual who has demonstrated exceptional leadership capabilities throughout his or her career to advance the chiropractic profession on a national and/or international level . The invitee will have demonstrated a consistent history of selfless work and high ethical standards . The invitee will have contributed to advancing the chiropractic profession in areas of clinical expertise, research, policy, and/or politics on a broad level . The invitee embodies the values of the aca, which include collaborating with others, promoting a more effective us healthcare system, supporting high professional and educational standards for the chiropractic profession, and contributing to healthier and more productive lives for the american people . The lecture is designed to provide inspiration, to stimulate thought, and to develop future leaders . Jerome f. mcandrews, dc, served as aca s vice president for professional affairs for 4 years, retiring in november 1995 while continuing to advance the profession as an official aca media spokesperson and an expert advisor on managed health care and related issues . In 2006, he was posthumously awarded aca s highest honor, chiropractor of the year, in light of more than 50 years of service to the chiropractic profession . His service beyond aca included a variety of leadership positions such as president of palmer college of chiropractic, president of the association of chiropractic colleges, vice president of the council on chiropractic education, ncmic board member, and executive vice president and director of the international chiropractors association . At the ceremony held at aca s hod meeting in scottsdale, az, his son patrick mcandrews, dc, accepted the award on his behalf . Dr jerry mcandrews enthusiastic presence in many and varied leadership roles entitles him to this honor . George p. mcandrews, jd, came from a family of chiropractic physicians, including his father, sister, brother (jerome), and daughter . George mcandrews may be best known throughout the chiropractic profession for his role as lead counsel in the wilk v american medical association (ama) et al case . This case was brought against the ama and other parties for their attempt to contain and eliminate the chiropractic profession . It is important to note that george mcandrews took the case against the ama when no other attorneys were willing to go against the powerful entity of the ama . Of great and ongoing importance to the chiropractic profession, according to george mcandrews, is that when ama was found guilty in wilk v ama of restraint of trade, a permanent injunction order was issued to the ama . The ama was permanently enjoined from restricting the freedom of any ama member or hospital from associating with chiropractors or chiropractic institutions . The wilk court order was required to be published in jama and a copy sent to each member of ama . It was common for the ama hod resolutions to suppress various groups, in addition to chiropractic physicians, which could possibly compete for patients . Evidence of the ongoing importance of the wilk v ama case is mentioned in lectures of significance to the chiropractic profession . The association for the history of chiropractic at its 20th annual conference on chiropractic history held in san antonio, tx, march 17 - 18, 2000, presented its lee - homewood award to george mcandrews . That award is given each year to an individual who has made a lifetime contribution to the chiropractic profession . George mcandrews has taken on many other lawsuits of importance to the chiropractic profession and has been a tireless advocate on chiropractic s behalf while running his patent law firm in chicago . The decision by aca to honor george with this pronouncement is based on his courage and strong will in the face of such a formidable foe as the ama . The aca is proud of the history of our profession in the united states and recognizes the legacy of the mcandrews brothers who helped to fight the battles necessary for our inclusion as a viable conservative health care delivery system . We hope that the annual mcandrews leadership lecture will inspire others to lead our profession to new heights.
Obesity is the most common metabolic disorder and results from the combined effects of excess energy intake and reduced energy expenditure . Obesity is associated with a variety of clinical disorders, including hypertension, insulin resistance, glucose intolerance, and dyslipidemia . It is well known that an oversupply of fat is associated with the development of obesity in mice . Long - term feeding with a high - fat diet can induce obesity with hyperlipidemia, insulin resistance, hyperphagia, and hypergluconemia [4, 5]. Nonalcoholic fatty liver disease (nafld) is present in up to one - third of the general population and affects all ages and ethnic groups . Nafld is the second leading cause of death in the general population [6, 7]. At present, there is no pharmacological agent known to reverse nafld and effective medical interventions have focused on the modification of risk factors, such as weight reduction and diet . Mistletoe is a hemiparasitic plant growing all over the world on various deciduous trees, such as oak . Korean mistletoe (km, viscum album cololatum) extract (kme) has been shown to have anticancer, antioxidant, antidiabetes and antidementia effects, and it enhances immune system function . Cellular power plants because they generate most of the cellular supply of adenosine triphosphate (atp) which is used as a source of chemical energy . Studies have implicated mitochondria in several human diseases, including metabolic diseases, cardiac dysfunction, mental disorders, and the aging process . Decreased mitochondrial respiration rates and reduced expression of genes involved in mitochondrial oxidative capacity have been reported in diet - induced obese rats . Because kme improves mitochondrial function, we asked whether it has beneficial effects on obesity . It is well known that adipocyte differentiation and the extent of subsequent fat accumulation are closely related to the occurrence and advancement of various diseases, such as coronary artery disease and obesity [1, 22, 23]. 3t3-l1 cells have served as a useful in vitro model for adipocyte differentiation and function . The differentiation of preadipocytes into adipocytes requires a variety of effectors that activate a cascade of transcription factors, such as ccaat / enhancer - binding protein- (c / ebp-), peroxisome proliferator - activated receptor (ppar-), and sterol regulatory element binding element protein-1c (srebp-1c). This cascade begins with the ccaat enhancer - binding protein- (c / ebp-) and, which induces the expression of c / ebp- and ppar- [2527]. These transcription factors coordinate the expression of genes involved in creating and maintaining the adipocyte phenotype, including genes for adipocyte fatty acid - binding protein, glucose transporter 4, lipoprotein lipase (lpl) and leptin [28, 29]. Activated srebp-1c accelerates adipogenesis through the overexpression of adipogenic enzymes, such as fatty acid synthase(fas), acyl - coa synthase (acc), and acyl - coa carboxylase (acs). Lpl is the major enzyme that hydrolyzes triglyceride (tg) molecules of chylomicrons and vldl particles . The released free fatty acids are either oxidized to generate atp in muscle or stored in adipose tissue . We measured the inhibitory effect of kme on adipogenic factors in 3t3-l1 cells and on the development of obesity and nafld in mice fed a high - fat diet . Our data show that kme suppresses adipocyte differentiation through down - regulation of adipogenic factors and could ameliorate obesity and nafld in mice fed a high - fat diet . Our results indicate the great potential of kme as a potential metabolic regulator of adipocyte differentiation and a potential therapeutic agent for preventing or treating obesity and nafld . Mistletoe growing on oak was harvested from gangwon - do, republic of korea, in february . The mistletoe was 1 or 2 years old, and the leaves, stems, and fruits were cut into 2 joints from the end of a branch, washed with distilled water (dw), and dried . . Then mistletoe leaves, fruits, and stems were freeze - dried, crushed and ground in approximately 10 volumes of dw for 30 seconds . After being washed, they were ground in a mixer for 2 minutes and stirred for 16 hours at 4c . To obtain fine mistletoe extract, the homogenized mistletoe was centrifuged at 8,000 rpm for 30 minutes at 4c, and the resulting supernatant was successively filtered through different pore sizes (0.9 and 0.45 m). The mistletoe extract was freeze - dried and resuspended in dw at an appropriate dilution factor . Lean, male c57bl/6 mice (7 weeks old) were purchased from central lab animal inc . All mice were housed for 1 week under a 12/12-hour light / dark cycle in a temperature(22 1c) and humidity(55 5%) controlled room and fed standard laboratory chow and water ad libitum . Kme was mixed with either powdered chow (m07, feedlab, republic of korea) or high - fat (d12327, research diets, inc ., new brunswick, nj, usa) feed at a concentration of 4 g / kg of food to provide a 3000 mg / kg / day (mpk) dose . These dietary amounts represent the maximum amount of chow that these animals were able to consume during a 24-hour period . Seven c57bl/6 mice on the above ad libitum diets had their daily average caloric intake and weekly body weight measurement during the course of the study . At the conclusion of the in vivo experiment, the mice were sacrificed and the epididymal pads were collected and weighed . The adaptive thermogenic response was measured in a cold test, during which the animals were individually housed at 4c for 6 hours . Liver tissues were isolated immediately after sacrifice . For hematoxylin and eosin (he) staining, tissues were fixed in 10% formalin and processed and embedded in paraffin prior to sectioning (10 m .) And staining . Liver biopsies for electron microscopy were cut into 1 mm pieces, fixed immediately after collection in karnovsky fixative (glutaraldehyde in cacodylate buffer), and kept at 4c . The second step involved postfixation with 1% osmium tetraoxide in 0.1 m cacodylate buffer for 1 hour at 4c . Tissues were then dehydrated through successive baths of graded alcohol, followed by a propylene oxide bath and a treatment with propylene oxide and resin mix before being embedded in a pure epoxy resin (araldite, epon 812) that solidified after 48 h at 60c . Semithin sections were cut at 2 m, stained with toluidine blue, and then analyzed by light microscopy . Ultrathin sections were cut at 70 nm and examined with an electron microscope (bx 50, olympus, japan). Electron micrographs (400x magnification) were digitized and analyzed using acd see, version 4.0 . The liver of 1 mouse from each group (control, high - fat diet (hfd), hfd + kme 3000 mg / kg) was measured . In brief, the following criteria were used for scoring hepatic steatosis: grade 0, grade 1, hepatocytes occupying <33% of the hepatic parenchyma; grade 2, fatty hepatocytes occupying 3366% of the hepatic parenchyma; and grade 3, fatty hepatocytes occupying> 66% of the hepatic parenchyma . Mice were subjected to an endurance test using a variable - speed belt treadmill enclosed in a plexiglass chamber with a stimulus device consisting of a shock grid attached to the rear of the belt (columbus instruments oxymax system, columbus, oh, usa). The shock grid was set to deliver 0.2 ma, which caused an uncomfortable shock but did not physically harm or injure the animals . Mice were run at 16.2 meter / minutes for 10 minutes with a 5 incline . For high - fat- (hf-) fed animals, the speed was gradually increased from 10.8 to 24.6 meter / minutes and then maintained until exhaustion . Exhaustion was determined to have been reached if the mice were unable to run for 10 seconds, at which point the electric shock was discontinued . 3t3-l1 preadipocytes purchased from atcc (american type culture collection, manassas, va, usa) were cultured in dulbecco's - modified eagle's medium (dmem, hyclone, logan, ut, usa) containing 10% bovine calf serum (bcs, hyclone, logan, utah, usa) at 37c in a humidified atmosphere of 5% co2 . After 3 or 4 days, the cells had reached 90% confluence and were collected with 0.05% trypsin/0.53 mm edta treatment . After centrifugation (1300 rpm, 5 minutes), the cells were plated in 6-well plates at a concentration of 3 10 cell / well . One day after confluence (designated day 0), cell differentiation was induced with a mixture of methylisobutylxanthine (0.5 mm), dexamethasone (0.25 mm), and insulin (5 mg / ml) in dmem containing 10% fetal bovine serum (fbs). On day 2 and thereafter, dmem supplemented with 10% fbs and 5 mg / ml insulin was replaced every 2 days . 3t3-l1 adipocytes, 7 to 8 days after differentiation, were treated with kme 6 g/l or phosphate - buffered saline (pbs) for 24 hours . Total rna was isolated from 3t3-l1 adipocytes using trizol reagent (invitrogen, carlsbad, ca, usa). Cells were homogenized by 4 - 5 passages through a 23-gauge needle . After a 5-minute reaction, samples were mixed with chloroform by vortex machine for 15 seconds . Total rna was precipitated by mixing with isopropyl alcohol and centrifuging at 12,000 rpm . The resulting rna pellets were washed with 75% ethanol and dissolved in rnase - free water . Total extracted rna (1 g) mixed with annealing oligo dt primer and rnase - free water was incubated in a thermocycler for denaturing rna . Reactant was mixed with 5x first strand buffer, 20 mm dtt, 10 mm dntp mix, rnase - free water, and superscript ii reverse transcriptase . The mixtures were incubated in a thermocycler (42c for 1 hour and 72c for 7 minutes) to generate cdna . Real - time rt - pcr analysis was performed with an ab 7500 real - time pcr system (applied biosystems, foster city, ca, usa). Samples containing 2x sybr green pcr master mix (applied biosystems, foster city, ca, usa), 0.5 mol each of appropriate primers, and cdna were incubated in the ab 7500 real - time pcr system for an initial denaturation at 94c for 10 minutes, followed by 40 pcr cycles . Each cycle -actin was used as an internal housekeeping control . To confirm amplification of specific transcripts, melting curve profiles were produced at the end of each pcr cycle by cooling the sample to 65c for 15 seconds and heating slowly to 95c, with continuous fluorescence measurement . Data (means se) were analyzed using graphpad prism (version 5.04, graphpad software, usa). Unpaired two - tailed student's t - tests were used to evaluate differences between means, as indicated . During the 15-week experiment, body weight and food intake were measured weekly . As shown in figure 1, the kme - treated group had a slight decrease in body weight compared to the control group . However, this effect was significantly pronounced in animals that were fed an hf diet with kme . The difference between the kme - treated and control animals became significant at week 9 . At the end of the experiment, the body weight of mice fed kme was 20% lower (p <0.05) than that of the control group, whereas hf diet plus kme mice weighed almost the same as the mice that ate normal chow (figure 1(a)). These effects of kme on body weight were not due to decreased food intake, as the amount of kcal consumed per mouse over a 24-hour period remained unchanged (figure 1(b)). The data indicate that kme might have antiobesity effects in vivo, without affecting food intake . To test whether body weight loss was caused by decreased adiposity this analysis revealed that epididymal white adipose tissue was significantly reduced in the hf diet plus kme group (figure 1(c)). We performed a cold test to assess the effect of kme on adaptive thermogenesis capacity . Kme - treated animals maintained higher body temperatures than nontreated animals (figure 2(a)), suggesting that it improved this capacity . To test whether thermogenesis is accompanied by changes in the expression of genes involved in thermogenesis, we examined mrna levels of ucp1, the major contributor to heat production, by real - time rt - pcr . Figure 2(b) shows that the mrna level of ucp1 was remarkably increased in kme - treated mice . Because kme reduced body weight (figure 1(a)) and increased thermogenesis (figure 3), we evaluated the effect of kme administration on a treadmill endurance test . The mice were trained to run at 16.2 meter / minutes for 10 minutes at a 5 incline the day before the running test, according to the procedure used for our previous report . The experiment was initiated at 10 meter / minutes at a 0 incline with a gradual increase in speed . The mice were run until exhaustion, which was defined as remaining on the shock grid for longer than 10 consecutive seconds . Surprisingly, kme - treated mice ran twice as far as high - fat diet mice (figure 3). One of the most common characteristics among people with obesity is the development of fatty liver [33, 34]. Histologic evaluation is regarded as the gold standard for assessing the presence and severity of nafld . We histologically evaluated liver sections to determine the extent to which kme attenuated hepatic steatosis development . As shown in figure 4, the control group exhibited little histologic evidence of hepatic steatosis . However, a marked reduction in the degree of steatosis was seen in livers from high - fat diet mice treated with kme . Furthermore, hepatic steatosis scores were dramatically lower in the high - fat diet mice fed kme (grade 1). To demonstrate the inhibitory mechanism of kme on adipocyte differentiation, we measured the expression levels of the transcription factors ppar-, c / ebp-, and srebp-1c in 3t3-l1 cells treated with 6 g/l of kme . Our results show that ppar-, c / ebp-, and srebp-1c mrna levels were decreased by 64%, 60%, and 32%, respectively, (figure 5(a)). We also measured the mrna expression levels of adipogenic enzymes, such as fas, acs, and acc . We found that they were reduced by 69%, 55%, and 22%, respectively (figure 5(b)). Lpl expression is not directly associated with lipid synthesis but is strongly associated with adipogenesis . Therefore, we measured the expression level of lpl mrna and found that the level of expression in kme - treated 3t3-l1 cells was decreased by 89% compared to that of untreated cells (figure 5(a)). Our results showed that body weight gain in groups fed a diet supplemented with kme was reduced compared to control hfd mice . This study also provides evidence that dietary supplementation of kme protects against hepatic steatosis development . We have considered the possibility that the effect of kme may be mediated by food intake because decreased food intake would be expected to significantly affect body weight, which influences hepatic steatosis . In this study, however, food intake did not differ between the groups . This suggests that kme directly protected against obesity and hepatic steatosis independent of food intake . Brown adipose tissues (bats) use stored triacylglycerols (tg) to maintain body temperature . These cells produce energy from fatty acid metabolism to generate heat through the action of uncoupling protein 1 (ucp1), a mitochondrial protein found only in brown adipose tissue . Brown adipocytes contain less tg and more mitochondria than white adipocytes, resulting in their unique color . Kme treatment increased ucp1 expression levels in bat, which poised the mitochondria for respiration uncoupling . This effect is consistent with the observed enhancement of cold tolerance and helps explain the increase in energy expenditure and resistance to weight gain . This is most evident in muscle, a metabolically flexible tissue that switches between lipid and carbohydrate substrates to fulfill energy requirements . One study found that mitochondrial oxphos activity within the oxidative soleus muscle, which is resistant to fatigue and is dependent on mitochondrial activity for atp production, was more affected than in the glycolytic muscle tibialis anterior . The main changes included a reduction in respiratory chain activity with a concomitant decrease in mitochondrial atp production . In line with this finding and previously, kme significantly improved muscle oxidative capacity, resulting in enhancing the endurance . To further consolidate our hypothesis that kme inhibits obesity, we evaluated the effects of kme treatment in vitro using an adipocyte differentiation development system, which is regulated by transcriptional activators such as ppar-, c / ebp-, and some transcriptional regulators at the molecular level [39, 40]. Ppar- is a member of the nuclear receptor superfamily of transcription factors and is dominantly expressed in adipose tissue . Srebp1 is known to critically cross - activate a ligand binding domain of ppar- and activate the production of an endogenous ppar- ligand . Adipogenesis can be induced through the action of some enzymes, such as fas, acc, and acyl - coa synthetase (acs). The expressions of these genes are regulated by transcription factors, including ppar-, c / ebp-, and srebp1, which are known to be crucial activators for adipogenesis and showed early changes in gene expression during adipocyte differentiation [44, 45]. In this study, we showed that kme treatment significantly decreased srebp-1c, c / ebp-, and ppar- mrna expression in cultured 3t3-l1 adipocytes and inhibited expression of adipocyte - specific proteins (fas, acc, acs, and lpl). Collectively, these observations suggest that kme suppresses adipocyte differentiation by regulating the expression levels of adipogenic transcription factors . Further investigation is necessary to define specific mechanisms by which kme protects against obesity - mediated hepatic steatosis . To elucidate the mechanism associated with the antiobesity effects of kme kme had marked inhibitory effects on the development of obesity and nafld in mice fed high - fat diets . Activating endurance capacity and increasing thermogenesis are two possible mechanisms for the antiobesity effect of kme . This finding suggests that kme may be a potential dietary strategy for preventing obesity and nafld.
We report a male child diagnosed with bilateral choroidal osteoma, high myopia and secondary choroidal neovascularization (cnv) membrane in one eye . Co - existence of posterior staphyloma made the clinical diagnosis of choroidal osteoma difficult due to the osteoma filling the depression of the posterior staphyloma . On review of the literature we were unable to find a similar case of co - existant choroidal osteoma and posterior staphyloma reported earlier . This case highlights the need for careful examination when presented with a case of choroidal osteoma filling the depression of a posterior staphyloma thereby making it appear flat rather than a typical elevated lesion . We report a case of bilateral choroidal osteoma - macular in the right eye and peripapillary with macular involvement in the left eye along with high myopia and posterior staphyloma in a male child associated with a cnv membrane in the left eye treated with the off - label use of intravitreal bevacizumab (avastin; genentech, san francisco, ca, usa). A 10-year - old male patient presented with the complaints of decreased vision in the left eye for the last 4 days . On examination, the best corrected visual acuity (bcva) in the right eye was 20/40, n6 and in the left eye 20/100, n9 . Anterior segment examination was normal and the fundus examination of both eyes showed myopic discs with a posterior staphyloma and an extensive orangish - yellow flat lesion in the macular area of the right eye and in the peripapillary and macular area with submacular hemorrhage in the left eye [figure 1a and b]. (a) color fundus photo of the right eye showing an orangish - yellow lesion in the macular area suggestive of a choroidal osteoma (b) color fundus photo of the left eye showing the choroidal osteoma with submacular hemorrhage and choroidal neovascularization ultrasound b - scan of both eyes showed a high reflective echo with shadowing at the posterior pole of both eyes . Patient underwent fundus fluorescein angiography (ffa) for both eyes which showed early hypofluorescence with late hyperfluorescence in the macular area of both eyes along with the peripapillary area in the left eye suggestive of the choroidal osteoma . There was an area of early hyperfluorescence which increased in size and intensity in the macular area of the left eye suggestive of an active cnv [figure 2a]. The indocyanine green angiography showed macular choroidal hypofluorescence in both eyes with an ill - defined hyperfluorescence plaque seen in the delayed frames of the left eye suggestive of a cnv [figure 2b]. Spectral domain optical coherence tomography of the left eye showed a steep curvature of the retinal layers due to posterior staphyloma along with a subfoveal cnv [figure 3]. (a) fundus fluorescein angiography of the left eye showing an area of blocked fluorescence due to submacular hemorrhage and an area of hyperfluorescence within it suggestive of choroidal neovascularization (b) indocyanine green angiography of the left eye showing macular choroidal hypofluorescence with an ill - defined hyperfluorescent plaque seen in the delayed frames of the left eye suggestive of a choroidal neovascularization optical coherence tomography of the left eye showing increased curvature of the retinal layers due to posterior staphyloma along with subfoveal choroidal neovascularization he was diagnosed to have bilateral choroidal osteoma with an active subfoveal cnv in the left eye . He underwent a loading dose of three intravitreal injections of 1.25 mg/0.05 ml bevacizumab (off - label use) delivered to the inferotemporal quadrant of the left eye under strict aseptic precautions and short general anesthesia after an informed consent was signed by the parents . At 1 year follow - up after the last injection showed a bcva of 20/60, n6 in the left eye . Fundus examination of the left eye showed choroidal osteoma in the macula with no evidence of submacular hemorrhage . Ffa of the left eye showed staining of the cnv with no evidence of dye leakage . Choroidal osteoma is a relatively rare benign intraocular tumor composed of mature bone that typically replaces the full thickness of the choroid . This tumor classically manifests as an orange - yellow plaque deep to the retina in the juxtapapillary or macular region.1 nearly 80% are unilateral though gass has described unilateral osteomas becoming bilateral.2 these tumors are said to show signs of evolution - growth of tumor, decalcification and cnv formation causing profound visual loss . Our patient has an osteoma involving the peripapillary and macular area, which is rare . We missed the diagnosis of the osteoma initially as the lesion appeared flat though the tumor is usually a little elevated . This was due to the tumor filling the depth of the co - existent posterior staphyloma . Based on our experience we recommend other clinicians to remain suspicious for a similar presentation when examining a myopic fundus with a posterior staphyloma . There was a large area of submacular hemorrhage overlying the osteoma, which may predispose to the formation of cnv along with an irregular surface of the tumor.3 the possible reasons for the development of the cnv in these cases includes thin degenerated retinal pigment epithelium (rpe) overlying the osteoma allowing the growth of new blood vessels or an extension of the osteoma itself due to the presence of osteoclasts on histopathology.4 the various treatments for cnv secondary to a choroidal osteoma include laser photocoagulation, photodynamic therapy (pdt), transpupillary thermotherapy (ttt) and surgical removal of the cnv . Laser photocoagulation especially for an extrafoveal cnv with osteoma, was found to be poorly responsive to laser photocoagulation due to depigmentation of the rpe and degenerated rpe / bruch's membrane complex, resulting in the reduced uptake of laser energy.5 pdt requires multiple treatments and there is a risk of a drop in vision after the treatment.6 visual results have been poor with ttt and surgical removal of the cnv associated with osteoma.47] in our case, the osteoma induced cnv was treated with a loading dose of three intravitreal bevacizumab injections (off label use) resulting in rapid regression of the cnv with a marked improvement in visual acuity . Bevacizumab may have better penetration in these cases due to a very thinned and degenerated rpe / bruch's membrane complex.8 there are reports of cnv secondary to choroidal osteoma in adult patients treated with intravitreal bevacizumab.89 however to the best of our knowledge this is the first report of a choroidal osteoma induced cnv treated with bevacizumab in a young child.
Point - of - care testing (poct) is an increasingly popular means of delivering laboratory tests close to the patient . Poct allows for rapid diagnostics and turnaround of test results to provide for faster medical decision - making and improved patient outcomes (1). However, if inappropriate samples are collected, specimen is mislabeled, analysis is performed incorrectly, or test is misinterpreted, wrong results may be reported and acted on by the clinician . Studies have indicated that for central laboratory testing, most errors occur in the preanalytical phase, prior to the sample arriving in the lab (2). For poct, the majority of errors occur in the analytic phase of testing (3). In fact, errors can occur in any phase of laboratory testing whether performed in a central laboratory or at the point - of - care . As laboratory directors, we should know our processes and take steps to detect and prevent errors before those mistakes reach the clinician and affect patient care . Risk management is a way to reduce errors with poct . Risk management is defined as the systematic application of management policies, procedures, and practices to the tasks of analyzing, evaluating, controlling, and monitoring risk (4). Risk is generally assumed from the patient s perspective, but risk can also apply to the operator of the poct device, the laboratory administration and even the hospital and its reputation . Risk is the chance of suffering harm or loss, and risk can be estimated through a combination of the probability of occurrence of harm and the severity of that harm(5). Errors that occur more frequently have greater risk, and errors that lead to greater harm also present greater risk . Staff troubleshoots control failures and followsup on complaints from clinicians . When errors are detected, the harm to patients is estimated and actions are taken to prevent recurrence in the future . So, risk management is simply a formal term for many of the activities that laboratories are already doing . Quality control is a means of detecting and preventing errors . Besides frequency and severity, detectability is a third factor in the risk estimation equation . As products are constructed on a factory line, the quality of the product is inspected to ensure that it meets manufacturer specifications . If problems are noted, the line can be stopped and production corrected to ensure the quality of the final product . These industrial risk management principles have application for reducing errors in laboratory testing as well . In the central laboratory, laboratories analyze liquid quality control, a stable sample with predefined acceptability ranges, in order to detect reagent problems before they affect the test result . Traditionally, liquid controls are analyzed at two concentration levels each day of testing or more frequently as required by the stability of the test system . These are errors that affect the patient sample in the same manner as the quality control sample . Reagent degradation, calibration errors, dilution and pipetting errors are examples of systematic errors that quality control can effectively detect and prevent before the errors affect a patient result . Quality control, however, does a poor job at detecting random errors which uniquely affect individual samples . Bubbles, clots, drugs, hemolysis and other sample specific errors are not detected by liquid quality control . Other mechanisms, like bubble and clot detection, or analyzer hemolysis indices must be utilized to detect random errors . So, analyzing two levels of liquid quality control each day of testing does not entirely eliminate risk, and laboratories have still produced bad results despite analyzing liquid quality control . Analysis of liquid quality control consumes the entire test in the process, and there is no guarantee that the next test will perform identically . Alternative control processes must be used for these devices in addition to liquid controls in order to optimize the quality of these tests . Many poct kits have built - in biologic and chemical controls to ensure the performance of individual tests . Fecal guaiac occult blood cards have a positive and negative control area on each card to ensure the reactivity of the card and developer . Urine pregnancy tests have a control line on each test to verify test storage and viability of the antibodies on the test . Drug, rapid strep, hiv and other poct unitized tests have similar control lines or regions that guarantee the quality of the test kit and result with each test . These control lines are control processes that act as an alternative to traditional liquid controls in order to detect the risk of specific errors when using those tests . Some tests, like bilirubinometers, cannot even accept a liquid sample, so how does a laboratory effectively control the quality of these tests? It is neither economical nor possible to analyze two levels of liquid controls for every reaction on this test each day . The effective way to ensure quality would consider risk of those errors that are most likely to occur or cause greatest severity of harm from an incorrect result . The amount and quality of specimen, the reactivity of the replicating enzyme, and the thermocycling of the device are key failure points, and those are the steps that should be monitored by the quality processes . Although the practice of analyzing two levels of liquid quality control have given laboratories some degree of assurance that results are valid, newer devices have built - in electronic controls, and on - board chemical and biological controls, no single quality control procedure can cover all devices, since devices may differ in design, technology, function, and intended use (6). Quality control information from the manufacturer increases the user s understanding of device overall quality assurance requirements, so that informed decisions can be made regarding suitable control procedures . Manufacturers understand their devices and the limitations of those devices, while laboratories know how the device will be utilized and test results applied for patient care . A quality control plan identifies the weaknesses in the testing process and defines the roles of the manufacturer built - in control processes and laboratory actions required to maintain risk to an acceptable level . The clinical and laboratory standards institute (clsi) document ep23-a introduces the industrial risk management principles to the clinical laboratory (7). Ep23 describes good laboratory practice for developing a quality control plan based on manufacturer s information, applicable regulatory and accreditation requirements, and the individual healthcare and laboratory setting . This guideline recommends collecting information about a test system and processing that information through a risk assessment to develop a quality control plan . Weaknesses in the testing process are identified and for each hazard identified, the laboratory defines a control process which will detect and prevent that error, controlling risk to a clinically acceptable level . Some hazards, like use of expired reagent, may be effectively controlled through a manufacturer built - in process such as barcoding which prevents the operator from utilizing expired reagents . Other hazards may require the laboratory to take an action, like instrument maintenance or operator training / competency . A quality control plan is essentially a summary of all the hazards considered and laboratory actions required to minimize risk . If errors continue to occur, the laboratory is encouraged to troubleshoot, reassess their risk and modify the quality control plan as required . A unit - use blood gas device may be used as an example of the risk management process . The first step let s consider a generic poct blood gas and electrolyte analyzer intended for use in a same - day surgical center . The need for testing is low, only 1 2 tests per day . At a cost of $10 20 per test, the requirement to perform two levels of liquid control each day of testing will increase the cost of testing significantly and add to the turnaround time of results since control results will need to be evaluated before patient testing can be conducted . The use of alternative control processes provided by the manufacturer will improve cost, test and labor efficiency . Review of the package insert allows the laboratory to determine intended use, test system operation and test limitations . The system is a portable clinical analyzer for the in vitro quantification of various analytes in whole blood . The test system consists of the portable clinical analyzer, test cartridges sealed in a foil pouch for protection during storage, quality assurance materials (liquid control and calibration verification solutions), and a data management system with a server class computer, data management software, wireless connectivity, and laboratory and hospital information system interfaces . The unit - use cartridge contains all the components to perform testing including; a calibrant solution, reagents, sample handling system, and sensors . The analyzer automatically controls all steps of the testing process such as fluid movement, calibration, fluid mixing, and thermal control . The cartridges are standardized to plasma core - laboratory methods using multi - point calibration curves stored in the device memory that are stable over many lots . Upon insertion, signals produced by the sensor responses to the calibrant solution are measured, and a one - point calibration adjusts the sensor offset to the stored multi - point calibration curve . The analyzer then moves the sample over the sensors and the signal of the sensor responses to the sample are measured from the adjusted calibration curve . Examination of the manufacturer, internal control processes allows an understanding of how the process functions and what errors can be detected and prevented with that process . The blood gas and electrolyte analyzer contains simulated internal control processes that check the edge connector, internal electronics and analyte circuitry . An isolated region of the internal circuit board sends a range of simulated sensor signals through the cartridge measurement channels . Next, conductivity out of the connector pins is measured, insuring no contamination is present on the edge connector which would interfere with the test . The internal simulated control is performed automatically every 8 hours or if there has been a significant change in analyzer temperature, from cold to hot, since this can cause condensation on the connectors . The internal control can also be performed manually whenever the performance of the device is in question . Internal simulated controls are never intended to entirely replace liquid quality control, and the manufacturer recommends analyzing liquid controls with each shipment of cartridges, new lots of cartridges, whenever cartridges experience a temperature shift> 8c, or as required by the laboratory . Temperature is monitored continuously during each test, but a temperature verification cartridge is recommended at least annually . The information about the test system and the function of the internal control processes can now be processed through a risk assessment . Risk assessment is best started by mapping the testing process to look for weaknesses and steps that could lead to error . Follow the sample from order to specimen collection, analysis, and reporting of results . Areas of focus should include the sample, the reagents, the operator, the analyzer, and the environment . Examine those hazards of greatest risk first including errors that occur frequently or lead to greater severity . For compliance with federal and state regulations, testing should only be conducted based on a physician order . With poct so, operators must be trained to only conduct a blood gas or electrolyte test with this system based on an existing physician order . With appropriate training and demonstration of ongoing operator competency, the laboratory can conclude that risk of this error is reduced to a clinically acceptable level . Inappropriate collection or use of the wrong specimen additive can affect blood gas and ionized calcium results . Failure to adequately mix or over - mixing the sample can further lead to clots or hemolysis of the sample . Whole blood samples continue to metabolize after collection, so prompt analysis, no more than 15 30 minutes after collection, is important . These are additional elements that should be added to the operator training and competency program to reduce risk of these errors . Operator technique can impact poct results, so the effect of operator technique is critical to assessing risk with poct . Operator lock - out features on poct devices require a personal identification number to unlock the device and perform patient testing . This feature ensures that only those trained and competent operators are conducting testing . Adding too much or too little sample can affect test results by flooding the cartridge or contaminating the connector pins, and insufficient sample failing to adequately contact the sensors in the cartridge . This analyzer has volume detection and will not allow overfilling or start a test until an adequate amount of sample has been added . The analyzer also automates all steps of the testing process, preventing incorrect timing, misinterpretation, or other procedural steps common for poct . The analyzer also detects the expiration date of the cartridge through barcoding, preventing use of expired reagents . Documentation of results into the patient s medical record presents an additional step for operators, so there is a risk of manual test results not being documented . The test system wireless connectivity and data management system ensure documentation of results without need for operator intervention or requiring additional operator actions . Poct devices can transmit nosocomial infections between patients, so cleaning and disinfection between patients is important . Training and reminders for staff on proper cleaning will effectively reduce risk of this error . Exposure of cartridges to temperatures outside of manufacturer specifications during shipping and lot - to - lot variation can affect test results . Analysis of liquid quality control upon receipt of new shipments and lots of cartridges can prove the viability of the cartridge prior to use for patient samples . However, cartridges can also degrade during storage, so temperature monitoring of storage conditions is required to ensure continued viability through the life of the cartridges . Temperature monitoring of liquid control sample storage is also important to ensure control viability . Periodic analysis of liquid quality control will further ensure cartridge and control stability . At what frequency should control samples the manufacturer recommends testing liquid control samples upon receipt of each shipment, with new lots of cartridges, and periodically to verify cartridge stability during storage . To determine the frequency of liquid control testing during storage, laboratories can perform side - by - side testing of daily liquid controls with internal control processes to document shelf stability for a period of several weeks . Once stability is documented for several weeks, the laboratory will have data to decrease the frequency of liquid control to every few days, and eventually weekly or monthly, depending on the life - span of cartridges after receipt . The analyzer does not require water, works on battery power and internally detects the electrical circuitry and sensor connector pins . So, these risks are not a consideration with this device . Once the testing process has been mapped, hazards recognized and control processes identified, the third step of the risk management process is summarizing the quality control plan . The quality control plan summarizes all of the hazards recognized during the risk assessment and the error mitigations selected, both those internal control processes from the manufacturer and the actions from the laboratory . If risk is not reduced to an acceptable level, then the laboratory must take additional mitigation steps to control the risk . The final step of the risk management process is implementing the quality control plan and monitoring the effectiveness of the plan . Benchmarks for this blood gas and electrolyte analyzer could include trends in quality control, internal controls as well as liquid quality controls, analyzer error codes, physician complaints, or any other unexpected trends . When errors do arise, the laboratory should troubleshoot to determine the source, correct the process and reassess risk in light of the new information, modifying the quality control plan as required . The us centers for medicare and medicaid services (cms) recently implemented new clinical and laboratory improvement amendments interpretive guidelines in january 2014 (8). Risk management principles have been incorporated into the new interpretive guidelines in the form of individualized quality control plans (iqcp). Cms will begin inspecting for laboratory iqcps beginning in 2016 . At that time, laboratories will have two quality control options: 1) perform two levels of liquid quality control each day of testing or 2) develop an iqcp in order to reduce the frequency of liquid quality control . The laboratory cannot reduce frequency below manufacturer recommendations, and the laboratory must perform liquid quality control at some frequency (i.e., performing no liquid quality control is not an option . ). Although iqcp will initially only apply to clia moderate complexity devices, any laboratory will benefit from mapping their processes and assessing weaknesses in their tests . Since the chemistry of the test reaction is in the unit - use test cartridge, facilities with dozens of the same device can select a subset of devices and rotate the analysis of liquid controls, since all devices share the same lot and supply of unit - use cartridges . For laboratory - developed tests, most importantly, by developing an iqcp the laboratory will embrace industrial risk management principles and learn how to better detect and control risks with their test systems.
Macroautophagy (hereafter autophagy) is a conserved pathway for the degradation of cytoplasmic material and the recycling of nutrients . During autophagy, a double - membrane organelle called autophagosome is formed in a de novo manner . Autophagosomes originate from a crescent - shaped structure termed the isolation membrane or phagophore (fig ., membranes are likely nucleated at a specific site in the perivacuolar region referred to as phagophore assembly site (pas), . When they close to give rise to an autophagosome, this cytoplasmic material is trapped in its lumen and is ultimately degraded upon fusion of the autophagosome with the lysosomal compartment (or the vacuole in yeast). The formation of autophagosomes requires a set of conserved factors that are recruited to the site of autophagosome formation in a hierarchical manner . These factors can be grouped into functional units including the atg1/ulk1 kinase complex, the class iii phosphatidylinositol 3-phosphate kinase complex 1 (pi3kc1) containing the atg14/atg14 subunit, the atg9/atg9a cycling system, the wipis and the two conjugation systems for atg12/atg12 and the atg8 family members . One of the most downstream effects of the combined action of these proteins is the covalent attachment of the ubiquitin - like atg8-family proteins to the membrane lipid phosphatidylethanolamine in the isolation membrane . Autophagy was initially characterized as a bulk degradation pathway induced by glucagon and nutrient deprivation,,,, . So - called bulk autophagy serves to recycle building blocks to compensate for the lack of nutrients and is thought to be rather non - selective toward its substrates (referred to as cargos), . It has, however, become clear that autophagy also contributes to intracellular homeostasis in non - starved cells by selectively degrading cargo material such as aggregated proteins, damaged mitochondria, excess peroxisomes and invading pathogens (reviewed in refs .,,). The crucial role of selective autophagy for cellular homeostasis is emphasized by the fact that tissue - specific knockout of autophagy genes in mice results in neurodegeneration or liver cancer,,, . Additionally, it has been shown that cells with defective autophagy are unable to clear certain intracellular pathogens (reviewed in refs .,). Emerging evidence has also shown that selective autophagy plays an important role in the homeostasis of intracellular free iron, by controlling the levels of the iron - chelating protein ferritin (referred to as ferritinophagy),,, . Finally, in yeast, the cytoplasm - to - vacuole targeting (cvt) pathway exploits the autophagic machinery for the delivery of the ape1, ams1 and ape4 enzymes to the vacuole via autophagosome - like vesicles called cvt vesicles,, . The cvt pathway also mediates the degradation of retrotransposon particles and thereby serves to protect the saccharomyces cerevisiae genome from retrotransposition . Unlike bulk autophagy, selective autophagy and the cvt pathway need to meet at least three criteria for an efficient process to happen: first, the cargo has to be specifically recognized; second, the cargo has to be effectively tethered to a nascent autophagosome; and third, non - cargo material has to be excluded from the autophagosome . In fact, cvt vesicles are smaller in diameter than starvation - induced autophagosomes, and in contrast to autophagosomes, their membrane is tightly apposed to the cargo preventing unrelated material from being engulfed,,, . Selectivity in autophagy is conferred by cargo receptor proteins, which are able to tether a cargo to a nascent autophagosomal by simultaneously binding the cargo and atg8-family proteins on the isolation membrane (see below). While some cargo receptors bind their cargos directly, in mammalian cells several receptor proteins recognize poly - ubiquitin chains attached to the surface of cargos for selective autophagy . In the following sections, we will discuss the biochemical principles of cargo sorting from other cellular material that often contains the same binding sites . We will further discuss how cargo receptors bind membrane - localized atg8-family proteins as opposed to their soluble forms . Finally, we will discuss the position of the cargo receptors in the autophagic hierarchy dependent on the stimulus for autophagosome formation . Prape1, the major cargo of the cvt pathway, is synthesized in the cytoplasm as a zymogen with an n - terminal propeptide . Prape1 monomers assemble in the cytoplasm into dodecamers which further aggregate into higher order particles in a propeptide - dependent manner,, . The cargo receptor atg19 specifically binds to the prape1 propeptide with high affinity via its coiled - coil domain,,, . Atg19 can also bind to ams1 and thereby include it into the so - called cvt complex, . Subsequently, atg19 mediates the recruitment of the cvt complex to the pas via an interaction with atg11,, . Finally, the interaction of atg19 with atg8 on the isolation membrane mediates the engulfment of the cvt complex by a cvt vesicle (see below), . The propeptide region of prape1 is essential for the formation of the prape1 complex and its transport into the vacuole,, . It has recently been shown that a mutation that prevents the formation of prape1 dodecamers also prevents its delivery into the vacuole, even though this mutant still retains an intact propeptide . Conversely, the fusion of a propeptide to unrelated oligomeric particles is sufficient to drive their efficient delivery into the vacuole . Intriguingly, these data suggest that several propeptides, and by implication several atg19 cargo receptors clustered on a larger structure, are required and sufficient for efficient transport into the vacuole via the cvt pathway . In mammalian cells, ncoa4 has been recently identified as the cargo receptor for ferritin during a process referred to as ferritinophagy,, . Ferritin forms complexes consisting of 24 subunits and therefore also represent multimeric particles . Under conditions of low intracellular iron, ncoa4 receptors bind specifically to ferritin heavy chains (fth1) and mediate the delivery of the ferritin complexes into the lysosomal compartment via selective autophagy,, . Degradation of ferritin within the lysosomes ultimately leads to the release of chelated iron, which is subsequently transported back into the cytosol, . The human cargo receptor p62/sqstm1 mediates the degradation of ubiquitinated cargo material such as aggregated proteins or cytosolic bacteria,,, . This low affinity is at least in part due to homo - dimerization of the uba domain, which is mutually exclusive with mono - ubiquitin binding,,, . The affinity of p62 for ubiquitin can be increased by phosphorylation of serine 403 within the uba domain . The n - terminal pb1 domain of p62 drives homo - oligomerization,, and in vitro p62 oligomers were shown to assemble into long helical structures . Oligomerization of p62 confers high avidity to the interaction with clustered ubiquitin and, thus, stabilizes binding to the cargo material on which ubiquitin is concentrated . It was proposed that the interaction of multiple uba domains in oligomeric p62 with clustered ubiquitin counteracts the self - association of uba domain . Thus, oligomerization of p62 and dimerization of uba domain might cooperate to achieve selectivity for highly ubiquitinated cargos, . The preferential binding of p62 to certain ubiquitin chain types might constitute a further level of regulation during selective autophagy . Indeed, it is known that different ubiquitin chains trigger different cellular processes . While k48-linked chains are recognized by the proteasome and therefore mediate proteasomal degradation of their substrates, k63-linked chains have also been associated with autophagy, . Interestingly, it has been shown that oligomeric p62 preferentially binds to linear and k63-linked chains, as well as to mono - ubiquitin, compared to k48-linked chains,, . Furthermore, it has been reported that binding to ubiquitin chains partially disrupts p62 oligomers in vitro, and that this effect is most evident in the presence of k48-linked chains, . Their presence on a substrate might therefore prevent the accumulation of p62 and thereby counteract its degradation by autophagy . The cargo receptor calcoco2/ndp52 acts during selective autophagy of intracellular bacteria and damaged mitochondria,,,, . Ndp52 contains a predicted coiled - coil region that mediates homo - dimerization and a c - terminal ubiquitin binding zinc - finger domain,, . Recently, it was shown that the isolated zinc - finger domain of ndp52 binds to single - ubiquitin moieties and to poly - ubiquitin chains, regardless of their linkage type . Interestingly, in the context of full - length dimeric ndp52, the zinc - finger domains bound to two different ubiquitin chains rather than to two ubiquitin molecules within the same chain . Thus, similar to p62, full - length ndp52 might preferentially accumulate on cargos that are modified with multiple chains and therefore represent regions with high local ubiquitin concentrations . Furthermore, unlike the isolated zinc - finger domain, full - length ndp52 showed a slightly reduced binding affinity for k48-linked di - ubiquitin chains compared to linear or k63-linked di - ubiquitin chains, . Therefore, it might be possible that, in analogy to p62, self - association confers chain specificity to ndp52 . Similarly, the uba domain of the dimeric nbr1 cargo receptor has been reported to bind to single - ubiquitin moieties with a slight preference for k63-linked chains over k48-linked chains . In contrast to the rather weak interactions of p62, ndp52 and nbr1 with single ubiquitin, atg19 binds with high affinity to its prape1 cargo . Prape1 is synthesized in the cytoplasm and must be efficiently delivered into the vacuole by the cvt pathway in order to function . The ncoa4 cargo receptor could act according to similar principles . On the other hand, the targets of p62, ndp52 and nbr1 are not normally destined to be transported into the lysosome but become a target for selective autophagy only after their ubiquitination . One way to distinguish ubiquitinated autophagic cargo material from other cellular structures with ubiquitin tags may thus be the presence of clustered ubiquitin on the cargo material that is read out by high - avidity interactions of the cargo receptors with ubiquitin . Indeed, while ubiquitination per se could be sufficient to render any cellular material a substrate for autophagy, the higher the local concentration of ubiquitin on a cytoplasmic material, the more efficient the recruitment of cargo receptors will be . An additional level of regulation may be provided at the level of ubiquitin chain linkages . In particular, k48-linked ubiquitin chains may be less preferred targets for receptor binding, allowing proteins marked with these chains to preferentially undergo proteasomal degradation . However, it should be noted that all ubiquitin chain types have been found enriched in insoluble inclusions of autophagy - deficient mice . Therefore, the relative contribution of ubiquitin clustering and of specific ubiquitin chains in cargo receptors recruitment has still to be elucidated . During autophagy and the cvt pathway in s. cerevisiae, the small ubiquitin - like modifier atg8 becomes conjugated to the headgroup of phosphatidylethanolamine on the isolation membrane . First, atg8 undergoes a proteolytic cleavage mediated by atg4, which exposes the c - terminal glycine employed for the subsequent conjugation reaction . The reaction proceeds similarly to the conjugation of ubiquitin to lysine residues in target proteins and requires atg7 as the activating enzyme (e1), atg3 as the conjugating enzyme (e2) and the atg12atg5atg16 protein complex as the e3-like enzyme,, . In mammalian cells, at least six functional atg8 homologues are found: the three members of the microtubule - associated protein 1 light chain 3 (map1lc3) subfamily (lc3a, lc3b and lc3c, respectively) and the three members of the gamma - aminobutyric receptor - associated protein (gabarap) subfamily (gabarap, gabarap - l1 and gabarap - l2/gate-16),,, . Cargo receptors bind to atg8-family proteins via a conserved lc3-interacting region (lir) motif, also known as atg8-interacting motif (aim), . The lir motif consists of the consensus sequence xx, where is an aromatic residue (w / f / y) and is hydrophobic (l / i / v), while x is any other residue, . However, some non - canonical lir motifs, which do not apparently match this consensus, are also found,, . The presence of negatively charged residues in the near proximity of the core lir sequence has been shown to contribute to the binding to atg8-family proteins,,,, . For instance, phosphorylation of a serine adjacent to the core lir motif in cargo receptor optineurin increases the affinity toward lc3b by a factor of 5 (and table 1). However, the overall affinity of the lir motif for atg8 family members remains relatively low, that is, in the micromolar range, even upon phosphorylation (table 1),,,, . Furthermore, the modification of atg8-family proteins with phosphatidylethanolamine is not essential for the interaction of atg8-family proteins with cargo receptors,, . This raises the question as to how cargo receptors can be specifically recruited to isolation membranes in vivo, a question that has recently been addressed for the atg19 cargo receptor . Atg19 contains multiple lir motifs in its c - terminal domain [30, 90]. Each lir motif binds to the same site in atg8, enabling atg19 to bind simultaneously to multiple atg8 proteins . Therefore, multiple lir motifs in tandem allow atg19 to select for atg8-decorated membranes, by establishing high - avidity interactions with concentrated atg8 (c. abert et al ., unpublished results). Mammalian p62 contains only one functional lir motif,, but oligomerization clusters the lir motifs and thereby increases the avidity of p62 toward lc3b concentrated on a surface . In contrast, oligomerization has no effect on the binding of each lir to free lc3b . Furthermore, introducing multiple lirs in a non - oligomerizing version of p62 restores avid binding to clustered lc3b, in analogy to the situation in atg19, . Therefore, atg19 and p62 both exploit avidity - driven interactions to select for atg8-decorated membranes, albeit employing different molecular mechanisms to achieve them (tandem lir motifs versus oligomerization, respectively). Given that many cargo receptors form dimers or multimers and/or contain multiple lir motifs, avidity - driven interactions with membrane - localized atg8-family proteins are likely to be a more general property of cargo receptors,,,,,,, . In yeast, the pas is defined as a peri - vacuolar, dot - like structure in which the prape1 cargo and atg proteins co - localize, and it is likely that autophagosomes form there . In mammalian cells, autophagosomes are generated at multiple sites throughout the cytoplasm (for detailed reviews, see refs . How these processes are coordinated is still unclear . In principle, it is possible that the cargo is recruited to pre - existing isolation membranes . Another intriguing possibility is that during selective autophagy, the presence of the cargo induces the formation of isolation membranes,, . Recent literature insights point in the direction that the nature of the stimulus for autophagosome formation determines whether the generation of the isolation membrane is upstream or downstream of cargo recruitment (fig . Autophagosome formation can be massively induced by starvation or inhibition of the mechanistic target of rapamycin complex 1 (mtorc1) using rapamycin . Genetic and imaging approaches have shown that the main protein complexes required for this process are recruited in a hierarchical manner . Although there is some discrepancy between the genetic and temporal hierarchies, these analyses have placed the atg1/ulk1 complex most upstream, followed by the atg9a system, pi3kc1, the wipis, the atg12atg5atg16l complex and finally the atg8-family proteins,, . There is substantial evidence that even starvation- or rapamycin - induced autophagy has some degree of selectivity . For instance, in s. cerevisiae, the vacuolar transport of the prape1 cargo is increased upon starvation and the protein is tethered to the membrane of larger autophagosomes that also contain other cytoplasmic material . In addition, ams1 can be selectively delivered into the vacuole upon starvation by the atg34 cargo receptor . Similarly, rapamycin treatment promotes atg39- and atg40-mediated autophagic degradation of the endoplasmic reticulum (er - phagy). In mammalian cells fam134b, the functional counterpart of atg40 mediates er - phagy during nutrient starvation . Furthermore, acetaldehyde dehydrogenase (ald6), leucine aminopeptidase iii (lap3), aspartyl aminopeptidase (ape4), ubiquitinated proteins, proteasomes and mature ribosomes are all preferentially targeted to the vacuole via autophagy during nitrogen starvation in s. cerevisiae,,,,, . In mammalian cells that were stimulated with wnt ligand and subsequently starved, the wnt - signaling effector dishevelled (dvl2) forms ubiquitinated aggregates that are selectively incorporated into autophagosomes in a p62-dependent manner . Rapamycin treatment promoted the clearance of aggregated proteins by autophagy in mammalian cell lines and drosophila melanogaster . In arabidopsis thaliana starvation cargo recruitment is likely downstream of isolation membrane formation and might coincide or follow atg8-family protein conjugation (fig . Indeed, in starved mammalian cells, the accumulation of the p62 cargo receptor to punctate structures followed the accumulation of the other components of the autophagic machinery and coincided with the recruitment of lc3b . However, in an earlier study, it was shown that p62 recruitment to pre - autophagosomal structures does not absolutely depend on the interaction with atg8-family proteins . Therefore, the actual mechanism of p62 recruitment to the site of autophagosome biogenesis might be more complex . During autophagic events that are independent of starvation or global mtorc1 inhibition, in fact there is increasing evidence that the cargo is upstream of isolation membrane formation . For instance, it was shown that in s. cerevisiae, in the absence of starvation, the deletion of the cvt pathway cargo prape1 prevents the recruitment of several core autophagy proteins to the pas, including atg1 and atg8 . However, during starvation the localization at the pas of these factors is not affected by the deletion of ape1 . These data strongly suggest that in the absence of starvation, the cvt complex has a crucial role in recruiting the autophagic machinery . Recently, it was shown that under normal growth conditions, the activation of the atg1 kinase is triggered by atg11 bound to the atg19 cargo receptor on the cvt complex . The amount of active auto - phosphorylated atg1 is strongly reduced in ape1 cells, as wells as in cells lacking atg19 and atg11, or in cells expressing an atg11 mutant incapable of binding to atg19 . In contrast, the activity of atg1 under starvation is not affected by the deficiency of these proteins . Collectively, these results suggest that the cargo is required for atg1 kinase activation, which is one of the most upstream signals during autophagosome formation . In contrast, upon starvation the requirement for the prape1 cargo is bypassed . Employing salmonella typhimurium or transfection reagent - coated latex beads as models for selective autophagy in mammalian cells, it was observed that both these substrates were associated with ubiquitin - positive structures after rupture of the endosomal membrane . Interestingly, the localization of the p62 cargo receptor preceded the recruitment of the machinery for autophagosome formation and in particular that of lc3b, which was recruited downstream of all the other components . Similarly, core components of the autophagy machinery including ulk1 and atg9a have been found to localize around damaged mitochondria independent of atg8-family proteins conjugation . Recently, it was reported that cargo receptors recruit the autophagic machinery to damaged mitochondria during mitophagy . The recruitment of the autophagic machinery to mitochondria upon induction of mitochondrial damage was monitored in hela cells lacking five cargo receptors (p62, nbr1, optineurin, ndp52 and taxbp1/calcoco3), . Strikingly, ulk1, wipi1 and dfcp1 were not efficiently recruited to damaged mitochondria in the cargo receptors ko cells . In addition, trim20 and trim21 that act as cargo receptors to degrade components of the inflammasome and the interferon response recruit the autophagic machinery to their cargo material . Collectively, these data suggest that during autophagic events that are triggered in the absence of starvation and that are commonly referred to as selective autophagy, the presence of cargo material triggers autophagosome formation (fig . The usually bulky cargo materials such as protein aggregates, mitochondria, bacterial pathogens or the prape1 complexes serve as template to recruit the autophagic machinery . The recruitment of this machinery is at least in part mediated by cargo receptors and culminates in local lipidation of atg8-family proteins . Additionally, ubiquitinated cargos may also contribute directly to the recruitment of the autophagic machinery . These membrane - localized atg8 proteins in turn are avidly bound by the cargo receptors resulting in close apposition of the autophagosomal membrane and the cargo and therefore exclusion of non - cargo material from its sequestration within autophagosomes (fig . This process may occur only at permissive sites that are capable of donating membrane to the process such as er - related structures in mammalian cells and plants or the pas in s. cerevisiae,, . In contrast, during starvation isolation membranes are generated independently of bulky cargo material, (fig . The high local concentration of lipidated atg8-family proteins in turn will recruit cargo receptors and/or cargo receptor cargo complexes to the isolation membranes,, which in turn bring with them cargo material such as ubiquitinated proteins or prape1 . As a result, cargo material is tethered to the isolation membranes, but the engulfment of random material would not be prevented . Given advances in the field, it is becoming increasingly confusing to group autophagic events into selective and starvation - induced autophagy, as some degree of selectivity may be a universal property of autophagosome formation . It may therefore be more appropriate to classify autophagic events into cargo - independent and cargo - induced autophagosome formation (fig . 2). The model of cargo - induced autophagosome formation could help to explain how isolation membranes are generated in non - starved cells and how they can be efficiently tethered to cargos . However, several links are still missing in order to have a more thorough understanding of the whole process . First, the hypothesis of high - avidity interactions between cargo receptors and clustered target molecules should be tested for other cargo receptor proteins . Second, a more detailed molecular understanding of the interaction networks between cargo receptors, ubiquitin, the autophagic machinery and membranes will be required . The model of cargo - induced autophagosome formation could help to explain how isolation membranes are generated in non - starved cells and how they can be efficiently tethered to cargos . However, several links are still missing in order to have a more thorough understanding of the whole process . First, the hypothesis of high - avidity interactions between cargo receptors and clustered target molecules should be tested for other cargo receptor proteins . Second, a more detailed molecular understanding of the interaction networks between cargo receptors, ubiquitin, the autophagic machinery and membranes will be required.
The study design and the primary results of the ascend - hf trial have been published.5,13 in brief, the trial was conducted, with ethical approval, beginning may 18, 2007 through august 2010 at 398 centers globally . Patients were included in ascend - hf if they were hospitalized for adhf occurring within 24 hours before they received their first intravenous treatment for hf or if they had received a diagnosis of adhf <48 hours after hospitalization for another cause and underwent randomization <24 hours after intravenous treatment for hf . Other criteria for inclusion included: dyspnea at rest or with minimal activity; 1 or more accompanying signs (respiratory rate 20 breaths per minute or pulmonary congestion or edema with rales one third of the way or more up the lung fields); and 1 or more additional measures of hf (evidence of congestion or edema on chest radiography, a b - type natriuretic peptide [bnp] level 400 pg / ml or an n - terminal pro - bnp [nt - pro - bnp] level 1000 pg / ml, pulmonary capillary wedge pressure> 20 mm hg, or left ventricular (lv) ejection fraction [lvef] <40% in the previous 12 months). Complete eligibility and exclusion criteria are described elsewhere.13,14 patient with or without af were allowed into the study, and there was no inclusion or exclusion criteria related to heart rate . Given that no treatment effect on mortality or rehospitalization with the study drug nesiritide was demonstrated, patients who did not receive study drug were excluded from this analysis (n=134; 1.9%). Demographic, clinical, laboratory, and diagnostic data were collected at randomization . In addition to this, further blood pressure (bp), laboratory, and medication data, as well as subsequent diagnostic testing, were collected up to, and including, hospital discharge . Patients were followed by a clinical visit at 30 and 180 days after discharge . Patients were classified as having af if it was identified on the case report form in the baseline medical history section (ie, a history of or current af). Where appropriate and possible, this has been referred to as patients with af . No core lab reading of electrocardiogram (ecg) was performed, but patients in ascend - hf were recommended to have appropriate standard of care, which includes an ecg when presenting with ahf.15 a total of 5378 (77%) patients had information from a baseline ecg entered on the case report form; however, this did not include reporting of sinus rhythm or atrial fibrillation from the ecg as a specific variable . For this analysis, we examined the ascend - hf coprimary endpoint of 6- and 24-hour dyspnea relief (measured using a 7-point likert scale) and the coprimary composite of 30-day all - cause mortality or hf rehospitalisation, its individual components and 180-day all - cause mortality . We measured adherence rates to quality - of - care measures (qcm) based on the class i american college of cardiology / american heart association (acc / aha) recommendations for care of patients with hf,16 as used in the get with the guidelines (gwtg)-hf program.17 specifically, we examined the use of oral anticoagulation for patients with atrial fibrillation at hospital discharge, barring documented contraindications, intolerances or specific reasons for nonuse, or documented plan for initiation at a later date . The use of antithrombotic medication (ie, an antiplatelet or an anticoagulant) was also evaluated according to the chads2 score.18 because patients in ascend - hf were admitted with ahf, all had a chads2 score 1 . We also examined control of the ventricular (heart) rate in patients with af over the course of their admission . Baseline patient characteristics and clinical outcomes are reported according to whether or not patients had a history of af at baseline . Data for continuous variables are presented as medians with 25th and 75th percentiles, and categorical variables are presented as frequencies and percentages . Wilcoxon s rank - sum tests were used to measure differences for continuous variables, and the chi - square or fisher s exact tests were used for categorical variables . Multivariable models, which were previously developed in the ascend - hf trial, were used to adjust the associations of af with 30-day outcomes (all - cause death and hf rehospitalization), and with 31- to 180-day outcomes (all - cause death). These models included the following characteristics at the time of randomization for 30-day outcomes: age; blood urea nitrogen (bun); serum sodium; dyspnea severity; and systolic blood pressure (sbp). Resultant odds ratios (ors) are presented with 95% confidence intervals (cis) comparing patients with af to patients without af (reference group). The adjustment model for 31- to 180-day mortality included age, weight, bun, sbp, sodium, dyspnea severity, and elevated jugular venous pressure; previous history of cerebrovascular disease, chronic obstructive pulmonary disease (copd)/chronic respiratory disease; and hf rehospitalization 1 year before admission . Resultant hazard ratios (hrs) are presented with 95% cis comparing patients with af to patients without af (reference group). Clinically meaningful 2-way interactions between age, dyspnea severity, and chads2 score and af status were also evaluated; none were significant . All analyses were performed using sas statistical analysis software (version 9.2; sas institute inc, cary, nc). The study design and the primary results of the ascend - hf trial have been published.5,13 in brief, the trial was conducted, with ethical approval, beginning may 18, 2007 through august 2010 at 398 centers globally . Patients were included in ascend - hf if they were hospitalized for adhf occurring within 24 hours before they received their first intravenous treatment for hf or if they had received a diagnosis of adhf <48 hours after hospitalization for another cause and underwent randomization <24 hours after intravenous treatment for hf . Other criteria for inclusion included: dyspnea at rest or with minimal activity; 1 or more accompanying signs (respiratory rate 20 breaths per minute or pulmonary congestion or edema with rales one third of the way or more up the lung fields); and 1 or more additional measures of hf (evidence of congestion or edema on chest radiography, a b - type natriuretic peptide [bnp] level 400 pg / ml or an n - terminal pro - bnp [nt - pro - bnp] level 1000 pg / ml, pulmonary capillary wedge pressure> 20 mm hg, or left ventricular (lv) ejection fraction [lvef] <40% in the previous 12 months). Complete eligibility and exclusion criteria are described elsewhere.13,14 patient with or without af were allowed into the study, and there was no inclusion or exclusion criteria related to heart rate . Given that no treatment effect on mortality or rehospitalization with the study drug nesiritide was demonstrated, patients who did not receive study drug were excluded from this analysis (n=134; 1.9%). Demographic, clinical, laboratory, and diagnostic data were collected at randomization . In addition to this, further blood pressure (bp), laboratory, and medication data, as well as subsequent diagnostic testing, were collected up to, and including, hospital discharge . Patients were followed by a clinical visit at 30 and 180 days after discharge . Patients were classified as having af if it was identified on the case report form in the baseline medical history section (ie, a history of or current af). Where appropriate and possible, this has been referred to as patients with af . No core lab reading of electrocardiogram (ecg) was performed, but patients in ascend - hf were recommended to have appropriate standard of care, which includes an ecg when presenting with ahf.15 a total of 5378 (77%) patients had information from a baseline ecg entered on the case report form; however, this did not include reporting of sinus rhythm or atrial fibrillation from the ecg as a specific variable . For this analysis, we examined the ascend - hf coprimary endpoint of 6- and 24-hour dyspnea relief (measured using a 7-point likert scale) and the coprimary composite of 30-day all - cause mortality or hf rehospitalisation, its individual components and 180-day all - cause mortality . We measured adherence rates to quality - of - care measures (qcm) based on the class i american college of cardiology / american heart association (acc / aha) recommendations for care of patients with hf,16 as used in the get with the guidelines (gwtg)-hf program.17 specifically, we examined the use of oral anticoagulation for patients with atrial fibrillation at hospital discharge, barring documented contraindications, intolerances or specific reasons for nonuse, or documented plan for initiation at a later date . The use of antithrombotic medication (ie, an antiplatelet or an anticoagulant) was also evaluated according to the chads2 score.18 because patients in ascend - hf were admitted with ahf, all had a chads2 score 1 . We also examined control of the ventricular (heart) rate in patients with af over the course of their admission . Baseline patient characteristics and clinical outcomes are reported according to whether or not patients had a history of af at baseline . Data for continuous variables are presented as medians with 25th and 75th percentiles, and categorical variables are presented as frequencies and percentages . Wilcoxon s rank - sum tests were used to measure differences for continuous variables, and the chi - square or fisher s exact tests were used for categorical variables . Multivariable models, which were previously developed in the ascend - hf trial, were used to adjust the associations of af with 30-day outcomes (all - cause death and hf rehospitalization), and with 31- to 180-day outcomes (all - cause death). These models included the following characteristics at the time of randomization for 30-day outcomes: age; blood urea nitrogen (bun); serum sodium; dyspnea severity; and systolic blood pressure (sbp). Resultant odds ratios (ors) are presented with 95% confidence intervals (cis) comparing patients with af to patients without af (reference group). The adjustment model for 31- to 180-day mortality included age, weight, bun, sbp, sodium, dyspnea severity, and elevated jugular venous pressure; previous history of cerebrovascular disease, chronic obstructive pulmonary disease (copd)/chronic respiratory disease; and hf rehospitalization 1 year before admission . Resultant hazard ratios (hrs) are presented with 95% cis comparing patients with af to patients without af (reference group). Clinically meaningful 2-way interactions between age, dyspnea severity, and chads2 score and af status were also evaluated; none were significant . All analyses were performed using sas statistical analysis software (version 9.2; sas institute inc, cary, nc). Of the 7007 patients in the ascend - hf trial, 2677 (38.2%) had a history of af at baseline and 4330 (61.8%) did not have a history of af at baseline . Patients with af were older, more likely to be white, have a higher bmi, and had more comorbidities, including diabetes, coronary artery disease, previous, and copd (tables1 and 2). There was no difference in gender or vital signs (heart rate, sbp, and respiratory rate) between those with and without af . Patients with af had a higher ejection fraction (31% vs. 27%; p<0.001) and lower bnp level (945 vs. 1051 pg / ml; p=0.005), compared to those without af; nt - probnp levels were not, however, different (af: 4496 pg / ml; no af: 4238 pg / ml; p=0.406). Af indicates atrial fibrillation; bnp, b - type natriuretic peptide; bp, blood pressure; cabg, coronary artery bypass graft; cad, coronary artery disease; copd, chronic obstructive pulmonary disease; crt - d, cardiac resynchronization defibrillator; cva, cerebrovascular disease; hf, heart failure; icd, implantable cardioverter - defibrillator; jvp, jugular venous pulse; nt - probnp, n - terminal pro - bnp; pci, percutaneous coronary intervention; pvd, peripheral vascular disease; s3, third heart sound . Baseline and discharge medications antiarrhythmic agents included amiodarone, dronedarone, or sotalol . Ace / arb indicates angiotensin - converting enzyme / angitensin receptor blocker; af, atrial fibrillation; ccb, calcium - channel blocker; hf, heart failure; mra, mineralocorticoid antagonist . Heart rate at baseline was similar between groups (83 bpm in patients without af vs. 81 bpm with af; p=0.138). Patents with current or a history of af had a lower heart rate at 24 hours (78 vs. 80 bpm; p=0.012) and at discharge (75 vs. 80 bpm; p<0.001; figure 1). There was no difference in dyspnea improvement between patients with or without af at 6 hours (p=0.087). However, patients with af had less improvement in dyspnea during the first 24 hours (p<0.001; figure 2). Patients with af had a longer median in - hospital length of stay (7 vs. 6 days; p<0.001). Compared to patients without af, patients with af had a higher in - hospital all - cause mortality rate (2.6% vs. 1.7%; p=0.015), a higher 30-day all - cause mortality rate (4.7% vs. 3.3%; p=0.005), a higher 31- to 180-day mortality rate (11.1% vs. 8%; p<0.001), and a higher 180-day mortality rate (15.3% vs. 11.1%; p<0.001). Patients with af also had a higher 30-day hf rehospitalisation rate (7.2% vs. 5.3%; p=0.001). Hence, more patients in the af group experienced the coprimary composite outcome of death or readmission at 30 days than in the group without af (8.6% vs. 11.6%; p<0.001). This difference persisted after adjustment for the predefined prognostic variables (adjusted or=1.19; 95% ci, 1.02 to 1.38; adjusted p=0.029; table 3). However, after adjustment, neither the difference in 30-day mortality (adjusted or=1.20; 95% ci, 0.91 to 1.58), or in 31- to 180-day mortality was significant (adjusted hr, 1.15; 95% ci, 0.93 to 1.43). Clinical event rates of patients with or without af af indicates atrial fibrillation; hf, heart failure . For 30-day outcomes, associations are presented as an odds ratio (95% confidence interval). For 31- to 180-day mortality, hazard ratios (95% confidence interval) adjusted for age, systolic blood pressure, blood urea nitrogen, sodium, and dyspnea severity . In patients with current or a history of af, anticoagulation rates according to chads2 score at discharge were 61.5%, 59.1%, 58.7%, and 57.5% for scores of 1, 2, 3 or 4, and 5, respectively (trend, p=0.782; table 2; figure 3). Antiplatelet use in patients with current or a history of af according to chads2 score was 36.1%, 51.6%, 58.3%, and 61.1% for scores of 1, 2, 3 or 4, and 5 respectively (trend, p<0.001). Patients with current or a history of af had a higher use of anticoagulants and a lower use of antiplatelet agents across all chads2 scores (all comparisons, p<0.001). Patients without af had higher use of antiplatelet across all chads2 scores (all comparisons, p<0.001), but anticoagulants use was only higher in chads2 (5). The percentage of patients with current or a history of af discharged on neither an antiplatelet nor anticoagulant was 17.6%, 14.2%, 12.3%, and 11.1% for chads2 scores of 1, 2, 3 or 4, and 5 respectively . The percentage of patients with or without af discharged on any antiplatelet / anticoagulant was 86.7% and 76.9%, respectively . Use of anticoagulation or antiplatelet therapy at discharge among patients with and without atrial fibrillation according to chads2 score . Of the 7007 patients in the ascend - hf trial, 2677 (38.2%) had a history of af at baseline and 4330 (61.8%) did not have a history of af at baseline . Patients with af were older, more likely to be white, have a higher bmi, and had more comorbidities, including diabetes, coronary artery disease, previous, and copd (tables1 and 2). There was no difference in gender or vital signs (heart rate, sbp, and respiratory rate) between those with and without af . Patients with af had a higher ejection fraction (31% vs. 27%; p<0.001) and lower bnp level (945 vs. 1051 pg / ml; p=0.005), compared to those without af; nt - probnp levels were not, however, different (af: 4496 pg / ml; no af: 4238 pg / ml; p=0.406). Af indicates atrial fibrillation; bnp, b - type natriuretic peptide; bp, blood pressure; cabg, coronary artery bypass graft; cad, coronary artery disease; copd, chronic obstructive pulmonary disease; crt - d, cardiac resynchronization defibrillator; cva, cerebrovascular disease; hf, heart failure; icd, implantable cardioverter - defibrillator; jvp, jugular venous pulse; nt - probnp, n - terminal pro - bnp; pci, percutaneous coronary intervention; pvd, peripheral vascular disease; s3, third heart sound . Baseline and discharge medications antiarrhythmic agents included amiodarone, dronedarone, or sotalol . Ace / arb indicates angiotensin - converting enzyme / angitensin receptor blocker; af, atrial fibrillation; ccb, calcium - channel blocker; hf, heart failure; mra, mineralocorticoid antagonist . Heart rate at baseline was similar between groups (83 bpm in patients without af vs. 81 bpm with af; p=0.138). Patents with current or a history of af had a lower heart rate at 24 hours (78 vs. 80 bpm; p=0.012) and at discharge (75 vs. 80 bpm; p<0.001; figure 1). There was no difference in dyspnea improvement between patients with or without af at 6 hours (p=0.087). However, patients with af had less improvement in dyspnea during the first 24 hours (p<0.001; figure 2). Patients with af had a longer median in - hospital length of stay (7 vs. 6 days; p<0.001). Compared to patients without af, patients with af had a higher in - hospital all - cause mortality rate (2.6% vs. 1.7%; p=0.015), a higher 30-day all - cause mortality rate (4.7% vs. 3.3%; p=0.005), a higher 31- to 180-day mortality rate (11.1% vs. 8%; p<0.001), and a higher 180-day mortality rate (15.3% vs. 11.1%; p<0.001). Patients with af also had a higher 30-day hf rehospitalisation rate (7.2% vs. 5.3%; p=0.001). Hence, more patients in the af group experienced the coprimary composite outcome of death or readmission at 30 days than in the group without af (8.6% vs. 11.6%; p<0.001). This difference persisted after adjustment for the predefined prognostic variables (adjusted or=1.19; 95% ci, 1.02 to 1.38; adjusted p=0.029; table 3). However, after adjustment, neither the difference in 30-day mortality (adjusted or=1.20; 95% ci, 0.91 to 1.58), or in 31- to 180-day mortality was significant (adjusted hr, 1.15; 95% ci, 0.93 to 1.43). Clinical event rates of patients with or without af af indicates atrial fibrillation; hf, heart failure . For 30-day outcomes, associations are presented as an odds ratio (95% confidence interval). For 31- to 180-day mortality, hazard ratios (95% confidence interval) adjusted for age, systolic blood pressure, blood urea nitrogen, sodium, and dyspnea severity . In patients with current or a history of af, anticoagulation rates according to chads2 score at discharge were 61.5%, 59.1%, 58.7%, and 57.5% for scores of 1, 2, 3 or 4, and 5, respectively (trend, p=0.782; table 2; figure 3). Antiplatelet use in patients with current or a history of af according to chads2 score was 36.1%, 51.6%, 58.3%, and 61.1% for scores of 1, 2, 3 or 4, and 5 respectively (trend, p<0.001). Patients with current or a history of af had a higher use of anticoagulants and a lower use of antiplatelet agents across all chads2 scores (all comparisons, p<0.001). Patients without af had higher use of antiplatelet across all chads2 scores (all comparisons, p<0.001), but anticoagulants use was only higher in chads2 (5). The percentage of patients with current or a history of af discharged on neither an antiplatelet nor anticoagulant was 17.6%, 14.2%, 12.3%, and 11.1% for chads2 scores of 1, 2, 3 or 4, and 5 respectively . The percentage of patients with or without af discharged on any antiplatelet / anticoagulant was 86.7% and 76.9%, respectively . Use of anticoagulation or antiplatelet therapy at discharge among patients with and without atrial fibrillation according to chads2 score . In this retrospective analysis of patients with ahf enrolled in the ascend - hf study, patients with current or a history of af had a 19% higher rate of the coprimary endpoint at 30 days, and a lower rate of dyspnea improvement at 24 hours . Second, we found that patients with current or a history of af had a lower heart rate on admission as well as discharge, when compared to patients without af . Third, the overall anticoagulation or antiplatelet therapy rate was lower than expected given the evidence base for the use of these agents in patients with af and provides an opportunity for improvement . Dyspnea is a key symptom in acute cardiovascular disease and has been the focus as a primary endpoint for ahf clinical trials.4,5 interestingly, patients with current or a history of af in the ascend - hf trial had a trend to less dyspnea improvement by 24 hours, when compared to patients without af . This occurred on the background of greater baseline dyspnea at rest than patients without af, and with> 60% of both groups (af and not af) having a moderate or marked improvement in dyspnea . Whether or not therapies targeted at the underlying pathophysiology of af would provide greater dyspnea relief than diuretics or vasodilators is uncertain, as is the underlying mechanism of dyspnea for many patients . Given that dyspnea is a patient - reported symptom, as opposed to an objective measure, such as peak expiratory flow rate,19 it remains uncertain whether other patient features, other than heart rate, may be feeding into their perception of breathlessness . Although there was no difference in heart rate at presentation between patients with or without af, heart rate was significantly lower at 6 and 24 hours and at discharge in patients with af and less dyspnea relief at 24 hours for patients with current or a history of af . There are likely multiple plausible explanations for this finding, including choices for therapy, aggressiveness of heart rate as a clinical target for therapy, underlying differences in patient characteristics, activation of the sympathetic nervous system, and features related to the conduct of a clinical trial . Patients with current or a history of af in ascend - hf had high rates of digoxin use as well as antiarrhythmic medications, which may reflect local variation in practice in a global trial, or clinicians using these medications to actively lower the heart rate or improve symptoms (noting the rate of beta - blocker use was similar in patients with or without a current or a history of af). Additionally, we noted that bnp levels (but not nt - probnp levels) were lower in patients with af, in contradistinction to other studies . This may be explained, in part, by trial entry criteria, which allowed for patients with an elevated bnp to be enrolled without distinction of af, as is now done in other trials.4 this may be further compounded by an older age, worse renal function, and higher rates of coronary artery disease burden, which may be related and increase levels of nt - probnp.20,21 in this study, there was greater usage of anticoagulation therapy among patients with current or a history of af than those without af across all chads2 scores; however, there is still undertreatment with anticoagulation . In a previous study from ascend - hf, significant global variation was noted when anticoagulation for patients with current or a history of af was assessed by quality performance metrics, with only 33% of asia - pacific and 76% of western europe meeting the quality performance metric.12 whether this finding impacts longer - term clinical outcomes is not elaborated in the current study; however, other studies of patients with af and concomitant hf or lv dysfunction have identified this as a risk factor for stroke (and thus a target for anticoagulation).18,22 though there are geographical differences in the hf or af guidelines during the years of conduct of the ascend - hf trial, in patients with a chads2 score 2, guidelines universally recommend oral anticoagulation with or without concomitant antiplatelet agents . Hospitalization provides an ideal time for patients and clinicians to assess risks and benefits of choices for oral anticoagulation or antiplatelet strategy . We found that patients with current or a history of af differed from those not in af in their age, lvef, comorbidity burden, and other clinical features . Patients with current or a history of af had greater heart rate reduction by the day of discharge, but less dyspnea relief and higher rates of readmission and mortality . Finally, we also identified that many patients with current or a history of af do not receive appropriate stroke prevention strategies despite the opportunity while in the hospital to assess the risks and benefits of oral anticoagulants or antiplatelet agents . The ascend - hf trial was funded through its sponsor, johnson & johnson inc . Dr ezekowitz reports consulting fees from pfizer and research support from amgen, bayer, and johnson & johnson . Dr hernandez reports research funding from johnson & johnson, bayer, amylin, and portola and honorarium from corthera and cytokinetics . Dr starling is a consultant for medtronic, novartis, biocontrol, and cardiomems and received research funding from johnson & johnson . Dr califf reports research funding from amylin, johnson & johnson - scios, merck / schering plough, novartis, and bristol - myers squibb foundation; consulting from johnson & johnson - scios, novartis, bayer, roche, pfizer, and bristol - myers squibb foundation; and equity in nitrox llc . Dr oconnor reports consulting fees / honoraria from amgen and actelion pharmaceuticals ltd; ownership / partnership / principal from biscardia, llc; and research grants from otsuka, astellas, gilead, bg medicine, roche diagnostics, critical diagnostics, and resmed . Full financial disclosures for hernandez, califf, and oconnor can be found at http://www.dcri.duke.edu/about-us/conflict-of-interest.
In recent years, morbidity and mortality of nosocomial infections have increased, at least partly due to the increased frequency of antibiotic resistance in both gram - positive and gram - negative organisms . In the past, gram - negative aerobes were the most frequently reported pathogens, but staphylococcus aureus has been increasing in frequency, particularly methicillin - resistant s. aureus (mrsa) in intensive care units.1 over 70% of s. aureus isolates are resistant to methicillin in the middle east and with> 85% of s. aureus pneumonia cases being mrsa pneumonia in the arabian peninsula.2 in saudi arabia, 76% of health - care workers have tested positive for mrsa colonization despite being asymptomatic.3 on a separate note, a systematic review on the epidemiology of s. aureus in the middle east indicates high incidence of community - acquired mrsa carried by healthy individuals, with positive mrsa samples obtained from random wound, throat, and nasal specimens.4 glycopeptide antibiotics, vancomycin and teicoplanin, have been considered drugs of choice in treatment for infections due to suspected or documented mrsa.58 burgeoning glycopeptide prescribing is believed to have led to the emergence of vancomycin - resistant enterococci and, more recently, s. aureus with reduced glycopeptide susceptibility . Treatment resistance of clinical mrsa has also been shown in this region against both -lactam and non--lactam antibiotics (erythromycin, clindamycin, ciprofloxacin, and tetracycline), chloramphenicol, gentamicin, and sulfamethoxazole trimethoprim.4 even though there is a recent debate surrounding the use of vancomycin,911 it is still a common antibiotic for the treatment of nosocomial pneumonia and is recommended in the most recent guideline endorsed by the american thoracic society and the infectious disease society of america.1 despite guidelines, real - world practice patterns are known to vary substantially from those recommended . Furthermore, little is known in low- and middle - income countries of the middle east regarding practice patterns and resource associated with mrsa pneumonia . The objectives of this study were to describe the microbiologic profile of mrsa pneumonia in lebanon and saudi arabia and assess the treatment received by these patients, in terms of drug selection, dosing, timing, and sensitivity of the infection pathogen, and to document hospital resource utilization and clinical outcomes among patients with this infection . This study is a retrospective, observational medical chart review that took place at a total of five study sites in saudi arabia (two sites) and lebanon (three sites). Per the study protocol, the study investigator (local physician responsible for data collection) assessed the need for ethics / irb application at each site, irb waiver or approval was completed at all sites based on local requirements . Specifically, approval was obtained from national guard health affairs, king abdalaziz medical city- jeddah and riyadh, kingdom of saudi arabia; ain wazein hospital irb; and makassed general hospital, riad el - solh beirut, lebanon . In order to be included, patients had to have suspected or culture - proven mrsa pneumonia with appropriate respiratory symptoms . In the absence of culture results, suspected cases needed to have at least two of the following symptoms for inclusion: fever (body temperature 38.5c), hypothermia (body temperature 35.5c), respiratory rate (> 30 breaths / min), systolic hypotension (<90 mmhg), heart rate (> 120 beats / min), elevated peripheral white blood cell count (> 10,000/mm), leukopenia with total white blood cell count (<4,500 cells / mm), and elevated inflammatory markers (eg, procalcitonin or c - reactive protein). Patients were excluded if they had inadequate data on the treatments and outcomes of interest or hospital resource utilization for the study, as judged by the investigators . Data were collected from patient medical records by hospital - based infectious disease specialists, medical microbiologists, internal medicine specialists, surgeons, and intensivists . All data collection instruments were translated into local language and, prior to roll out, pilot tested to ensure relevance and clarity of data points to be collected . Extracted chart data included demographics and comorbid conditions; antibiotic treatment patterns, including initial and subsequent inpatient antibiotic regimens prescribed and antibiotic therapy prescribed at discharge; health - care resource utilization, including diagnostic procedures, laboratory tests, and length of hospital stay; and clinical outcomes, including clinical response at discharge and rehospitalization . Pneumonia was classified into one of the four categories by the abstracting physician as follows: community - acquired pneumonia: symptom onset prior to hospital admission but with no known patient history of 1) another hospital visit, 2) visit to another health - care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admissionhealth - care - associated pneumonia (hcap): symptom onset during 1) another hospital visit, 2) visit to another health - care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admissionnosocomial pneumonia: symptom onset at least 48 hours after hospital admissionunknown community - acquired pneumonia: symptom onset prior to hospital admission but with no known patient history of 1) another hospital visit, 2) visit to another health - care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admission health - care - associated pneumonia (hcap): symptom onset during 1) another hospital visit, 2) visit to another health - care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admission nosocomial pneumonia: symptom onset at least 48 hours after hospital admission antibiotics received during hospitalization were categorized as mrsa active and not mrsa active . Mrsa - active antibiotics as defined by guidelines included vancomycin (intravenous only), teicoplanin (intravenous only), clindamycin, doxycycline, trimethoprim / sulfamethoxazole, linezolid, fusidic acid, rifampin, and tigecycline.12 antibiotics categorized as not mrsa active included beta - lactams (by definition), and in the absence of institution - specific antibiograms, fluoroquinolones and carbapenems were only categorized as mrsa active if culture results indicated sensitivity to the respective antibiotic . Unless otherwise listed, binary and categorical end points were summarized in terms of percentages in each category, and continuous variables were presented using number of observations (when less than the entire sample), arithmetic mean, and standard deviation (sd). This study is a retrospective, observational medical chart review that took place at a total of five study sites in saudi arabia (two sites) and lebanon (three sites). Per the study protocol, the study investigator (local physician responsible for data collection) assessed the need for ethics / irb application at each site, irb waiver or approval was completed at all sites based on local requirements . Specifically, approval was obtained from national guard health affairs, king abdalaziz medical city- jeddah and riyadh, kingdom of saudi arabia; ain wazein hospital irb; and makassed general hospital, riad el - solh beirut, lebanon . In order to be included, patients had to have suspected or culture - proven mrsa pneumonia with appropriate respiratory symptoms . In the absence of culture results, suspected cases needed to have at least two of the following symptoms for inclusion: fever (body temperature 38.5c), hypothermia (body temperature 35.5c), respiratory rate (> 30 breaths / min), systolic hypotension (<90 mmhg), heart rate (> 120 beats / min), elevated peripheral white blood cell count (> 10,000/mm), leukopenia with total white blood cell count (<4,500 cells / mm), and elevated inflammatory markers (eg, procalcitonin or c - reactive protein). Patients were excluded if they had inadequate data on the treatments and outcomes of interest or hospital resource utilization for the study, as judged by the investigators . Data were collected from patient medical records by hospital - based infectious disease specialists, medical microbiologists, internal medicine specialists, surgeons, and intensivists . All data collection instruments were translated into local language and, prior to roll out, pilot tested to ensure relevance and clarity of data points to be collected . Extracted chart data included demographics and comorbid conditions; antibiotic treatment patterns, including initial and subsequent inpatient antibiotic regimens prescribed and antibiotic therapy prescribed at discharge; health - care resource utilization, including diagnostic procedures, laboratory tests, and length of hospital stay; and clinical outcomes, including clinical response at discharge and rehospitalization . Pneumonia was classified into one of the four categories by the abstracting physician as follows: community - acquired pneumonia: symptom onset prior to hospital admission but with no known patient history of 1) another hospital visit, 2) visit to another health - care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admissionhealth - care - associated pneumonia (hcap): symptom onset during 1) another hospital visit, 2) visit to another health - care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admissionnosocomial pneumonia: symptom onset at least 48 hours after hospital admissionunknown community - acquired pneumonia: symptom onset prior to hospital admission but with no known patient history of 1) another hospital visit, 2) visit to another health - care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admission health - care - associated pneumonia (hcap): symptom onset during 1) another hospital visit, 2) visit to another health - care institution, or 3) residence in a nursing home within 90 days prior to the present hospital admission nosocomial pneumonia: symptom onset at least 48 hours after hospital admission antibiotics received during hospitalization were categorized as mrsa active and not mrsa active . Mrsa - active antibiotics as defined by guidelines included vancomycin (intravenous only), teicoplanin (intravenous only), clindamycin, doxycycline, trimethoprim / sulfamethoxazole, linezolid, fusidic acid, rifampin, and tigecycline.12 antibiotics categorized as not mrsa active included beta - lactams (by definition), and in the absence of institution - specific antibiograms, fluoroquinolones and carbapenems were only categorized as mrsa active if culture results indicated sensitivity to the respective antibiotic . Unless otherwise listed, binary and categorical end points were summarized in terms of percentages in each category, and continuous variables were presented using number of observations (when less than the entire sample), arithmetic mean, and standard deviation (sd). Chart - level data were collected for 93 patients with mrsa pneumonia, 50 in saudi arabia and 43 in lebanon . Of the 93 patients in the sample, 20% of the mrsa infections were community acquired, 32% health - care associated, 35% nosocomial, and 12% of unknown origin . However, the most common comorbidities among these patients were diabetes (39%) and congestive heart failure (30%). Ninety of the 93 patients in the sample (97%) had at least one culture taken, and 82 of these (91%) had at least one culture positive for mrsa . As would be expected, only one - third of the isolates tested were sensitive to ciprofloxacin and only 15% were sensitive to beta - lactams . Patients most frequently had positive cultures from pulmonary (87%) and blood (27%) samples; nasal sites were also culture positive in 16% of patients . Table 2 shows the susceptibility profile for mrsa isolates from patients with pneumonia . All isolates tested were sensitive to vancomycin (n=80) and teicoplanin (n=32). Only ten isolates were tested for linezolid sensitivity, and all were found to be sensitive . Drug selection for mrsa pneumonia, expressed as the percentage of patients receiving each antibiotic (with potential for multiple antibiotics per line of therapy), is summarized in figure 1 . Overall, 79 patients (85%) received at least one antibiotic considered to be mrsa active . The 93 mrsa pneumonia patients received a total of 140 antibiotic orders designated by study investigators to be first - line therapy (as some regimens consisted of multiple antibiotics). Of these, 28% were beta - lactams (thus representing inactive therapy), and the most frequently prescribed mrsa - active antibiotic was vancomycin (22% of orders). Among the 48 patients (52%) who were prescribed a total of 66 second - line antibiotic orders, vancomycin remained the most commonly prescribed antibiotic (39% of orders), followed by teicoplanin (12%) and linezolid (9%). Of the eleven third - line drugs received by eight patients, linezolid was the most commonly used (36% of orders), followed by vancomycin (27%) and ciprofloxacin (18%). Overall, the mean total length of stay (los) was 32.4 days, with the majority (20.4 days) spent in general wards (figure 2). Thirty - six patients (39%) required mechanical ventilation, for a mean sd of duration of 18.724.4 days per patient . Four patients (4%) required hemodialysis, for a mean of 9.8 11.9 days per patient . Of these patients, two patients were reported to have moderate - to - severe renal disease on admission, but whether these patients were receiving dialysis prior to admission is unknown . Other interventions required among patients with mrsa pneumonia included peripheral catheters (52%), central venous catheters (52%), and peripherally inserted central catheters (6%). Over one - fourth of the patients were considered treatment failures at the time of discharge (table 3). The overall in - hospital mortality rate was 30%, with nearly one - third of deaths being mrsa related . Of the 28 patients who died, treatment response at discharge was failure in 24 (86%) cases, improved in one (3.5%), cure in two (7%), and indeterminate in one (3.5%). Relapse of mrsa pneumonia (3%) and rehospitalization for mrsa pneumonia (2%) were rare; however, all - cause rehospitalization occurred in 20% of patients . Chart - level data were collected for 93 patients with mrsa pneumonia, 50 in saudi arabia and 43 in lebanon . Of the 93 patients in the sample, 20% of the mrsa infections were community acquired, 32% health - care associated, 35% nosocomial, and 12% of unknown origin . However, the most common comorbidities among these patients were diabetes (39%) and congestive heart failure (30%). Ninety of the 93 patients in the sample (97%) had at least one culture taken, and 82 of these (91%) had at least one culture positive for mrsa . As would be expected, only one - third of the isolates tested were sensitive to ciprofloxacin and only 15% were sensitive to beta - lactams . Patients most frequently had positive cultures from pulmonary (87%) and blood (27%) samples; nasal sites were also culture positive in 16% of patients . All isolates tested were sensitive to vancomycin (n=80) and teicoplanin (n=32). Only ten isolates were tested for linezolid sensitivity, and all were found to be sensitive . Only one - third of the isolates tested were sensitive to ciprofloxacin . Drug selection for mrsa pneumonia, expressed as the percentage of patients receiving each antibiotic (with potential for multiple antibiotics per line of therapy), is summarized in figure 1 . Overall, 79 patients (85%) received at least one antibiotic considered to be mrsa active . The 93 mrsa pneumonia patients received a total of 140 antibiotic orders designated by study investigators to be first - line therapy (as some regimens consisted of multiple antibiotics). Of these, 28% were beta - lactams (thus representing inactive therapy), and the most frequently prescribed mrsa - active antibiotic was vancomycin (22% of orders). Among the 48 patients (52%) who were prescribed a total of 66 second - line antibiotic orders, vancomycin remained the most commonly prescribed antibiotic (39% of orders), followed by teicoplanin (12%) and linezolid (9%). Of the eleven third - line drugs received by eight patients, linezolid was the most commonly used (36% of orders), followed by vancomycin (27%) and ciprofloxacin (18%). Overall, the mean total length of stay (los) was 32.4 days, with the majority (20.4 days) spent in general wards (figure 2). Thirty - six patients (39%) required mechanical ventilation, for a mean sd of duration of 18.724.4 days per patient . Four patients (4%) required hemodialysis, for a mean of 9.8 11.9 days per patient . Of these patients, two patients were reported to have moderate - to - severe renal disease on admission, but whether these patients were receiving dialysis prior to admission is unknown . Other interventions required among patients with mrsa pneumonia included peripheral catheters (52%), central venous catheters (52%), and peripherally inserted central catheters (6%). Over one - fourth of the patients were considered treatment failures at the time of discharge (table 3). The overall in - hospital mortality rate was 30%, with nearly one - third of deaths being mrsa related . Of the 28 patients who died, treatment response at discharge was failure in 24 (86%) cases, improved in one (3.5%), cure in two (7%), and indeterminate in one (3.5%). Relapse of mrsa pneumonia (3%) and rehospitalization for mrsa pneumonia (2%) were rare; however, all - cause rehospitalization occurred in 20% of patients . This real - world burden and treatment patterns study of mrsa pneumonia in the middle east indicates a resource - intensive disease with a high los and mortality, consistent with other studies in high - income countries . Inappropriate / inactive therapy for mrsa nosocomial pneumonia was prescribed more than a quarter of the time over the entire course of therapy, and in first - line treatment was prescribed 42% of the time . The relatively high mortality rate in this study may have been influenced by the high proportion receiving suboptimal therapy as first - line treatment . In a multinational observational study in asia and a randomized clinical trial in canada, the frequency of prescribing inadequate initial antibiotic therapy in pneumonia may be> 50%, and this significantly increases mortality rates.13,14 mrsa - targeted antibiotics were more frequently prescribed in second - line (vancomycin 54%) and third - line (linezolid 51%) treatments . Potential reasons for the administration of inappropriate antibiotics may be due to lack of physician knowledge, delay in receiving culture results, or availability of medications that cover mrsa . While the exact reason for prescribing inappropriate antibiotics cannot be determined from this study, a body of literature has focused on inadequate initial therapy as a driver of added morbidity and mortality.14,15 administration of inappropriate empiric antibiotic therapy can increase mortality in patients with hospital - acquired pneumonia (defined in this study as occurring within 48 hours of admission) and ventilator - associated pneumonia (vap, defined as occurring within 48 hours of endotracheal intubation). Results from a prospective surveillance study conducted through the asian network for surveillance of resistant pathogens (ansorp) found that of the 15.8% of patients with s. aureus hospital - acquired pneumonia or vap, 50.4% received discordant therapy.13 patients receiving discordant therapy had higher all - cause (44% vs 35%) and pneumonia - related (32% vs 21%) mortality than patients who received concordant therapy . Multivariate analysis results showed that discordant therapy was a risk factor for pneumonia - related mortality (or, 1.542; 95% ci, 1.1272.110; p=0.007). Administration of inappropriate empiric antibiotic therapy can also increase mortality in patients with hcap, defined as pneumonia in a patient admitted from a nursing home or other long - term care setting, who was hospitalized within the last 12 months, receiving dialysis or infusion therapy requiring regular visits to a hospital - based clinic, or who is immunocompromised . In a us - based study, patients with hcap compared to community acquired pneumonia had higher mortality (24.6% vs 9.1%, p<0.001) and were more likely to receive inappropriate antimicrobial treatment (28.3% vs 13.0%, p<0.001).16 mortality was higher for patients who received inappropriate compared to appropriate antibiotic therapy (32.2% vs 15.7%, p<0.001). Inappropriate initial antimicrobial therapy was identified as an independent risk factor for hospital mortality (or, 2.19; 95% ci, 1.273.78; p<0.005). Results from separate studies conducted at the same institution found that treatment escalation did not decrease mortality in patients with hcap treated with inappropriate empiric antibiotic therapy17 and that initial therapy for hcap should include an antibiotic that targets mrsa.16 the resource use findings were quite disparate between countries in this study where the mean los was 30 days longer in saudi arabia than in lebanon . It is unclear whether this difference results from an underlying disparity in the severity of infection between groups, or a difference in health - care infrastructure . A surveillance study in saudi arabia looking at 2,800 patients admitted to the medical - surgical intensive care unit suggests that long los is not uncommon for patients that are severely ill . They found that patients with vap had a longer mean los of 85 days versus 61 days in those without vap.18 this population had primarily gram - negative pneumonias, but 20% were attributed to gram - positive infection . The study is naturally limited by its retrospective design and reliance on data that are documented in the medical chart . Since inclusion criteria required that the patients were diagnosed with mrsa pneumonia, we do not have details on other patients that may have been colonized and not developed active infection . However, the information gathered from the charts represents a snapshot of the real - world treatment patterns and supplements the limited data we have in this indication and region of the world . Mrsa pneumonia in saudi arabia and lebanon may be treated with antibiotics that are inactive against mrsa in a majority of patients for first - line therapy, resulting in an added resource use burden and mortality . The information gathered in this middle east population provides important real - world perspectives on the current treatment patterns and opportunities to improve care . Future studies should more closely examine the appropriateness of initial therapy in mrsa pneumonia patients.
Therefore, to increase the effectiveness of health education, information given to patients verbally should be supplemented and reinforced with written materials . Some advantages of written materials are consistency of message, ready availability of the required information, and ability to share this information . These materials can be used to underpin the information obtained verbally from health care providers . However, to influence health behaviors, it is important to ensure that health education materials are suitable for their target audience . To maximize their suitability, a number of factors, such as content, layout, and cultural appropriateness of these materials should be taken into account . In spite of a range of benefits of written material, the means of acquiring health information are changing in this era of advancing electronics; people rely more on electronic devices such as television and internet . Therefore, printed materials should be made to suit the audience by making them attractive and useful . The development of effective health education materials requires formative research as well as evaluation, which systematically obtains information to be used to improve these materials . Written education materials in the form of posters and brochures are common in the kingdom of saudi arabia (ksa). However, we were unable to find any study conducted to determine the suitability of written health education materials disseminated in ksa in the literature review . Consequently, it is expected that there is a lack of awareness of the factors that make written education material suitable . However, the evaluation of these brochures for their suitability is not conducted in a systematic manner . The purpose of our study was to formally evaluate the available health education brochures, designed and disseminated by government health care institutions in the qassim province, ksa . This was a cross - sectional review of health education brochures developed by the ministry of health (moh) institutions in the qassim province . Qassim, a central province of ksa, has 174 health care facilities belonging to saudi moh . The majority of these health care facilities carry out health education activities, including the distribution of written health education materials . We invited all health care institutions under the moh, in the qassim province to send written health education materials available in their facilities for evaluation . All health education materials received were classified as leaflets, booklets, brochures and posters . We defined a health education brochure as a single page, two - sided, folded document designed to communicate health - related information to the community or patients . The brochures were further classified as two - fold, three - fold and four - fold . Only brochures in arabic structured evaluation form composed of two sections, was designed and used as a research instrument . The first section of the evaluation form asked for general information while the second section was the suitability assessment of materials (sam) score sheet . This information included the title of the brochure and credentials of the authors, publication date, and citation for the sources of the contents . Sam is used by researchers for the evaluation of printed materials, as well as web - based ones . The sam uses six factors to assess the suitability of materials: content, literacy demands, graphics, layout and typography, learning stimulation / motivation and cultural appropriateness . Each criterion is scored on a maximum of two points and a minimum of zero point . A score of two points is rated as superior, one point as adequate, zero point as not suitable . If the criterion does not apply to the material, it is labeled as not applicable . Scored items are summed up and the percentage (excluding n / a items) is calculated . The material is then classified according to percentage as either superior (70 - 100%), adequate (40 - 69%) or not suitable (0 - 39%). The sam criteria use readability formulas to assess the reading level of written material for the factor on literacy demand. As readability formulas are not available for arabic, this item was omitted . Amendments were also made in the criteria for typography . The criteria of uppercase and lowercase serif or sans serif, and no all caps for long headers or running text were omitted, as they are not applicable to arabic typography . Font size of at least 14 points was used as a criterion, instead of size 12 standard set by sam criteria . Leonard doak and cecilia doak, authors of the original sam, were consulted through e - mails about the modifications in the sam instrument . Two qualified consultant family physicians from the local community who were trained in a workshop arranged for this purpose did the evaluation . Each brochure was independently evaluated and scored by each evaluator . Data were entered and analyzed using epi info version 3.4 statistical package (centers for disease control and prevention, atlanta, georgia, usa). The mean of percentages by the two evaluators, of all six sam factors and overall sam percentage were used to designate the categories of superior, adequate and not suitable to each brochure . For individual criterion, the scores assigned by the two evaluators were summed up and the categories re - defined . The criteria which attained four points were labeled as superior, while those attained two or three points were labeled, adequate and those with zero or one point were labeled as not suitable . A total of 110 brochures were evaluated, out of which 59 were three - fold, 45 were two - fold and six were four - fold . Of the brochures evaluated, 75 (68.2%) were designed and disseminated by hospitals while 35 (31.8%) were made by various departments in the public health administration, qassim . The most common subject written on was mental health (21.8%), followed by chronic diseases (12.7%), and the least common addressed topic was ophthalmology (1.8%). Although the institution's name was mentioned in all (100%) brochures, the names of the authors (36.4%) and reviewers (5.5%) were mentioned in only a small proportion of the brochures . Salient features of evaluated health education brochures (n=110) seventy - four (67.3%) brochures scored as adequate, 34 (30.9%) were not suitable and two (1.8%) were rated as overall, the average sam score for all brochures was 44.3% (range: 20.0% to 70.0%), which is considered adequate based on the sam ratings . The suitability rating, across the six sam factors, are displayed in table 2, while the suitability rating for individual criteria of the sam factors are shown in table 3 . Suitability rating of the health education brochures in qassim (n = 110) rating of sam factors for health education brochures in qassim (n=110) rating of individual criterion of sam factors for health education brochures in qassim (n=110) the content factor in the majority (74.5%) of the brochures was categorized as adequate . A large proportion (92.7%) of the brochures stated their purpose, which warranted the classification as superior or adequate according to sam scores . A succinct summary to emphasize important take home messages was absent in almost all brochures resulting in the rating of 107 (97.3%) brochures as not suitable . Overall, the average sam score for the content factor of all brochures was 48.9% (range: 13.0 - 75.0%), which placed them in adequate category . The literacy demand factor in 54.5% of the brochures was categorized as adequate; only one (0.9%) brochure was rated as overall, the average sam score for the literacy demand factor of all brochures was 42.0% (range: 7.0 - 75.0%), which put them into the adequate category . The brochures were largely written in a passive, third - person using medical terms, thus rendering almost half (50.9%) of them not suitable with regard to style of writing . Approximately, two - thirds (68.2%) of the brochures were rated as not suitable in graphics . Overall, the average sam score for the graphics factor of all brochures was 25.9% (range: 0.0 - 75.0%), which fell into the not suitable category . Only 3 (2.7%) brochures had covers with graphics that were rated as superior . A sizable proportion of brochures (63.7%) had illustrations that made them not suitable . Approximately, two - thirds (64.5%) of the brochures were categorized as adequate in layout and typography . Overall, the average sam score for this factor was 48.7% (range: 17.0 - 92.0%), which fell into the adequate category . Most brochures used an appropriate font size (14 point or larger). However, in some brochures pages were text - dense, and watermarks used as background illustrations made the text difficult to read . More than one - third (41.0%) of the brochures were categorized as not suitable in the learning stimulation and motivation factor . Overall, the average sam score for this factor was 39.9% (range: 0.0 - 75.0%), which fell into the upper limit of the not suitable category . None of the brochures used active interaction, and very few used passive interaction such as question and answer format for headings and subheadings . Few brochures focused on practical, behavior - modifying steps for the reader; 11 (10.0%) brochures scored superior in this category . The cultural appropriateness of the majority (82.7%) of the brochures was adequate . Overall, the average sam score for the factor for cultural appropriateness of all brochures was 48.3% (range: 25.0 - 75.0%), which fell into the adequate category . On the whole, the brochures in this study addressed a variety of topics including mental health, chronic diseases and infectious diseases . Some topics such as mental health and cardiac health were addressed more frequently, as the institutions with these specialties had specific budgets for health promotion activities . Author's credentials are considered important to assure readers that the information provided was credible since the authors had the appropriate expertise in the field . The majority of our brochures (63.6%) did not give the name of the authors or their credentials . However, all of them displayed the institution's name as well as logo of the moh . This display of the name of the institution and logo of moh endorsed the information as credible . A record of the date of publication is important to assure readers that the information provided was up - to - date . Brochures published within the past 2 - 3 years are considered up - to - date . However, a study conducted in the united kingdom found that some general practices were using information leaflets that were 6 - 13 years old for the education of patients . In our study, only a small proportion (15.5%) of brochures mentioned the date of publication, which made it difficult to determine if they were current . In this study, the average sam score for all brochures was 44.3%, which is considered adequate on the sam ratings . In an evaluation of the written education materials for patients with prostate cancer, the mean overall sam rating was however, average scores were higher (63.3%) in comparison to our study . Our study found that approximately two - thirds (67.3%) of brochures fell into the adequate category . This finding is similar to an evaluation of 31 information leaflets, 64.5% of which were rated as adequate ., the researchers found that 22 (75.8%) scored adequate for their overall suitability, while sam suitability scored six as superior (20.6%) and one (3.4%) as not suitable . In an evaluation of 21 printed educational materials on human papillomavirus (hpv), the average sam score was 37.4%, which is considered not suitable . In another assessment of 35 brochures and eight worldwide web education materials for neurology patients, 14% of the materials were superior, in that study, well - known organizations had brochures with an average rating in the adequate category . A concise summary, interactive format, and motivation content help people to adopt the desired behavior . In our study, few brochures (13.6%) most of the brochures mainly had scientific information, but no practical guidance for behavior change . Other studies have also found similar limitations in the written health education materials . In our study, 97.3% of brochures had no summary of the contents which is more than was found in another study in which 86% of the materials were without a summary statement . An evaluation of 101 written documents for novel h1n1/09 influenza published on the cdc website, showed that not a single document had a summary . Many of our brochures needed to be more focused on the information which was in the category of needed to know, rather than another area of considerable weakness in our brochures was the absence of interactive techniques such as question and answer format . Evaluation of the use of common vocabulary in the brochures showed that our study had only 2 (1.8%) that could be rated superior in comparison to 4 (14%) that were superior in another study . Several studies have found that written materials with graphics are well - received by the target audience because they can help to reinforce messages . In our study, approximately 68% brochures were rated as not suitable compared to 82% of the brochures in another study that required improvements to their illustrations . In evaluating the cover graphics for each brochure, only 3 (2.7%) were rated superior . This is in contrast to another study in which the researchers rated the majority of cover graphics as superior . Captions are helpful in conveying the message of the graphics . In our study, only 10 brochures (9.1%) had captions to explain the information in the graphics, in contrast to another study in which there were captions to the illustrations of 24% of the written materials . Though the brochure must be visually appealing and well - formatted, the main message can be obscured with the use of several bright colors and stylish borders . In our study, 76.4% were adequate in typography, but in another study, the font used in 51% of the brochures was appropriate . In our study, 25.5% of the brochures needed improvement in layout, while in the study by arnold et al . Subheadings were required in 45.5% of our brochures to shorten the paragraphs and make them easier to understand, compared to 51% in another study that needed shortening of paragraphs . Other researchers also had issue with the paragraphs, text - dense pages and the lack of subdivision of complex information into smaller pieces of written materials . In evaluating content for behavior modeling, adequate in another study . In that same study, evaluation of the materials for motivation showed that 72.3% of the materials were adequate or not suitable, while all of our brochures (100%) fell into these two categories . In our study, as was also found by weintraub et al . Our brochures were culturally sensitive with no offensive illustrations or examples, as was also noted in other studies . This study was limited to the evaluation of health education brochures designed by government institutions . The brochures from private health care institutions, and those written in languages other than arabic, were not evaluated . As the brochures were selected from qassim province only, the findings of this study may not be generalized to cover those produced and disseminated in other provinces of saudi arabia . Therefore, the evaluator's attitudes, cultural and professional background may have influenced the outcome of this evaluation . This study was limited to the evaluation of health education brochures designed by government institutions . The brochures from private health care institutions, and those written in languages other than arabic, were not evaluated . As the brochures were selected from qassim province only, the findings of this study may not be generalized to cover those produced and disseminated in other provinces of saudi arabia . Therefore, the evaluator's attitudes, cultural and professional background may have influenced the outcome of this evaluation . To sum up, this study showed that although two - thirds of the health education brochures were adequate in terms of their overall suitability the majority needed improvements to be made to their graphics, learning stimulation and motivation . The lack of a summarized statement, and interactive features, insufficient use of graphics, the absence of instructions to model desired behavior, and a crowded layout were some of the weaknesses also found in other studies that used sam to evaluate written materials . Moreover, health care workers who prepare the health education brochures should be properly trained for the purpose . The health care professionals should strive to ensure that the quality of the written education materials is appropriate for the target audience.
Therefore, the clinical difference in myocardial infarction (mi) between men and women could affect the diagnosis and time of decision making for treatment and hence disease outcomes . Knowledge of clinical symptoms and risk factors contribute to the prognosis of mi mortality in men and women differently . Documentation of these findings could be helpful in the management of patients treatment and/or outcomes . Accumulating evidence over the last several decades regarding the treatment and outcomes for coronary artery disease reveals disparities that have a clear relationship to gender and the number of people above 85 years of age is growing faster than any other age group . Gender differences in the clinical outcome of patients with mi may be partially explained by the women condition . Several conditions specific to women represent differences in the pathophysiology of coronary heart disease (chd) between men and women . Such female - specific conditions include early menopause, gestational diabetes, peripartum vascular dissection, preeclampsia and eclampsia, polycystic ovarian syndrome, low - birth - weight infants, and hypothalamic hypoestrogenemia . Some of these states, which occur at a younger age, lead to an increased risk for later development of chd in life . The main source of estrogen before menopause is estradiol . After menopause, low level of estrogen is produced mainly from the conversion of androgens into estrone in adipose tissue . These variations could be associated with the differences in ischemic and chd between men and women and are concurrent with the increased risk in women, which occurs after menopause . It is confirmed by the fact that younger women with endogenous estrogen deficiency have a higher than 7-fold increase in coronary artery risk . A number of studies have reported that women with mi have higher mortality rates than men . However, the women with mi history have higher rates of mortality during hospitalization or the first 30 days after mi . Epidemiology of mi has been well - explained and gradually updated in the developed countries . This epidemic has been reported from some asian countries, as well . The prevalence of cardiovascular disease risk factors including hypertension, type 2 diabetes, smoking, and high cholesterol as well as access to therapies including percutaneous coronary intervention (pci), coronary artery bypass graft (cabg), and thrombolytic therapy in the patients in iran were reported differently from those in the patients in persian gulf countries . For example, the prevalence of hypertension, diabetes, and high cholesterol in study of acute coronary syndrome in six arab countries (kuwait, bahrain, the uae, yemen, oman, and qatar) was reported as 68%, 36%, and 63%, respectively . Clinical epidemiology of mi in men and women has not been yet studied in iran, one of the largest countries in middle east, and no relevant report is available . This study was to determine the clinical epidemiology of mi in men and women in iran . This research is a hospital - based cross - sectional study, which used the data of 20,750 mi patients in iran in 2012 . The study enrolled the patients registered in iranian mi registry of iran health and medical education ministry (department of cardiovascular disease prevention) commissioned in the hospitals equipped with a cardiology ward across 31 provinces of iran using the date at arrival in and discharge from the hospital . Inclusion criteria were defined based on international classification of diseases (the codes i22, i21, i24, and i25.2). Echocardiography (ecg) is used to differentiate between two types of mi, st - segment elevation mi (stemi) and nonstemi, by the shape of the tracing . An st section of the tracing higher than the baseline is considered stemi which usually necessitates more invasive treatment . In stemi, the ecg must show new st elevation in two or more adjacent ecg leads . Left bundle branch block (lbbb) and right bundle branch block were defined in accordance with recently published criteria with the exception that, for lbbb, q waves in ecg leads i, v5, and v6 were included . Lbbb criteria included qrs duration of 120 ms; rs or qs deflection in ecg leads v1 and v2; and slurred broad r waves in ecg leads i, avl and v6 . An r wave amplitude of 1 mm in lead v1 was determined as a readily measurable standard . The patients with no definite diagnosis of mi by the cardiologist were excluded from the study . The demographic data of the patients including age, gender, education, place of residence, and individual, clinical, and laboratory risk factors such as the date at mi incidence, duration of hospitalization, type 2 diabetes, hypertension, smoking, dyslipidemia, type of diagnosis, treatment, the place of mi, and pain pattern were gathered . The above variables were measured based on national protocol and guidelines, and world health organization (who) standards and registered by the cardiologist and matron of the cardiology ward in electronic medical records of the patients . The registry's data accuracy is acceptable, and several articles using these data have been so far published in authentic journals which have been referenced to in references . Iran's mi registry was developed in 1389 h.s and has been being implemented in all coronary care unit (ccu)-equipped hospitals affiliated to medical universities in iran . By this registry, the data of in - patients in ccu with confirmed mi diagnosis are recorded and saved in the respected software at discharge from ccu and could be used by cardiology department of iran's ministry of health and medical education . All hospitals affiliated to medical universities and some nonaffiliated (social insurance, humanitarian, private, and military) hospitals have run this registry in ccus from which the data used in this study were obtained . The access to these data was provided by an official permission within a letter of understanding to conduct a phd thesis of epidemiology . All continuous values are expressed as mean standard deviation (sd) and categorical variables are presented as a percentage . Univariate comparisons used chi - square tests for categorical variables and t - test for continuous variables . The software used for data analysis was stata statistical software: release 12 statistical software statacorp lp, college station, tx, usa . P <0.05 was considered as significant . This research is a hospital - based cross - sectional study, which used the data of 20,750 mi patients in iran in 2012 . The study enrolled the patients registered in iranian mi registry of iran health and medical education ministry (department of cardiovascular disease prevention) commissioned in the hospitals equipped with a cardiology ward across 31 provinces of iran using the date at arrival in and discharge from the hospital . Inclusion criteria were defined based on international classification of diseases (the codes i22, i21, i24, and i25.2). Echocardiography (ecg) is used to differentiate between two types of mi, st - segment elevation mi (stemi) and nonstemi, by the shape of the tracing . An st section of the tracing higher than the baseline is considered stemi which usually necessitates more invasive treatment . In stemi, the ecg must show new st elevation in two or more adjacent ecg leads . Left bundle branch block (lbbb) and right bundle branch block were defined in accordance with recently published criteria with the exception that, for lbbb, q waves in ecg leads i, v5, and v6 were included . Lbbb criteria included qrs duration of 120 ms; rs or qs deflection in ecg leads v1 and v2; and slurred broad r waves in ecg leads i, avl and v6 . An r wave amplitude of 1 mm in lead v1 was determined as a readily measurable standard . The patients with no definite diagnosis of mi by the cardiologist were excluded from the study . The demographic data of the patients including age, gender, education, place of residence, and individual, clinical, and laboratory risk factors such as the date at mi incidence, duration of hospitalization, type 2 diabetes, hypertension, smoking, dyslipidemia, type of diagnosis, treatment, the place of mi, and pain pattern were gathered . The above variables were measured based on national protocol and guidelines, and world health organization (who) standards and registered by the cardiologist and matron of the cardiology ward in electronic medical records of the patients . The registry's data accuracy is acceptable, and several articles using these data have been so far published in authentic journals which have been referenced to in references . Iran's mi registry was developed in 1389 h.s and has been being implemented in all coronary care unit (ccu)-equipped hospitals affiliated to medical universities in iran . By this registry, the data of in - patients in ccu with confirmed mi diagnosis are recorded and saved in the respected software at discharge from ccu and could be used by cardiology department of iran's ministry of health and medical education . All hospitals affiliated to medical universities and some nonaffiliated (social insurance, humanitarian, private, and military) hospitals have run this registry in ccus from which the data used in this study were obtained . The access to these data was provided by an official permission within a letter of understanding to conduct a phd thesis of epidemiology . All continuous values are expressed as mean standard deviation (sd) and categorical variables are presented as a percentage . Univariate comparisons used chi - square tests for categorical variables and t - test for continuous variables . The software used for data analysis was stata statistical software: release 12 statistical software statacorp lp, college station, tx, usa . P <0.05 was considered as significant . In this study, 20750 patients (15,033 males and 5,717 females) with age range of 13106 (mean: 61.2 13.4) years were enrolled . Mean sd of age was 59.6 13.3 years in men and 65.4 12.6 in women . Mean age at mi incidence in men was lower than women with a significant difference (p = 0.001). By who definition since the number of patients under 18 years (nine individuals), as adolescent subgroup, was negligible, all patients were enrolled and studied . However, as the respectful reviewer suggested, two independent analyses were run with inclusion and exclusion of these patients . Then, two independent analyses were run with inclusion and exclusion of the patients under 30 years . Therefore, as the data were gathered from patients in all provinces across iran and the total sample size depicts mi epidemiology more soundly; all patients were included in the analysis . The analysis results of under 18-year - old patients data have been reported, as well . Only 9 (0.04%) patients (8 men and 1 woman) were 18 years (mean age: 14.8 1.9), of whom two men were diabetic . Age and risk factors of coronary artery disease in individuals with acute mi are shown in table 1 . 22.2% (18% men and 33.4% women) of the patients had diabetes, 26.2% (28.5% men and 20.1% women) were smoker, 35.5% (28.6% men and 53.7% women) had hypertension, and 17.8% (15%% men and 25.3% women) had hypercholesterolemia . In - age and risk factors in mi patients in - hospital status and complications of acute mi in this study, epidemiologic status of mi incidence in men and women was reported for the first time in iran through a hospital - based study . The findings of the present study indicated that the mi incidence pattern, mortality from mi, and risk factors prevalence were significantly different between men and women in iran . In kuwait, 87% of mi patients were male, and the mean age was reported to be 55 years, which is lower than in our study . According to registries of acute coronary events in 65 hospitals in six arab countries (yemen, oman, bahrain, kuwait, qatar, and united arab emirates), our study indicates that the mean age at incidence is higher in the islamic republic of iran than in these arab countries (except for yemen with a mean age at incidence of 61 years). The difference in mean age across different studies could be attributed to different population age distributions, differences in life expectancies and lifestyles and variations in the distribution of and coping with cardiovascular diseases risk factors . There was a significant difference in chest pain between men and women . However, some studies reported no difference in this regard and some others reported a higher prevalence of chest pain in women than men . Our findings indicated that different factors, including chest pain, could be used as a prognosis for mi, and hence patients delayed referring to emergency centers and mortality could be partially prevented . In a similar study, 73% of the patients were male, and their mean age was 61.8 years, which is similar to our study . History of cabg, pci, and diabetes was reported, respectively, 4.4%, 12.5%, and 25.3%, which are higher compared to our study . In a study in india, mean age of the patients was reported 57.5 years, which is lower compared to iran . In india, 30.4% of the patients had type 2 diabetes, 37.7% had hypertension, and 40% were smoker; the corresponding figures in our study are, respectively, 22.2, 35.5, and 26.2% . In korea, 19.2% of mi patients had diabetes, 67.3% were smoker, and 61.2% had stemi . In our study, furthermore, the prevalence of type 2 diabetes and other risk factors including hypertension and smoking in mi patients was lower in iran than in india . The prevalence of type 2 diabetes in mi patients was higher in iran than in korea, and lower than in japan . In this study, men / women frequency was 2.62 . In the framingham study, this ratio was 10:1 in - patients under 45 years old, declining progressively to approximately 2:1 in - patients over 75 years old . Finally, approximately 11.6% and 6.5% of men and women, respectively, in our study had a family history of early coronary artery disease, with a significant difference . Our results indicated that the familial history of early coronary artery disease was an important cardiovascular risk factor in both male and female patients . Our findings are consistent with several studies in other countries . However, in some studies, the prevalence of hypertension and diabetes was similar in male and female patients, which is not consistent with our study . In contrast, smoking and family history of cardiovascular disease were reported higher in men than women . The findings of this study confirm the higher relative risk of hypercholesterolemia, diabetes, and hypertension and mi development in women . The mi incidence pattern, mortality from mi, and risk factors prevalence are significantly different between men and women in iran . More emphasis on these issues in training people and healthcare professionals seems to contribute partially to patients timely referring to health care centers and preventing mi - associated mortalities . The key message of the present study addresses the difference in mi incidence pattern and risk factors between men and women in iran . Therefore, the approach to managing, preventing, and controlling mi as the leading cause of death in iran must be different for men and women . Informing patients appropriate with their risk factors and preventing delayed referral to emergency in case of mi prognosis is the first step to incorporate the findings of this study into health system . Further empowerment of health system staff in dealing with and triaging mi patients and adoption of appropriate care and treatment with male and female patients characteristics could be the second step.
The patient had a 25% tbsa burn, which included a mixture of second - degree, deep second - degree, and third - degree burns involving the anterior aspect of the right lower extremity, the medial aspect of the left lower extremity, and the dorsum of both feet (figure 1a). B. patient s burns demonstrate conversion of second - degree burns to third - degree burns after 5 days . After 5 days, the patient s burns demonstrated progression from second - degree burns to deeper second - degree and third - degree burns (figure 1b). Seven days postinjury, the patient underwent tangential excision of necrotic tissue of her lower extremities, which led to wounds clinically assessed to require fbc matrix and subsequent stsg based on our burn treatment protocol . A total of 1100 cm and 2400 cm of fbc was meshed 3:1 and 4:1 and applied to the debrided areas of the left and right lower extremity, respectively . The wounds were dressed with bridal veil, xeroform, softsorb soaked in an antibiotic solution containing neosporin g.u . The patient s postoperative course was benign, and during her first dressing change (3 days postoperatively), the fbc was noted to be intact and in the early stages of engraftment (figure 2a). Postoperative evaluation after 10 days demonstrated tissue regeneration (figure 2b), and the wounds were deemed suitable for skin grafting . When the patient was taken to the operating room 2 days later for skin grafting, islands of reepithelialization were noted in the center of the wound bed covering areas where fbc was applied . Punch biopsies were taken at this time in an effort to elucidate pertinent microscopic activity . A. after 3 days, fbc was adherent to wound bed with granulation tissue beginning to fill the interstices of the fbc . Two punch biopsies were taken 12 days post - fbc application as noted in figures 3 and 4 . Biopsies were placed in 10% buffered formalin and processed by strata pathology services (lexington, ma). Hematoxylin and eosin (h&e; polyscientific, bay shore, ny) staining was performed to assess the dermis . To distinguish between human dermal collagen fibers and bovine collagen fibers, tissue sections were stained with an antibody specific to bovine collagen type i and a fluorescein isothiocyanate conjugated secondary antibody (millipore, billerica, ma). A fluorescent cellular counterstain was used to visualize nuclei within tissue sections (hoeschst 34580, life technologies, grand island, ny). Keratinocytes were identified using antibodies to cytokeratin 5, a fibrous structural protein, and p63, an essential transcription factor for epithelial development and proliferation . Cd31a was used to evaluate endothelial cell borders, and ki-67 was used as a cellular marker for proliferation . A. a punch biopsy was taken from the right leg as indicated in a1 by the dotted circle . Tissue was processed and stained with (b, c) h&e, (d, e) bovine collagen type i (green) and hematoxylin to color cellular nuclei (blue), (f) cd31a, (g) ki-67 . A. a punch biopsy was taken from the left leg as denoted by the dotted circle . Tissue was processed and stained with (b, c) h&e, (d) bovine collagen type i (green) and hematoxylin to color cellular nuclei (blue), (e) cd31a, (f) ki-67, (g) ck5, and (h) p63 . Bv, blood vessels; ec, epidermal cells; fbc, fetal bovine collagen; h&e, hematoxylin and eosin . H&e staining of the biopsy obtained from the right lower extremity demonstrated a full thickness wound containing dermal collagen fibers (figure 3a e). Immunohistochemical staining of serial sections from this biopsy revealed the presence of bovine collagen type i repopulated with the patient s cells (figure 3e). The thickness of this tissue was approximately 1 mm and contained histotypic dermal collagen fibers populated with cells at a similar density to that of uninjured human dermis . Proliferating cells and vasculature were found in the fbc, suggesting the repopulation of the dermis with the patient s cells (figure 4f, g). These results represent the early stages of wound healing after fbc application . The biopsy obtained from the left lower extremity revealed islands of reepithelialization that could be seen clinically as well as histologically (figure 4a c). Staining indicated the biopsy contained irregular mixtures of regions with remnants of dermal and epidermal components (figure 4c, d). Immunohistochemical staining of serial sections from this biopsy revealed the presence of a 0.75-mm thick region of bovine collagen type i (figure 4d). Proliferating cells and vasculature were found throughout the biopsied tissue (figure 4e, f). A stratified squamous epithelium was present on top of the bovine collagen dermal fibers as well as within the interstices of the fbc (figure 4h, i). Approximately 1 mm and 2 mm from the surface of the wound, small clusters of keratinocytes were detected adjacent to adipocytes visible by cytokeratin 5 staining . A percentage of the epidermal cells also stained positive for p63 (figure 4). This biopsy demonstrated the capacity for the fbc to be repopulated and revascularized with the patient s own cells and provided a supportive environment for epidermal cell migration and reepithelialization . During the next few days, wounds covered with fbc continued to undergo reepithelialization, and as a result, on postoperative day 18, a final decision was made not to perform skin grafting . On postoperative day 26, significant reepithelialization of her wounds was observable (figure 5a), and the patient was discharged on postoperative day 30 . A. after 26 days, (b) 53 days, (c) 80 days, (d) 102 days, (e) 6 months, (f) 9 months, (g) 12 months, (h) 26 months after fbc application . On postoperative day 53, the patient displayed additional reepithelialization and repigmentation (figure 5b). Examination of the patient s skin on postoperative day 80 and day 102 revealed complete wound closure and additional repigmentation (figure 5c, d). Subsequent follow - up of the patient after 6 months, 9 months, 12 months, and 26 months revealed healthy, soft, and supple skin, without any visible signs of hypertrophy or fibrosis (figure 5e h). After 5 days, the patient s burns demonstrated progression from second - degree burns to deeper second - degree and third - degree burns (figure 1b). Seven days postinjury, the patient underwent tangential excision of necrotic tissue of her lower extremities, which led to wounds clinically assessed to require fbc matrix and subsequent stsg based on our burn treatment protocol . A total of 1100 cm and 2400 cm of fbc was meshed 3:1 and 4:1 and applied to the debrided areas of the left and right lower extremity, respectively . The wounds were dressed with bridal veil, xeroform, softsorb soaked in an antibiotic solution containing neosporin g.u . The patient s postoperative course was benign, and during her first dressing change (3 days postoperatively), the fbc was noted to be intact and in the early stages of engraftment (figure 2a). Postoperative evaluation after 10 days demonstrated tissue regeneration (figure 2b), and the wounds were deemed suitable for skin grafting . When the patient was taken to the operating room 2 days later for skin grafting, islands of reepithelialization were noted in the center of the wound bed covering areas where fbc was applied . Punch biopsies were taken at this time in an effort to elucidate pertinent microscopic activity . A. after 3 days, fbc was adherent to wound bed with granulation tissue beginning to fill the interstices of the fbc . Two punch biopsies were taken 12 days post - fbc application as noted in figures 3 and 4 . Biopsies were placed in 10% buffered formalin and processed by strata pathology services (lexington, ma). Hematoxylin and eosin (h&e; polyscientific, bay shore, ny) staining was performed to assess the dermis . To distinguish between human dermal collagen fibers and bovine collagen fibers, tissue sections were stained with an antibody specific to bovine collagen type i and a fluorescein isothiocyanate conjugated secondary antibody (millipore, billerica, ma). A fluorescent cellular counterstain was used to visualize nuclei within tissue sections (hoeschst 34580, life technologies, grand island, ny). Keratinocytes were identified using antibodies to cytokeratin 5, a fibrous structural protein, and p63, an essential transcription factor for epithelial development and proliferation . Cd31a was used to evaluate endothelial cell borders, and ki-67 was used as a cellular marker for proliferation . A. a punch biopsy was taken from the right leg as indicated in a1 by the dotted circle . (d, e) bovine collagen type i (green) and hematoxylin to color cellular nuclei (blue), (f) cd31a, (g) ki-67 . A. a punch biopsy was taken from the left leg as denoted by the dotted circle . Tissue was processed and stained with (b, c) h&e, (d) bovine collagen type i (green) and hematoxylin to color cellular nuclei (blue), (e) cd31a, (f) ki-67, (g) ck5, and (h) p63 . Bv, blood vessels; ec, epidermal cells; fbc, fetal bovine collagen; h&e, hematoxylin and eosin . H&e staining of the biopsy obtained from the right lower extremity demonstrated a full thickness wound containing dermal collagen fibers (figure 3a e). Immunohistochemical staining of serial sections from this biopsy revealed the presence of bovine collagen type i repopulated with the patient s cells (figure 3e). The thickness of this tissue was approximately 1 mm and contained histotypic dermal collagen fibers populated with cells at a similar density to that of uninjured human dermis . Proliferating cells and vasculature were found in the fbc, suggesting the repopulation of the dermis with the patient s cells (figure 4f, g). These results represent the early stages of wound healing after fbc application . The biopsy obtained from the left lower extremity revealed islands of reepithelialization that could be seen clinically as well as histologically (figure 4a c). Staining indicated the biopsy contained irregular mixtures of regions with remnants of dermal and epidermal components (figure 4c, d). Immunohistochemical staining of serial sections from this biopsy revealed the presence of a 0.75-mm thick region of bovine collagen type i (figure 4d). Proliferating cells and vasculature were found throughout the biopsied tissue (figure 4e, f). A stratified squamous epithelium was present on top of the bovine collagen dermal fibers as well as within the interstices of the fbc (figure 4h, i). Approximately 1 mm and 2 mm from the surface of the wound, small clusters of keratinocytes were detected adjacent to adipocytes visible by cytokeratin 5 staining . A percentage of the epidermal cells also stained positive for p63 (figure 4). This biopsy demonstrated the capacity for the fbc to be repopulated and revascularized with the patient s own cells and provided a supportive environment for epidermal cell migration and reepithelialization . H&e staining of the biopsy obtained from the right lower extremity demonstrated a full thickness wound containing dermal collagen fibers (figure 3a e). Immunohistochemical staining of serial sections from this biopsy revealed the presence of bovine collagen type i repopulated with the patient s cells (figure 3e). The thickness of this tissue was approximately 1 mm and contained histotypic dermal collagen fibers populated with cells at a similar density to that of uninjured human dermis . Proliferating cells and vasculature were found in the fbc, suggesting the repopulation of the dermis with the patient s cells (figure 4f, g). The biopsy obtained from the left lower extremity revealed islands of reepithelialization that could be seen clinically as well as histologically (figure 4a c). Staining indicated the biopsy contained irregular mixtures of regions with remnants of dermal and epidermal components (figure 4c, d). Immunohistochemical staining of serial sections from this biopsy revealed the presence of a 0.75-mm thick region of bovine collagen type i (figure 4d). Proliferating cells and vasculature were found throughout the biopsied tissue (figure 4e, f). A stratified squamous epithelium was present on top of the bovine collagen dermal fibers as well as within the interstices of the fbc (figure 4h, i). Approximately 1 mm and 2 mm from the surface of the wound, small clusters of keratinocytes were detected adjacent to adipocytes visible by cytokeratin 5 staining . A percentage of the epidermal cells also stained positive for p63 (figure 4). This biopsy demonstrated the capacity for the fbc to be repopulated and revascularized with the patient s own cells and provided a supportive environment for epidermal cell migration and reepithelialization . During the next few days, wounds covered with fbc continued to undergo reepithelialization, and as a result, on postoperative day 18, a final decision was made not to perform skin grafting . On postoperative day 26, significant reepithelialization of her wounds was observable (figure 5a), and the patient was discharged on postoperative day 30 . A. after 26 days, (b) 53 days, (c) 80 days, (d) 102 days, (e) 6 months, (f) 9 months, (g) 12 months, (h) 26 months after fbc application . Long - term follow - up of the patient after discharge was uneventful . On postoperative day 53 examination of the patient s skin on postoperative day 80 and day 102 revealed complete wound closure and additional repigmentation (figure 5c, d). Subsequent follow - up of the patient after 6 months, 9 months, 12 months, and 26 months revealed healthy, soft, and supple skin, without any visible signs of hypertrophy or fibrosis (figure 5e h). In this case study, we report on a patient with a 25% tbsa burn treated with fbc matrix postexcision that healed without subsequent autologous skin grafting . Long - term evaluation after 26 months post - fbc application indicated healthy skin, without any visible signs of disfigurement . Moreover, repigmentation was visible over 85% of the treated areas . Immunohistological analysis of biopsies obtained 12 days post - fbc application demonstrated that the fbc was capable of generating a dermal component approximately 0.75 mm to 1 mm in thickness with the capacity to support reepithelialization . Additionally, it was noted that the wound bed contained a mixture of full thickness and deep partial thickness zones . Staining for human keratinocyte markers indicated small islands of remaining epidermal cells found adjacent to the deep reticular dermis . Keratinocytes located next to the intradermal epithelial structures are hypothesized to have migrated through the fbc matrix and contribute to the reepithelialization of the wound . Although biopsies were not obtained at later time points, it can be inferred that a fully stratified layer of keratinocytes formed and that a basement membrane was established between the epidermal and dermal components of the skin, based on the continued durability of the skin and the lack of recidivism observed during follow - up . The results from this case demonstrate a potential treatment strategy to reduce the amount of donor skin needed to reconstruct wounds during surgical intervention, especially wounds with indeterminate depth . The burn injury may be excised, treated with fbc to regenerate dermal tissue, and reassessed to determine areas requiring skin grafting based on the amount of reepithelialization . If reepithelialization is not noted within 2 to 3 weeks, there is no delay in treatment regimen, and autologous skin grafting can proceed over the newly generated dermal tissue as originally planned . However, if areas of reepithelialization are visible, close monitoring of the rate of reepithelialization is necessary to determine whether the reepithelialization may result in the closure of the wound without stsg . Long - term follow - up indicated excellent functional and cosmetic outcomes for an injury of this extent and size that healed without autologous grafting . Consistent with previous reports that use fbc, the outcomes of this study suggest that the regenerated dermal tissue aided by the fbc contributed to the durability, functionality, and appearance of the healed wound . In summary, these results encourage further use of fbc to treat patients with deep partial thickness and full thickness burns for possible wound coverage.
Strategies for cancer prevention necessarily focus on eliminating unhealthy lifestyle habits such as alcoholism or cigarette smoking or improving both diet and exercise patterns which are believed to contribute to about one - third of annual cancer deaths worldwide [14]. Over the last decades, accumulating epidemiological evidence and animal investigations have suggested that consumption of a diet rich in food plants significantly reduces the risk of several types of cancers and recent recommendations point to plant - based diets [57]. This raises the theoretical possibility that such protective effects could be attributed to specific micronutrient or phytochemical constituents of food plants and that such components might have beneficial effects in the field of cancer chemoprevention either as naturally occurring dietary constituents / pharmaceuticals or in functional foods [810]. It has been speculated that they could manipulate the activity of metabolic enzymes that break down chemical mutagens and carcinogens to reduce lifetime cancer risk . It is indeed widely believed that the postoxidative enzymes (also, i.e., phase ii enzymes), such as glutathione s - transferase, udp - glucuronosyl transferase, sulphotransferase, and acetyl transferase, are able to promote health by detoxifying xenobiotics . On the contrary, the oxidative enzymes (e.g., phase i), represented mainly by the superfamily of cytochrome p450 (cyp) and fad - containing monooxygenases, raise cancer risk by the bioactivation of ubiquitous mutagenic compounds [1117]. This rather simplistic dichotomy has in turn suggested that food plant - derived nutrients or phytochemicals might be employed to reduce the risk of cancer through two enzyme - based strategies such as boosting the good detoxifying phase ii enzymes (using, for example, representative phytochemical - containing fruits and vegetables such as grapes, cauliflower, kale, and broccoli), or inhibiting the bad activating phase i enzymes (using those contained in garlic, tea, and onion). We must remember here that these strategies were extrapolated from epidemiological observations on populations consuming diets varying in both quantity and type of food plant containing thousands of chemical agents which are able to modulate the specific activity of the metabolizing enzyme battery in a very complex way . They have been popularized by the media and exploited by marketers of supplements of phytochemicals and desiccated vegetables labelled as containing suitable amounts of detoxifying enzyme modulators . However, this approach totally fails to address the complexity of the multiple interactions between dietary components and xenobiotic metabolism simultaneously generating health benefits or harmful outcomes, depending on circumstances that cannot yet be predicted . Consequently, the potential effects of whole - food plant - derived single constituents on xenobiotic metabolism and cancer risk are also uncertain . This modulation strategy foresees large - scale induction of postoxidative phase ii enzymes that detoxify xenobiotics by means of single green constituents, thereby accelerating the clearance of mutagens and protecting cells against cancer . The potential benefits of this strategy have stimulated active in vitro and in ex vivo studies on the molecular mechanism and specificity of such chemical compounds [1823]. Particular attention has been devoted to cruciferous vegetables of the brassica genus, such as kale, cabbage, broccoli, brussels sprouts, and cauliflower . These vegetables contain considerable amounts of glucosinolates which are the precursors (via the enzymatic conversion by the enzyme myrosinase) of isothiocyanates [2426], which are phase ii enzyme inducers [2730]. Some researchers have actually created hybrid plants specifically to produce higher amounts of single phytochemical inducers . Resveratrol, a phytoalexin found in grapes and other food products, is also able to boost postoxidative - linked activities, but many other compounds contained in plants could be cited . An alternative anticancer approach is to inhibit the oxidative bioactivating this hypothesis is emphasized by both the scientific literature and the media, as exemplified by numerous reports urging regular consumption of green or black tea containing catechins as well as onion and garlic rich in diallyl sulfide [3234]. Finally, both proposed strategies also must be considered in the context of genetic metabolic polymorphisms, which may differentially, per s, modulate the effects of any one dietary factors on individuals . We would like to point out that the main difficulty with these strategies is that they totally ignore the complexity of metabolizing enzymatic machinery . Indeed, if on one hand the consumption of food plants, which contain thousands and thousands of phytochemicals (an apple, e.g., seems to contain more than 700 chemical compounds, and a simple fruit salad?) Is linked to a reduced cancer risk, on the other, the induction of xenobiotic metabolism by one specific food component may also stimulate the unwanted formation of highly reactive mutagens [35, 36]. The use of single naturally occurring dietary constituents such as isothiocyanates or individual drugs such as disulfiram, oltipraz, or food additives such as bha [2(3)-tert - butyl-4-hydroxyanisole], for example, also elicits unhealthy effects [3740]. It should be pointed out that in addition to the increase in xenobiotic clearance, each postoxidative (phase ii) enzyme is also involved in electrophilic species generation and, therefore, must be considered as a bioactivating system for specific chemical classes such as halogenated hydrocarbons by glutathione s - transferases, for example, or polycyclic aromatic hydrocarbons (pahs) by sulphotransferases [4164]. So, the activation or inactivation of a compound depends on the chemical nature of the compound itself and not on the metabolic enzyme involved . More in general, the manipulation of the activity of one or more phase ii enzyme can either increase or reduce the bioactivation of specific compounds . Whereas induction increases the detoxification of some promutagens, thereby favoring chemoprevention, it also increases the bioactivation of countless other foreign chemicals to which humans are simultaneously exposed . As the population is exposed to a myriad of potentially harmful molecules, any modification of the activity of these enzymes could actually lead to unexpected dangerous effects . For example, cruciferous isothiocyanates such as the sulforaphane, widely considered as a beneficial phase ii inducer, turn out to be genotoxic or a strong promoter of urinary bladder and liver carcinogenesis, also inducing cell cycle arrest and apoptosis [6567]. Similarly, engineered salmonella typhimurium ta1535 transfected with the plasmid vector pkk233 - 2 containing rat glutathione s - transferase 5 - 5 cdna has been shown to activate many genotoxicants, whereas the nontransfected counterpart does not; in addition, heterologous expression of mammalian theta class glutathione transferases in s. typhimurium and escherichia coli systems has been used to demonstrate the role of glutathione conjugation in the genotoxicity of dihalomethanes [61, 69]. Paradoxically, liver metabolic s9 fractions isolated from rodents treated with the monofunctional postoxidative inducer bha have been proposed as a complementary s9 metabolizing system to bioactivate pro - mutagens in typical short - term mutagenicity bioassays . Similar considerations should be made for the inhibitory strategy, a hypothesis that has stimulated recommendations to increase, for example, consumption of green and black teas, as they contain phytochemicals such as catechins able to inhibit the oxidative (phase i) enzymes thus reducing the production of mutagens and carcinogens such as n - nitroso compounds [13, 71]. The inhibition of dimethylhydrazine - induced colon cancer by diallyl sulfide, a flavour component of garlic (allium sativum), has encouraged garlic consumption increase [72, 73]. Moreover, the flavonoid naringin, present in grapefruit and related citrus fruits, has been found to inhibit aflatoxin b1 activation by cyp3a4 in cells and animal models supporting the general idea that green - based metabolism inhibition may reduce carcinogenesis risk . The concept of metabolic manipulation, however, ignores the simple fact that the institutional role of cyp superfamily toward xenobiotics is to promote their detoxification from the cells and body as well: it is our misfortune that some of them are bioactivated . In other words, due to the dual beneficial and detrimental nature of any of the p450 enzymes, the reduced activation of certain toxins occurs simultaneously with the reduced detoxification of other environmental toxicants to which humans are exposed daily, a phenomenon clearly unpredictable . Once again, it should be noted that the bioactivation of chemicals by phase i apparatus, as occurs for phase ii ones, depends on the nature of the substance itself and not on the involved enzyme . Of course, as it is impossible to select safe, personalized human exposure levels to environmental toxins in such a way as to systematically avoid harmful compounds, the regular inhibition of oxidative enzymes might actually lead to an increase toxicological risk . In addition, it should take into account that a selective inhibitor of one cyp enzyme (and due to the existence of many cyp isoforms this strategy should foresee the use a cocktail of multiple inhibitors) may be an inducer of other cyps; for example, phenethyl isothiocyanate derived from brassica and diallyl sulfide from garlic are able to inhibit cyp2e1 but also induce cyp2b1 and cyp1a2 . Paradoxically, in 1985 when wattenberg proposed chemoprevention strategies, including the ones that foresee the above mentioned manipulation of metabolic (according to the belief that they are classified as bad - phase i and good - phase ii) enzyme activities, guengerich published a comprehensive review on the dual bioactivating and detoxifying nature of each metabolic enzyme regardless of whether belongs to the phase i or ii battery (figure 1). Not least, the use of enzyme activity modulators can lead to other serious unhealthy consequences stemming from the alteration of endogenous metabolism where these catalysts are involved (e.g., arachidonic acid derivatives, nitric oxide, aldosterone, cholesterol, or vitamins) as well as alteration of fundamental physiological functions (growth, differentiation, apoptosis, homeostasis, and neuroendocrine functions); the effects on the pharmacokinetics of coadministrated drugs should not be overlooked as well . The illogical effects of single daily consumed dietary constituents on xenobiotic metabolism are further complicated by genetic (metabolic) polymorphisms that lead to the occurrence of high- or low - metabolizer phenotypes in the population, each at increased toxicological risk from exposure to specific chemicals [78, 79]. The multiple polymorphisms (e.g., occurrence of high or low (or intermediate in some cases) metabolizers for any oxidative or postoxidative isoforms) characterizing the so called individual metabolic fingerprint further complicate the issue . Constitutive up- or down - regulation of any phase i or ii dependent enzyme . In other words, the infinite number of possible combinations of human genetic metabolic polymorphisms constitutes another set of variables in the xenobiotic metabolism . Thus, it appears even more clear that the possibility of manipulating enzyme activity, which in its constitutive diversity already may determine genetic disorders as well as perturbations on the chemical biotransformation (including drugs), raises further questions about the effectiveness of the chemical - based enzymatic modulation of cancer risk [81, 82]. In our opinion, these considerations suggest the need for considerable caution before allowing for any form of enzyme - activity manipulation for a generalized prevention, particularly in healthy individuals . What is the clinical significance of the perpetual manipulation of such enzymatic systems by single nutrient or phytochemicals? Summarizing the various aspects depicted above, the scenario that arises shows how both oxidative and postoxidative enzymes are highly multifunctional and can be induced or inhibited or both by a great number of dietary components . Noteworthy, is the often ignored existence of the dual activating and detoxicating nature of these enzymatic systems . So, the impressive number of chemical compounds that can modulate them, the presence in greens of chemicals that induce both activation and inhibition of mutagenesis, the genetically determined interindividual variability that may moderate (increasing and/or inhibiting) the effects of specific dietary factors on any metabolic enzyme, and the complexity of the interactions among food constituents and enzyme systems have fed the ongoing debates as to whether phytochemicals can alone explain the anticancer ability of plants [9, 83]. It is very difficult to imagine how a single phytochemical, today selected as representative of this or that green, such as lycopene in tomato, resveratrol in grapes, sulforaphane in broccoli, and beta - carotene in carrots, used as a food supplement would offer an advantage, since a variety of fruit and vegetables seems necessary to provide the mixture of vitamins and minerals that appear to favour protection against neoplasia . How can we imagine that the beneficial effects of consuming entire fruits and vegetables, in which enzyme modulating components appear in varying amounts and proportions, and in which unpredictable synergistic and antagonistic (or both depending on the enzyme involved) interactions occur among thousands of different chemicals in their natural matrix, could be just reproduced by supplements of single representative phytochemicals? The beneficial or harmful outcomes of a single compound (portio facit venenum, paracelso) can be quite different from those elicited by the same compound within complex mixtures (portio and interactiones faciunt venenum). Components failed to reproduce the beneficial effects of the plants from which they were derived should not be underestimated . Unexpected results of cancer chemoprevention trials of antioxidant provitamins and vitamins which we believe can constitute an exemplary warning about the vulnerability of single - nutrient strategies [8790]. Beta - carotene administered alone or in combination with vitamins a, e, or c for the prevention of lung cancer and other cancers in heavy smokers or asbestos workers failed to reduce cancer risk and, in some cases, actually increased the risk, raising the suspicion that single chemical supplements may have harmful and beneficial effects as well [9193]. It has been documented that the deleterious effect of beta carotene can be linked to its ability to stimulate the metabolizing machinery, such as activators of polycyclic aromatic hydrocarbons, and to generate an oxidative stress . In addition, supplementation with commercial doses of vitamin c for 6 weeks is enough to induce dna damage in human lymphocytes, probably by means of its ability to generate oxidative stress connected to phase i upregulation [96, 97]. In the field of cancer prevention, the idea of producing the so called magic - bullet, as conceived by paul ehelich for antibacterials, too easily evokes the long - life elixir on a molecular level capturing the imagination of both the public and researchers . From the standpoint of cancer research policy, the possible role of single dietary constituents is of pivotal interest in cancer research but basic information about the role of metabolizing apparatus, however, makes it clear that the role of any single anticarcinogenic phytochemical cannot be understood except in the context of broader dietary patterns . The ongoing scientific controversy surrounding the effects of single molecules on cancer risk seems to provide a salutary warning for health policymakers . Considering that unhealthy lifestyle factors are also taken into account, educational campaigns encouraging the consumption of fruit, fibres, and greens should be encouraged.
It does not only affect the couples' life, but it affects the healthcare services and social environment . A male factor is solely responsible in about 20% of infertile couples and contributory in another 3040% . If a male infertility factor is present, it is almost always defined by the finding of an abnormal semen analysis . Clinicians usually rely on the results of semen analysis for the assessment of male fertility . Standard semen analysis is widely considered to be part of the routine assessment in evaluating male fertility and the surrogate measure of male fecundity in clinical practice [4, 5]. There is little consensus as to which parameter within a conventional semen analysis is the best predictor of male fertility . A correlation between sperm count and pregnancy has been reported in previous studies [68], showing evidence of a predictive value of sperm count . Therefore, who adopted the threshold of 20 10/ml as reference for minimal value of sperm count . Routine semen analysis also includes the evaluation of sperm morphology and motility; both of these parameters are considered to be related to male fertility [68, 10, 11]. However, since who reference values were adopted, it has become evident that a basic semen analysis is insufficient to determine the fertility status of the male since previous who reference values were made considering a normal population of healthy men, and not a population of men with proven fertility . Due to this, serious concerns are raised about the possibility of conceiving with semen parameters below who cutoff values [7, 13, 14]. Additional sperm function tests, such as swelling and/or eosine test, do not provide further information to traditional semen parameters in the assessment of the fertility status of infertile male patients . It is very difficult to identify a semen parameter and/or test capable in itself of predicting male fertility . This difficulty can lead to inaccurate diagnosis, inappropriate treatment, and unnecessary anxiety on the part of the patient . Therefore, a correct interpretation of male subfertility could reduce the number of patients requiring assisted reproduction . The role of the female partner must also be considered in the assessment of potential male fertility . Minor degrees of fertility impairment are not necessarily associated with couple infertility when present in only one partner, because the fertility status of the woman may compensate for the partner's subfertility . We report a large retrospective study in a population of infertile couples who conceived after aetiological therapies . This study provides additional information regarding the relationship between semen parameters and spontaneous conception and regarding the indication for etiological treatments also in presence of severe reduction in semen parameters . 2935 infertile couples were evaluated at the infertility clinic of a tertiary - care university hospital [a . University general hospital, rome (italy)] between january 2004 and june 2009 . Each couple underwent evaluation by gynaecologists with expertise in infertility, supported by andrologists and endocrinologists . Physical examination, hormonal assessment of ovulatory function, screening for cervical and vaginal infections, transvaginal sonogram, evaluation of tubal patency, and genetic assessment (when indicated) were carried out in the female patients . Standard semen analysis was performed periodically (every two months) in trusted laboratories, according to who guidelines . Etiological treatments were performed in men that showed identified causes of infertility, whereas empirical treatments (such as gonadotropins, antioxidants, carnitine, and aspartic acid) were performed in men with no identified causes of infertility . Assisted clinical and laboratory data, including standard semen analysis, were collected from all couples . Semen parameters, obtained by semen analysis within 60 days before the cycle prior to conception, were evaluated in order to define seminal status at conception . Recent studies demonstrated that the intraindividual variation in seminal parameters has no clinical value and that there is no indication that intraindividual variation in semen parameters is more pronounced among men from infertile couples when compared with healthy controls . Therefore, one ejaculate is a sufficient indicator of semen quality in a group of patients . Patients were divided in different groups according to sperm count, motility, and morphology . Subsequently, we classified them in different groups according to the predictor value for pregnancies, as previously reported (oligospermia, asthenospermia, teratospermia, and oligoasthenoteratospermia) [15, 7]. Patients with a reduction in sperm count were also classified into 3 subgroups: severe (<5 mil / ml), moderate (515 mil / ml), and mild (1520 mil / ml) oligospermia . For each group, we evaluated the mean sd of seminal parameters . In order to define the role of treatment in male subfertility, we also evaluated sperm parameters at admission and after the therapies, the last carried out before conception, in the group with oligoasthenoteratospermia (oat). The average age of the male population was (mean sd) 36.05 5.73 yrs, and of the female population was 33.62 4.65 yrs, in the 453 couples that conceived . All the pregnancies occurred within 48 months after the first evaluation at our institute (table 1). Known causes of male infertility were identified in 314 patients (69% of the pregnancies) (table 2). Two causes of infertility were present simultaneously in 39 patients . Amongst genital tract infections, ureaplasma urealyticum, mycoplasma hominis, and gram - negative bacteria were the most recurrent (80% of the species identified). Varicocele was present in 115 patients (25% of patients), and 52 patients underwent varicocele repair (surgical varicocelectomy or scleroembolization). Normal semen analysis was present only in 35% of patients (158 pts), with sperm count 74.93 40.16 10/ml, linear motility 63.18 12.51%, and normal morphology 53.74 20.41% . 295 subfertile patients (65%) showed alterations in at least one seminal parameter, as reported in figure 1 . A sperm count lower than who reference values, isolated or associated with motility or morphology alterations, was present in 116 patients (26% of the total pregnancies). When stratified according to the grade of oligospermia, 13 patients presented mild oligospermia, 71 had moderate oligospermia, and 32 presented severe oligospermia (table 3). Isolated asthenospermia was the most frequent semen abnormality and was present in 123 patients (27% of the total pregnancies). Semen parameters in the asthenospermic group were sperm count 61.23 34.79 10/ml, linear motility 33.09 11.25%, and normal morphology 55.51 20.40% . Isolated teratospermia was present in 19 patients (4% of the total pregnancies) and was associated with other seminal alterations in 80 patients (18% of the total pregnancies). Normal sperm morphology in isolated teratospermia was 22.29 2.22%, and in associated teratospermia, it was 15.28 10.40% . 41 pregnancies were obtained by men with oat (9% of the total pregnancies). Sperm count in oat group was 8.93 5.12 10/ml, linear motility was 24.36 12.67%, and normal morphology was 13.00 8.38% . In the oat group, aethiological causes of male infertility were identified, isolated or associated, and treated in 31 patients . Male genital infections were appropriately treated in 28 patients, whereas hormonal disfunctions involving dysthyroidism, hypogonadism, and pituitary disfunctions were treated in 8 patients . Varicocele was treated in 3 patients . When comparing seminal analysis before and after aethiological therapies in the oat group, an increase in sperm count was observed in 3 patients, while an increase in linear motility was present in 15 patients . In 10 patients with idiopathic oat, empirically different kinds of pharmacological treatments were tried: hmg / hcg, antioxidants, carnitine, and aspartic acid . The group of 52 patients who underwent varicocele repair, sperm count (mean sd) at admission was 9.06 4.34 10/ml, linear motility was 21.13 7.12%, and normal morphology was 18.74 3.93% . Semen analysis after surgery in the varicocele group showed a significative increase in sperm concentration (14.24 3.12 10/ml) and normal morphology (22.62 3.05%) while a nonsignificative increase was observed in sperm motility (25.31 8.27%). Subfertility is a common condition affecting at least 15% of couples during their reproductive lives, and in half of these, a male factor is involved . Recent evidence has questioned the clinical value of who criteria for basic semen analysis in the prediction of fecundity . We report 453 pregnancies which occurred between january 2004 and june 2009 among 2935 infertile couples evaluated at our institute . In the infertile couples, we applied a standard protocol in terms of investigation and therapeutical management of couples affected by infertility, performed in both partners in a sequential and parallel way . This was achieved by a unified clinical management of the couple, which includes gynecologists, endocrinologists, and andrologists, with specific interest in the field of human reproduction . Our data supports the possibility of spontaneous conception with semen parameters below the who reference values, as documented by the 65% of pregnancies that occurred in our center . A sperm count reduction was, in fact, observed in 116 patients (26% of the total pregnancies). 32 patients (27%) within the oligospermic group presented severe oligospermia showing a sperm count of 3.58 1.54 10/ml . An overlap between fertile and infertile patients in sperm concentration was previously reported [4, 6, 7]. Even though bonde reported a predictive value for sperm concentration with increase up to 40 10/ml, we present a group of severe oligospermic men in couples who conceived spontaneously . Astenospermia was the most frequent semen abnormality, both when evaluated as isolated or associated with other semen abnormalities . Previous studies on sperm motility as a predictor of infertility have proven contradictory [4, 6, 10, 11, 20]. This data seems to indicate that astenospermia may be a surmountable condition in infertile couples . A predictive value for normal morphology was previously reported [4, 6, 7, 10, 20] although only moderate predictive value for spontaneous pregnancy can be given . Our data underscore the possibility of spontaneous conception also with reduction in normal sperm morphology . When we consider the percentage of normal morphology, it was near the lower end of reference values (22% for isolated teratospermia and 15% for associated teratospermia). A conception with severe teratospermia might be improbable; therefore, we confirm that morphology could be considered the best predictive parameter for male fertility . We reported 41 pregnancies which occurred in men affected by oligoastenoteratospermia, even though bonde and ombelet described an increase in predictive value when altered seminal parameters were considered in combination with each other [7, 8]. These data highlight that seminal parameters, even when considered together as a whole, cannot exclude the possibility of a spontaneous conception . For these reasons, we sustain that semen parameters cannot lead to a clear - cut discrimination between fertile and infertile men . Our data call into question the predictive value of who reference values as a diagnostic test, which are not always predictive of fertility . The who reference values were drawn up considering a population of healthy men, disregarding the absolute minimal values of the semen parameter necessary in order for conception to occur . As a consequence, none of these parameters, considered alone or in combination, can be considered diagnostic of infertility . In order to improve the clinical value of seminal parameters, who recently revised its laboratory manual for the examination and processing of human semen . In this edition, the lower reference limit for seminal parameters is given at the 5th percentile in a population of men in couples who conceived within 12 months . The new who manual, considering as normal population the male of proven fertility, reduces previous reference values and is in agreement with our evidence of spontaneous conception by men with reduced seminal parameters . To date, there is no threshold in semen parameters which detects and predicts male fertility . New molecular insights into sperm properties which make it capable of fertilizing the egg are recently emerging . Increased knowledge of sperm proteome will allow us to have new predictors of molecular index in male fertility . According to medical literature, 3040% of patients do not receive care based on available scientific evidence . On top of this, an estimated 2025% of provided health care is unnecessary . In the case of subfertility, this could mean the use of unnecessary and expensive diagnostic tests and assisted reproductive techniques (arts). It is debated how long patients should be treated with medical and surgical therapies, strictly according to current clinical guidelines, when they present a significant range of uncertainty currently due to an incomplete understanding of etiology and physiopathology of a clinical condition . Reported in a multicenter randomized trial significantly higher live birth rates per in vitro fertilization (ivf)/intracytoplasmatic sperm injection (icsi) cycle when compared with no treatments for three months in women with fallopian tube patency . Another trial compared the effectiveness of immediate ivf with six - month delayed ivf in couples with all causes of infertility . Patients in the control group were permitted to have any form of fertility treatment other than ivf . Significant differences were observed in pregnancy rates per couple (17% with immediate ivf versus 8% with delayed ivf) and live birth rates per couple (12% with immediate ivf versus 5% with delayed ivf). No details of the fertility treatment received by the control group were presented . A further randomized trial compared early ivf with late ivf (after six months) in couples with all causes of infertility . Intention to treat analysis of all causes of infertility showed no significant differences in clinical pregnancy rates per couple (10% with immediate ivf versus 7% with delayed ivf), nor in live birth rates per couple (9% with immediate ivf versus 5% with delayed ivf). According to these evidences, uk - nhs national institute for health and clinical excellence (nice) clinical practise guidelines (cpgs) for fertility assessment and treatment for people with fertility problems (2004) reported that the decision to recommend ivf treatment should take into consideration the likelihood of spontaneous pregnancy without treatment, in particular in cases where significant spontaneous pregnancy rates may be expected . As a consequence, according to these cpgs, severe alterations in seminal plasma might be addressed immediately to ivf treatments . In this paper, we report a conception in 453 couples with infertility, treated with only medical and surgical therapies; some of these presented severe alterations in seminal parameters . The mild improvement of seminal parameters in 18/31 patients affected by severe combined alterations in sperm count, motility, and morphology, via etiological treatments, suggests that no patient should be excluded from a complete diagnostic evaluation and etiological therapeutic opportunity, even in presence of severe alterations in seminal parameters . Therefore, the significant improvement in sperm concentration and morphology in selected patients who underwent varicocele repair underlines that varicocele repair may represent an effective option to the fertility . To date, unique cpgs have not yet been developed regarding the indication and the timing for ivf techniques in relation to seminal parameters . These data should be therefore taken into consideration in evaluating current cpgs for couple infertility . The present analysis raises two levels of considerations: the epistemology of the cpg in male infertility / subfertility treatment and the ethical issues related to this matter . It is well known that the cpgs are a set of recommendations based on evidence as much as possible, able to change the behavior in specific clinical scenarios . Particularly, the literature about male infertility shows that few clinical trials with pregnancy as the main outcome measure have been published . Consequently, who - cpg for male infertility are limited and in this area clinical practice is merely dominated by authority - based guidelines, even though who reference clinical parameters are the most widely used criteria to define a male factor . Therefore, some points to be taken into consideration with regard to ethical issues concerned in male infertility treatment are as follows . Firstly, the actual uncertainty of cpg on male infertility treatment (mit): it is ethically correct to reach the infertile / subfertile patient / couple's best interest not in one - way only, that is, through the automatic shift towards art, but also utilizing other convenient remedies, according to a clinical logic of graduality since it is sometimes possible to conceive a baby without recurring to art . Secondly, the dutifulness of an adequate counselling setting: in fact, the more recent literature [3032] shows the need for psychological and ethical counselling for infertile / subfertile (naive and treated) couples . 40/2004 supplies the couple with realistic information to face all the issues involved (benefits, risks, costs, clinical alternatives, and psycho - social / emotional aspects), ethical issues enclosed, because it is strictly linked to both patients' personal values / preferences and physicians' personal / professional values . At this condition, it would be possible to create and safeguard an adequate relationship, and we could contribute in increasing the autonomy of patient's mature and responsible choices, without leaving him / her alone . Thirdly, the need for a further development of alternative remedies to solve / overcome fertility problems other than art . Finally, the ethical reflection on mit also pursued through an appropriate use of clinical ethics committees could contribute to give meaning to objective limitations of cpg, granting further value to the human subject . This is one more reason to not immediately address the choices towards art, without first considering the relevant issues on distributive justice, fairness, and prioritization criteria of such expensive procedures [3436]. The role of the endocrinologist / andrologist is primary and necessary for a complete diagnostic evaluation and etiological therapeutic opportunity, as reported in a large series of pregnancies achieved in a single tertiary - care center experience . We confirmed previous data about the impossibility for semen parameters to lead to a clear - cut discrimination between fertile and infertile men . As a consequence, no patient should be automatically shifted towards art, even in presence of severe alterations in seminal parameters . Art techniques should be considered after a sufficient time of etiological therapies, also according to ethical criteria of distributive justice, fairness, and prioritization . Therefore, unique cpgs have not yet been developed regarding the indication and the timing for ivf techniques in relation to seminal parameters . These data should be therefore taken into consideration in evaluating current cpg for couple infertility . In this context, further studies are therefore needed in this topic in order to identify new molecular predictors of male fertility, which might solve this ancient dilemma about clinical decision in male infertility.
Perennially frozen and glacierized high - alpine areas react very sensitively to changes in air temperature and even small differences in temperature determine their frozen status . As a consequence, glaciers in the european alps, for example, have lost 3040% of surface area since the end of the little ice age (middle of 19th century), and the lower permafrost limit is estimated to have risen by 1 m per year . Discharge from melting glaciers and thawing permafrost can be highly enriched in solutes due to prolonged interaction with fine - grained rocks and freshly exposed mineral surfaces . Solute outflows from catchments characterized by cryospheric features can cause serious changes in water chemistry downstream . A survey of remote high altitude lakes in the european alps has provided strong evidence of a substantial increase in base cation and sulfate concentrations in lake water over the past few decades, especially in lakes from glaciated areas . The increase of solutes in lake water was attributed to an enhanced weathering resulting from climate warming . Until now, very little research has examined how changes in the mountain cryosphere affect downstream water quality, including the threats to ecosystem health and human use . Recent studies that investigated water quality in alpine watersheds draining mineralized areas with sulfide - bearing lithologies demonstrate that retreating glaciers and degrading permafrost may adversely affect downstream ecosystems receiving meltwater runoff . The exposure of fresh surfaces of sulfide - rich rocks to air and oxygenated water by retreating glaciers or degrading permafrost increases the oxidation of sulfide minerals, which is responsible for the generation of natural acid rock drainage (ard). Ard typically produces acid - sulfate waters enriched in fe, al, mn, and various trace elements such as cu, ni, pb, zn, and others . Concentration of the dissolved minor and trace elements in natural ard systems generated by retreating glaciers or degrading permafrost can be comparable to ard associated with mining activity . Some of the mobilized trace elements associated with ard pose a potential hazard to environmental quality and human health . Although ice loss in permafrost can be orders of magnitude slower than in a glacier, the total permafrost area usually exceeds the glacier - covered area in mountain environments and can contribute substantially to the hydrology of alpine catchments . For example, the perennially frozen area in the european alps is approximately three times larger than the glacier - covered area . Despite the fact that rock glaciers are one of the most common geomorphological expressions of mountain permafrost, still little is known about the geochemical content, especially about the trace - element composition of supra- and subpermafrost meltwater that drains rock glaciers . Until recently, most of the research conducted on rock glaciers primarily focused on their movement, origin, internal structure, and hydrology . However, the impacts of rock glacier solute fluxes on lakes downstream still remain largely obscure even though more than half of the world population relies on fresh water from mountain areas . Unexpected high nickel and manganese concentrations exceeding the appropriate eu limits for drinking water by more than an order of magnitude have recently been reported in a study investigating temporal trends in water chemistry of a remote high - mountain lake, lake rasass, situated at the toe of an active rock glacier the european alps, italy . The high concentrations of mn and ni were observed in the lake water enriched in sulfates (more than 4400 equivl). In contrast, negligible concentrations of these metals were recorded in the adjacent pond without a rock glacier in the catchment . In an earlier study, application of paleoecological techniques provided strong evidence of acidic conditions in lake rasass (ph 5.86.5) during at least the past two centuries . The overall results of the previous studies focused on the lake give evidence of naturally occurring ard generated by the rock glacier in the lake rasass catchment . The present study aims to explore the effects of rock glacier solute fluxes on a high alpine lake within a crystalline - rock watershed . We applied comparative research design in the study of two neighboring water bodies, lake rasass and the adjacent pond, with and without a rock glacier in their mineralized catchments having similar soil conditions and underlying geology . In addition, we used chironomid - based paleolimnological techniques to track long - term temporal trends in the ecotoxicological state of both water bodies and to establish their baseline conditions . Depth = 9.3 m) and the adjacent pond (rpd; surface area = 0.08 ha, max . Depth = 1.6 m) are remote, fishless water bodies situated just 50 m away from each other, above the actual and historical timberline in the upper vinschgau valley (2682 m a.s.l .) In the central eastern alps, italy (figure 1). The bedrock geology of the valley belongs to the austroalpine tztal - stubai crystalline complex, and the average mineral composition of the bedrock is quartz (50%), feldspar (27%), muscovite (15%), chlorite (6%), and dolomite (2%). The chemical composition of the bedrock is characterized by sources of disseminated water - soluble sulfate (180 g g dry weight (dw); total sulfur is 590 g g dw) and metals, including trace elements mn (597 g g dw), ni (24 g g dw) and zn (90 g g dw). The concentrations of mn and zn are comparable with their earth crustal abundances, while ni content is more than three times lower . Soil cover is sparse and bedrock is exposed in 80% of the ras and rpd catchment areas . The active rock glacier extends on a north - facing slope down to the shore of ras and occupies 18.5% of its catchment area which adjoins the permafrost - free catchment of rpd . Both water bodies lacking inflow streams have well - developed outflows . Near the rpd shoreline, there are visible sources of groundwater that infiltrates onshore discharges . Since the rock glacier is located only a few meters upslope from ras, its internal drainage system, a common feature of rock glaciers, is most likely connected to the lake, and meltwater seeps from the rock glacier into ras through cracks and fractures in the bedrock . Map showing the location of the study site (a) and photograph of lake rasass (ras) and the adjacent pond (rpd) and their catchments (b). The map of italy is reproduced from http://d - maps.com/. Since 1980, summer and cold - season (october may) temperatures in the european alps show a simultaneous, strong increase, which is unprecedented over the last millennium . A multifold increase in concentrations of the dominant ions (mg, so42 +, and ca) and electrical conductivity in the ras water over the last two decades (19862005), recorded by thies et al ., may reflect current permafrost degradation in the rock glacier under the recent warming . Monnier and kinnard assume that indications of degrading permafrost in a rock glacier may also be the near - melting - point (0 c) thermal regime of permafrost year - round and a high water content (> 40%) in deeper parts of a rock glacier . As solutes concentrate and the freezing point decreases during fluid freezing, it is most likely that highly concentrated salt solutions seep from the rock glacier into ras through the cold season . Both water bodies were sampled for limnochemical analyses at least twice during the open - water seasons of 20102012 . Water samples were taken with a patalas - schindler sampler (uwitec, austria) at 2-m intervals along a vertical profile in ras and from 0.5 m water depth in the shallower rpd . The physicochemical parameters of water, such as conductivity, ph, alkalinity, and concentrations of major ions and nutrients were determined following standard methods and analytical quality control procedures as described in mosello and wathne . Metals (ag, al, as, ba, be, cd, co, cr, cu, fe, mn, ni, pb, sr, ti, v, zn) in water samples were assayed by inductively coupled plasma - optical emission spectrometry (icp - oes) with standard addition methods . Sampling strategy was designed to compare metal concentrations in surface sediments and biota of the water bodies as follows: the ras and rpd shallow waters against the ras deep water, and between shallow waters of ras and rpd . To provide background information for the sampling in the relatively deep ras, bathymetric distribution and substratum associations of the most abundant macroinvertebrates were studied in the lake along a depth transect (supporting information (si), figure s1). Twenty macroinvertebrate samples were collected with an ekman grab sampler (in deep water) and a kick net (in the upper littoral) at 0.51.0 m depth intervals along the east west transect across the deepest point in the lake . Surface sediment (01 cm) samples were collected using an ekman grab sampler with a lining of plastic inside from three random sites in shallow waters (0.51.5 m depth) of both water bodies and in deep water (68 m depth) of ras . The sediment and biota sampling for subsequent multielement analysis was conducted in the early open - water season of 2012 . For multielement analysis of body tissues of aquatic organisms, larvae of the chironomid midge pseudodiamesa nivosa (diptera: chironomidae), the oligochaete tubifex tubifex (oligochaeta: tubificidae), and the aquatic moss warnstorfia exannulata (hypnales: amblystegiaceae) were collected with a biological bottom dredge (efe & gb nets, uk) in the ras deep - water habitats (68 m depth). Larvae of the chironomid p. nivosa were also collected in rpd (0.51.5 m depth). The predatory aquatic beetle agabus bipustulatus (coleoptera: dytiscidae) was collected with a kick net in shallow - water habitats (0.51.5 m depth) of both water bodies . Invertebrates were held in lake water at field temperatures for transport to the laboratory where they were sorted according to species, cleaned with distilled water, placed in petri dishes and kept at 4 c for 24 h in order to purge the guts . Thereafter they, as well as the aquatic moss w. exannulata, were rinsed with distilled water and frozen pending analyses . Only plastic storage vessels and tools (tweezers, scapulae, etc .) Were used . All plastic labware was soaked in 1% nitric acid for at least 24 h and rinsed with doubly deionized water to leach out metal ions . All sediment and biota samples were dried in an oven at 25 c to a constant weight (34 days) and homogenized with an agate mortar and pestle, and triplicate 0.5 g aliquots of every sample were used for the analysis . The samples were dissolved with concentrated nitric acid using the microwave digestion technique . A total of 42 elements (ag, al, as, ba, bi, br, ca, cd, cl, cr, cu, fe, ga, ge, hf, i, k, mg, mn, mo, na, nb, ni, p, pb, rb, s, sb, se, si, sn, sr, ta, th, ti, tl, u, v, w, y, zn, zr) were assayed by energy dispersive x - ray fluorescence (edxrf) analysis using calibration models based on fundamental parameter methods . In the sediment samples, ten major elements, si, al, fe, mn, mg, na, ca, k, ti, and p, were measured as their oxides . Analytical accuracy was verified by the use of ccrmp (canadian certified reference materials project) reference samples . Sediment cores were taken using uwitec gravity and piston corers (uwitec, austria) in the deepest point of both water bodies in the summer of 2011 . The 119.2 cm long sediment sequence from ras was sectioned contiguously into 156 samples: at 0.220.50 cm resolution within the top 24 cm and at 1 cm resolution from 24 cm to the bottom . The 15.5 cm long sediment sequence from rpd was sectioned contiguously every 0.5 cm, resulting in a total of 31 samples . Chronological control for the sediment sequences was provided by accelerator mass spectrometry (ams) radiocarbon dates derived from terrestrial plant macrofossils: six ams c dates from the ras sequence and four dates from the rpd sequence . The bayesian statistical method of the oxcal software package version 4.2.1 was used to produce age - depth models (si figure s2). According to these estimates, the ras and rpd sediment cores span time periods of 10 200 years and 3200 years, respectively . The ages are expressed in calibrated years before present (cal yr bp = calibrated years before ad 1950) (see si 1 for further details). Incidence of mouthpart abnormalities in chironomid larvae was used as measures of toxic stress in the water bodies . Occurrence of mouthpart deformities in chironomids is a sublethal effect of larval exposure to environmental stress factors . Since mouthpart deformities may also be induced by toxic elements and their compounds not included in routine chemical analyses, they may better reflect environmental quality than chemical analysis alone . Assessment of these morphological abnormalities offers an effective bioindication of toxic stress in modern freshwater ecosystems and over previous time periods . The mentum and other mouthparts of chironomid larvae damaged during the cleaning and mounting process usually have abrupt breaks that are clearly visible and easily distinguishable from deformed structures . The chironomid - based paleolimnological study was designed to track and compare past changes in incidence of larval mouthpart deformities in identical chironomid taxa from ras and rpd . To provide background information on the structure of the subfossil chironomid assemblages, sediment core samples from both water bodies were analyzed for species composition of the assemblages following standard procedures outlined in brooks et al . Larval head capsules of the two most common chironomid taxa, pseudodiamesa nivosa and micropsectra radialis - type, found in both dredge and sediment core samples (live specimens and their subfossil remains, respectively) from ras and rpd (si figures s1 and s3) were inspected for deformities of the mentum, a mouth part which is typically well preserved in subfossil material . Three biometric variables, head length, head width, and mentum width, were measured for each head capsule . These variables were used to assign each head capsule to a specific larval stage of development (instar). A minimum of 100 third- and fourth - instar head capsules of each of the two chironomid taxa, p. nivosa- and m. radialis - type, were inspected for mentum deformities in each sediment sample at 250400 magnification using a compound microscope . The severity of mentum deformities was categorized into three classes according to lenat: class i slight deformities, such as fused teeth; class ii more conspicuous, moderate deformities, such as extra teeth, missing teeth, large gaps, and distinct asymmetry; and class iii severe deformities, including at least two class ii characteristics . Head capsules of live larvae were treated and inspected for mentum deformities in the same manner as their subfossil remains . One - way analysis of variance (anova), followed by bonferroni post hoc test, was used to determine whether there are any significant differences in the elemental composition of water, sediment, and biota between the two adjacent water bodies, as well as between the shallow and deep - water habitats of ras . All data were square root transformed to reduce the biasing effect of extremely low or high element concentrations . The nonparametric mann whitney u - test was used to detect significant differences in the frequency of chironomid deformities between different time intervals in the holocene . Chemical analysis of water samples revealed that the ratios of dissolved inorganic nitrogen (din = no3-n + nh4-n) to total phosphorus (tp) exceed 100 by mass in ras and rpd (see si 3 for more details). This suggests that phytoplankton communities in both water bodies are phosphorus - limited, defined by bergstrm as din: tp> 3.4 by mass . Physico - chemical analysis of water taken from ras and rpd in 20102012 indicated that the striking differences in limnochemistry of these two neighboring water bodies, recorded in 2005, persisted to the present day (table 1). Although the water in both water bodies is dominated by the same ions (ca, mg, and so42), the total ion concentration and electrical conductivity in ras (10 500 equiv . L and 510 s cm, respectively) are more than an order of magnitude greater than in rpd (780 equiv . The concentrations of many dissolved minor and trace elements (al, cu, mn, ni, sr, zn) are negligibly small (one - way anova, bonferroni post hoc test, p <0.05) in the circumneutral rpd (ph 6.196.64) compared to the acidic ras (ph 4.935.86) (table 1). However, in the acidic ras, an extensive area of the lake bottom at 5.08.5 m water depth is coated with orange - colored iron oxyhydroxides (si figures s1). It is known that under mildly acidic conditions in surface waters affected by ard, the soluble ferrous iron oxidizes to ferric iron, which precipitates as a ferric oxyhydroxide . Ras data for 20112012, and rpd data for 20102012 . For each parameter, values (mean se) that share common letters (k, m, or n) do not differ significantly (one - way anova and bonferroni post hoc test, p <0.05). Values are for the upper (05 m depth) and lower (59 m depth) parts of the ras water column . Na: not available, i.e. Parameter is not regulated by the european drinking water directive; parameter values exceeding the appropriate european union limit values for drinking water are shown in bold . Nh4-n = ammonium - nitrogen . In the ras catchment dominated by crystalline basement rocks with an inherently low acid buffering capacity, enhanced cryogenic weathering attributed to ice of the active rock glacier is the most likely mechanism causing groundwater inputs of acidic, metal- and sulfate - enriched water to the lake . Cryogenic weathering of the local bedrock, which involves a plethora of physical, chemical and biological processes (ice segregation, freeze thaw cycles, volumetric expansion of water, hydration etc . ), results in the exposure of fresh bedrock surfaces to air and water in a complex rock glacier system and leads to oxidation of sulfide minerals and the generation of ard . A solute - rich inflow sinks, as a rule, down the water column due to its high density . This effect can result in physical and chemical stratification of the water column which reduces water circulation and aeration of deep - water layers . Our data show that strong stratification develops in ras during periods of prolonged ice cover (89 months). After the ice breakup, the concentrations of major ions (ca, mg, so42 +) and minor and trace elements (al, mn, ni, sr, zn) in the upper water layers (05 m) are four to seven times smaller than in the deeper waters (59 m), and the ph decreases from 5.48 near the surface to 5.07 at depth (table 1 and figure 2). Depth profiles of selected physical and chemical properties of the ras water . Electrical conductivity (a), ph (b), total ion (c), calcium (d), magnesium (e), and sulfate (f) profiles in the ras water column on fourth july 2012 (closed circles; the lake was 70% ice covered) and 29th august 2012 (open circles; 1.5 months prior to lake ice freeze - up). Thus, the short growing season in ras begins under extremely unfavorable conditions for aquatic life, especially in the deep water, and the lake water may be dangerous for human consumption . In fact, the concentrations of manganese, nickel, aluminum, and sulfate in the deep water (59 m) exceed the appropriate eu limits for drinking water 32, 23, 7, and 2 times, respectively (table 1). Although solutes become more homogeneously distributed in the water column by late august / early september due to destratification by wind - induced mixing and convection (figure 2), concentrations of manganese, nickel, and aluminum along a water depth gradient remain considerably higher (520820, 140220, and 440620 g l, respectively) than the eu maximum permissible levels in drinking water . Total metal concentration, however, does not necessarily correspond with metal bioavailability and toxicity to aquatic organisms . The proportion of the total metal content that is available for uptake by biota depends greatly on the chemical form in which the metals are present . Metals in aquatic systems can exist in different chemical forms, including the dissolved inorganic complexes with dissolved anions (e.g., hydroxides, sulfates, carbonates) where metals occur as free hydrated metal ions, organic complexes with dissolved organic matter, and variety of particulate forms incorporated into the matrix of solid organic or mineral particles . The free ionic form of metals is the most relevant for uptake by biota and hence the most toxic to aquatic life . Hydrogen ion activity (ph) and redox potential greatly affect metal availability because of their strong influences on metal partitioning and speciation . Numerous studies have shown that the free metal ion concentrations increase with decreasing ph as well as upon oxidation of initially reduced environments . Thus, both processes, namely winter stratification resulting in low ph in the deep waters (ph 5.1), and oxygenation of the metal - rich hypolimnion and sediment during the open - water season, may result in greater bioavailability and toxicity of metals to biota in ras . The concentrations of most metals in the sediments of ras and rpd are comparable with their abundances in the earth s continental crust (figure 3). Rather high concentrations of lead and zinc in sediments of both water bodies may be attributed to the site - specific geologic conditions, whereas elevated nickel and copper concentrations (244 and 179 g g, respectively) in the ras deep - water sediments are likely to be caused by enhanced accumulation of fine - grained particles in the deep water . These particles provide a large surface area for the sorption of metals at the water - sediment interface . Concentrations (mean se; n = 3) of copper (a), zinc (b), lead (c), nickel (d), chromium (e), aluminum (f), iron (g), titanium (h), manganese (i), and vanadium (j) in surface sediments and biota of rpd and the ras shallow and deep waters . For each metal, means followed by the same letter are not significantly different (one - way anova, bonferroni post hoc test, p <0.05). The abundances of the chemical elements in the earth s continental crust are shown with green lines . Concentrations of 10 elements (cu, zn, pb, ni, cr, al, fe, ti, mn, and v) were significantly higher (one - way anova, bonferroni post hoc test, p <0.05) in the larvae of pseudodiamesa nivosa, omnivores that feed on a wide variety of foods from detritus to small invertebrates, collected from the ras deep water than from rpd unaffected by the rock glacier solutes (figure 3). This suggests a high bioavailability of these metals in the ras deep - water habitats . The aquatic moss warnstorfia exannulata, which prefers mildly acidic conditions, have colonized an extensive area of the ras bottom at 58 m water depth (si figures s1). Increased concentrations of nickel and aluminum in the w. exannulata tissues were comparable to those recorded in the chironomid larvae from the ras deep - water habitats (figure 3). They are tolerant of metal contamination, long - lived, and have large cationic exchange properties within the cell wall . Owing to the lack of roots, metal uptake occurs straight from the water where they develop . Among all benthic organisms analyzed from the ras deep water, the highest concentrations of cu, zn, and pb were recorded in the body tissues of the sediment - ingesting oligochaete tubifex tubifex (one - way anova, bonferroni post hoc test, p <0.05). The cu concentrations in the t. tubifex tissues were 2.5 times greater (one - way anova, bonferroni post hoc test, p <0.05) than that in surrounding sediments (figure 3). Ingestion of contaminated sediment has been shown to be the major source of particle - sorbed metals to sediment - feeding organisms . The cu concentrations in the predatory beetle agabus bipustulatus, which is common in shallow - water habitats of both water bodies, were also significantly higher (one - way anova, bonferroni post hoc test, p <0.05) in ras than those from rpd (figure 3). Results of a recent study demonstrate that predatory aquatic beetles are capable of reflecting trace elements bioaccumulation in habitats with different disturbance levels, although their innate ability to take up / excrete metals may vary widely between species . Inspection of modern pseudodiamesa nivosa and micropsectra radialis - type larvae, represented by live specimens and their recent remains in surface sediments, for deformities of mentum revealed the morphological abnormalities in the ras p. nivosa population . No mentum deformities were noted upon inspection of p. nivosa from rpd and m. radialis - type from both water bodies . Although mentum deformities in micropsectra larvae have been recorded at some metal - contaminated sites, it is likely that the p. nivosa larvae are more susceptible to the harmful effects of acidic, metal - rich environments than the m. radialis - type larvae . Incidence of mentum deformities in the live p. nivosa larvae from ras varied from 81% to 87% (n = 3) and was comparable with the frequency of mentum deformities among recent remains of p. nivosa in the ras surface sediments (7887%, n = 3). The observed deformities were categorized into two classes following lenat: class i fused median tooth (3034%, n = 6), and class ii large median gap (4855%, n = 6) or extra teeth (<2%, n = 6) (figure 4). Mentum abnormalities are generally related to a physiological disturbance during the molting process, as a somatic response to a wide range of environmental stress factors, but not to a genetic heritability of larvae . In many polluted sites, the total incidence of deformities in chironomid populations usually varies between 20% and 50%, but the incidence of abnormalities in the ras p. nivosa population (7887%) was as high as that recorded in a population of chironomus spp . (83%), another chironomid taxon, in a site heavily polluted by radionuclides and trace metals . Micrographs (200 magnification) of larval menta of the chironomid pseudodiamesa nivosa from the ras modern and subfossil material: normal mentum (a) and deformed menta (b e). The severity of the observed deformities was categorized into two classes following lenat: class i (slight deformities)fused median tooth (b, c), and class ii (more conspicuous deformities)large median gap (d) and extra teeth (e). The analysis of chironomid remains in core sediment samples showed that a high total incidence of p. nivosa mentum deformities of 70% persisted in ras during the last 2.5 millennia and was significantly lower (mann whitney u - test, p <0.0004), around 40%, in the early holocene (10 1509800 cal yr bp) (figure 5). The incidence of the class ii mentum deformities (median gap or extra teeth) in p. nivosa from ras was also significantly higher (mann whitney u - test, p <0.0008) in the late holocene (49%) than in the early holocene (21%). Unfortunately, the cold - stenothermal p. nivosa was absent in ras, like other high alpine lakes, through a relatively warm interval of the early and mid - holocene (figure 5, si figure s3), and this species cannot be used for reconstruction of temporal trends in the ecotoxicological state of ras over the entire holocene . In rpd, the frequency of p. nivosa mentum abnormalities did not exceed 2% (only the class i mentum deformities was recorded) through the last 3.2 millennia, or, in other words, it did not exceed the frequency of chironomid deformities found in natural unstressed environments (up to 1214%). Relative abundance of pseudodiamesa nivosa in chironomid assemblages and the total incidence of p. nivosa mentum deformities in the sediment records from rpd (a) and ras (b), and holocene july air temperatures (tjuly) reconstructed for the eastern alps (c). The results provide evidence that the present adverse conditions in the lake are not unprecedented in its holocene history and cannot be associated exclusively with global warming and enhanced thawing of the rock glacier in recent years . It is likely that the development of the rock glacier started at the onset of the holocene, when the area occupied by the modern rock glacier became deglaciated . The formation of ras at the toe of the rock glacier most probably began at the onset of acidic drainage in the freshly deglaciated area . Overall, our findings illustrate that natural ard systems generated by active rock glaciers in mineralized areas with sulfide - bearing lithologies may seriously disturb lake ecosystems and endanger the quality of drinking water in pristine alpine environments . More adverse effects may be expected in lakes situated at the toe of greater and more active rock glaciers than the one in the ras catchment.
Allergy to grass pollen is one of the most common inhalant allergies in the western world . In an unselected healthy population it has been found that 8 to 21% of children and 13% of adults are sensitized to grass pollen . In selected populations of allergic subjects 44% are allergic to grass pollen . Allergic rhinoconjunctivitis has been identified as one of the main reasons for visits to primary care clinics, and although usually not regarded as a severe disease it significantly limits the social life of the subject and affects school learning performance and work productivity . Allergen - specific immunotherapy is the practice of administering to allergic patients increasing amount of allergen in order to obtain hyposensitization . Allergen - specific immunotherapy is considered the only causal treatment of allergic diseases such as allergic rhinitis asthma and insect venom allergy . The purpose of allergen - specific immunotherapy is to expose the patient to the allergen that causes the allergic symptoms, in order to increase the tolerance to this allergen and reduce symptoms . Mechanisms of action of allergen - specific immunotherapy are not so far clearly identified; however, data are available that allergen - specific immunotherapy can induce an increased production of allergen - specific igg4 and il-10 . Alternative mechanisms include immune deviation in favour of th1 responses and apoptosis and/or anergy of antigen - specific t cells . Allergen - specific immunotherapy exerts its beneficial effects over long periods (i.e., weeks or months), and 3 - 4 years of treatment are required to obtain a favourable clinical and immunological response . The treatment with allergen - specific immunotherapy is currently administered as subcutaneous (s.c . Allergy vaccination program requires an updosing period followed by a maintenance period of 35 years . This implies that only a fraction of allergic subjects are actually offered allergy vaccination despite the fact that allergen - specific immunotherapy is the only treatment modality that changes the natural cause of the allergic disease and thereby prevents its exacerbation . Allergen - specific sublingual immunotherapy (slit) has gained wide acceptance in many european countries and has raised the level of interest in immunotherapy among practicing allergists and primary care physicians . Large pivotal double - blind, placebo - controlled, randomized clinical trials have confirmed the efficacy and safety of slit . Allergen - specific sublingual immunotherapy with grass products has also had a widespread application especially in southern europe throughout the past 30 years . The general recommendation today is to apply a higher than the accumulated dosage applied subcutaneously . Grass allergy immunotherapy tablet (ait) (grazax; alk denmark) has been developed for allergen - specific immunotherapy . Grazax is formulated as an orodispersible tablet for sublingual use and contains a standardised allergen extract derived from extraction and purification of the source material, phleum pratense timothy grass pollen . To obtain an optimal therapeutic response with immunotherapy maximal compliance can improve the patient's condition and also result in a reduction in drug costs . Conversely, poor compliance may result in the physicians adding in more medications to treat the patient's condition, which may make the problem worse . Compliance to allergen - specific immunotherapy could be negatively influenced by several factors such as: duration of treatment, side effects, especially in the initiation phase, and need to take medication also outside the pollen season period when the patient in general does not have any symptoms . Specific allergen slit is a long - lasting home treatment that is directly managed by patients and parents . Therefore, as allergen - specific slit is self - managed at home without direct supervision, adequate compliance with this administration route is important . The primary objective of the trial was to evaluate if compliance of once daily dosing with grass ait in adult subjects with grass - pollen - induced allergic rhinoconjunctivitis could be increased by providing patients with compliance device (memozax) (figure 2) given from the beginning of immunotherapy in comparison with patients without the memozax . Secondary endpoints of the trial were to evaluate safety and tolerability of grass ait treatment and finally to evaluate the tolerability of the first dose intake of ait and to evaluate after 48-week treatment with grass allergy tablet tablets the impact on symptom score and patient's acceptance in comparison with previous pollen seasons . This was a 23-centre, single - dose, randomized parallel - group, open - label, controlled trial . Enrolled patients were adult (> 18 years), men or women, suffering from mild or moderate / severe grass - pollen - induced allergic rhinoconjunctivitis . A total of 261 patients were screened, enrolled, and randomized to 48-week treatment with ait using the compliance aid device (memozax) or 48-week treatment with ait without the compliance device . Inclusion criteria were as follows: subjects, men and women> 18 years of age and <65 years; suffering from mild or moderate / severe grass - pollen - induced rhinoconjunctivitis (according to aria guidelines) and with a positive spt for phleum pratense extract (3 mm); every patient should give a written informed consent to participate in the trial . Exclusion criteria at randomization were as follows: current symptoms of, or treatment for, upper respiratory tract infection, acute sinusitis, acute otitis media, or other relevant infectious processes; history of emergency visit or admission for asthma in the previous 12 months; use of an investigational drug within 30 days prior to screening; previous treatment by immunotherapy with grass pollen allergen; previous treatment by immunotherapy with other allergen than grass pollen allergen; within the previous 5 years; history of anaphylaxis, including anaphylactic food allergy, bee venom anaphylaxis, exercise anaphylaxis, or drug - induced anaphylaxis; or history of angioedema . The treatment used was grazax oral lyophilisate 75,000 sq - t tablets (phleum pratense grass pollen allergen extract). The daily dose was one tablet, which should preferably be taken in the morning . The tablet was placed under the tongue and swallowing should be avoided for one minute . Eating and drinking was not allowed within five minutes after trial medication intake . The same drug taking instructions were given to both study groups (randomized to memozax or not). Concomitant medications were all medications (including rhinoconjunctivitis medications and asthma treatments) being continued by a subject on entry to the trial and all medications given in addition to the treatment during the trial . All concomitant medications should have been documented in the crf (trade name as appropriate). Further, each change in concomitant treatment (e.g., new treatment, discontinuation of treatment, and change in dosage / routine) during the trial must be documented in the same way . At each visit, any concomitant medication was recorded in the subject's notes as source data documentation and in the crf . The investigators were instructed to store the drug in an appropriate, secure area (e.g., locked cabinet) and to store it according to the conditions specified on the labels . The investigator should have to maintain an accurate record of the shipment and dispensing of trial medication in a drug accountability log, a copy of which was given to alk - abell at the end of the trial . An accurate record of the date and amount of trial medication dispensed to each subject was available for inspection at any time . The first dose was taken in the clinic and the subject stayed in the clinic for 60 minutes for observation . The blister cards were packed in visit specific boxes . Between october 2007 and february 2008 a total of 240 subjects the subjects were randomized (1: 1) using a randomization list with half the subjects planned to receive the compliance device and half not to receive the device . Subjects were identified by ascending 2-digit randomization numbers, plus 2-digit referring to the centre and entered in the case report form . When a subject was randomized in the trial he / she had to be assigned the lowest available randomization number for that centre . The randomization number was a 5-digit number where the two first digits gave a center code . Grass allergen tablet treatment was provided at the screening / randomization visit together with the memozax, according to the randomization list . In all enrolled patients treatment started at least 3 months before the pollen season of 2008 . The primary outcome of the study was the evaluation and comparison of compliance in the two groups (with memozax and without memozax) evaluated with pill count at visits 3 (week 2), 4 (week 24), and 5 (week 48) calculated in the following manner: number of pill effectively taken / number of pill to be taken 100 . Subjects were instructed to return all residual and unused trial medications and all empty packaging at every visit . Other efficacy assessment was to evaluate after 48 weeks of treatment with grazax tablets the impact on quality of life, symptom score, and patient's acceptance in comparison with previous pollen seasons . This was evaluated, globally, through a 10 cm vas scale (0 = big improvement, 5 = not improvement, and 10 = worsening of symptoms). The safety assessments included recording of all adverse events (aes) and serious adverse events (saes) findings from physical examinations and vital signs . The trial was monitored according to sponsor company standard operating procedures for the monitoring of clinical studies and other trial - specific procedures . The trial was monitored by the sponsor or its delegate by means of on - site visits, telephone calls, and regular inspection of the crfs with sufficient frequency (every 812 weeks) to verify the following: subject enrolment; compliance with the protocol; the completeness and accuracy of data entered in the crfs by verification against original source documents; compliance in the use of imp; drug accountability; recording of adverse events . The following analysis set was defined in the protocol: full - analysis set (fas), this consists of all subjects randomized following the intent - to - treat (itt) ich principle . A scientific publication has shown that compliance to slit treatment> 90% without any device system was registered in 75% of treated patients . In this study it was hypothised that the group of patients with the memozax device should have a better compliance (a relative increase of 15% or more) in comparison with the group without the aid device (86% of patients with a compliance of 90% or more in the memozax group versus a 75% in the group without the memozax). A minimum of 120 subjects per group, with an alfa error of 0.05 and a power of 80%, therefore should be enrolled in the trial . The comparison of percentage of patients with a compliance <90% and> 90% between the two groups was performed with the fisher exact test . The subject demographic values, smoking history, and allergy disease history at baseline are summarised in table 1 . It is to note that 73% of the enrolled patients suffered from moderate / severe allergic rhinoconjunctivitis . A total of 50 patients (25 in both groups) also reported asthma (19% of fas population). Monosensitive patients (subjects with spt positive only for grass extracts) were 68 (26%) and multisensitive patients (subjects with also at least one positive spt toward nongrass allergens) were 193 (74%). The overall mean compliance rate was 91.3% (median 97%) for the subjects with complete compliance data from visit 3 to visit 5 . The primary endpoint in this trial was a comparison of the degree of compliance in the two groups (memozax and non - memozax). For this purpose compliance was categorised as excellent (90%) or less excellent (<90%). The proportion of subjects with excellent compliance in the memozax group was similar (79%) to that in the non - memozax group (78%) (table 2). Other efficacy assessment was to evaluate after 48 weeks of treatment with grazax tablets the impact on quality of life, symptom score, and patient's acceptance in comparison with previous pollen seasons . This was evaluated, globally, through a 10 cm vas scale (0 = big improvement, 5 = not improvement, and 10 = worsening of symptoms) which was performed by the patient . At visit 5 the mean the percentage of patients with a vas score = or> 5 (no - difference / worsening in comparison with the previous season) was 19% . Therefore 81% of patients reported a clinical improvement of symptoms after treatment with grazax in comparison with the previous year . Clinical efficacy was comparable both in monosensitized patients (68 out of 261: 26%) and in multisensitized subjects (193 out of 261: 74%). Investigator global clinical evaluation of 48-week treatment with grazax was good / very good: 85%; sufficient: 9%; not good: 6% . The percentage of subjects who reported adverse events (aes) in each of the two groups was almost similar, 14% in the memozax group and 11% in the non - memozax group . A total of 63 aes (78%) reported were judged as probably or possibly related to immunotherapy by the investigator, while 14 reported aes were judged as unlikely or not related to allergy tablet treatment . For 2 aes, the majority of aes (71 out of 79: 90%) were either mild or moderate, with only 4% (absolute number: 3) of ae reported as severe ae (mouth itching). The main feature of this therapeutic approach is its capacity to modify the natural history of the disease, reducing the development of asthma and new sensitizations after 3 - 4 years of treatment . Adequate compliance to allergen - specific slit is, however, mandatory in order to obtain these results . This study trial investigated the compliance of 48-week grass allergy tablet treatment in two groups of subjects, one issued with the memozax compliance device and the other not issued with the device . The compliance rate in the memozax group was slightly higher (91.7%) than that in the non - memozax group (90.3%), but the difference was not statistically significant . A total of 79% of the patients in the memozax group who completed the trial had a compliance> 90% . In the group without memozax this percentage was 78% . In the global evaluation, a total of 81% of patients reported an improvement of symptoms after treatment with grass ait, evaluated through a 10 cm vas in comparison with the previous season . Investigators evaluated the efficacy of treatment as good or very good in 85% of patients . In this is a strong indication that treatment with grass is effective in relieving these symptoms, and it is in line with results from previous grass ait trials . The safety profile seen in this trial reflects the overall good tolerance to grass allergen tablet treatment . In this study no serious adverse events or deaths were observed . In conclusion compliance to the treatment with clinical setting, the use of electronic devices is not associated with a greater compliance . In addition this trial supports the safety, tolerability, and efficacy profile observed in previous trials of this grass allergy immunotherapy tablet in grass allergic patients.
The following probe oligonucleotides were made for nf-b - dse - fret (f, 6fam; d, dabcyl; lowercase letters, single - stranded tail; underline, nf-b binding site): 5-fag ttg agg gga ctt tcc cag gcg act cac tat agg cgg tgt ctc gct cgc-3 (nf-01f), 5-agt tga ggg gac ttt ccc agg cga ctc act ata ggc acc aca cca ttc cc-3 (nf-13), 5-ggg aat ggt gtg gtg cct ata gtg agt cgc ctg gga aag tcc cct caa ctd-3 (nf-14d), 5-gcg agc gag aca ccg cct ata gtg agt cgc ctg gga aag tcc cct caa ct-3 (nf-02). To prepare probes, 0.5 m of each oligonucleotide was mixed in annealing buffer (10 mm hepes - naoh [ph 7.9], 50 mm kcl, 0.1 mm edta), denatured for 2 min at 95c, and then cooled gradually to allow annealing . Duplexes were stored at 4c and diluted with binding buffer (10 mm hepes naoh [ph 7.9], 50 mm kcl, 0.1 mm edta, 2.5 mm dtt, 10% glycerol, 0.05% nonidet p40) before use . All other chemicals were reagent grade or better and were obtained from wako (tokyo, japan). The gst - fusion recombinant human nf-b proteins (p50, p52) were expressed and purified(13) and glutathione was removed by dialysis . In general, dna - binding experiments were carried out in the following manner: 5 l nf - d1 (consisting of nf-01f and nf-14d) and 5 l protein in binding buffer were incubated in a 96-well (half - area) microplate for 30 min at room temperature . Next, 40 l nf - d2 (consisting of nf-02 and nf-13) was added and allowed to incubate for another 60 min at room temperature . Fluorescence was measured at 535 nm after excitation at 485 nm using an envision microplate reader (perkinelmer, waltham, ma, usa). In experiments with inhibitor, 0.5 l inhibitor, 5 l nf - d1, and 4.5 l protein in binding buffer were incubated before addition of 40 l nf - d2 . To obtain an ic50, the following equation was solved using the solver add - on in microsoft excel: where y is the observed fluorescence, x is the inhibitor concentration, a is the lowest fluorescence, b is the highest fluorescence, and c gives the largest absolute value of the slope of the curve . S / b was obtained by the following formula: where a is the mean fluorescence of four wells in the absence of protein and p is the mean florescence of four wells in the presence of protein . Z-factors were obtained as follows: where a is the mean fluorescence value of four wells without protein, p is the mean florescence of four wells with protein, sda is the standard deviation of a, and sdp is the standard deviation of p. the following probe oligonucleotides were made for nf-b - dse - fret (f, 6fam; d, dabcyl; lowercase letters, single - stranded tail; underline, nf-b binding site): 5-fag ttg agg gga ctt tcc cag gcg act cac tat agg cgg tgt ctc gct cgc-3 (nf-01f), 5-agt tga ggg gac ttt ccc agg cga ctc act ata ggc acc aca cca ttc cc-3 (nf-13), 5-ggg aat ggt gtg gtg cct ata gtg agt cgc ctg gga aag tcc cct caa ctd-3 (nf-14d), 5-gcg agc gag aca ccg cct ata gtg agt cgc ctg gga aag tcc cct caa ct-3 (nf-02). To prepare probes, 0.5 m of each oligonucleotide was mixed in annealing buffer (10 mm hepes - naoh [ph 7.9], 50 mm kcl, 0.1 mm edta), denatured for 2 min at 95c, and then cooled gradually to allow annealing . Duplexes were stored at 4c and diluted with binding buffer (10 mm hepes naoh [ph 7.9], 50 mm kcl, 0.1 mm edta, 2.5 mm dtt, 10% glycerol, 0.05% nonidet p40) before use . All other chemicals were reagent grade or better and were obtained from wako (tokyo, japan). The gst - fusion recombinant human nf-b proteins (p50, p52) were expressed and purified(13) and glutathione was removed by dialysis . In general, dna - binding experiments were carried out in the following manner: 5 l nf - d1 (consisting of nf-01f and nf-14d) and 5 l protein in binding buffer were incubated in a 96-well (half - area) microplate for 30 min at room temperature . Next, 40 l nf - d2 (consisting of nf-02 and nf-13) was added and allowed to incubate for another 60 min at room temperature . Fluorescence was measured at 535 nm after excitation at 485 nm using an envision microplate reader (perkinelmer, waltham, ma, usa). In experiments with inhibitor, 0.5 l inhibitor, 5 l nf - d1, and 4.5 l protein in binding buffer were incubated before addition of 40 l nf - d2 . To obtain an ic50, the following equation was solved using the solver add - on in microsoft excel: where y is the observed fluorescence, x is the inhibitor concentration, a is the lowest fluorescence, b is the highest fluorescence, and c gives the largest absolute value of the slope of the curve . S / b was obtained by the following formula: where a is the mean fluorescence of four wells in the absence of protein and p is the mean florescence of four wells in the presence of protein . Z-factors were obtained as follows: where a is the mean fluorescence value of four wells without protein, p is the mean florescence of four wells with protein, sda is the standard deviation of a, and sdp is the standard deviation of p. two partially double - stranded dna probes, named d1 and d2, are used . Each probe has a double - stranded region containing a protein binding site and two single - stranded tails . One strand of d1 is labeled with a fluorophore at the 5-end and the other is labeled with a quencher at the 3-end . The fluorophore and quencher are placed at the same end of the double - stranded region; therefore, the fluorescence of the fluorophore is quenched . The tails of d1 are complementary to those of d2, so that d1 hybridizes with d2 to form a four - way structure . As the double - stranded regions of the two probes have identical sequences, the junction of the structure migrates spontaneously, followed by irreversible dissociation to give two fully double - stranded duplexes . In other words, strand exchange occurs between d1 and d2 . As a result, the quencher - labeled strand of d1 is exchanged for its non - labeled counterpart in d2; therefore, fluorescence is restored . Dna - binding proteins bind to the duplex and block strand exchange, thereby suppressing the fluorescence elevation (a) in the absence of target protein, strand exchange between d1 and d2 will occur and fluorescence will be elevated . (b) in the presence of target protein, strand exchange will not occur and fluorescence will remain quenched . To illustrate the method, we attempted to detect the nf-b (p50) interaction with dna . Nuclear factor-b plays a pivotal role in the coordinated transcription of multiple inflammatory genes and is a probable drug target. (1416) two probes, nf - d1 and nf - d2, were prepared to test quantitative detection of p50 binding to dna . Their double - stranded regions are identical and include an nf-b binding sequence, d(gggactttcc). These probes interact with each other through their single - stranded tails and are then involved in a strand exchange reaction . Each strand of nf - d1 was labeled with 6fam and dabcyl at the double - stranded terminus . Nf - d1, various concentrations of recombinant p50, and nf - d2 were mixed in a half - area 96-well microplate and changes in fluorescence were measured . The fluorescence signal of nf - d1 increased rapidly within 30 min after addition of nf - d2 and was fivefold higher than that of nf - d1 alone at 60 min in the absence of p50 . Fluorescence elevation was suppressed in a p50 concentration - dependent manner by half at 40 nm p50 and almost completely at 320 nm p50 . (a) five microliters of 40 nm nf - d1 was mixed with 5 l of 0 nm (open circles), 400 nm (open squares), 800 nm (closed squares), 1600 nm (open triangles), or 3200 nm (closed triangles) p50 and incubated for 30 min . Then, 40 l of 10 nm nf - d2 was added and fluorescence was measured; thus, the final concentrations of p50 were 0, 40, 80, 160, and 320 nm, respectively . Closed circles with a dotted line represent nf - d1 alone . (b) fluorescence at 60 min for a positive control completely exchanged product (nf-01f + nf-02, named nf-01f02), an exchanged product (nf - d1 + nf - d2), and an unexchanged product (nf - d1 alone) with 0 or 320 nm p50 . Duplex nf-01f02, which consists of nf-01f and nf-02, was prepared as a positive control as a completely exchanged product . Varied concentrations of nf-1d (1, 2, and 4 nm) were mixed with twofold or fourfold amounts of nf-2d in the presence (40 nm) or absence of p50 . As shown in table 1, the combination of 2 nm nf - d1 and 8 nm nf - d1 showed the highest z-factor (0.93). S / b values (ratio in the absence to presence of p50) were 1.92.3 and were not influenced by the dna concentration . Therefore, we used 2 nm nf - d1 with 8 nm nf - d2 in the following assays . Optimization of probe concentration we examined the specificity of the assay by using double - stranded competitor dna . Nf - cpt2, nf - cpt3, nf - cpt4, and nf - cpt5 are non - specific competitors with one to four nucleotides substituted in the nf-b binding site . The restoration level decreased as the number of substituted nucleotides in non - specific competitors increased and nf - cpt5, with four substituted nucleotides, had little effect on p50 binding . For further evaluation of specificity, we prepared non - specific probes, nfs - d1 and nfs - d2, with nucleotide substitutions as in nf - cpt5 . Hence, we concluded that p50 suppressed strand exchange in a sequence - specific manner . (a) 2.5 l of 2000 nm competitor was mixed with 2.5 l of 800 nm p50 and incubated for 30 min . Then, 5 l of 20 nm nf - d1 was added and incubated for a further 30 min . Finally, 40 l of 10 nm nf - d2 was added and fluorescence was measured: nf - d1 and nf - d2 without p50 (white bar), without competitor (black bar), and with competitors (gray bars). Horizontally and diagonally striped bars indicate non - specific probes (nfs - d1 + nfs - d2) without and with p50, respectively . (b) sequences of the competitor upper strand are shown: underline, nuclear factor-b (nf-b) binding site; bold type, substituted nucleotide in binding site . Error bars represent 3 sd (n = 4). A 10-point dose response experiment with evans blue (eb) was also carried out . One hundred m evans blue inhibits nf-b binding to dna by emsa and has been suggested to bind non - covalently to the p50 dna binding region by molecular modeling. (17) in dse - fret, 10 m eb showed little effect on p50, but 30 m eb inhibited p50 completely (fig . The ic50 values of eb for p50 and p52 inhibition were 12.9 and 12.8 m, respectively . We also showed that our method can be used for evaluation of an uncompetitive inhibitor, ()-dhmeq, which binds covalently to a specific cys residue of rel family proteins to inhibit their dna binding. (18,19) we detected an inhibitory effect of ()-dhmeq on p50 and p52 by dse - fret (fig . 5). As shown previously,(19) ()-dhmeq was less potent against p52 (ic50 62.5 m) compared to p50 (ic50 8.8 m). Half a microliter of various concentrations of eb was mixed with 4.5 l of 890 nm p50 (a) or p52 (b) and incubated for 30 min . Then, 5 l of 20 nm nf - d1 was added and incubated for a further 30 min . Finally, 40 l of 10 nm nf - d2 was added and fluorescence was measured . Horizontal axes show the concentration of eb in incubations with protein and nf - d1 . Half a microliter of various concentrations of ()-dhmeq was mixed with 4.5 l of 440 nm p50 (a) or p52 (b) and incubated for 30 min . Then, 5 l of 20 nm nf - d1 was added and incubated for a further 30 min . Finally, 40 l of 10 nm nf - d2 was added and fluorescence was measured . Horizontal axes show the concentrations of ()-dhmeq in incubations with protein and nf - d1 . The dse - fret technique can detect protein dna interaction quantitatively and specifically using a simple procedure; just mix and measure . It detected p50 binding with a high z-factor and a dose response analysis was carried out with two types of inhibitor (eb and ()-dhmeq). Moreover, the differential effect of ()-dhmeq on p50 and p52 was well displayed . Yamamoto et al . Found that ()-dhmeq inhibited p50 more strongly than p52 in emsa analysis. (19) the same preference of the inhibitor was shown by dse - fret, with the ic50 for inhibition of p50 being sevenfold lower than that for p52 . However, the amount of ()-dhmeq required to inhibit the proteins differed between the two methods . A ()-dhmeq concentration fourfold that of p50 (8020 m) inhibited protein binding completely in emsa,(19) whereas a ()-dhmeq concentration 44-fold that of p50 (8.80.2 m) was required to inhibit half of the p50 binding in dse - fret . In contrast, inhibition of ()-dhmeq to p52 was shown only by dse - fret . Although we carried out the assay in a 96-well (half area) format, a 384-well format can be used with appropriate specialized equipment . The probes used in dse - fret consist of a double - stranded moiety and two single - stranded tails . Inadequate intra- and intermolecular hybridization of tails interferes with appropriate formation of a four - way junction and markedly reduces the efficiency of strand exchange . These tails were also confirmed to not have protein binding sequences using the transcription factor analysis tool tfsearch (version 1.3 online, http://mbs.cbrc.jp/research/db/tfsearch.html). The double - stranded moiety should have a sequence that allows protein binding, but is otherwise not restricted in sequence . Taken together, these features make design of a probe for dse - fret as easy as that for emsa . To our knowledge, this is the first report to show that nf-b blocks spontaneous strand exchange . Several proteins, including histone octamer, p53, trf1, and trf2, have also been found to block strand exchange. (1012) however, this blockage can be explained based on particular features of these proteins: thus, the histone octamer forms a large protein dna complex and the other proteins have junction - binding activity . Panyutin and hsieh(20) found that spontaneous strand exchange is also blocked by a mismatch, which suggests that this process may be blocked easily by a small energy barrier . In this paper nuclear factor-b is a desirable target for therapy in various cancers and inflammatory diseases. (21,22) in most cancer cells, nf-b is localized in the nucleus and is constitutively active . Therefore, anticancer drug targeting of nf-b may have great therapeutic value by inhibiting cell growth or increasing the sensitivity of conventional chemotherapy . We believe that the dse - fret assay will be a powerful tool to isolate novel nf-b inhibitors that inhibit dna binding activity and cancer growth . We also found that dna binding of transcription factors sp1 and ap1 (c - jun) can be detected by dse - fret (data not shown). Therefore, dse - fret may be applicable to detection of many dna - binding proteins.
F - fluoro - deoxy - glucose (fdg), an f - labeled glucose analog, is the most common radiotracer for positron emission tomography (pet) in cancer diagnosis . However, fdg - pet has several limitations in practice, for example, fdg cannot distinguish tumor tissues from inflammatory or normal brain tissues . Therefore, f - labeled amino acid - based radiotracers have been reported as an alternative, which is based on the fact that tumor cells take up and consume more amino acids to maintain their sustained fast growth . Among those radiotracers, f - labeled alpha - methyl tyrosine (amt) has shown high tumor uptake and great ability to differentiate tumor tissue from inflammatory sites in brain tumors and squamous cell carcinoma . F - amt enters the tumor cells via l - type amino acid transporters (lat), which is the only system that can transport large neutral amino acids with aromatic rings . Lat, especially its subtype lat1, was reported to be highly expressed in many cancer cell lines and positively correlates with tumor growth [4, 5]. So far, f - amt is the most suitable amino acid transporter - targeting radiotracer for tumor imaging, regardless of low synthesis yield and requirement of an on - site cyclotron to produce f. although pet has emerged as an advanced imaging tool for cancer diagnosis, only limited facilities around the world can afford complete armamentarium of pet and cyclotron for the local production of short - lived positron - emitting radionuclides such as c and f. therefore, mature technologies, that is, single photon emission computed tomography (spect) or its combination with computed tomography (ct), still play important and irreplaceable roles in nuclear imaging area . (tc, t1/2 = 6.02 h), which is produced by in - house generator and does not require the cyclotron . Due to their similar chemistry, the diagnostic radioisotope tc and the therapeutic radioisotope rhenium-188 (re) could be labeled to the same ligand, which leads to the diagnostic / therapeutic matched pair . Unlike most of the cyclotron - produced radionuclides that utilize the covalent chemistry for labeling, tc requires a the nitrogen, oxygen, and sulfur combinations have been shown to be stable chelators for tc such as n4 (e.g., dota, cyclam-14), n3s (e.g., mag-3), n2s2 (e.g., ecd), ns3, s4 (e.g., sulfur colloid), diethylenetriamine pentaacetic acid (dtpa), o2s2 (e.g., dmsa), and hydrazinonicotinamide (hynic) [813]. Here, we report the synthesis of precursor o-[3-(1,4,8,11-tetraazacyclotetradecan)-propyl]--methyl tyrosine (n4-amt) and its radiolabeling with tc . In vitro cellular uptake kinetics and planar scintigraphic imaging of tc - n4-amt all chemicals of analytical grade and solvents of hplc grade for compound synthesis were purchased from sigma - aldrich (st . Louis, mo). H-, c - nmr spectra were performed on bruker 300 mhz spectrometer in cdcl3, cd3od, and d2o . Sodium pertechnetate (natco4) was obtained from mo / tc generator in mallinckrodt (houston, tx). Thionyl chloride (10 ml; 137.42 mmol) was added to a solution of -methyltyrosine 1 (10.00 g; 51.22 mmol) in anhydrous ethanol (60 ml) at 0c, and then heated at 78c for 4 h while stirring . After cooling, the reaction mixture was reduced to 20 ml, and then 10 ml of triethylamine was added into it . H - nmr (cdcl3) = 7.02 (d, 2h, j = 8.4 hz), 6.70 (d, 2h, j = 8.4 hz), 4.22 (dd, 2h, j = 7.2 hz, j = 7.8 hz), 3.14 (dd, 2h, j = 13.5 hz, j = 13.5), 1.42 (s, 3h), 1.33 (t, 3h, j = 16.2 hz) ppm . C - nmr = 175.47, 156.36, 130.66, 126.39, 114.87, 61.05, 58.65, 45.29, 24.08, 13.07 ppm, ms: m / z = 224.23 [m]. Compound 2 (2.09 g; 9.36 mmol) was dissolved in 40 ml of anhydrous dmf under nitrogen and treated with triethylamine (2.78 ml; 20 mmol) while stirring . Ditertiarbutyl dicarbonate (3.27 g; 15 mmol) was added to the reaction mixture and stirred over night at room temperature . The solvent was removed under reduced pressure to yield a residue, which was extracted with ethyl acetate and dried with anhydrous mgso4 . The extraction was filtered and evaporated to give yellow oil which was purified by column chromatography on silica gel and eluted with hexane: ethyl acetate (5: 1.5 v / v). H - nmr (cdcl3) = 6.97 (d, 2h, j = 8.4 hz), 6.75 (d, 2h, j = 8.7 hz), 4.22 (dd, 2h, j = 2.7 hz, j = 7.2 hz), 3.32 (dd, 2h, j = 15.0 hz, j = 13.5), 1.55 (s, 3h), 1.48 (s, 9h), 1.32 (t, 3h, j = 18.0 hz) ppm . C - nmr = 174.16, 155.04, 154.51, 131.16, 128.50, 115.14, 77.25, 61.64, 60.54, 40.99, 28.39, 23.55, 14.77 ppm, ms: m / z = 324.36 [m]. Sodium metal (0.09 g; 14.02 mmol) was dissolved in 30 ml of anhydrous ethanol with stirring under nitrogen . Compound 3 (1.00 g; 3.09 mmol) was dissolved in 50 ml of anhydrous ethanol and treated with sodium ethoxide solution and refluxed for 2.5 h at 70c . 3-bromopropanol (0.56 ml; 6.18 mmol) was added and continued heating over night . The ethanol was removed under reduced pressure and replaced with 40 ml of ethyl acetate, washed with water, and dried over mgso4 . After removal of the solvent, it was purified by silica gel column chromatography (hexane: ethylacetate 2: 1), giving 0.83 g (2.17 mmol, 71% yield) of the product as clear yellow oil . H - nmr (cdcl3) = 7.01 (d, 2h, j = 6.3 hz), 6.81 (d, 2h, j = 6.6 hz), 4.20 (dd, 2h, j = 5.1 hz, j = 5.1 hz), 4.12 (t, 2h, j = 15.0 hz), 3.86 (t, 2h, j = 10.2 hz), 3.16 (dd, j = 13.5 hz, j = 12.9 hz), 2.06 (m, 2h), 1.54 (s, 3h), 1.47 (s, 9h), 1.31 (t, 3h, j = 12.3 hz) ppm . C - nmr = 174.01, 171.91, 157.73, 154.35, 131.07, 128.65, 114.14, 79.39, 65.65, 61.54, 60.40, 60.24, 40.79, 32.01, 28.39, 23.58, 14.15 ppm, ms: m / z = 381.033 [m]. A solution of 4 (5.44 g; 16.82 mmol) and 1,3-dibromopropane (136.74 g; 677.30 mmol) in 100 ml acetone was purged with nitrogen for 15 min . Potassium carbonate (22.62 g; 163.69 mmol) was added to the reaction mixture, the mixture was then refluxed for 12 h at 75c . After the removal of solvents and excessive reagents under reduced pressure, the residue was dissolved in chloroform, washed with water, and dried in anhydrous magnesium sulfate . A yellow liquid was purified by silica gel flash chromatography (hexane: ethylacetate, 2: 1) to furnish compound 5 as pale yellow liquid . H - nmr (cdcl3) = 7.02 (d, 2h, j = 9.00 hz), 6.82 (d, 2h, j = 9.00 hz), 4.23 (dd, 2h, j = 27.00), 4.09 (t, 2h, j = 12.00 hz), 3.61 (t, 2h, j = 12.00 hz), 3.32, 3.16 (d, 2h, j = 15.00, 12.00 hz), 2.34 (dd, j = 24.00 hz), 1.54 (s, 3h), 1.47 (s, 9h), 1.32 (t, 3h, j = 15.0 hz) ppm . C - nmr = 173.96, 157.63, 154.32, 131.09, 128.78, 114.18, 77.48, 65.24, 61.51, 60.35, 40.80, 30.00, 28.40, 23.60, 14.19 ppm . Ms: m / z = 446.3 [m]. 1,4,8,11-tetraazacyclotetradecane (cyclam) (15.00 g; 74.88 mmol) was dissolved in 150 ml of anhydrous ethanol, and diethyl oxalate (10.94 g; 74.88 mmol) was added . The solvent was rotary evaporated, and the crude product was recrystallized in acetone: ethanol to yield white crystals of n, n - dioxylyl-1,4,8,11 - 1,5,8,12-tetraazabicyclotetradecane (n, n - cyclooxamide) 6 . H - nmr (cdcl3) = 4.35 (m, 2h), 375 (m, 2h), 3.40 (m, 2h), 2.77 (m, 2h), 2.68 (m, 2h), 2.54 (m, 2h), 2.43 (m, 4h), 1.75 (m, 2h), 1.24 (m, 2h)) ppm . C - nmr = 158.55, 49.92, 49.38, 47.73, 44.13, 25.42 ppm . Ms: m / z = 255.33 [m]. Compound 6 (1.00 g; 3.93 mmol) was dissolved in 20 ml of anhydrous dmf and treated with a solution of n - t - butoxycarbonyl - o-[3-br - propyl]--methyl tyrosine ethylester 5 (1.74 g; 3.93 mmol) in 40 ml of dmf under nitrogen atmosphere . The reaction mixture was heated to reflux for 18 h at 75c and then allowed to cool down to room temperature . The residue was dissolved in chloroform (30 ml), washed with water and 1 m na2co3, (12 ml), and then separated for the organic layer . The crude compound was purified by silica gel column chromatograph (chloroform: methanol 9: 1). H - nmr (cdcl3) = 7.00 (d, 2h, j = 9.00 hz), 6.79 (d, 2h, j = 9.00 hz), 4.56 (m, 2h), 4.22 (t, 2h, j = 12.0 hz), 3.95 (m, 2h), 3.67 (m, 2h), 3.55 (t, 2h, j = 15.0 hz), 3.30 (m, 4h), 2.81 (m, 12h), 1.88 (m, 6h), 1.54 (s, 3h), 1.47 (s, 9h), 1.32 (t, 3h, j= 15.0 hz) ppm . C - nmr = 173.97, 158.65, 158.06, 157.91, 154.31, 131.01, 128.34, 114.04, 79.38, 77.27, 66.01, 61.50, 60.26, 53.76, 52.76, 49.20, 48.41, 47.54, 46.15, 45.83, 44.50, 42.60, 40.84, 28.40, 26.39, 23.57, 23.36, 23.07, 14.14 ppm . Ms: m / z = 618.38 [m]. Compound 7 (0.25 g; 0.40 mmol) was dissolved in dichloromethane (10 ml), and trifluoroacetic acid (1.0 ml) was added to it . The solution was stirred over night at room temperature, and volatiles were removed in vacuo . The crude compound was purified by chromatography over silica gel (chloroform: methanol 9: 1) as white solid . H - nmr (cd3od) = 7.14 (d, 2h, j = 9.0 hz), 6.94 (d, 2h), 4.33 (m, 4h), 4.05 (t, 2h, j = 9.0 hz), 3.85 (m, 2h), 3.71 (m, 2 h), 3.26 (m, 13h), 2.07 (m, 6h), 1.58 (s, 3h), 1.33 (t, 3h, j = 15.0 hz) ppm . C - nmr = 170.65, 161.87, 161.42, 159.70, 159.02, 158.67, 131.01, 125.14, 118.79, 114.65, 65.34, 62.47, 60.33, 56.92, 52.16, 51.26, 45.64, 45.31, 44.50, 43.20, 42.05, 24.31, 22.00, 21.39, 21.03, 16.96, 12.90 ppm, ms: m / z = 517.63 [m]. To a solution of compound 8 (0.20 g; 0.39 mmol) in a 5 ml of water, 10 n naoh (2 ml) was added, stirred, and refluxed over night at 90c . The solvent was evaporated under vacuum, giving white solid, which was dissolved in 5 ml of water and neutralized with 5 n hcl solution to ph = 7 . The solid was stirred in 25 ml anhydrous methanol, filtered, and evaporated to afford an off white solid . H - nmr (d2o) = 7.21 (d, 2h, j = 9.00 hz), 7.00 (d, 2h, j = 9.00 hz), 4.14 (t, 2h, j = 12.0 hz), 3.10 (m, 5h), 2.94 (m, 2h), 2.84 (m, 13h), 1.95 (m, 6h), 1.33 (s, 3h) ppm.c - nmr = 180.69, 163.42, 163.16, 162.88, 162.60, 157.06, 131.40, 129.19, 117.57, 115.26, 115.10, 112.93, 66.25, 60.73, 53.69, 51.46, 49.88, 49.26, 48.04, 47.84, 46.60, 45.42, 45.07, 43.94, 24.71, 24.32, 23.62, 22.55 ppm . Ms: m / z = 436.327 [m]. Briefly, radiosynthesis of tc - n4-amt was achieved by adding a required amount of sodium pertechnetate into a vial containing precursor n4-amt and sncl2 (100 g). Radiochemical purity was assessed by high - performance liquid chromatography (hplc), equipped with nai and uv detector (274 nm), and was performed using a c-18 reverse column with a mobile phase of acetonitrile: water (7: 3) at a flow rate of 0.5 ml / min . Rat mammary tumor cell line 13762 was obtained from american type culture collection (rockville, md). The same cell line was used to create the animal model for in vivo evaluation . Cells were maintained at 37c in a humidified atmosphere containing 5% co2 in dulbecco's modified eagle's medium and nutrient mixture f-12 ham (dmem / f12; gibco, grand island, ny). Cells were plated to 6-well tissue culture plates (2 10 cells / well) for two days before the study, and incubated with tc - n4-amt (0.05 mg / well, 8 uci / well) or tc - n4 chelator itself (0.025 mg / well, 8 uci / well) for 15 min4 h. after incubation, cells were washed with ice - cold pbs twice and detached by adding 0.5 ml of trypsin . Cells were then collected and the radioactivity was measured with gamma counter (cobra quantum; packard, mn). All animal work was carried out in the small animal imaging facility (saif) at university of texas md anderson cancer center under the protocol approved by institutional animal care and use committee (iacuc). Tissue distribution studies of tc - n4-amt (study i, n = 9) or tc - n4 (study ii, n = 9) were conducted by using normal female fischer 344 rats (150 25 g, n = 18) (harlan sprague - dawley, indianapolis, in). For each radiotracer, nine rats were divided into three groups for three time intervals (0.5, 2, 4 h). The injection activity was 25 0.5 ci / rat intravenously . At each time interval, the rats were sacrificed, and the selected tissues were excised, weighed, and measured for radioactivity by gamma counter . Data from each sample were represented as the percentage of the injected dose per gram of tissue wet weight (% id / g). Female fischer 344 rats were inoculated subcutaneously with 0.1 ml of 13762 rat mammary tumor cell suspension (10 cells / rat) into the right posterior legs using 22-gauge needles . Imaging studies were performed 14 to 17 days after inoculation when tumors reached approximately 1 cm in diameter . The anesthetized rats were injected intravenously with tc - n4-amt (0.3 mg / rat, 300 ci / rat; n = 3) or with tc - n4 (0.15 mg / rat, 300 ci / rat; n = 3), respectively . Planar scintigraphic images were obtained using m - cam (siemens medical solutions, hoffman estates, il) equipped with a low - energy high - resolution collimator at 30120 min . The intrinsic spatial resolution was 3.2 mm and the pixel size was from 19.18 mm (32 32, zoom = 1) to 0.187 mm (1024 1024, zoom = 3.2). Computer outlined regions of interest (roi) (counts per pixel) of tumors, and normal muscle tissues at symmetric sites were used to calculate tumor - to - muscle (t / m) ratios . Commercially available -methyl tyrosine 1 was converted into corresponding acid chloride, then to ethyl ester by reacting with thionyl chloride in ethanol . The amine in -methyl tyrosine ethyl ester 2 was protected as its boc - derivative n - t - butoxycarbonyl--methyl tyrosine ethylester 3 with triethylamine and di - t - butyldicarbonate in dmf . The chain at the oh group was extended when compound 3 was treated with 1, 3-dibromopropane and obtained as n - t - butoxycarbonyl - o-[3-bromopropyl]--methyl tyrosine ethylester 4 . Acylation of 1,4,8,11-tetraazacyclo - tetradecane (cyclam) with diethyloxalate led to n, n - dioxalyl 1,4,8,11-tetraazacyclotetradecane (n, n - cyclamoxamide) 5 . Under sn condition 4 and 5 were efficiently converted to alkylated compound n - t - butoxycarbonyl - o-[3-(n, n - dioxalyl-1,4,8,11-tetraazacyclotetradecane)-propyl]--methyl tyrosine ethylester 6 . Exposure of 6 to trifluoroacetic acid in ch2cl2 caused qualitative de - t - butoxycarboxylation to yield o-[3-(n, n - dioxalyl 1,4,8,11-tetraazacyclotetradecane)-propyl]--methyl tyrosine ethylester 7 . 10 n naoh in water at 75c promoted the hydrolysis of ester to acid and a simultaneous deoxalation of 7 to yield the final compound o-[3-(1,4,8,11-tetraazacyclotetradecan)-propyl]--methyl tyrosine (n4-amt) 8 . The structure and purity of the compounds at each step were validated by h- and c - nmr, mass spectra, and hplc . As shown in figure 2, precursor n4-amt could be labeled with tc successfully in a high radiochemical purity (> 96%). The retention time of tc - n4-amt was 6.899 min . Given that tc - n4-amt is a kit - product and labeled without any further purification, the radiochemical yield was assumed to be identical to its radiochemical purity . In this study, we labeled amino acid -methyltyrosine amt with tc using cyclam n4 as the chelator because of its stable chelating ability and fast renal clearance . Tc was selected as the radionuclide due to its favorable physical characteristics, suitable half - life, and cost - effectiveness . The cellular uptake kinetics of tc - n4-amt and tc - n4 in rat breast tumor cell line 13762 is shown in figure 3 . There was a drastically increased uptake for tc - n4-amt in the tumor cells at 15240 min, but not for tc - n4 chelator . These findings suggest that by adding the amino acid amt, tc - n4-amt can enter and accumulate into tumor cells effectively and rapidly . To further investigate the transport mechanisms of tc - n4-amt, the competitive inhibition study using various types of transporter inhibitors will be conducted in the future . The result of the in vivo biodistribution studies in the normal fischer rats at 0.5, 2, and 4 hours after intravenous administration of tc - n4-amt is shown in table 1 . Planar scintigraphic images of tc - n4-amt and tc - n4 at 30, 60, and 120 min in breast tumor - bearing rats are shown in figure 4 . T / m ratios of tc - n4-amt were 2.34.0, whereas those of tc - n4 were 1.92.5, respectively . Tumors could be clearly visualized by tc - n4-amt, but not by tc - n4 . In addition, the rat kidneys showed intense activity of tc - n4-amt in the planar images, which was consistent with the results from the in vivo biodistribution studies in the normal rats . This may be due to the nature of amt, an inhibitor of tyrosine hydroxylase that cannot be excreted from kidneys and hence crystallized in the proximal tubules because of its poor solubility at the hydrogen ion concentrations of body fluids (ph 58). In the future, in vivo uptake blocking study using the unlabeled amt will be performed to ascertain whether accumulation of tc - n4-amt in the kidney is attributed to amt module . In conclusion, efficient synthesis of n4-amt was achieved . In vitro cellular uptake and in vivo imaging findings collectively suggest that tc - n4-amt is a potential radiotracer for breast cancer imaging . In compliance with the chelating capability of n4, n4-amt could be labeled with positron emitting radionuclides such as gallium-68 or with short - ranged beta emitters for internal radiotherapeutic purposes hereafter.
Though thousands of transition - metal hydride complexes are known, relatively few of them have unpaired electrons . Even fewer have metals with a high - spin electronic configuration, since hydride is a strong - field ligand and since hydride complexes are often supported by strong - field ancillary ligands . In a series of publications, we described exceptional di(-hydrido)diiron(ii) complexes supported by bulky -diketiminate ligands (chart 1). In these complexes, the iron(ii) ions had high - spin electronic configurations because of the low metal coordination number and the -donor character of the anionic -diketiminate . All of the iron(ii) examples were dimers in the solid state, and the two pseudotetrahedral metal centers were bridged by hydride ligands (chart 1, upper left). With the bulkiest -diketiminate (l), the dimer dissociated in solution to give a three - coordinate monomer, as shown by a combination of magnetic, spectroscopic, and kinetics studies . The high - spin electronic configuration of the complexes presents characterization challenges that are distinctive to paramagnetic species . Namely, the resonances in the h nmr spectra are broadened and highly shifted, and these chemical shifts do not correlate with structure in the manner that is familiar from diamagnetic complexes . The relaxation of h nuclei directly bonded to the paramagnetic metal is particularly rapid, and to our knowledge no metal - bound h nuclei have been detected in nmr spectra of hydride complexes with a paramagnetic ground state . Another challenge is that the paramagnetic hydride complexes in chart 1 are highly reactive: for example, they cleave b c bonds and reductively eliminate h2 with light or with added ligands including n2 . Though the high reactivity of the hydrides makes them difficult to handle, their reactivity is also an opportunity to form new c h bonds, because the m h bond weakening as a result of the partial population of fe h * orbitals in the high - spin d electronic configuration . One of the important reactions of coordinatively unsaturated metal - hydride complexes is the exchange of the hydride hydrogens with free h2 . This reaction has biological relevance because of the h / d exchange of h2 protons with solvent protons in nitrogenase enzymes, which occurs only in the presence of n2 . This specificity has been used to suggest that hydride species are key intermediates during n2 reduction . Very recently, deuterium atoms from d2 were incorporated into ethylene produced from acetylene reduction by nitrogenase . However, the mechanisms are difficult to evaluate without model studies on well - characterized synthetic hydride complexes, particularly with iron . Unfortunately, the aforementioned inability to observe resonances for h and d bound to a metal has so far prevented the monitoring of h / d exchange in paramagnetic hydride complexes . In this article, we describe synthetic and characterization advances for two high - spin iron(ii)-hydride complexes, supported by the -diketiminate ligands l and l (chart 1). In an interesting twist, these studies benefit from unusually large (up to 5.7 ppm) isotope effects on the h nmr chemical shifts of the distant protons upon hydride deuteration, a phenomenon that occurs only in the dimeric hydride complexes in our system . This tool for differentiating isotopologues enables the revision of the h nmr assignments in two previously reported hydride complexes . In addition, this is the only way to distinguish hydride from deuteride isotopologues, and this discovery enables us to monitor h / d exchange in this system for the first time . The results show that the rates of h / d exchange between hydride complexes, and between these hydride complexes and h2, are greatly influenced by the size of the supporting ligand . Mechanistic considerations lead to new insights into the distinctive reactivity of high - spin hydride complexes . The synthesis of [lfe(-d)]2 (1-d2) was reported previously, and initial characterization of 1-d2 by h nmr spectroscopy in c6d6 suggested that the deuteride and the hydride complexes had identical h nmr spectra . However, closer investigation has shown that the resonances have different chemical shifts in the different isotopologues . A h nmr spectrum of an equimolar mixture of 1 and 1-d2 in c6d6 showed that the differences were not from temperature or medium effects (figure 1). Close examination of the h nmr spectrum revealed several envelopes of nearby resonances, and the components of each envelope had a 1:2:1 ratio of integrations . For example, resonances at 37.2 (resonance assigned to 1), 40.1, and 43.0 (resonance assigned to 1-d2) ppm were observed in a 1:2:1 ratio, rather than the single resonance at 37.2 ppm in 1 . When this experiment was repeated, starting from a different mixture of isotopologues that contained more 1 than 1-d2, the h nmr spectrum showed the same number of resonances, but the integrations were no longer 1:2:1 and favored the hydride resonance at 37.2 ppm . The resonances located between 1 and 1-d2 are most reasonably assigned to {lfe}2(-h)(-d) (1-d). These experiments also indicate that mixing of 1 and 1-d2 rapidly yields an equilibrium mixture of 1, 1-d, and 1-d2 . The difference between the chemical shifts of distant protons in different isotopologues has been described previously in a number of paramagnetic complexes, and theopold has termed this paramagnetic isotope effect on chemical shift (piecs). This particular sample has a 7% impurity of the oxo complex {lfe}2(-o). Peaks are marked as follows: dimer d, monomers m, oxo impurity i, and solvent and solvent impurities s. (b) h nmr spectrum from mixing equimolar amounts of [lfe(-h)]2 (1) and [lfe(-d)]2 (1-d2) in c6d6 for 45 min . All three isotopologues of the dimer (h / h, h / d, and d / d) are visible in (b), as several groups of three nearby peaks in a statistical 1:2:1 ratio . Only the parts of the spectra from 45 to 70 ppm are shown for clarity . The discovery that 1, 1-d, and 1-d2 exhibited piecs enabled the use of h nmr spectroscopy to accurately determine the amount of deuterium incorporation into 1-d2 . This was done by comparing the integrations of the three isotopologues in the h nmr spectrum . Compound 1-d2 typically had greater than 90% deuterium incorporation into the hydride positions, which is consistent with the level of deuteration previously reported . (in earlier studies this determination was done indirectly, using mass spectrometric analysis of 3-hexene - d1 generated from treating 1-d2 with 3-hexyne and then acid .) Note that piecs is not observed for some of the peaks in the spectrum . Seven of these peaks are assigned to monomeric lfeh, which is in equilibrium with 1, as previously shown . This is the number of resonances expected for l in an environment having c2v symmetry, when the n - aryl bonds have hindered rotation that makes the two methyl groups of the isopropyl substituents inequivalent . By process of elimination, the piecs of the peaks of 1 enabled the assignment of 18 resonances each to the 1, 1-d, and 1-d2 isotopologues of the dimers, giving 54 resonances in total . Therefore, the h nmr spectrum of isotopically pure 1 contains 25 resonances, where 7 peaks may be assigned to lfeh and 18 peaks may be assigned to the dimer . It is notable that h nmr spectra of a mixture of the three isotopologues always showed some additional resonances that neither exhibit piecs nor can be assigned to the monomer lfeh . Comparison with literature h nmr spectra indicated that these additional resonances were associated with a persistent impurity, {lfe}2(-o), which can come from the reaction of 1 with trace h2o to give {lfe}2(-o). We were not able to completely avoid or eliminate the oxo impurity, but careful handling gave samples of 1 that were below 10% oxo impurity, with typical samples between 4 and 8% . The oxo impurity in 1-d2 was typically higher (around 20%), due to the multiple d2 additions necessary for full deuterium incorporation . The zero - field mssbauer spectrum of solid 1 at 80 k was previously reported to have = 0.59 mm / s and \|eq\| = 1.58 mm / s . However, the published data were reexamined after the discovery of the persistent oxo impurity . The oxo impurity was modeled using parameters that were fixed at literature values of = 0.64 mm / s and \|eq\| = 1.42 mm / s, while the amount of the oxo impurity and the parameters of the major component were refined to give the best fit to the data . Complex 1 was determined to be 71% of the earlier sample, and the major component had = 0.57 mm / s and \|eq\| = 1.63 mm / s . A new sample of 1 with <5% oxo impurity by nmr spectroscopy yielded a zero - field mssbauer spectrum at 80 k with parameters = 0.58 mm / s and \|eq\| = 1.62 mm / s, which are the same within the experimental uncertainty of 0.02 mm / s . The zero - field mssbauer spectrum of solid 1-d2 at 80 k is shown in figure 2 . The slightly asymmetric two - line pattern was modeled with a two - component fit, of which the major component was found to have = 0.58 mm / s and \|eq\| = 1.74 mm / s, accounting for 79% of the sample . We assign this subspectrum to compound 1-d2, whereas the second doublet was fixed to the properties of the oxo impurity (= 0.64 mm / s and \|eq\| = 1.42 mm / s). The isomer shifts of 1 and 1-d2 are identical, but there was a variation of 0.12 mm / s in the quadrupole splitting values . The reason for the difference in the quadrupole splitting is unknown at this time . Unfortunately, attempts to fit the variable - field mssbauer spectra of 1 and of 1-d2 did not yield a comprehensible model that gave additional insight . The isomer shifts of 1 and 1-d2 lie in the range of 0.470.90 mm / s observed in other high - spin fe(ii) diketiminate complexes . Zero - field mssbauer spectrum of [lfe(-d)]2 (1-d2) recorded at 80 k. the signal with = 0.58 mm / s and \|eq\| = 1.74 mm / s accounted for 79% of the sample . The blue line represents the contribution of the oxo impurity, the green line represents the contribution of 1-d2, the red line represents the sum, and the black circles are the data . We now shift to iron(ii) complexes of the smaller diketiminate ligand l, which we have previously derived from the iron chloride precursors lfe(-cl)2li(thf) (thf = tetrahydrofuran) and [lfe(-cl)]2 . However, the relative ease of removing br salts encouraged us to prepare lfebr(thf) (2) in 82% yield from lk and febr2 . During the preparation of our work, compound 2 was reported by tonzetich and lippard, using a very similar prepatory method with lna . Our characterization of 2 by nmr and x - ray diffraction (supporting information) is indistinguishable from the literature . The zero - field mssbauer spectrum of 2 at 80 k (figure s-1, supporting information) has a doublet with = 0.89 mm / s and \|eq\| = 2.36 mm / s, which is nearly identical to that of lfe(-cl)2li(thf). Thf was removed from 2 by dissolving it in noncoordinating solvents; this gave [lfe(-br)]2, which precipitated from solution as an orange powder . Tonzetich and lippard also reported this behavior, and they reported that lfebr(thf) had different electronic absorption spectra in thf versus toluene, which resulted from the formation of [lfe(-br)]2 . Here, the mssbauer spectrum of orange [lfe(-br)]2 derived from 2 was recorded at 80 k (figure s-2, supporting information). One quadrupole doublet with = 0.91 mm / s and \|eq\| = 2.64 mm / s was observed . The isomer shift of [lfe(-br)]2 is the same as 2, but the quadrupole splitting is larger, consistent with a slightly different geometry at iron . The successful synthesis of [lfe(-h)]2 from addition of h2 to an iron(i) source prompted us to use this synthetic method for an improved synthesis of [lfe(-h)]2 and [lfe(-d)]2 . However, the order of addition of reagents was important, as reduction of 2 with kc8 in et2o followed by h2 addition yielded many unidentified resonances in the crude h nmr spectrum . This suggests that the transient iron(i) species formed by kc8 reduction rapidly decomposes in the absence of a trap . Therefore, a degassed solution of 2 in thf was exposed to 14 equiv of h2 gas prior to addition of kc8 . After 3 h, volatile materials were removed from the brown reaction mixture, and 3 was isolated in 56% yield following workup . The identity of 3 was established by comparing its h nmr spectrum to the spectrum reported in the literature . Again, the iron - bound hydrogen atoms are not visible by h nmr spectroscopy due to close proximity to the paramagnetic iron atoms . The deuterated isotopologue 3-d2 was synthesized using the above method with d2 gas in 58% yield . The h nmr spectrum of 3-d2 (figure 3) revealed that 3 and 3-d2 exhibited piecs as with the hydride dimers described above . Thus, h nmr spectroscopy could similarly be used to determine the amount of deuterium incorporated into the hydride ligands . The bridging ligands in samples of 3-d2 were typically greater than 90% deuterated, as judged by h nmr spectroscopy . H nmr spectra of (bottom) [lfe(-h)]2 (3) and (top) [lfe(-d)]2 (3-d2) in c6d6 . In the h nmr spectrum of 3 at room temperature, only four paramagnetically shifted peaks were observed, a number that is well short of the seven resonances expected for 3 in a dimeric structure with d2d or d2h symmetry . In addition, the integrations of the peaks are inconsistent with the original assignments for the h nmr spectrum of 3, so further investigations were pursued . H nmr spectra of 3 between 26 and 85 c in c6d6 are shown in figure 4a . The peak at 13 ppm at 26 c corresponds to the backbone methyl and meta - aryl protons, two resonances that are only distinct above 60 c (a close - up is shown in figure s-3, supporting information). A previously undetected resonance for isopropyl methyl groups integrating to 24 protons (which had been hidden under the residual benzene at room temperature) became visible above 40 c . The other resonance for isopropyl methyl protons, a broad peak at 25 ppm in the 26 c spectrum, sharpened at elevated temperatures . Finally, a new broad resonance for the isopropyl methine protons at 1.5 ppm was observed above 70 c . This resonance is broadened into the baseline at room temperature, explaining why it had not been identified in previous studies . The remaining resonances at 24 and 56 ppm corresponded to the para - aryl protons and the backbone protons, respectively, completing the catalogue of resonances with the expected integrations (see experimental section). (a) variable - temperature h nmr spectra of [lfe(-h)]2 (3) between 26 and 85 c in c6d6 . (b) variable - temperature h nmr spectra of 3 between 90 and 20 c in toluene - d8 . The asterisks indicate the resonance that splits with a coalescence temperature of 0 c . The appearance and sharpening of resonances in the high - temperature h nmr spectra of 3 suggested the possibility of a fluxional process in solution . Therefore, low - temperature h nmr spectra were measured between 90 and 20 c in toluene - d8 (figure 4b). The broad isopropyl methyl resonance that appeared at 25 ppm in the h nmr spectrum of 3 at 20 c split into two broad resonances at 27.6 and 48.3 ppm in the h nmr spectrum at 75 c . These resonances moved together and became broader as the sample was warmed, with a coalescence temperature of 0 c . The barrier for this fluxional process, assuming that the resonances at 75 c are in the slow - exchange limit, is g = 10.5 kcal / mol . Decoalescence of other peaks was not observed within this temperature range, likely because there was a smaller difference between the frequencies in the slow - exchange limit . The purity and electronic structure of solid 3 and 3-d2 were evaluated using mssbauer spectroscopy . The zero - field mssbauer spectra of solid 3 and 3-d2 at 80 k are shown in figure 5 . Compound 3 had = 0.51 mm / s and \|eq\| = 2.05 mm / s . The deuterated isotopologue 3-d2 had an indistinguishable mssbauer spectrum, with = 0.51 mm / s and \|eq\| = 2.10 mm / s . There were impurities of 13 and 6%, respectively, which are discussed in detail in the supporting information . One impurity doublet in each case has parameters similar to those in the literature iron(i) benzene compound lfe(-c6h6), which has = 0.70 mm / s and \|eq\| = 0.74 mm / s (figure s-4, supporting information). We also note that this nearly nmr - silent impurity was the major species in the mssbauer spectrum previously attributed to 3 . The black circles are the data, and the red lines represent the sums of a major doublet for 3 (green) and impurities (blue, purple) that are discussed in the supporting information . As reported above, a 1:2:1 mixture of 1, 1-d, and 1-d2 was obtained upon mixing an equimolar mixture of 1 and 1-d2 in c6d6, and piecs enabled all three isotopologues to be distinguished in the paramagnetic h nmr spectrum . Equilibrium was established within 45 min at room temperature . An analogous experiment was performed using an equimolar solution of 3 and 3-d2 maintained at 30 c in c6d6 . A 1:2:1 mixture of 3, {lfe}2(-h)(-d) (3-d), and 3-d2 was observed after 2 h. this shows that the inability of 3 to form monomeric lfeh in solution does not hinder hydride exchange between isotopologues . The exchange of hydride and deuteride ligands with d2 and h2 was also examined . Compound 3 was treated with an excess of d2 (1 atm) in c6d6 to give a mixture of 3-d and 3-d2 . When this mixture was treated with fresh d2, the equilibrium was pushed all the way to the fully deuterated isotopologue, 3-d2 (figure 6b). Two treatments with h2 caused the sample to revert to 3 in quantitative yield (figure 6c, d), showing that the exchange is reversible . In contrast to the immediate exchange in 3, treatment of 1 in c6d6 with d2 (1 atm) produced the deuterated isotopologue only after much longer amounts of time . Compound 1-d2 was finally observed in quantitative yield after 50 h at room temperature . Hydride exchange between h2 and 1-d2 is reversible, as treatment of 1-d2 with h2 (1 atm) produced 1 in quantitative yield under the same conditions and time . Treatment of 1 with 8 atm of d2 was faster but required 24 h to yield 1-d2 in quantitative yield . H nmr spectra of [lfe(-h)]2 (3), {lfe}2(-h)(-d) (3-d), and [lfe(-d)]2 (3-d2) isotopologues in c6d6 during gas exchange . The columns on the right indicate the order and type of gas that was added to give the observed spectrum . This compound is much less reactive than the iron analogues described above; for example, it does not react with alkenes or lewis bases . The low reactivity was attributed to the greater stability of the dimer and/or to the decreased lability of the co h bonds . Therefore, it was interesting to evaluate the analogous cobalt compounds for intermolecular h / d exchange . A sample of [lco(-d)]2 was prepared from [lco(-f)]2 and et3sid, using a method analogous to that used to synthesize the protiated analogue . Several peaks were shifted by 0.20.5 ppm from those in [lco(-h)]2, as verified by spiking the sample with an equimolar amount of [lco(-h)]2 (figures s-6s-8, supporting information). Thus, this dimer also exhibits piecs, though the shifts are not as pronounced as in the iron species described above . Heating the mixture of [lco(-h)]2 and [lco(-d)]2 to 80 c for 12 h gave no change in the nmr spectrum, indicating that there is no significant exchange of hydrides between complexes in this time frame . This contrasts with the iron analogues (table 1) and is consistent with the idea that the cobalt(ii) hydride dimer does not break up in solution . The mssbauer spectra of 1 and 3 have similar isomer shifts, = 0.59 and 0.51 mm / s, which are consistent with the values observed in other high - spin iron(ii) -diketiminate complexes . High - spin, tetrahedral iron(ii) sites in iron - sulfide clusters have similar isomer shifts (= 0.60.7 mm / s). Low - spin octahedral iron(ii) sites have very different isomer shifts in the range of = 0.30.45 mm / s . The intermediate - spin (s = 1) iron(ii) hydride complex [fe(dppe)2h] has = 0.23 mm / s . The much higher isomer shifts in 1 and 3 strongly support the assignment of 1 and 3 having high - spin fe(ii) subsites and are consistent with the paramagnetic shifts in the h nmr spectra . We note that our assignment of the mssbauer spectrum of 3 here replaces an incorrect assignment we gave in an earlier paper; the previous spectrum actually corresponds to lfe(arene), which can be formed during the synthesis of 3 when arenes are present . The h nmr spectra of 1 and 3 were found to exhibit significant piecs, as all the resonances shifted upon deuterium substitution . The term piecs was coined by heintz and theopold and has been reported in a number of complexes . For example, deuteration of the hydrides in (cp)4cr4(3h)4 (cp = -c5me4et) gives changes in the chemical shifts of the cp protons, though they are far removed from the bridging hydride ligands . Most explanations for piecs are based on the shorter bond lengths to d versus h. the heintz / theopold study is particularly relevant to our complexes because of the presence of bridging hydrides: shorter m m distance and the exchange coupling, which in turn would influence the magnetic susceptibility and thus the chemical shift of the protons . However, other explanations have been advanced for other cases of piecs: for example, differential m h / m d bond energies could influence the ligand - field splitting slightly, which in turn could influence the paramagnetic shift . However, we saw no evidence for any sizable change in the ligand field of iron upon deuteration, because the quadrupole splittings were the same within error between 3 and 3-d2, and only slightly different (eq = 0.12 mm / s) between 1 and 1-d2 . The piecs in the h nmr spectra of 1 and 3 varied from a negligible change in some resonances up to 5.7 ppm in others . Importantly, piecs was observed only for the dimeric form of 1, as the monomer lfe(h / d) had the same chemical shifts in the h nmr spectrum for both isotopologues . This observation supports the hypothesis that the piecs is connected to changes in the fe a significant finding of this study is that variable - temperature h nmr spectroscopy, together with piecs, was essential for defining the correct h nmr assignments of 1 and 3 . Compound 3 was particularly vexing, because almost half of the seven expected resonances were masked . One resonance was hidden under the residual solvent, another apparent resonance was actually two resonances with the same chemical shift, and one was broadened into the baseline at room temperature and was broad even at elevated temperatures . However, variable - temperature studies enabled us to finally assign the resonances for 3 with confidence . Examination of the variable - temperature h nmr data for 3 also revealed a fluxional process in solution with a barrier of g = 10.5 kcal / mol at 0 c . We tentatively assign the low - temperature structure to be similar to the solid - state structure, in d2 symmetry with the diketiminate planes perpendicular to one another . In this case, the barrier would correspond to the energy required to reach the d2h symmetric conformation in which the n2c3fe planes are coplanar (scheme 1) and through which two d2 isomers of different chirality can interconvert . Meanwhile, piecs was vital to the assignment of all resonances in the h nmr spectrum of 1, which is complicated by the presence of both monomer and dimer . In addition, the crystallographic symmetry of the dimer in 1 is lower than that in 3, because crowding gives a boat conformation of the n2c3fe rings that lowers the symmetry to idealized c2 . We observed 18 resonances in dimeric 1, which is somewhat less than the 21 predicted from c2 symmetry in solution, implying that three of the resonances are lost to overlap or broadness . However, the number of peaks is significantly larger than in 3, indicating that the increased steric bulk of the l ligand in 1 (from a buttressing effect) prevented the fluxional process that was observed in 3 . The h nmr spectra imply that the hydride ligands are oriented such that there is no plane of symmetry relating the two l ligands . Overall, these studies show the usefulness of variable - temperature h nmr spectroscopy and piecs to decipher paramagnetic spectra and solution structure despite multiple overlapping resonances . Compounds 1 and 3 undergo intermolecular hydride exchange with their deuterated isotopologues . Dissolving 1 and 1-d2 in c6d6 produced an equilibrium mixture containing dimeric 1, 1-d, and 1-d2, along with monomeric lfeh and lfed . In the iron(ii) complexes, formation of 1-d most likely results from equilibrium between monomeric and dimeric 1, which is slow on the nmr time scale but rapid on the chemical manipulation time scale . (the rate of dissociation has been estimated to be 5 10 s at 288 k, based on kinetics studies of the reaction of 1 with alkynes .) Consistent with this rate regime, equilibrium between the isotopologues of 3 is reached within 45 min at room temperature, as judged by h nmr spectroscopy . The hydride isotope exchange between 3 and 3-d2 cannot follow this process because 3 does not interconvert with monomer in solution, as previously shown using kinetics studies on the reaction with boranes . Two possible mechanisms for hydride isotope exchange between 3 and 3-d2 are shown in scheme 2 . The first is a concerted process, while the second requires rate - limiting cleavage of one of the bridging hydrides to give a single terminal hydride . The hydride ligands in the terminal position could attack another dimer to give exchange . Partial breaking of the dimer of 3 to give a terminal hydride, as in scheme 2b, was previously found to be the most reasonable mechanism for the reaction of 3 with trialkylboranes, and so we consider this to be the most likely possibility that is consistent with the combined studies on 3 . It is also notable that the cobalt analogue, [lco(-h)]2, did not undergo h / d exchange even with heating to 80 c . Its low reactivity in general may be attributable to its inability to open one of the bridges, as in scheme 2b for 3, or both bridges, as in 1 . Dissociation of 3 into monomers is inconsistent with earlier kinetics studies on the reaction of boranes with 3 . The exchange of hydride complexes with h2 and d2 has been studied in detail in the literature . It is well - established in mononuclear iron - hydride complexes . In each of these cases, an open coordination site is required to bind h2 in an binding mode . The oxidative addition of the h2 is not necessary, because there can be direct h transfer from coordinated h2 to the hydride without changing the oxidation state at the metal . It is interesting that the inability of 3 to form monomeric lfeh in solution did not hinder hydride ligand exchange with d2 . Equilibrium was established in less than a minute after treatment of 3 with d2 . Though we cannot determine the mechanism unambiguously, we can advance two possible mechanisms . First, the opened form of the dimer has an open coordination site on one iron that could coordinate h2 or d2 to make a transient side - on d2 complex that is well - situated to exchange with the hydride on the other metal (scheme 3a). Another potential mechanism involves oxidative addition of h2, either to one metal (giving a transient iron(iv) on one side) or to both metals (giving a diiron(iii) species). The latter possibility is shown in scheme 3b . It is also relevant that compound 1 does not undergo facile hydride exchange with d2, despite its ability to form monomer in solution . This required over 2 d at room temperature under 1 atm of d2 and 1 d under 8 atm of d2 . The hindered reaction of 1 with d2 supports the contention that h2/d2 exchange in these hydride species does not proceed through a transient monomer . It is possible that the reaction of 1 with d2 proceeded via the monomer at a significantly slower rate . Another possibility is that the exchange goes through the dimeric form of 1, but that the bulkier ligands hinder its ability to access the reactive conformation . Dinuclear iron sites with bridging hydride ligands have been studied extensively as [fefe]-hydrogenase models . These diiron hydride complexes have been reported to undergo h / d hydride ligand exchange with d2/h2 via photolysis . In these systems, photolysis opens a coordination site by dissociating co or cleaving a hydride bridge, and the h / d hydride exchange requires days (which contrasts with exchange in the 3/3-d2 system that occurs in seconds). H / d hydride ligand exchange has also been reported using d2/h2o for hydrogenases, diiron complexes, other metal complexes, along with d as a deuterium source . In addition, nitrogenase can exchange d from d2o into h2, but only does so in the presence of n2 . We suggest that the mechanisms advanced above for hydride / d2 exchange in 3 should be considered in nitrogenase: specifically, bridging hydride species may play key roles in h2/d2 exchange in the femoco cluster . D2 was utilized as a deuterium source to enable the isolation and characterization of the low - coordinate iron deuteride complexes [lfe(-d)]2 and [lfe(-d)]2 . The mssbauer spectra of these hydride complexes indicated that the metal centers are high - spin iron(ii). This effect was observed only in the dimeric complexes, implicating the slightly smaller size of the m2(-d)2 core as the main cause of piecs . Piecs also enabled the correct h nmr assignments of the hydride complexes, as well as the study of intermolecular hydride exchange . The exchange of hydrides between hydride complexes of the largest supporting ligand is likely to occur through dissociation of the dimers into monomers . However, exchange of the hydrides with added h2 occurs most rapidly with the smaller supporting ligand, implicating diiron(ii) hydrides as the key intermediates . More generally, these studies show that h nmr spectroscopy can be a powerful tool for the study of paramagnetic iron hydride complexes: not despite the paramagnetism, but because of the paramagnetism through the piecs effect . All manipulations were performed under a nitrogen atmosphere (or argon atmosphere where specified) by schlenk techniques or in an m. braun glovebox maintained at or below 1 ppm of o2 and h2o . Glassware was dried at 150 c overnight, and celite was dried overnight at 200 c under vacuum . Pentane, hexane, benzene, diethyl ether, and toluene were purified by passage through activated alumina and q5 columns from glass contour co. (laguna beach, ca). All solvents were degassed by removing a small amount of solvent under reduced pressure prior to argon glovebox entry . Toluene - d8 and thf - d8 were vacuum - transferred from sodium benzophenone ketyl solutions and were stored over 3 molecular sieves . Before use, an aliquot of each solvent was tested with a drop of sodium benzophenone ketyl in thf solution . Ultrahigh - purity h2 was purchased from air products, and d2 (99.8% d) was purchased from sigma - aldrich or cambridge isotope laboratories . Lfecl, potassium graphite, [lfe(-h)]2, [lco(-h)]2, and lh were prepared by published procedures . Lk was prepared using the published procedure, except et2o was used as the solvent instead of toluene . All resonances in the h nmr spectra are referenced to residual protiated solvents: benzene (7.16 ppm), toluene (2.09 ppm), or thf (3.58 or 1.73 ppm). Resonances were singlets unless otherwise noted . Ir data were recorded on a shimadzu ftir spectrophotometer (ftir-8400s) using a kbr pellet . Uv vis spectra were recorded on a cary 50 spectrophotometer using schlenk - adapted quartz cuvettes with a 1 mm optical path length . Gc - ms was performed using a shimadzu qp2010 system with electron impact ionization . Elemental analyses were obtained from the centc elemental analysis facility at the university of rochester . Microanalysis samples were weighed with a perkinelmer model ad-6 autobalance in a vac atmospheres glovebox under argon, and their compositions were determined with a perkinelmer 2400 series ii analyzer . Dimeric [lfe(-h)]2: 67.3, 21.8, 19.8, 14.3 (18h, bu), 12.0 (18h, bu), 5.4, 2.6, 7.5, 9.7, 10.6, 14.5, 15.4, 22.0, 27.8, 31.6, 37.2, 51.8, 57.5 ppm . Resonances in the dimers could not be assigned to specific proton environments, because of overlap that prevented accurate integration . Monomeric lfeh: 115 (1h, -h), 41.7 (18h, bu), 11.7 (4h, pr - ch or aryl m - h), 26.5 (12h, pr ch3), 109 (4h, pr - ch or aryl m - h), 113 (2h, aryl p - h), 122 (12h, pr ch3) ppm . The synthesis of [lfe(-d)]2 relied on the same procedure as the synthesis of 1 from h2 . After 16 h, the headspace gases were removed, and fresh d2 was added; this process was repeated twice . H nmr (c6d6, 25 c): [lfe(-d)]2: 73.0, 23.0, 21.0, 14.3 (18h, bu), 13.0 (18h, bu), 6.4, 1.7, 3.9, 8.0, 10.6, 16.9, 17.5, 25.1, 29.6, 33.0, 43.0, 50.2, 55.0 ppm . Lfed: 115 (1h, -h), 41.7 (18h, bu), 11.7 (4h, pr - ch or aryl m - h), 26.5 (12h, pr ch3), 109 (4h, pr - ch or aryl m - h), 113 (2h, aryl p - h), 122 (12h, pr ch3) ppm . The monodeuterated hydride dimer, 1-d, was also observed in solution as described above . H nmr (c6d6, 25 c): 70.2, 22.4, 20.5, 14.3 (18h, bu), 12.5 (18h, bu), 5.9, 2.1, 6.6, 7.7, 10.6, 15.7, 16.5, 23.6, 28.7, 32.3, 40.1, 51.0, 56.2 ppm . Lk (3.215 g, 7.039 mmol) was added to a flask with a teflon pin closure and was dissolved in thf (75 ml) to give a light yellow solution . Anhydrous febr2 (1.532 g, 7.104 mmol, 1.01 equiv) was added to the solution, which produced a red reaction mixture . The flask was sealed, and the mixture was heated at 70 c for 16 h. the reaction mixture turned yellow in color upon heating . The yellow solution was concentrated to 20 ml, and pentane (100 ml) was added to precipitate additional insoluble material (presumably kbr), which was removed by filtration through celite . The yellow solution was concentrated to 5 ml, which resulted in the formation of a large amount of yellow crystalline solid . The supernatant was decanted, and the crystalline yellow solid was washed with pentane (12 ml). Additional product (1.328 g) was collected from subsequent crystallizations of the supernatant by layering with pentane and cooling to 45 c . H nmr (thf - d8, 22 c): 18.6 (4h, aryl m - h), 4.9 (12h, pr ch3), 8.7 (12h, pr ch3), 12.3 (br, 4h, pr - ch), 39.9 (2h, aryl p - h), 67.3 (6h, backbone ch3), 78.7 (1h, -h) ppm . Ir (kbr): 3058 (w), 2964 (s), 2928 (s), 1529 (s), 1459 (s), 1437 (s), 1388 (vs), 1316 (s), 1261 (m), 1176 (m), 1100 (m), 1057 (w), 1022 (m), 935 (m), 899 (w), 872 (m), 855 (m), 795 (s), 758 (s) cm . Vis (thf): 333 (21.2 mmcm), 433 (sh, 0.9 mmcm) nm . Calcd for c33h49n2febro: c, 63.36; h, 7.91; n, 4.48 . Found: c, 63.13; h, 8.10; n, 4.31% . In an ar - filled glovebox, lfebr(thf) (703 mg, 1.12 mmol) was dissolved in thf (20 ml) to give a yellow solution, which was added to a small three - neck round - bottom flask with a stir bar . On a vacuum line, h2 (1 atm) was added to a bulb (297.5 ml, 12.4 mmol, 11 equiv), and the bulb was brought into the glovebox . Kc8 (182 mg, 1.35 mmol, 1.2 equiv) was added to a solid addition tube . The three - neck flask was attached to the volume bulb, a vacuum adapter, and the solid addition piece . The reaction apparatus was degassed under reduced pressure until a small amount of thf had been removed . Then, the apparatus was backfilled with h2 by slowly opening the stopcock of the volume bulb . Kc8 was added to the stirring solution by inverting the solid addition piece, which immediately produced a dark green reaction mixture . After 3 h, the mixture was filtered through celite, and the volatile components were removed under reduced pressure . The resulting brown residue was dissolved in toluene (35 ml) and was filtered through celite to remove additional insoluble material . Toluene was removed under reduced pressure to give a brown powder, which was washed with cold pentane (45 c, 10 ml). The solid was dried under reduced pressure to give 263 mg of brown powder . The pentane wash was concentrated to 3 ml and was layered with hexamethyldisiloxane (4 ml). Cooling to h nmr (c6d6, 25 c): 13.0 (12h + 8h, backbone ch3 and aryl m - h), 7.1 (24h, pr ch3), 24.0 (4h, aryl p - h), 24.8 (br, 24h, pr ch3), 55.9 (2h, -h) ppm . The pr - ch protons were not observed at this temperature (see text). The deuterated isotopologue of 3, 3-d2, was synthesized using the same method with d2, in 58.1% yield . A wilmad 522-pv-7 pressure nmr tube with a 5 mm outer diameter (od) and a maximum pressure rating of 200 psi was used for all high - pressure gas experiments . The tube comes equipped with a wilmad pv - anv valve that is capable of accepting a swagelok 1/8 tubing nut and ferrule . Poly(tetrafluoroethylene) (ptfe) tubing (od = 1/8) and swagelok 1/8 tubing nuts and ferrules were used for all the connections . Ptfe tubing was used to connect the gas regulator to a t - shaped splitter, which provided two paths . One path connected through ptfe tubing a high - pressure gas gauge and the pv - anv valve on the nmr tube . The second path connected the ptfe tubing to a swagelok valve, which was connected to a metal o - ring joint . The other part of the o - ring joint was equipped with a metal - to - glass flange that had a glass 14/20 female joint . The minimum experimental line width was 0.24 mm / s (full width at half - height). Isomer shifts are quoted relative to iron metal at 300 k. the zero - field spectra were simulated by using lorentzian doublets.
Statins (3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors) were introduced in denmark in 1990 and are currently recommended as a preventive treatment for men and women of all ages at high risk of cardiovascular events.1 during the past two decades, statins have become one of the most commonly used groups of drugs worldwide . Between 1995 and 2012, 842,484 danish individuals (the current population of denmark is 5.6 million) filled at least one statin prescription at a community pharmacy, according to data from the danish national board of health.2 in the 1990s, there was a four- to fivefold increase in numbers of new patients who received statins.3 the prevalence of statin use in denmark was predicted to increase by over 300% between 2006 and 2012,4 based on a continuous rise in new users after 2005 . Compliance with statin treatment was rated high in denmark in the 1990s,5 but others have raised concerns regarding persistence of use, with reports of a large number of dropouts in short - term studies.6 while clinical studies often focus on middle- and low - risk populations, population - based studies may focus on real - life data, including high - risk populations.710 clinical trials have demonstrated that the most widely prescribed statins (ie, simvastatin, atorvastatin, rosuvastatin, and pravastatin) for both primary and secondary prevention purposes reduce total plasma cholesterol and low - density lipoprotein (ldl) cholesterol; reductions of 0.52.26 mmol / l for total and ldl cholesterol levels, respectively, were reported in a meta - analysis of 181 randomized controlled trials of statin.11 on the other hand, the cholesterol level target attainment in routine clinical care is less well described, with somewhat inconsistent results . Among 2,227 elderly medicare advantage members in the us, 67%94% reached the ldl cholesterol goal, depending on statin type prescribed.7 in their study of 2,303 patients starting statins in the netherlands, heintjes et al8 stratified cholesterol target attainment by patients risk for cardiovascular disease; among high - risk patients, target attainment varied from 40% for patients on pravastatin to 81% for patients on rosuvastatin . In a population - based cohort study in spain, 74% of patients did not attain their ldl cholesterol treatment goal after 3 years of follow - up.9 in contrast, among 1,103 patients from community - based practices in canada who filled prescriptions for statins, 73% achieved the target ldl cholesterol level.10 in the current population - based study, we examined the rate of patients initiating statin use and the prevalence of use in northern denmark during 200410, a setting in which national guidelines recommend using simvastatin as first - line drug . In addition, we aim to examine changes in lipid values over time after statin initiation and determined the percentage of patients attaining treatment goals for cholesterol levels, stratified by risk groups, and whether statin type was switched . Denmark provides tax - funded universal access to primary and secondary health care, with no substantial out - of - pocket expenses and with partial reimbursement for most prescribed medications, including statins . Individual - level data from all danish registries can be linked via the unique personal identifier (cpr number) assigned at birth by the danish civil registration system (crs).12 in denmark, statins are available to outpatients only by prescriptions filled at community pharmacies . We retrieved all entries for redemption of a first prescription (index date) of a statin (atc classification system: c10aa statins, c10ba statin + ezetimibe) in the aarhus university prescription database . This is a research database of all reimbursed prescriptions filled at community pharmacies in northern denmark since 1998.13 the study included all patients initiated on statins in 200410, excluding patients with a statin treatment 15 months prior to 2004, who were aged between 18 and 86 years and who filled a first statin prescription between january 2004 and december 2010 at any pharmacy in northern denmark . We excluded patients with a previous cancer diagnosis (international classification of diseases [icd], eighth edition: 140208, excluding 172173 and icd-10: c00c99) through linkage with the danish national registry of patients . This registry contains discharge diagnoses from all inpatient admissions to danish hospitals since 1977 and hospital outpatient clinic diagnoses since 1995.14 we obtained information about doses, such as the defined daily dose (ddd), and adherence to statin treatment, including medication possession ratio (mpr), switches among statin types, and persistence . Ddd is given to each atc code and is the assumed daily average maintenance dose when used for a main indication . Nonpersistence was defined as no reimbursements for new statin prescriptions during the time corresponding to all ddds in the last prescribed package plus the following 6 months (180 days). The crs contains electronic records of age, sex, vital status, and place of residence (address) for the entire danish population since 1968, updated daily.12 from the crs, we retrieved information on sex, age, place of residence (urban / rural), marital status (married, never married, widowed / divorced), and vital status (alive, dead, emigrated). From the danish national registry of patients we obtained information on each patient s comorbidities, assessing the presence of major conditions based on the complete hospital contact history before start of statin use . From the aarhus university prescription database we obtained information on comedications, including oral glucose - lowering drugs, antithrombotics, and oral anticoagulants . The clinical laboratory information system (labka) research database contains information on blood samples obtained by primary care and hospital physicians and analyzed at hospitals in northern denmark since 2000.15 from the labka database we collected information on total cholesterol, ldl cholesterol, high - density lipoprotein (hdl) cholesterol, sample dates, and results during 200410 . In total, 76% (123,743/161,646) of patients had one or more cholesterol measurements before and one after statin initiation . Based on international and national guidelines1619 we defined three risk groups, based on availability of information in the hospital contact and prescription registries, and determined the achievement of target cholesterol levels in each group . The first group contained a very high - risk group of patients with a hospital - based diagnosis of cardiovascular disease, whose targets were <1.8 mmol / l for ldl cholesterol and <4 mmol / l for total cholesterol . The second group consisted of a high - risk group of patients with diabetes (receiving antidiabetic medications or with a history of hospitalization or outpatient care for diabetes), hypertension, or a diagnosis of familial dyslipidemia, whose targets were <2.5 mmol / l for ldl cholesterol and <4.5 mmol / l for total cholesterol . The third group contained the remaining patients with low to moderate risk, whose targets were <3 mmol / l for ldl cholesterol and <5 mmol / l for total cholesterol . This study was approved by the danish data protection agency (record number 2009 - 41 - 3866). We examined the rate and prevalence of statin use annually during 200410 and overall; annual person - years at risk in northern denmark for individuals aged 1886 years (denominator) were obtained from statistics denmark . We examined the starting dose (mg) among new statin users by calendar year and type of statin . To measure adherence, we calculated average daily use in ddds and mpr . We calculated the mpr as the ratio of days statin pills are supplied to days in the time interval to the next statin prescription (eg, 30 pills prescribed/40 days mpr of 0.75)20 and the persistence of use (see previous definition of nonpersistence). We calculated the proportion of patients with different demographic and clinical characteristics at the time they filled their first statin prescription (index date). We created similar contingency tables for the subgroup of patients who switched to another statin type, at the time of their switch . We also compared the characteristics of statin initiators with and without cholesterol measurements recorded in the aarhus university prescription database . For the 123,743 patients with at least one cholesterol measurement recorded prior to and at least one after their first statin prescription, we examined cholesterol values before and after start of statin treatment, graphically examined cholesterol values over time, and calculated percentage reduction . We examined median and quartiles of total, ldl, and hdl cholesterol at each measurement, focusing particularly on changes between the last cholesterol measurement before commencement of statin treatment and the first measurement after treatment initiation, and on cholesterol level treatment goals . These analyses were conducted for all new statin users, stratified by switching (yes / no). We also examined the proportion of patients reaching their target levels for ldl and total cholesterol at any time within the study period, stratified by level of risk for cardiovascular events (very high, high, and low to moderate). A sensitivity analysis was conducted to examine cholesterol target attainment at the very first measurement after the date of statin initiation . Denmark provides tax - funded universal access to primary and secondary health care, with no substantial out - of - pocket expenses and with partial reimbursement for most prescribed medications, including statins . Individual - level data from all danish registries can be linked via the unique personal identifier (cpr number) assigned at birth by the danish civil registration system (crs).12 in denmark, statins are available to outpatients only by prescriptions filled at community pharmacies . We retrieved all entries for redemption of a first prescription (index date) of a statin (atc classification system: c10aa statins, c10ba statin + ezetimibe) in the aarhus university prescription database . This is a research database of all reimbursed prescriptions filled at community pharmacies in northern denmark since 1998.13 the study included all patients initiated on statins in 200410, excluding patients with a statin treatment 15 months prior to 2004, who were aged between 18 and 86 years and who filled a first statin prescription between january 2004 and december 2010 at any pharmacy in northern denmark . We excluded patients with a previous cancer diagnosis (international classification of diseases [icd], eighth edition: 140208, excluding 172173 and icd-10: c00c99) through linkage with the danish national registry of patients . This registry contains discharge diagnoses from all inpatient admissions to danish hospitals since 1977 and hospital outpatient clinic diagnoses since 1995.14 we obtained information about doses, such as the defined daily dose (ddd), and adherence to statin treatment, including medication possession ratio (mpr), switches among statin types, and persistence . Ddd is given to each atc code and is the assumed daily average maintenance dose when used for a main indication . Nonpersistence was defined as no reimbursements for new statin prescriptions during the time corresponding to all ddds in the last prescribed package plus the following 6 months (180 days). The crs contains electronic records of age, sex, vital status, and place of residence (address) for the entire danish population since 1968, updated daily.12 from the crs, we retrieved information on sex, age, place of residence (urban / rural), marital status (married, never married, widowed / divorced), and vital status (alive, dead, emigrated). From the danish national registry of patients we obtained information on each patient s comorbidities, assessing the presence of major conditions based on the complete hospital contact history before start of statin use . From the aarhus university prescription database we obtained information on comedications, including oral glucose - lowering drugs, antithrombotics, and oral anticoagulants . The clinical laboratory information system (labka) research database contains information on blood samples obtained by primary care and hospital physicians and analyzed at hospitals in northern denmark since 2000.15 from the labka database we collected information on total cholesterol, ldl cholesterol, high - density lipoprotein (hdl) cholesterol, sample dates, and results during 200410 . In total, 76% (123,743/161,646) of patients had one or more cholesterol measurements before and one after statin initiation . Based on international and national guidelines1619 we defined three risk groups, based on availability of information in the hospital contact and prescription registries, and determined the achievement of target cholesterol levels in each group . The first group contained a very high - risk group of patients with a hospital - based diagnosis of cardiovascular disease, whose targets were <1.8 mmol / l for ldl cholesterol and <4 mmol / l for total cholesterol . The second group consisted of a high - risk group of patients with diabetes (receiving antidiabetic medications or with a history of hospitalization or outpatient care for diabetes), hypertension, or a diagnosis of familial dyslipidemia, whose targets were <2.5 the third group contained the remaining patients with low to moderate risk, whose targets were <3 mmol / l for ldl cholesterol and <5 mmol / l for total cholesterol . This study was approved by the danish data protection agency (record number 2009 - 41 - 3866). We examined the rate and prevalence of statin use annually during 200410 and overall; annual person - years at risk in northern denmark for individuals aged 1886 years (denominator) were obtained from statistics denmark . We examined the starting dose (mg) among new statin users by calendar year and type of statin . To measure adherence, we calculated average daily use in ddds and mpr . We calculated the mpr as the ratio of days statin pills are supplied to days in the time interval to the next statin prescription (eg, 30 pills prescribed/40 days mpr of 0.75)20 and the persistence of use (see previous definition of nonpersistence). We calculated the proportion of patients with different demographic and clinical characteristics at the time they filled their first statin prescription (index date). We created similar contingency tables for the subgroup of patients who switched to another statin type, at the time of their switch . We also compared the characteristics of statin initiators with and without cholesterol measurements recorded in the aarhus university prescription database . For the 123,743 patients with at least one cholesterol measurement recorded prior to and at least one after their first statin prescription, we examined cholesterol values before and after start of statin treatment, graphically examined cholesterol values over time, and calculated percentage reduction . We examined median and quartiles of total, ldl, and hdl cholesterol at each measurement, focusing particularly on changes between the last cholesterol measurement before commencement of statin treatment and the first measurement after treatment initiation, and on cholesterol level treatment goals . These analyses were conducted for all new statin users, stratified by switching (yes / no). We also examined the proportion of patients reaching their target levels for ldl and total cholesterol at any time within the study period, stratified by level of risk for cardiovascular events (very high, high, and low to moderate). A sensitivity analysis was conducted to examine cholesterol target attainment at the very first measurement after the date of statin initiation . In total, there were 161,646 statin patients initiated on statins in northern denmark between 2004 and 2010 . In 2004, there were 16.3 new users per 1,000 person - years (figure 1a). The annual number of patients initiated on statins increased until 2008 (19.9 per 1,000 person - years) and then decreased until 2010 (14.1 per 1,000 person - years). Figure 1b shows the resulting cumulative prevalence of statin use up to 2010 . In 2010, there were 133,979 adults, corresponding to 93.8 per 1,000 adults (aged between 18 and 86 years), in northern denmark who still used statins and who had initiated their statin use between 2004 and 2010 . At statin initiation among the 161,646 patients, 98% started with simvastatin . Demographic characteristics varied little between users of different statin types . Sixty percent of patients were aged between 55 and 75 years at statin initiation, and 51% were men (table 1). As expected, many patients had previous hospital - based diagnoses of heart disease and other chronic conditions (table 2). In general, patients initiated on statins during 200410 were on statin treatment for a median of 1,075 days, or close to 3 years . In this period, for example, the proportion of simvastatin users who started with 40 mg tablets increased from 40% in 2004 to 75% in 2010, and the proportion of rosuvastatin users who started with high doses (20 mg or 40 mg) increased from 12% in 2004 to 21% in 2010 . Overall, the average mpr for the entire period was 0.93 (interquartile range [iqr] 0.771.0). Of the statin initiators in this sample, 88% (142,807/161,646) did not switch statin type . Of these 142,807 nonswitchers, only 18% (24,998/142,807) altered their statin dose during the study period . Of the 18,839 (12%) persons who did switch one or more times, 13,138 (70%) switched only once . By far the most common initial switches were from simvastatin to atorvastatin (48%; 9,104/18,839), to rosuvastatin (35%; 6,644/18,839), or to pravastatin (9%; 1,603/18,839). For example, 21% versus 11% had chronic ischemic heart disease and 11% versus 8% had a previous myocardial infarction . In contrast, the prevalence of other important comorbidities (eg, chronic obstructive pulmonary disease) was similar to that in nonswitchers . The sex and age distributions among statin switchers and nonswitchers were similar (51% males among nonswitchers and 48% among switchers; 40% aged over 65 years among nonswitchers and 39% among switchers). The mpr for the nonswitchers was 0.93 (iqr 0.781.0), while the mpr for the patients who switched statin type was 0.90 (iqr 0.751.0) preswitch and 0.93 (iqr 0.701.0) postswitch . Among the statin initiators, 26,314 persons (16%) completely stopped their statin treatment: ie, were nonpersistent . Compared with all patients who started statins, nonpersistent patients were more likely to be very young (aged <45 years) or very old (aged> 75 years), more likely to live in small municipalities in rural areas, more likely to be divorced, and importantly had a slightly higher prevalence of almost all examined diagnoses of comorbidity, including cardiovascular disease (data not shown). Among the 133,856 patients with at least one cholesterol measurement recorded prior to their first statin prescription, the most recent cholesterol measurement was, on average, 9 days (quartiles 528 days) before redemption of a statin prescription . Furthermore, 123,743 patients had at least one cholesterol surveillance measurement (median 80 days) after starting to use statins . The demographic and clinical characteristics of patients with and without cholesterol measurements recorded in the database were very similar, except that more patients without measurement lived in large cities (29% vs 8%), and more patients without measurement (49% vs 38%) started statin treatment early in the period (200406), when the laboratory database may have been less complete . Median total cholesterol in new statin users decreased from 6.3 (quartiles 5.57.0) mmol / l at the most recent measurement before statin initiation to 4.5 (quartiles 3.95.1) mmol / l at the first measurement after initiation, and remained remarkably stable thereafter (figure 2a), corresponding to a reduction of 28% . Median ldl cholesterol decreased rapidly from 4.0 (quartiles 3.34.6) mmol / l to 2.3 (quartiles 1.92.8) mmol / l, corresponding to a reduction of 43% . Median hdl cholesterol remained stable at 1.4 mmol / l before and after statin initiation . Among patients who later switched statin type, there was a smaller initial decrease in cholesterol values compared with nonswitchers, and some increase in cholesterol levels after the first surveillance measurement (figure 2b). Among patients who switched statin type, we observed a modest reduction in cholesterol level after their switch (figure 2c). Table 3 shows attainment of target cholesterol levels by risk group . Among all statin users, 66% of patients in the very high - risk group reached their target value for total cholesterol, while corresponding figures were 74% and 80% for patients in the high - risk and low- to moderate - risk groups, respectively . When stratified by switching of statin type (yes / no), results for statin nonswitchers were comparable with the overall results . Among patients who switched statin type, the percentages attaining target cholesterol levels after the first switch and before the second were 46%, 56%, and 64% for the three risk groups, respectively . When focusing on target attainment at the very first measurement after statin initiation (averaging 3 months after statin start), 43% in the very high - risk, 49% in the high - risk, and 55% in the low- to moderate - risk groups reached total cholesterol target . In total, there were 161,646 statin patients initiated on statins in northern denmark between 2004 and 2010 . In 2004, there were 16.3 new users per 1,000 person - years (figure 1a). The annual number of patients initiated on statins increased until 2008 (19.9 per 1,000 person - years) and then decreased until 2010 (14.1 per 1,000 person - years). Figure 1b shows the resulting cumulative prevalence of statin use up to 2010 . In 2010, there were 133,979 adults, corresponding to 93.8 per 1,000 adults (aged between 18 and 86 years), in northern denmark who still used statins and who had initiated their statin use between 2004 and 2010 . Demographic characteristics varied little between users of different statin types . Sixty percent of patients were aged between 55 and 75 years at statin initiation, and 51% were men (table 1). As expected, many patients had previous hospital - based diagnoses of heart disease and other chronic conditions (table 2). In general, initial use of statins other than simvastatin was associated with higher baseline comorbidity . Patients initiated on statins during 200410 were on statin treatment for a median of 1,075 days, or close to 3 years . In this period, for example, the proportion of simvastatin users who started with 40 mg tablets increased from 40% in 2004 to 75% in 2010, and the proportion of rosuvastatin users who started with high doses (20 mg or 40 mg) increased from 12% in 2004 to 21% in 2010 . Overall, the average mpr for the entire period was 0.93 (interquartile range [iqr] 0.771.0). Of the statin initiators in this sample, 88% (142,807/161,646) did not switch statin type . Of these 142,807 nonswitchers, only 18% (24,998/142,807) altered their statin dose during the study period . Of the 18,839 (12%) persons who did switch one or more times, 13,138 (70%) switched only once . By far the most common initial switches were from simvastatin to atorvastatin (48%; 9,104/18,839), to rosuvastatin (35%; 6,644/18,839), or to pravastatin (9%; 1,603/18,839). For example, 21% versus 11% had chronic ischemic heart disease and 11% versus 8% had a previous myocardial infarction . In contrast, the prevalence of other important comorbidities (eg, chronic obstructive pulmonary disease) was similar to that in nonswitchers . The sex and age distributions among statin switchers and nonswitchers were similar (51% males among nonswitchers and 48% among switchers; 40% aged over 65 years among nonswitchers and 39% among switchers). The mpr for the nonswitchers was 0.93 (iqr 0.781.0), while the mpr for the patients who switched statin type was 0.90 (iqr 0.751.0) preswitch and 0.93 (iqr 0.701.0) postswitch . Among the statin initiators, 26,314 persons (16%) completely stopped their statin treatment: ie, were nonpersistent . Compared with all patients who started statins, nonpersistent patients were more likely to be very young (aged <45 years) or very old (aged> 75 years), more likely to live in small municipalities in rural areas, more likely to be divorced, and importantly had a slightly higher prevalence of almost all examined diagnoses of comorbidity, including cardiovascular disease (data not shown). Among the 133,856 patients with at least one cholesterol measurement recorded prior to their first statin prescription, the most recent cholesterol measurement was, on average, 9 days (quartiles 528 days) before redemption of a statin prescription . Furthermore, 123,743 patients had at least one cholesterol surveillance measurement (median 80 days) after starting to use statins . The demographic and clinical characteristics of patients with and without cholesterol measurements recorded in the database were very similar, except that more patients without measurement lived in large cities (29% vs 8%), and more patients without measurement (49% vs 38%) started statin treatment early in the period (200406), when the laboratory database may have been less complete . Median total cholesterol in new statin users decreased from 6.3 (quartiles 5.57.0) mmol / l at the most recent measurement before statin initiation to 4.5 (quartiles 3.95.1) mmol / l at the first measurement after initiation, and remained remarkably stable thereafter (figure 2a), corresponding to a reduction of 28% . Median ldl cholesterol decreased rapidly from 4.0 (quartiles 3.34.6) mmol / l to 2.3 (quartiles 1.92.8) mmol / l, corresponding to a reduction of 43% . Median hdl cholesterol remained stable at 1.4 mmol / l before and after statin initiation . Among patients who later switched statin type, there was a smaller initial decrease in cholesterol values compared with nonswitchers, and some increase in cholesterol levels after the first surveillance measurement (figure 2b). Among patients who switched statin type, we observed a modest reduction in cholesterol level after their switch (figure 2c). Table 3 shows attainment of target cholesterol levels by risk group . Among all statin users, 66% of patients in the very high - risk group reached their target value for total cholesterol, while corresponding figures were 74% and 80% for patients in the high - risk and low- to moderate - risk groups, respectively . When stratified by switching of statin type (yes / no), results for statin nonswitchers were comparable with the overall results . Among patients who switched statin type, the percentages attaining target cholesterol levels after the first switch and before the second were 46%, 56%, and 64% for the three risk groups, respectively . When focusing on target attainment at the very first measurement after statin initiation (averaging 3 months after statin start), 43% in the very high - risk, 49% in the high - risk, and 55% in the low- to moderate - risk groups reached total cholesterol target . Our population - based data show that the rate of new statin use in northern denmark seemed to reach a plateau and begin to decline starting in 2008, and the initiators were adherent in their use . The reduction in median total cholesterol and ldl cholesterol was similar to that observed in clinical trials . Following european guideline lipid goals, 66% in the very high - risk group reached target levels for overall cholesterol and 54% for ldl cholesterol . Corresponding percentages were about 80% among the high - risk and low- to moderate - risk groups . Previous reports have shown a large increase in population - wide statin use in the 2000s . Raymond et al21 reported a 1.7-fold increase in incident statin use based on prescription drug claims in british colombia during 19992004 . Our finding that new statin use levelled off starting in the late 2000s may be due to statin treatment saturation within a large pool of previously untreated individuals, or possibly more restrictive use after reports of potential adverse long - term effects of statins . As expected, many patients initiated on statins had previous hospital - based diagnoses of cardiovascular diseases and other comorbidities . The higher proportions of documented cardiovascular disease among patients initiated on the more potent statins may be related to guidelines recommending their use in patients with acute cardiovascular disease.19 furthermore, the increase in starting doses of statins during the observation period is also in accordance with newer treatment guidelines;22 further examination of the impact on the changed starting doses over time is warranted, as dose is related to efficacy in lowering cholesterol.11 we chose to examine attainment of cholesterol levels overall for the statins, since almost all new users start with simvastatin in denmark, in accordance with danish guidelines . Our finding that attainment of target cholesterol levels differs among patient risk groups is in line with previous real - world findings from the netherlands.8 additionally, the cholesterol target level attainment in this routine clinical care setting was similar to findings from randomized controlled trials.11 while danish and european guidelines contain lipid goals stratified on risk,17,18 the newest us guidelines have removed the use of lipid goals, and suggestions have been voiced that they ought to be refined rather than removed.23 given that the main desired outcome of increased statin therapy is a decrease in cardiovascular events, it is interesting to note that the incidence of new myocardial infarction has continously decreased over the past 20 years in denmark, as reported by schmidt et al24 based on population - based data . They only included follow - up until 2008; thus, further observation is warranted as to whether the continuing increase in statin use and the plateau from 2008 have been accompanied by a continuing decrease in cardiovascular events in denmark . Adherence was high in this study; both a high medication possession (mpr) and high treatment persistence were observed during our study period . Whereas others have reported clinically important declines in persistence over time,6 only 16% among our study participants appeared to quit statin use totally . Compared with previous studies of statin initiators, our study had the advantage of a rather long follow - up time and allowed for shorter gaps of treatment . The generally high adherence observed in this study is positive news, and further examinations of predictors of adherence are warranted . Danish guidelines state that simvastatin should be the first - line drug of choice, and it is apparent from our findings that danish physicians adhere to these guidelines.17 twelve percent of patients initiated on statins switched statin type, most often from simvastatin to a more potent statin . In clinical practice, reasons for switching may include adverse side effects and/or failure to attain target cholesterol levels . Accordingly, we observed that patients who later shifted statin type experienced less initial reduction of cholesterol levels . Unfortunately, our data did not allow us to determine whether insufficient cholesterol reduction was related to poor drug effectiveness, reduced compliance (eg, due to side effects), or choice of dose with the first statin prescribed, but we know that adherence (expressed as mpr) is comparable . Patients who later shifted statin type also had substantially more heart disease than all initiators, which may explain this group s greater difficulty in reaching their cholesterol targets . Strengths of this study include its large size, population - based design, and access to high - quality danish medical databases providing a complete hospital contact, prescription, and laboratory history . Our findings are based on data from a wide range of unselected patients in the community and may be transferrable to other population - based settings . Some statin - treated patients apparently did not present for surveillance measurements, some may have emigrated outside our data catchment area, some died, and some started statins late in the study period and were followed for only a short time . The patients without cholesterol measurement did not differ in demographic or clinical characteristics compared with the patients with cholesterol measurements . We focused on new statin users from 2004 onward, and patients who had already received statins before the start of the study were left - truncated . This may have excluded from our study early statin users in the background population with a strong medical indication for statins, and subjects who had earlier started and then entirely discontinued statins due to adverse events or lack of effect . On the other hand, having a short look - back period of only 15 months before 2004 may have artificially inflated new statin user rates in the earliest study years because of the inclusion of some previous statin users who restarted statin use during the first study years . In addition, filling a statin prescription is only a proxy for statin utilization . However, the calculated medication possession ratio in our patients was close to 1, suggesting a surprisingly high adherence to the medication in our region . Furthermore, diagnoses made in the primary care setting by patients general practitioners were not included in our data, and our estimates of comorbidity prevalence are likely to be underestimates, as can be identified by the discrepancy between diseases and marker drugs . Another concern is that lifestyle changes such as a healthier diet and increased physical exercise are the recommended first interventions to decrease high lipid levels . Unfortunately, we had no information on such and other (eg, smoking cessation) behavioral changes . Finally, our patient risk group classification had to be modified according to data availability . In particular, we lacked data on individual risk factors necessary to evaluate the indication for statin treatment in the low- to moderate - risk group . In conclusion, our population - based data from routine clinical care show that statin use is very prevalent in northern denmark . Cholesterol level target attainment in the community setting is similar to findings from clinical trials; however, a substantial proportion of patients, particularly those at high risk, did not reach target cholesterol levels.
The frequency of upper urinary tract urothelial carcinoma (utuc) is approximately 5% of urothelial malignancies and <10% of renal tumors . This cancer can be treated by radical nephroureterectomy with excision of the bladder cuff, but treatment is often problematic owing to difficulties in early diagnosis, the high incidence of tumor recurrence, and poor prognostic outcomes . Because systemic recurrences are common in this disease, it is reasonable to consider perioperative treatments that might reduce this risk . However, few studies have explored such perioperative treatments, including chemotherapy or radiation therapy, and these limited studies have yielded conflicting results . Some potential perioperative agents that might confer chemoprotective effects are vitamin a, vitamin e, vitamin c, selenium, nonsteroidal anti - inflammatory drugs, isoflavone, and statins (hydroxymethylglutaryl - coa - reductase inhibitors). Of these, statins have been used to treat high cholesterol in general owing to their efficacy and minimal side effects . Statins improve the blood lipid profile, treat a number of cardiovascular diseases, and specifically reduce mortality from coronary heart disease . Although vascular secondary end points are the main clinical target of these agents, statins also affect other conditions . In particular, a growing body of evidence suggests that statins provide a chemoprotective advantage against many cancers, likely by arresting cell cycle progression, suppressing angiogenesis, inducing apoptosis, and inhibiting tumor growth and metastasis . Currently, preclinical data show that statins induce cell cycle arrest and apoptosis and inhibit proliferation in human urothelial carcinoma cell lines . However, the impact of statin use has been investigated mainly in patients with lower urinary tract carcinomas owing to the infrequency of utuc . In previous studies, statin use showed conflicting effects on the prognosis of patients with lower utuc, and only one multi - institutional study did not suggest an influence of statin therapy on utuc prognosis . However, these studies were limited by heterogeneous groups of patients and variable follow - up schedules according to each institution . Therefore, we assessed the impact of statins on oncologic outcomes in patients with utuc after radical nephroureterectomy performed at a single center . A review of the medical records of our single institute identified 291 patients with primary utuc who underwent radical nephroureterectomy between january 2006 and december 2011 . Patients who had previous or synchronous invasive bladder cancer, who had distant metastasis at diagnosis, or who had received neoadjuvant therapy were excluded . Three patients with concomitant invasive bladder cancer and 11 patients with distant metastasis at diagnosis were excluded from analysis . Each patient underwent a preoperative evaluation, which included blood tests, urine cytology, cystoscopy, chest x - ray, abdominopelvic computerized tomography, and a bone scan . Information regarding medication use (statin use), the presence of comorbid illnesses, smoking history, and alcohol intake history was obtained from the electronic database . Because of the retrospective nature of data collection, we were unable to obtain the duration of statin use because this information was not available in the charts . The anesthesiologists used the american society of anesthesiologists score prior to surgery to assess the ability of patients to undergo surgery . In all cases, radical nephroureterectomy with bladder cuff excision was performed and lymphadenectomy was performed when enlarged lymph nodes were either identified on preoperative computed tomography or were palpable during surgery . Tumors were pathologically staged by using the 2010 american joint committee on cancer tnm staging system, and tumors were graded according to the 2004 world health organization classification . Lymphovascular invasion was defined as the presence of tumor cells within an endothelium - lined space without underlying muscular walls . Adjuvant therapy was selectively recommended for patients with locally advanced or node - positive disease on pathology, except for those who could not tolerate chemotherapy of refused treatment . Adjuvant therapy was administered to 42 patients (12.3%), including 31 who received adjuvant chemotherapy and 27 who received adjuvant radiation therapy . Follow - up consisted of chest radiography and abdominopelvic computed tomography every 6 months for the first 2 years and annually thereafter . Surveillance cystoscopy was generally performed every 3 months for the first 2 years after radical nephroureterectomy, every 6 months for the next 2 years, and annually thereafter . Tumor recurrence was defined as local failure in the tumor bed or regional lymph nodes or distant metastasis . Since bladder recurrence did not affect survival in the current patient population, this factor was not considered when calculating the rfs rate . The cause of death was determined by chart review and was corroborated by death certificates . The statin user and nonuser subgroups were compared in terms of clinicopathological factors by using pearson chisquare test or fisher exact test for categorical variables and student t - test or mann whitney u - test for continuous variables . Kaplan - meier survival curves were used to estimate rfs and css and were compared by using the log - rank test . A cox proportional hazards regression model was used to estimate the prognostic significance of each variable . Correlations between outcomes and variables were expressed as hazard ratios (hrs) with 95% confidence intervals (cis). All statistical analyses were performed by using the ibm spss statistics ver . 21.0 (ibm co., armonk, ny, usa). A review of the medical records of our single institute identified 291 patients with primary utuc who underwent radical nephroureterectomy between january 2006 and december 2011 . Patients who had previous or synchronous invasive bladder cancer, who had distant metastasis at diagnosis, or who had received neoadjuvant therapy were excluded . Three patients with concomitant invasive bladder cancer and 11 patients with distant metastasis at diagnosis were excluded from analysis . Each patient underwent a preoperative evaluation, which included blood tests, urine cytology, cystoscopy, chest x - ray, abdominopelvic computerized tomography, and a bone scan . Information regarding medication use (statin use), the presence of comorbid illnesses, smoking history, and alcohol intake history was obtained from the electronic database . Because of the retrospective nature of data collection, we were unable to obtain the duration of statin use because this information was not available in the charts . The anesthesiologists used the american society of anesthesiologists score prior to surgery to assess the ability of patients to undergo surgery . In all cases, surgery was performed with curative intent . Radical nephroureterectomy with bladder cuff excision was performed and lymphadenectomy was performed when enlarged lymph nodes were either identified on preoperative computed tomography or were palpable during surgery . Tumors were pathologically staged by using the 2010 american joint committee on cancer tnm staging system, and tumors were graded according to the 2004 world health organization classification . Lymphovascular invasion was defined as the presence of tumor cells within an endothelium - lined space without underlying muscular walls . Adjuvant therapy was selectively recommended for patients with locally advanced or node - positive disease on pathology, except for those who could not tolerate chemotherapy of refused treatment . Adjuvant therapy was administered to 42 patients (12.3%), including 31 who received adjuvant chemotherapy and 27 who received adjuvant radiation therapy . Follow - up consisted of chest radiography and abdominopelvic computed tomography every 6 months for the first 2 years and annually thereafter . Surveillance cystoscopy was generally performed every 3 months for the first 2 years after radical nephroureterectomy, every 6 months for the next 2 years, and annually thereafter . Tumor recurrence was defined as local failure in the tumor bed or regional lymph nodes or distant metastasis . Since bladder recurrence did not affect survival in the current patient population, this factor was not considered when calculating the rfs rate . The cause of death was determined by chart review and was corroborated by death certificates . The statin user and nonuser subgroups were compared in terms of clinicopathological factors by using pearson chisquare test or fisher exact test for categorical variables and student t - test or mann whitney u - test for continuous variables . Kaplan - meier survival curves were used to estimate rfs and css and were compared by using the log - rank test . A cox proportional hazards regression model was used to estimate the prognostic significance of each variable . Correlations between outcomes and variables were expressed as hazard ratios (hrs) with 95% confidence intervals (cis). Of the 277 patients, 62 (22.4%) reported being on statins at the time of radical nephroureterectomy, whereas 215 were not . The statins used were lipophilic (n=57, 91.9%) and hydrophilic (n=5, 8.1%). The two groups differed significantly in terms of age, body mass index (bmi), cardiovascular disease, and diabetes status . Compared to the statin nonusers, the statin users were older (66.88.8 years vs. 62.710.4 years, p=0.006), had higher bmis (25.63.7 kg / m vs. 24.53.2 kg / m, p=0.015), and had higher rates of cardiovascular disease (27.4% vs. 15.3%, p=0.039) and diabetes (32.3% vs. 14.9%, p=0.003). There were no significant differences in tumor stage, grade, lymphovascular disease, surgical margin status, or adjuvant therapy rate between the two groups . At a median follow - up time of 40 months, 58 patients (18.8%) had disease recurrence and 42 patients (15.2%) had already died from utuc progression . Of the patients who had died, 7 (11.3%) were statin users and 35 (16.3%) were statin nonusers . The 5-year rfs rates of statin users and nonusers were 78.5% and 72.5%, respectively (p=0.528) (fig . 1a), and the 5-year css rates were 85.6% and 77.7%, respectively (p=0.516) (fig . The 5-year os rates were 74.5% and 71.4%, respectively (p=0.945) (fig ., rosuvastatin was excluded) also had no significant effect on outcomes (data not shown). Table 2 presents the results of the univariate and multivariate analyses of rfs and css . Multivariate analysis revealed that the only statistically significant predictors of rfs and css were tumor grade, tumor stage, and adjuvant therapy . However, statin use (hr, 0.47; 95% ci, 0.22 - 1.02; p=0.056) was not an independent prognostic factor for rfs (hr, 0.47; 95% ci, 0.22 - 1.02; p=0.056) and css (hr, 0.46; 95% ci, 0.18 - 1.14; p=0.093). In the multivariate analysis of prognostic factors for os (data not shown), age (hr, 1.05; 95% ci, 1.02 - 1.07; p<0.001), tumor grade (hr, 1.97; 95% ci, 1.21 - 3.23; p=0.008), tumor stage (hr, 2.03; 95% ci, 1.20 - 3.42; p=0.001), surgical margin (hr, 2.12; 95% ci, 1.16 - 3.86; p=0.014), and lymph node status (hr, 2.39; 95% ci, 1.34 - 4.26; p=0.003) were independent prognostic factors for os in patients who underwent radical nephroureterectomy for utuc . Of the 277 patients, 62 (22.4%) reported being on statins at the time of radical nephroureterectomy, whereas 215 were not . The statins used were lipophilic (n=57, 91.9%) and hydrophilic (n=5, 8.1%). The two groups differed significantly in terms of age, body mass index (bmi), cardiovascular disease, and diabetes status . Compared to the statin nonusers, the statin users were older (66.88.8 years vs. 62.710.4 years, p=0.006), had higher bmis (25.63.7 kg / m vs. 24.53.2 kg / m, p=0.015), and had higher rates of cardiovascular disease (27.4% vs. 15.3%, p=0.039) and diabetes (32.3% vs. 14.9%, p=0.003). There were no significant differences in tumor stage, grade, lymphovascular disease, surgical margin status, or adjuvant therapy rate between the two groups . At a median follow - up time of 40 months, 58 patients (18.8%) had disease recurrence and 42 patients (15.2%) had already died from utuc progression . Of the patients who had died, 7 (11.3%) the 5-year rfs rates of statin users and nonusers were 78.5% and 72.5%, respectively (p=0.528) (fig . 1a), and the 5-year css rates were 85.6% and 77.7%, respectively (p=0.516) (fig . The 5-year os rates were 74.5% and 71.4%, respectively (p=0.945) (fig ., rosuvastatin was excluded) also had no significant effect on outcomes (data not shown). Table 2 presents the results of the univariate and multivariate analyses of rfs and css . Multivariate analysis revealed that the only statistically significant predictors of rfs and css were tumor grade, tumor stage, and adjuvant therapy . However, statin use (hr, 0.47; 95% ci, 0.22 - 1.02; p=0.056) was not an independent prognostic factor for rfs (hr, 0.47; 95% ci, 0.22 - 1.02; p=0.056) and css (hr, 0.46; 95% ci, 0.18 - 1.14; p=0.093). In the multivariate analysis of prognostic factors for os (data not shown), age (hr, 1.05; 95% ci, 1.02 - 1.07; p<0.001), tumor grade (hr, 1.97; 95% ci, 1.21 - 3.23; p=0.008), tumor stage (hr, 2.03; 95% ci, 1.20 - 3.42; p=0.001), surgical margin (hr, 2.12; 95% ci, 1.16 - 3.86; p=0.014), and lymph node status (hr, 2.39; 95% ci, 1.34 - 4.26; p=0.003) were independent prognostic factors for os in patients who underwent radical nephroureterectomy for utuc . Currently, statins are being researched in a number of studies for their ability to prevent a variety of cancers, but the results have been inconsistent . One recent study showed that cancer patients who used statins prior to diagnosis had reduced css compared with that of patients who had never taken statins . However, that study did not report on utuc patients specifically and the effects of statin use on the clinical treatment outcomes of urothelial cell carcinoma are unknown . Especially in urology, the effect of statins on prostate cancer incidence and disease progression has been investigated in many studies . Recent meta - analysis showed a 33% risk reduction for advanced high - grade or metastatic prostate cancer and that statin use protects against prostate cancer with poorer pathological characteristics . In our previous study, we showed that postoperative statin use decreased the risk of biochemical recurrence, especially in patients with high - risk disease . However, only few studies have investigated the effect of statins on urothelial carcinoma incidence and prognosis . In one previous study that examined the effects of atorvastatin on human bladder cancer cell lines, a significant antiproliferative effect was observed in the statin group compared with controls . In another study using mouse cells transfected with the h - ras oncogene from human bladder carcinoma, the researchers observed that statin treatment significantly inhibited ras oncogene - transformed cells ., only a few studies have focused their investigations on the clinical effect of statins on urothelial carcinoma incidence and prognosis, and these studies have primarily concentrated on bladder carcinoma, not utuc . Tsai et al . Suggested that statins may improve local control in patients who underwent concurrent chemoradiotherapy for muscle - invasive bladder cancer . However, this study showed that statin use was associated with local control only in univariate, not multivariate, analysis . Recently, one multi - institutional study found that statin use was not associated with rfs or css in patients treated with radical nephroureterectomy for utuc . Similarly, statin use was also not associated with rfs or css in patients treated with radical cystectomy for bladder cancer, and statins did not affect the oncologic outcome for non - muscle - invasive bladder cancer . Additionally, a recent meta - analysis study showed no significant association between statin use and the risk of bladder cancer . Furthermore, there have been 5 retrospective studies exploring the impact of statin use on patient responses to bacillus calmette - guerin (bcg) immunotherapy . Reported that concurrent statin therapy with bcg might reduce the clinical efficacy of bcg therapy . Statin users were more likely to proceed to more aggressive disease as well as to require radical cystectomy . However, subsequent studies failed to confirm that statin therapy affects patient responses to bcg . These studies found no differences between statin users and nonusers in the mean number of recurrences or incidence of disease progression, css, or os . Overall, these results are consistent with the findings in our study, in which statin use did not improve rfs, css, or os after radical nephroureterectomy for utuc . First, because the findings represent the clinical experience of a single center, the rate of patients taking statins was lower than in a previous study . However, the prevalence of statin use in patients 50 years or older was 22.8% in 2008 . Second, medical chart review can be an unreliable method for determining the duration and dosage of medications used by a study population, because much of this information may not be reported consistently in the medical records . It would be interesting to assess the correlation between the dosage and the duration of statin use with prognosis, but unfortunately, this information was not available in the patient records . Therefore, well - designed, prospective, randomized trials that include a large patient population are warranted . Despite the limitations of the present study, to the best of our knowledge, this is the first single - center study to investigate the potential impact of statin use on the clinical outcomes of patients with utuc being treated with radical nephroureterectomy . In patients undergoing radical nephroureterectomy for utuc, statin use was not associated with rfs, css, or os . Owing to the inherent limitations of this retrospective study, the potential antineoplastic effect of statins needs further investigation prospectively.
Knowledge of maxillary artery (ma) and its branches in the infratemporal region is of great importance in dental, oral, and maxillofacial surgeries . It arises behind the mandibular neck, then passes medial to the mandibular neck and superficial or deep to the lower head of lateral pterygoid to reach the pterygoid fossa . The inferior alveolar nerve (ian) is a mixed nerve that provides sensory innervation to the lower teeth, lower lip, and buccal mucosa located between the premolars and lower central incisor through the mental nerve, and the motor innervation to the mylohyoid muscle and anterior belly of the digastric muscle through the mylohyoid nerve . The lingual nerve (ln) carries sensory fibers to the mucosa of the floor of the mouth, the ventral side of the tongue and to the anterior 2/3 of the tongue and secretary fibres to the sublingual and submandibular glands . Literature is replete with variations in infratemporal region, in the form of communication between branches of mandibular nerve (mn), entrapment of nerves by bony bridges, abnormal course and branching pattern of arteries . We present a rare case of complex relation of ln and ian with second part of ma and discussed its clinical implications . The described variations were found in left infratemporal fossa of a 65-year - old male cadaver during routine dissection . The infratemporal fossa was exposed by resection of ramus of mandible, zygomatic arch, and lateral pterygoid muscle . Posterior root (pr) originated from the mn directly, while the anterior root (ar) was a branch of common trunk (ct). Ct originated from mn directed downward anterior to second part of ma and was about 2 cm in length . Ct was divided into two branches, ar of ln which was about 4 mm in length directed downwards and medially, and other branch continued downwards as ian [figure 1a]. (a) second part of maxillary artery (ma) passing through the nerve loop formed by mandibular nerve (mn), common trunk (ct), anterior root of lingual nerve (ar), and posterior root of lingual nerve (pr). (b) (inset) close view of the artery - nerve complex showing common trunk (ct) arise from mandibular nerve (mn), anterior root of lingual nerve (ar), posterior root of lingual nerve (pr), and maxillary artery (ma). Ma instead of being lateral (superior) to lateral pterygoid muscle was found medial (deep) to the muscle . Second part of ma passed through the nerve loop which was superiorly formed by mn, anteriorly by ct, posteriorly by pr and inferiorly by ar [figure 1b]. Posterior root (pr) originated from the mn directly, while the anterior root (ar) was a branch of common trunk (ct). Ct originated from mn directed downward anterior to second part of ma and was about 2 cm in length . Ct was divided into two branches, ar of ln which was about 4 mm in length directed downwards and medially, and other branch continued downwards as ian [figure 1a]. (a) second part of maxillary artery (ma) passing through the nerve loop formed by mandibular nerve (mn), common trunk (ct), anterior root of lingual nerve (ar), and posterior root of lingual nerve (pr). (b) (inset) close view of the artery - nerve complex showing common trunk (ct) arise from mandibular nerve (mn), anterior root of lingual nerve (ar), posterior root of lingual nerve (pr), and maxillary artery (ma). Ma instead of being lateral (superior) to lateral pterygoid muscle was found medial (deep) to the muscle . Second part of ma passed through the nerve loop which was superiorly formed by mn, anteriorly by ct, posteriorly by pr and inferiorly by ar [figure 1b]. Hussain et al . (2008) reported in a study that the relationship of the ma and lateral pterygoid muscle (lpm) is still a controversial topic . They reported that the ma was lateral (superficial) to the lpm in 68% (30) and medial (deep) in 32% (14) of cases . The communicating branches between the ian and ln are well described in the literature and these communications have been identified as a possible explanation for the inefficient mandibular anesthesia and also suggested that it will affect the mobility of the nerves . Erdogmus et al observed that the ln was divided into anterior and posterior parts by pterygospinous bony bridge . The anterior part passed medially and posterior part lateral to the bony bridge . In our case sandoval et al presented a similar case in which second part of maxillary artery passes through the nerve loop formed anteriorly by ian and posteriorly by ln . Arterial variations in infratemporal region cause failure of inferior alveolar nerve block and intravascular injection during the procedure which leads to serious systemic complications which may endanger the life of patient . Frangiskos et al reported arterial penetration during mandibular block in up to 20% of cases . The infratemporal fossa is a complex region on the skull base that is affected by benign and malignant tumors . This type of anatomical knowledge is essential in choosing the best approach to treat lesions in this area . As in the present case, a close approximation of ma to the ln and ian may lead to arterial penetration during ian anesthesia and cause systemic complications . Ma puncture during administration of local anesthetic can cause a hematoma which can exert a soft pressure in surrounding structures such as ln and the ian leading to sensory alterations which must be considered in the differential diagnosis of facial pain, hyperalgesia, and allodynia . If the artery is passing through the nerve loop, increased blood flow may irritate the surrounding nerves, leading to tingling and numbness in their area of distribution . Knowledge of such arterial and nervous complex may be helpful in radical neck dissection and in dental procedures to avoid untoward complications.
Brain abscesses arise from a contiguous focus of infection, a direct implantation due to trauma, or a hematogenous spread from a remote site . It was reported that the direct spread from surgery, trauma, meningitis, frontal sinusitis, and dental karies was seen in 50% of patients, and that the hematogeneous spread from congenital or other heart diseases, lung and abdominal abscesses was found in 32% of patients . However, 18% had abscesses of an unknown origin . Otitis media also was a direct source of infection, and septicemia or chronic bronchitis also were hematogeneous sources of infection . The majority of cases show some evidence of a systemic infection, such as peripheral leukocytosis, elevated c - reactive protein (crp) values, elevated erythrocyte sedimentation rate, and elevated body temperature . However, these indicators of an infective process are not prominent in the initial stage, and a correct diagnosis is often not considered in the outpatient department or on admission . Only progressive neurological symptoms frequently prompt an urgent referral for investigation with computed tomography (ct) scanning . The difficulties in making an early diagnosis are responsible for the residual high mortality, in spite of the advent of antibiotic treatment . The best result can only be achieved if the physician remains alert to the possibility of an intracranial abscess . The current series of 12 patients with brain abscesses retrospectively analyzed the levels and chronological changes of body temperature, leukocyte, and serum crp for the purpose of making an early and reliable initial diagnosis . Furthermore, sources of infection and infecting organisms were also analyzed to find more information about what antibiotics should be initially selected . Twelve patients with brain abscesses treated between 2006 and 2011 were retrospectively analyzed (table 1). The patients included 10 males and 2 females whose ages ranged from 33 to 79 years old . The underlying diseases were diabetes mellitus (2 patients), chronic glomerulonephritis treated with immunosuppressant (1 patient), subarachnoid hemorrhage (1 patient), cardiac failure and pulmonary fibrosis (1 patient), and acquired immunodeficiency syndrome (aids, 1 patient). The initial diagnoses in the outpatient department were brain abscess in 6 patients, cerebral infarction in 2 patients, metastatic brain tumor in 1 patient, alcohol intoxication in 1 patient, venous thrombosis in 1 patient, and simple headache in 1 patient (table 1). The time from onset to the final diagnosis of brain abscess was 04 days (mean 0.8 days) in the correctly diagnosed 6 patients and 215 days (mean 7.2 days) in the inaccurately diagnosed 6 patients . Abbreviations: penicillin (pcg, benzylpenicillin; abpc, ampicillin; ampc, amoxicillin; pipc, piperacillin), sbtpc, sultamicillin; sbt / abpc, sulbactam / ampicillin; sbt / cpz, sulbactam / cefoperazone), cephalosporin (cez, cefazolin; ctm, cefotiam; caz, ceftazidime; ctrx, ceftriaxone), oral cephem (cftm, cefteram; cdtr, cefditoren; cfpm, cefcapene), oxacephem (fmox, flomoxef), carbapenem (papm, panipenem; ipm, imipenem; mepm, meropenem), aminoglycoside (tob, tobramycin; amk, amikacin), macrolide (em, erythromycin; cam, clarithromycin; cldm, clindamycin), tetracycline (mino, minocycline), other antibiotic (vcm, vancomycin; fom, fosfomycin; cp, chloramphenicol; s / t, sulfamethoxazole / trimethoprim), quinolone (tflx, tosufloxacin; lvfx, levofloxacin; pzfx, pazufloxacin). The frontal lobe was the most common site of the abscess (5 cases), and the other lesions were in the parietal lobe in 3 cases, basal ganglia in 2 cases, thalamus in 1 case, and cerebellum in 1 case . A bacteriological culture was performed under an aerobic condition of 35c for 1824 hours with aerobic bacteria, under an anaerobic condition of 35c for 2 days with anaerobic bacteria, and under an aerobic condition of 35c for 3 days with nocardia . The identification of the bacterial strains was conducted using an identification kit, and the clinical isolates of nocardia were determined with a restriction fragment length polymorphism method using bacterial 16s rdna . . Minimum inhibitory concentration (mic) was measured with dry plate (eiken chemical co., ltd ., the initial therapy was conducted with intravenous infusion of antibiotics or antimicrobials (table 1). The secondary intravenous antibiotics or antimicrobials were administered according to the results of the microbial sensitivity test . Excision of abscess was performed in 6 cases, and aspiration of abscess with / without decompressive craniotomy or ventricular drainage was performed in 4 cases . Three patients developed ventriculitis and/or meningitis (ventriculitis group) and 9 cases did not (non - ventriculitis group). The inflammatory indices of body temperature, leukocyte counts, and crp levels were analyzed and compared within each group . The mean leukocyte count on admission was 9,694/mm in the non - ventriculitis group and 14,477/mm in the ventriculitis group (fig . The maximum leukocyte count was 12,708/mm in the non - ventriculitis group and 20,747/mm in the ventriculitis group . The leukocyte count increased significantly after admission in the non - ventriculitis group (wilcoxon signed - ranks test, p=0.028). The mean serum crp level on admission was 2.72 mg / dl in the non - ventriculitis group and 10.68 mg / dl in the ventriculitis group (fig . The maximum crp was 10.47 mg / dl in the non - ventriculitis group and 31.95 mg / dl in the ventriculitis group . The crp in the non - ventriculitis group increased significantly after admission (wilcoxon signed - ranks test, p=0.028). The maximum crp was significantly higher in the ventriculitis group in comparison to the non - ventriculitis group (welch s t test, p=0.001). The mean temperature on admission was 37.3c in the non - ventriculitis group and 37.9c in the ventriculitis group (fig . The maximum temperature was 38.6c in the non - ventriculitis group and 39c in the ventriculitis group . The temperature significantly increased after admission in the non - ventriculitis group (wilcoxon signed - ranks test, p=0.011). For leukocytes, the mean time for each of the indicators of infection to reach their maximum values was 10.8 days from the onset in the non - ventriculitis group and 6.7 days in the ventriculitis group . The respective values for crp were 12.6 days in the non - ventriculitis group and 6.7 days in the ventriculitis group . In respect to body temperature, the values were 17.1 days in the non - ventriculitis group and 7 days in the ventriculitis group (no significant differences between each groups, fig . These were analyzed separately in patients with ventriculitis and/or meningitis (ventriculitis group) and in cases without ventriculitis and meningitis (non - ventriculitis group). The leukocyte count increased significantly () after admission in the non - ventriculitis group (wilcoxon signed - ranks test, p=0.028). Levels of crp at the initial stage and their maximum levels . These were analyzed separately in patients with ventriculitis and/or meningitis (ventriculitis group) and in cases without ventriculitis and meningitis (non - ventriculitis group). The crp in the non - ventriculitis group increased significantly () after admission (wilcoxon signed - ranks test, p=0.028). The maximum crp was significantly () higher in the ventriculitis group in comparison to the non - ventriculitis group (welch s t test, p=0.001). Levels of body temperature at the initial stage and their maximum levels . These were analyzed separately in patients with ventriculitis and/or meningitis (ventriculitis group) and in cases without ventriculitis and meningitis (non - ventriculitis group). The temperature significantly () increased after admission in the non - ventriculitis group (wilcoxon signed - ranks test, p=0.011). Time (days) for each inflammatory index to reach their maximum values in the patients with ventriculitis and/or meningitis (ventriculitis group) and in cases without ventriculitis and meningitis (non - ventriculitis group). A hematogenous spread from a remote site was the most common cause of an abscess (11 cases). Dental caries were found in 4 cases, and the other lesions were lung and retroperitoneal abscess in 1 case and gluteal abscess in 1 case . Although the source of the infection was not recognized, hematogenous spread was suspected in another 5 cases . An abscess occurred following the infection of a ventriculoperitoneal shunt in one case of subarachnoid hemorrhage . The primary causative organisms were streptococcus viridians (2 cases), streptococcus milleri (1 case), and fusobacterium nucleatum (1 case) in 4 cases of abscesses arising from dental caries . The causative organism was nocardia farcinica in 1 case of retroperitoneal and lung abscess, and nocardia sp . In 1 case of gluteal abscess . Toxoplasma infection was suggested by the mri findings in the patient with aids, although no immunoserological test was performed . Carbapenem, tetracycline, and quinolone were generally effective for many types of bacteria, including nocardia (fig . 2). Penicillin, cephalosporin, sultamicillin and macrolide were effective for many kinds of bacteria other than nocardia . Imipenem was ineffective for propionibacterium sp ., and benzylpenicillin was ineffective for prevotella sp . Abbreviations: mic, minimum inhibitory concentration; penicillin (pcg, benzylpenicillin; abpc, ampicillin; ampc, amoxicillin; pipc;piperacillin), sbtpc, sultamicillin; cephalosporin (cez, cefazolin; ctm, cefotiam; ctx, cefotaxime; caz, ceftazidime; ctrx, ceftriaxone), oral cephem (cftm, cefteram; cdtr, cefditoren; cfpm, cefcapene), oxacephem (fmox, flomoxef), carbapenem (papm, panipenem; ipm, imipenem; mepm, meropenem), aminoglycoside (tob, tobramycin; amk, amikacin), macrolide (em, erythromycin; cam, clarithromycin; cldm, clindamycin), tetracycline (mino, minocycline), other antibiotic (vcm, vancomycin; fom, fosfomycin; cp, chloramphenicol), quinolone (tflx, tosufloxacin; lvfx, levofloxacin; pzfx, pazufloxacin). Nine patients had an independent gait at discharge, but one required assistance with walking (table 1). One patient was transferred to another hospital and required a wheelchair . The patient with aids three of the 6 incorrectly - diagnosed patients at discharge required help with walking, needed a wheelchair, or had consciousness disturbance (table 2). In contrast, all 6 correctly - diagnosed patients could move about independently at discharge (p=0.046, chi - square for independence test). The condition at discharge in patients with correct or incorrect initial diagnosis the initial incorrect diagnosis resulted in a significantly poor patient outcome . The mean leukocyte count on admission was 9,694/mm in the non - ventriculitis group and 14,477/mm in the ventriculitis group (fig . The maximum leukocyte count was 12,708/mm in the non - ventriculitis group and 20,747/mm in the ventriculitis group . The leukocyte count increased significantly after admission in the non - ventriculitis group (wilcoxon signed - ranks test, p=0.028). The mean serum crp level on admission was 2.72 mg / dl in the non - ventriculitis group and 10.68 mg / dl in the ventriculitis group (fig . The maximum crp was 10.47 mg / dl in the non - ventriculitis group and 31.95 mg / dl in the ventriculitis group . The crp in the non - ventriculitis group increased significantly after admission (wilcoxon signed - ranks test, p=0.028). The maximum crp was significantly higher in the ventriculitis group in comparison to the non - ventriculitis group (welch s t test, p=0.001). The mean temperature on admission was 37.3c in the non - ventriculitis group and 37.9c in the ventriculitis group (fig . The maximum temperature was 38.6c in the non - ventriculitis group and 39c in the ventriculitis group . The temperature significantly increased after admission in the non - ventriculitis group (wilcoxon signed - ranks test, p=0.011). For leukocytes, the mean time for each of the indicators of infection to reach their maximum values was 10.8 days from the onset in the non - ventriculitis group and 6.7 days in the ventriculitis group . The respective values for crp were 12.6 days in the non - ventriculitis group and 6.7 days in the ventriculitis group . In respect to body temperature, the values were 17.1 days in the non - ventriculitis group and 7 days in the ventriculitis group (no significant differences between each groups, fig . These were analyzed separately in patients with ventriculitis and/or meningitis (ventriculitis group) and in cases without ventriculitis and meningitis (non - ventriculitis group). The leukocyte count increased significantly () after admission in the non - ventriculitis group (wilcoxon signed - ranks test, p=0.028). Levels of crp at the initial stage and their maximum levels . These were analyzed separately in patients with ventriculitis and/or meningitis (ventriculitis group) and in cases without ventriculitis and meningitis (non - ventriculitis group). The crp in the non - ventriculitis group increased significantly () after admission (wilcoxon signed - ranks test, p=0.028). The maximum crp was significantly () higher in the ventriculitis group in comparison to the non - ventriculitis group (welch s t test, p=0.001). Levels of body temperature at the initial stage and their maximum levels . These were analyzed separately in patients with ventriculitis and/or meningitis (ventriculitis group) and in cases without ventriculitis and meningitis (non - ventriculitis group). The temperature significantly () increased after admission in the non - ventriculitis group (wilcoxon signed - ranks test, p=0.011). Time (days) for each inflammatory index to reach their maximum values in the patients with ventriculitis and/or meningitis (ventriculitis group) and in cases without ventriculitis and meningitis (non - ventriculitis group). A hematogenous spread from a remote site was the most common cause of an abscess (11 cases). Dental caries were found in 4 cases, and the other lesions were lung and retroperitoneal abscess in 1 case and gluteal abscess in 1 case . Although the source of the infection was not recognized, hematogenous spread was suspected in another 5 cases . An abscess occurred following the infection of a ventriculoperitoneal shunt in one case of subarachnoid hemorrhage . The primary causative organisms were streptococcus viridians (2 cases), streptococcus milleri (1 case), and fusobacterium nucleatum (1 case) in 4 cases of abscesses arising from dental caries . The causative organism was nocardia farcinica in 1 case of retroperitoneal and lung abscess, and nocardia sp . In 1 case of gluteal abscess . Toxoplasma infection was suggested by the mri findings in the patient with aids, although no immunoserological test was performed . Carbapenem, tetracycline, and quinolone were generally effective for many types of bacteria, including nocardia (fig . 2). Penicillin, cephalosporin, sultamicillin and macrolide were effective for many kinds of bacteria other than nocardia . Imipenem was ineffective for propionibacterium sp ., and benzylpenicillin was ineffective for prevotella sp . Abbreviations: mic, minimum inhibitory concentration; penicillin (pcg, benzylpenicillin; abpc, ampicillin; ampc, amoxicillin; pipc;piperacillin), sbtpc, sultamicillin; cephalosporin (cez, cefazolin; ctm, cefotiam; ctx, cefotaxime; caz, ceftazidime; ctrx, ceftriaxone), oral cephem (cftm, cefteram; cdtr, cefditoren; cfpm, cefcapene), oxacephem (fmox, flomoxef), carbapenem (papm, panipenem; ipm, imipenem; mepm, meropenem), aminoglycoside (tob, tobramycin; amk, amikacin), macrolide (em, erythromycin; cam, clarithromycin; cldm, clindamycin), tetracycline (mino, minocycline), other antibiotic (vcm, vancomycin; fom, fosfomycin; cp, chloramphenicol), quinolone (tflx, tosufloxacin; lvfx, levofloxacin; pzfx, pazufloxacin). Nine patients had an independent gait at discharge, but one required assistance with walking (table 1). Three of the 6 incorrectly - diagnosed patients at discharge required help with walking, needed a wheelchair, or had consciousness disturbance (table 2). In contrast, all 6 correctly - diagnosed patients could move about independently at discharge (p=0.046, chi - square for independence test). The condition at discharge in patients with correct or incorrect initial diagnosis the initial incorrect diagnosis resulted in a significantly poor patient outcome . Headache, vomiting, drowsiness and focal symptoms, sometimes accompanied by focal or generalized seizures, are the classic symptoms of brain abscess . In addition, papilledema is commonly found in drowsy patients that appear ill . However, many individuals frequently fail to show all of these symptoms . In such people, half of the patients in the current series were diagnosed inaccurately in the initial stage, and it took 7.2 days to achieve the final diagnosis of brain abscess for the misdiagnosed patients . Bibliographically, the average time between the onset of the symptoms and confirmed diagnosis was 9.6 days . It was reported that patients with brain abscess usually show high leukocyte counts (12,40032,300/mm) at the time of admission . The crp was elevated to a high level (221.6 mg / dl) in 69.271.4% of patients on admission, and 25.650% of abscess patients were febrile (37.839.8c) at the time of admission . However, the value of these inflammatory indices were generally lower than the ones reported in the non - ventriculitis group of the current series . The mean leukocyte count, crp level and body temperature on admission were 9,694/mm, 2.72 mg / dl, and 37.3c, respectively . This tendency made the initial diagnosis of brain abscess more difficult in the patients without ventriculitis . Body temperature is known to be associated with cytokine levels in serum and cerebrospinal fluid . In the current series, therefore, the inflammatory reaction was considered to be less prominent in the non - ventriculitis group than in the ventriculitis group . Microbial infection stimulates the production of cytokines, such as interleukin-1,6,8 and tnf - alpha, in the macrophage, dendrite cells, and glial cells . Interleukin-6 causes a biphasic neutrophilia, wherein the first peak results from the mobilization of polymorphonuclear leukocytes into the circulating pool from the marginated pool, and the second peak results from an accelerated bone marrow release of polymorphonuclear leukocytes . Interleukin-1 stimulates the production of crp in hepatocytes, and cytokines such as interleukin-1,6 and tnf - alpha act on the preoptic area of the hypothalamic thermoregulatory center and regulate body temperature . In the current series, the change of inflammatory indices began with increased leukocyte counts, followed by elevation of crp and body temperature . The mean time for each infectious indicator to reach its maximum value in the non - ventriculitis group was 10.8 days for leukocytes, 12.6 days for crp, and 17.1 days for body temperature . In an experiment with rabbit in which the endogeneous pyrogen was injected in to the cerebroventricular region, these inflammatory indices changed chronologically in the order of body temperature, leukocyte count, and crp . In the current series, the pattern of chronological change of the inflammatory indices was different from that of the rabbit experiment, but our results will help us diagnose patients correctly . The causative organisms were reported to be eventually identified in 5881% of cases . The majority of them were gram - positive bacteria . Anaerobes were present in 23% of patients, and methicillin - resistant staphylococcus aureus or nocardia were also identified in some patients . It was reported that the primary causative organisms were streptococcus milleri, staphylococcus aureus and bacterioides in dental caries, streptococcus milleri, b - haemolytic streptococcus, staphylococcus aureus and bacterioides in sinusitis, streptococcus and proteus mirabilis in otitis media, haemophillus influenza in chronic bronchitis, and streptococcus milleri and staphylococcus epidermidis in infections of an unknown origin . In the current series, the primary causative organisms from dental caries were streptococcus viridians, streptococcus milleri, or fusobacterium nucleatum . Nocardia farcinica or nocardia sp . Were involved when the abscess originated from other infections . The primary causative organisms were streptococcus milleri and propionibacterium sp . When the source of infection was unrecognized . Antibiotics must be selected during the initial stage before the bacteria are identified in a laboratory culture . These tendencies of this disease revealed in the current series will help us in the selection of appropriate antibiotics . Optimal management of brain abscesses requires intensive antibiotic therapy, for which the selection of an appropriate antibiotic is crucial . The treatment has usually been performed with penicillin (benzylpenicillin) or cephalosporin (cefotaxime or ceftriaxone) in combination with vancomycin or metronidazole . Although metronidazole is stable in abscesses and a high concentration of it can be obtained from cerebrospinal fluid, its usage is not covered by japanese national medical insurance . Cure was obtained in 100% of the patients with imipenem therapy, in contrast with 86.7% of patients with the conventional chemotherapy, including penicillin and metronidazole . Another report also mentioned the efficacy of carbapenems (imipenem or meropenem) versus standard chemotherapy (cefotaxime and metronidazole). Cure was obtained in 81.8% of those on imipenem, and 96.0% of those on meropenem, in contrast with 66.7% of those cured on standard chemotherapy . The microbial sensitivity test in the current series also showed that carbapenem therapy is a reasonable therapeutic choice for treatment of bacterial brain abscesses, including nocardia infection . Another antibiotic can be selected if there is little possibility that nocardia is the cause . Penicillin, cephalosporin, sultamicillin, and macrolide were effective for many types of bacteria other than nocardia in the current series . Nocardiosis is an opportunistic infection that has been frequently noted in patients with malignancies, systemic lupus erythematosis, long - term steroid usage, transplantation, and hiv infection . However, recent data demonstrated that central nervous system nocardiosis was common or more prevalent in patients with normal immune systems, as shown in our patient number 6 . At any rate, nocardial brain abscesses remain a clinical challenge associated with high mortality and morbidity rates, because nocardia is resistant to many antibiotics . The recommended treatment is usually imipenem with amikacin, ceftriaxone with amikacin, amoxicillin - clavulanate, minocycline, sulfamethoxazole / trimethoprim, sulfisoxazole, ciprofloxacin, clindamycin, linezolid, or vancomycin . In the current series, carbapenem (imipenem), tetracycline (minocycline), and quinolone (levofloxacin, pazufloxacin) proved effective for nocardia . A brain abscess has a lethal nature despite recent advances in its diagnosis and treatment . Poor prognostic factors were prominent consciousness disturbance at admission, an age below 2 years, and multiple abscesses . Fungus and aerobic bacteria were related to poor outcomes . In the current series, initial incorrect diagnoses resulted in significantly poor outcomes . The delay in making a diagnosis allows time for the rapid enlargement of the brain abscess and causes consciousness disturbance . Therefore, the early diagnosis of brain abscess, proper estimation of causative organisms from the source of infection, and appropriate usage of antibiotics are mandatory.
Acute pancreatitis (ap), a debilitating inflammation of the pancreas, is usually triggered by inappropriate intra - acinar activation of the trypsinogen . Although most cases of ap are mild or self - limiting, sometimes uncontrolled subsequent events including activation of immune cells and further systemic progression of inflammatory response are capable of converting ap to a severe form [1, 2]. Vigorous release of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species (ros) during systemic inflammatory responses results in a life - threatening condition with multiple - organ dysfunction, which carries substantial morbidity and mortality . Currently, much research attention has been focused on the involvement of ros and resultant oxidative stress in the inflammatory processes during the development of pancreatitis [35]. At the early stage of ap, several pathogenetic mechanisms, such as proteolytic damage, tissue ischaemia, and activation of polymorphonuclear leucocytes, stimulate the production of ros that act as the molecular trigger of various inflammatory processes . In general, the detrimental ros can be detoxified either by endogenous antioxidants like glutathione (gsh) and superoxide dismutase (sod) or when needed by exogenous dietary antioxidants like carotenoids, flavonoids, and vitamin e. however, once the production of ros is excessive and overwhelms endogenous antioxidants, oxidative stress can occur with increasing cellular injury . Therefore, the theory that attack of oxidative stress is responsible for not only the amplification of pancreatic damage but also the progression of the disease from a local damage to a systemic organ failure is generally accepted as fact . Therefore, supplementation with exogenous antioxidants is regarded as a beneficial therapeutic strategy, at least, a useful adjunctive option in the treatment of ap, as evidenced in some animal models [79]. Among numerous antioxidants, molecular hydrogen (h2) is emerging as a promising candidate in the therapeutic approaches of oxidative stress - implicated inflammatory diseases because this simple molecule is a selective scavenger of detrimental ros, such as hydroxyl radical and peroxynitrite [10, 11]. In some animal experiments, hydrogen gas can be given by inhalation or more conveniently administered by injection when it dissolves in normal saline to achieve the saturation level (called hydrogen - rich saline, hrs). Recent studies, including our previous work, have provided evidence that treatment of hrs relieved the severity of pancreatitis in rats, describing the potential of hrs in the management of ap caused by different pathogenetic mechanisms [12, 13]. Although the molecular mechanism underlying the protective effect of hrs against ap has not been fully elucidated, it is well considered that the direct ros elimination activity of hrs is mostly responsible for its therapeutic benefits due to the specific scavenging activity of cytotoxic ros . Interestingly, emerging evidence has been presented that the potential of hrs to suppress the proinflammatory cascade also contributes to the reduction of the severity of the disease . Recently, the finding that activation of an intracellular multiprotein complex, the nod - like receptor family, pyrin domain - containing 3 (nlrp3) inflammasome, is involved in the proinflammatory process in pancreas provides valuable insights into the pathogenesis of pancreatitis . As the most well - characterized inflammasome, the nlrp3 inflammasome has been shown to possess the function of regulating the maturation and release of proinflammatory cytokine interleukin- (il-) 1. in previous work, we found that augmented secretion of il-1 in rats suffering from trauma - induced pancreatitis was substantially depressed by the treatment of hrs, which implies a possibility that hrs is able to inhibit the activation of nlrp3 inflammasome, thereby contributing to the suppression of il-1 production . Thus, the herein study extended previous studies to evaluate the potential of hrs to inhibit nlrp3 inflammasome activation, which probably mediated the protective activity of hrs against experimental ap . Antibodies against the p65 subunit of nuclear factor-b (nf-b p65), i-b, phosphorylated - i-b (p - i-b), thioredoxin - interacting protein (txnip), nlrp3, p20 subunit of caspase-1 (casp-1-p20), -actin, and horseradish peroxidase conjugated goat anti - rabbit antibody were obtained from santa cruz biotechnology (santa cruz, ca, usa). Enzyme - linked immunosorbent assay (elisa) kits for il-1 and tumor necrosis factor- (tnf-) were obtained from r&d system (minneapolis, mn, usa). Hrs was prepared as described previously . In brief, h2 gas (0.4 mpa) was dissolved in normal saline (ns) for 6 hours to achieve a concentration of 0.6 mmol / l, and the concentration of h2 in ns was detected using a dissolved hydrogen analyzer (dh-35a; dkktoa, tokyo, japan). The hrs was freshly prepared every week and then stored under atmospheric pressure at 4c in an aluminum bag with no dead volume and sterilized by gamma radiation before use . Male balb / c mice (2025 g) were obtained from the experimental animal center of sichuan university (chengdu, sichuan, china). Experimental ap model was induced by hourly intraperitoneal injections of cerulein (50 g / kg) to mice for 6 hours . One hour after the final injection, animals received an intraperitoneal injection of hrs at various doses (2, 4, and 8 ml / kg, every 20 min for 3 times) or control saline (air dissolved in ns at a pressure of 0.4 mpa for 6 h), respectively . Tail vein blood samples were collected 2 h after the final administration of cerulein, and 24 h after the initiation of ap, all the animals were sacrificed by carbon dioxide asphyxiation followed by the collection of pancreatic tissue samples . All procedures involving the mice were carried out in accordance with the guidelines of the institutional animal care and use committee of sichuan university . Pancreas tissues were collected and homogenized in 20 mm phosphate buffer (ph 7.4) containing 0.5 mm butylated hydroxytoluene followed by a centrifugation at 4c at 1,500 g for 15 minutes . Then cytoplasmic proteins in tissue homogenate were extracted using cytoplasmic extraction reagents according to the manufacturer's instructions (pierce biotechnology, rockford, il, usa). Subsequently, the extracted proteins were separated by sds - polyacrylamide gels and then transferred to a polyvinylidene difluoride membrane (millipore, billerica, ma, usa). The membrane was incubated with blocking solution containing 5% skim milk for more than 1 h at room temperature and probed by 1: 1,000 dilutions of rabbit polyclonal antibodies against txnip, nlrp3, and casp-1-p20, respectively . After washing, membrane was incubated with secondary goat anti - rabbit antibody conjugated to horseradish peroxidase for 1 h at room temperature . Immunoreactive proteins were visualized by chemiluminescence using the western blotting detection system (amersham biosciences, piscataway, nj, usa). The nuclear and cytoplasmic proteins in pancreatic tissue homogenate were extracted using a protein extraction kit (pierce biotechnology, rockford, il, usa). The dna - binding activity of nf-b p65 in nuclear extracts was detected using a sandwich elisa kit (transam nf-b p65, active motif, carlsbad, ca, usa). Briefly, oligonucleotide containing the nf-b consensus - binding site (5-gggactttcc-3) was preimmobilized onto 96-well plates . The nuclear extract (1 g) diluted in complete lysis buffer was added to each well containing 30 l of complete binding buffer and incubated at room temperature for 1 h with mild agitation . After washing three times with wash buffer, the antibody against nf-b p65 subunit was added (1: 1,000), followed by 1 h incubation at room temperature . After washing, the wells were incubated with the secondary antibody conjugated to horseradish peroxidase (1: 1,000) for 1 h at room temperature . Then the dna - binding activity of nf-b p65 was quantified by measuring the absorbance at 450 nm with a reference wavelength of 655 nm, after the wells were incubated with developing solution for 5 min at room temperature . Moreover, the degradation of i-b caused by nf-b activation was evaluated by determining the phosphorylation of i-b in pancreatic extracts using western blotting assay as described above . A colorimetric assay kit purchased from cayman chemical company (ann arbor, mi, usa) was used to measure the activity of sod in pancreas, which uses a tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine . Briefly, the pancreatic tissue was homogenized at 4c in 20 mm hepes buffer (containing 1 mm egta, 210 mm mannitol, and 70 mm sucrose, ph 7.2) and then centrifuged at 1,500 g for 5 min at 4c . After mixed, the sample was incubated at room temperature for 20 min and the absorbance was read at 450 nm . One unit (u) of sod was defined as the amount of enzyme needed to produce 50% dismutation of superoxide radical . The total tissue protein concentration was determined by a commercial kit (jiancheng corp ., nanjing, china), and the activity of sod was expressed as u / mg of protein . Tissue mda level, a marker of lipid peroxidation, was detected using a commercial mda-586 assay kit (oxisresearch, portland, or, usa). In brief, pancreatic tissues were homogenized in 20 mm phosphate buffer (ph 7.4) containing 0.5 mm butylated hydroxytoluene followed by a centrifugation at 4c at 1,500 g for 15 minutes . After protein concentration measurement, equal amounts of proteins were used in triplicate to react with a chromogenic reagent n - methyl-2-phenylindole to form a stable carbocyanine dye with a maximum absorption at 586 nm . The level of mda in sample was then calculated with the standard curve obtained from the kit according to the manufacturer's instructions and expressed as mol / g of total protein . The level of gsh in pancreas tissue was measured according to the method reported by tietze . In brief, pancreatic homogenate was centrifuged at 1,500 g for 20 min and 1 ml of the supernatant was mixed with 1 ml of 5% trichloroacetic acid for 30 min . Subsequently, 0.5 ml of ellman's reagent (5,5-dithiobis-2-nitrobenzoic acid) and 3 ml of phosphate buffer (ph 8.0) were added . After mixed thoroughly, the absorbance was recorded at 412 nm . The gsh level in tissue was normalized against total protein (mol / g). Plasma samples were obtained from harvested blood specimens by centrifugation (1,800 g for 15 min at 4c) and the amylase or lipase activity was analyzed through colorimetric method using a commercial kit for amylase or lipase (jiancheng co., nanjing, china), respectively . The activity of amylase or lipase was expressed as units per liter (u / l). To determine the concentrations of proinflammatory cytokines in plasma, quantitative elisa kits for il-1 and tnf- . Then samples were sectioned and stained with hematoxylin and eosin, using a standard staining procedure . Histopathological evaluation was performed under light microscope by an experienced laboratory pathologist who was blinded to the group identity for the samples . Edema, inflammatory infiltration, hemorrhage, and necrosis were evaluated in accordance with the scoring scale reported by rongione et al . . Statistical comparisons among the groups were assessed by one - way analysis of variance (anova) using spss 13.0 software . The protein expression of nlrp3, a major component of this inflammasome, was remarkably increased in the progression of ap . Moreover, the development of cerulein - induced pancreatitis conspicuously stimulated the expression of txnip, the critical regulator of nlrp3 activity . Similar elevation was also found in the protein expression of p20 subunit of activated caspase-1, used as one of the markers for the nlrp3 inflammasome activation . However, treatment of hrs dose - dependently diminished expression levels of nlrp3, txnip, and casp-1-p20, exhibiting the significant inhibitory activity on the nlrp3 inflammasome activation (figure 1). It was known that nf-b is ubiquitous transcription factor that controls proinflammatory gene expression during the development of inflammation related diseases including ap . In present study, the dna - binding activity of nf-b p65 in pancreas measured by the elisa increased significantly in response to the induction of pancreatitis, indicating the activation of nf-b, whereas it was substantially depressed by the administration of hrs in a dose - response manner (figure 2(a)). In line with the elisa data, protein expression of cytoplasmic p - i-b in pancreatic homogenates from ap mice rose markedly, demonstrating the degradation of i-b . Likewise, hrs showed a dose - dependent inhibition on the degradation of i-b, which was in agreement with the inhibitory effect on nlrp3 activity (figure 2(b)). As shown in figures 3 and 4, experimental ap was induced by cerulein administration in mice with significant elevated plasma pancreatic enzymes activities and pathological damage in pancreas . There were no significant changes of plasma amylase and lipase activities in ap mice after the treatment of hrs at various doses (figure 3). Moreover, only at the higher doses (4 and 8 ml / kg 3), hrs conferred favorable histopathological changes in pancreatic tissues, mainly characterized by reduced cytoplasmic vacuole generation, alleviated inflammatory infiltration, and parenchyma necrosis, which was paralleled with lower histopathological scores (figures 4(a) and 4(b)). The proinflammatory cytokines tnf- and il-1 are known to mediate amplification of inflammatory process during pancreatitis . Here it was shown that plasma concentrations of tnf- and il-1 markedly increased in mice suffering from cerulein - induced ap, which were substantially abolished by the treatment of hrs (figure 5). In addition, a significant dose - dependent effect of hrs to decline the plasma il-1 level was observed; however, middle dose of hrs (4 ml / kg 3) failed to demonstrate superiority in reducing tnf- release in ap mice when compared with the low dose (2 ml / kg 3). In order to verify the potential of hrs to reduce oxidative stress during ap, we measured the changes of pancreatic mda level, an indicator of lipid peroxidation in oxidative stress process, as well as the endogenous antioxidants gsh and sod . As expected, enhanced oxidative stress in pancreas was observed in mice after the initiation of ap, which was characterized by increased amount of mda and decreased sod activity and gsh level . In contrast, administration of hrs dose - dependently diminished the mda production, accompanied with the elevated levels of sod and gsh (figure 6). Moreover, the capacity of hrs to help recruit endogenous antioxidants was observed only at the middle (4 ml / kg 3) or high dose (8 ml / kg over the past years, there has been continuing controversy about the benefits of supplementation with exogenous antioxidants for the management of oxidative stress related inflammatory diseases largely because once it was considered that these antioxidants only served as ros scavengers and thereby helped correcting the pro- or antioxidative imbalance . However, recent findings that elimination of ros by antioxidants suppressed activation of nlrp3 inflammasome and the resultant maturation of il-1 have shed new light on understanding the roles of antioxidants in pathogenesis of inflammation related diseases . Here we found that, although hrs failed to suppress plasma pancreatic enzymes activity, it significantly attenuated the pancreatic damage in mice in response to the induction of pancreatitis, together with the impaired nlrp3 inflammasome activity, suggesting that the potential of hrs to block the nlrp3 inflammasome activation was probably involved in the amelioration of pancreatic injury . Moreover, the data from this study have demonstrated the possible mechanism by which hrs inhibited the activation of nlrp3 inflammasome . Firstly, because the nf-b activation is shown previously to be the traditional priming signal to induce the nlrp3 expression, blockade of nf-b activity by hrs, with the following downregulation of expression of nlrp3, probably conferred the suppression of nlrp3 inflammasome activation . Furthermore, treatment of hrs promoted the elimination of ros and thereby reduced the expression of txnip, the ros - sensing protein required for nlrp3 activation, also resulting in the blockade of nlrp3 inflammasome activation . As an important component of innate immune system, the nlrp3 inflammasome was initially found to be activated by pathogens [2023]. In recent years, some endogenous molecules known as damage - associated molecular patterns (damps), such as extracellular glucose, uric acid, amyloid-, and hyaluronan, have been demonstrated to activate nlrp3 inflammasome, playing important roles in the metabolic disorders and sterile inflammatory responses including type ii diabetes, gout, alzheimer's disease, and tissue injury after trauma [2427]. As for ap, some known extracellular nlrp3 activators like dna, adenosine triphosphate (atp), high mobility group box 1 (hmgb1), and nucleotide binding oligomerization domain 2 (nod2) are all associated with the inflammation during the development of pancreatitis . Moreover, some researches, including our previous work, have revealed that heparan sulfate (hs), a degradation product of extracellular matrix known as an important member of damps, triggered intracellular proinflammatory responses, resulting in the aggravation of pancreatitis [2830]. Therefore, the above - mentioned findings hint at a possible fact that hs might act as a previously unknown agonist of nlrp3 inflammasome and thereby promote the progression of ap . In this context, there arises an interesting question required to be addressed by future work, that is, whether hrs possesses the ability to inhibit the presumed hs - induced activation of nlrp3 during the development of pancreatitis . Obviously, the elucidation of this question is of considerable significance to further understand the beneficial roles of hydrogen molecule in ap . In addition, although the mechanism underlying the therapeutic potential of hrs is probably shared by other antioxidants due to their ros scavenging capacity, hrs still exhibits significant advantages in the treatment of oxidative stress - implicated diseases . At first, it was known that mammalian species are lacking endogenous detoxification systems for the hydroxyl radical, which is considered as one of the strongest oxidant species having harmful reactions to nucleic acids, lipids, and proteins, further causing irreversible cellular damage . Compared with other known exogenous antioxidants, hydrogen molecule can selectively quench hydroxyl radical forming water due to the unique molecular structure and chemical property, meanwhile without excessively interfering the physiological roles of the beneficial ros like superoxide anions . Moreover, hrs can be easily injected or administered orally and even used as the dissolvent for other therapeutic agents . Therefore, the advantages of hrs mentioned above make it a superior candidate for the detoxification of oxidative stress in clinical application . In summary, our work has, for the first time, revealed that hrs can act as an antagonist of nlrp3 inflammasome resulting in attenuation of cellular inflammatory processes and consequently relieve the pancreatic injury during ap . This finding allows for a further understanding of the mechanism underlying the protective effects of hrs against ap and furthermore marks the nlrp3 inflammasome as a potential target for future therapeutic approaches of this dangerous disease.
The lysosome is a dynamic organelle that operates at an acidic ph and contains numerous enzymes that are critical for cellular degradation pathways . Uptake of extracellular material reaches the lysosome via the endocytic pathway, whereas intracellular cargo is delivered to the lysosome via autophagy . The lysosome can also play a role in secretion, membrane repair, and cell clearance through the process of lysosomal exocytosis [2, 3]. Recently, the importance of lysosomes in cell signaling pathways and nutrient sensing has also become apparent [4, 5]. Importantly, the regulation of lysosome structure and function is cell type dependent and is regulated by environmental stimuli . Macrophages are cells of the innate immune system that are important for organ homeostasis, inflammation, host defense, and tissue repair . Recently, there has been a renewed interest in macrophage lysosome biology . The importance of macrophage autophagy in several clinically relevant diseases has helped to fuel this renaissance [710]. Moreover, it has also come to light that lysosomal pathways activate il-1 release via the inflammasome in several important human diseases including atherosclerosis, gout, and alzheimer's disease [1113]. Also, of relevance, lipid overload and obesity can also induce lysosomal reprogramming in adipose tissue macrophages, which may contribute to the metabolic complications of nutrient excess . Together, these and many other studies indicate that additional cellular and molecular studies of lysosome function in macrophages will be critical to understand the role of this organelle in inflammatory diseases . Ex vivo analysis of primary macrophages will be important for mechanistic cell biology experiments investigating lysosome function in phagocytic cells . The most common sources of primary macrophages include bone marrow - derived macrophages (bmdms) and thioglycollate- (tg-) elicited peritoneal macrophages (pmacs). Although bmdms and pmacs are derived from very distinct environments they are often used interchangeably to model macrophage biology . Pmacs are monocyte derived cells that are typically isolated from the peritoneal cavity 35 days after tg administration . Thus, pmacs are actively involved in the process of inflammation resolution, which includes the uptake of dead cells and/or debris through efferocytosis or phagocytosis, respectively . In contrast, bmdms are derived from a myeloid precursor cell functionally nave and would be expected to have fewer demands on their endosomal system . Based on this, we hypothesized that the lysosomes would be phenotypically and functionally distinct in these subtypes of primary macrophages . In the current study, we investigated lysosome content, morphology, and function in pmacs and bmdms . Our data demonstrate that pmacs have a larger lysosome volume, increased cathepsin activity, and enhanced expression of several lysosomal genes and proteins . Moreover, using the example of the lipotoxic inflammasome, we provide evidence that these differences in the lysosomal compartment can influence macrophage inflammatory responses . Together, our results argue that the interpretation of data involving lysosome - dependent processes in primary macrophages must take the source of the cells into consideration . The cd107a (lamp-1) pe conjugated antibody was from ebiosciences (cat#12 - 1071). Ultrapure - bovine serum albumin (bsa) was from lampire and was tested for tlr ligand contamination prior to use . Peritoneal macrophages (pmacs) were isolated from c57bl/6 mice 4 days after intraperitoneal injection of 3.85% thioglycollate and plated at a density of 0.9 - 1 10 cells / ml in dmem containing 10% inactivated fetal serum (ifs), 50 u / ml penicillin g sodium, and 50 u / ml streptomycin sulfate (pen - strep), 2 mm l - glutamine, and sodium pyruvate . Bone marrow - derived macrophages (bmdms) were prepared by harvesting bone marrow from the femurs and tibias of 812-week - old c57bl/6 mice . The cells were seeded in 10 cm dishes and differentiated for 6 days in dmem media as above supplemented with 10% supernatant from cmg14.12 cells as a source of m - csf . On day 6, bmdms were plated at 1 10 cells / ml in media containing 5% supernatant from cmg14.12 cells . Stimulations were performed on the day after harvest in media containing 5% cmg1.12 media . For flow cytometry experiments, macrophages were cultured on low adherence plates (greiner bio - one) to facilitate cell harvest . Cells were removed from the plate by washing with pbs followed by 10 minutes with cell stripper (gibco) and then 10 minutes with edta / trypsin (sigma). Growth medium was supplemented with palmitate and bsa at a 2: 1 molar ratio as described previously and bsa - supplemented media were used as control . For cell stimulations, pbs or lps (50 ng / ml) were added to bsa or free fatty acid containing media . Flow cytometry was performed on a bd biosciences facscalibur machine and the data was analyzed using flowjo software . Wild type (wt) c57bl/6 mice were obtained from oriental bioscience and maintained in our mouse colony . The atg5flox x lysm - cre on the c57bl/6 background were kindly provided by skip virgin (washington university). Mice were maintained in a pathogen - free facility on a standard chow diet ad libitum (6% fat). All animal experiments were conducted in strict accordance with nih guidelines for humane treatment of animals and were reviewed by the animal studies committee of washington university school of medicine . Total cellular rna was isolated using qiagen rneasy columns and reversely transcribed using a high capacity cdna reverse transcription kit (applied biosystems). Real - time qrt - pcr was performed using sybr green reagent (applied biosystems) on an abi 7500 fast thermocycler . Relative gene expression was determined using the delta - delta ct method normalized to 36b4 expression . Mouse primers sequences were as follows (all 5-3): 36b4 (forward - atc cct gac gca ccg ccg tga, reverse - tgc atc tgc ttg gag ccc acg tt); ctsb (forward - gat caa gga cca cca cat cc, reverse - ctt agg agt gca cgg gag ag); ctsd (forward - gac agc tcc ccg tgg tag ta, reverse - caa cag aag ctg gtg gac aa); ctsk (forward - tgc cgt ggc gtt ata cat ac, reverse - cgg cta tat gac cac tgc ct); lamp1 (forward - tct tca gtg tgc agg tcc ag, reverse - atg agg acg atg agg accag); lamp2a (forward - cca aat tgg gat cct aac cta, reverse - tgg tca agc agt gtt tat taa ttc c); lamp2b (forward - ggt gct ggt ctt tca ggc ttg att, reverse - acc acc caa tct aag agc agg act): atp6v1h (forward - aga cag cca gca aca cac tg, reverse - tca cag aaa ctt cgt ggc ag); p62 (forward - gct gcc cta tac cca cat ct, reverse - cgc ctt cat ccg aga aac); lc3 (forward - cgt cct gga caa gac caa gt, reverse - att gct gtc ccg aat gtctc). Total cellular protein was isolated by lysing cells in 150 mm nacl, 10 mm tris (ph 8), triton x-100 1%, and 1x protease complete . Proteins were separated on a tgx gradient gel (420%; biorad) and transferred to a nitrocellulose membrane . Western blotting for cathepsin d and actin was performed using 40 g of total cellular protein . After the indicated stimulations, pmacs were removed from the plate as described above and fixed in 4% pfa for 15 minutes in the dark at room temperature . After fixation, cells were permeabilized (pbs + 0.1% triton x) for 15 minutes in the dark and washed with facs buffer + 0.1% triton x. cell were treated with fc receptor block (bd pharmigen) for 5 minutes on ice followed by incubation with cd107a (lamp1) pe antibody for 30 minutes in the dark on ice . After the indicated stimulations, cells were removed from the plate as described above and then stained with 500 nm lysotracker red in tissue culture media for 15 minutes at 37c . After staining, cells were washed three times with pbs, harvested as described above, and analyzed by flow cytometry . For fluorescent dextran experiments, macrophages were incubated with 500 g / ml tetramethylrhodamine- (tmr-) dextran for 2 h in regular media . Lysotracker red or tmr - dextran stained cells were fixed for 10 minutes in 4% paraformaldehyde followed by nuclear staining with hoechst 33342 dye . The cover slips were mounted on glass slides and imaged using a zeiss confocal microscope . After the indicated stimulations, pmacs were removed from low adherence plates as described above . The fluorescently labeled cathepsin b substrate was reconstituted per manufacturer's instructions and diluted in dmem+10% ifs . Macrophages were incubated in 150 l of staining solution for 45 minutes at 37c under 5% co2 with gentle mixing every 10 minutes . After the incubation, cells were washed 2 times in 1 ml of pbs and analyzed by flow cytometry (fl2). Il-1 was quantified using a duoset elisa kit (r&d systems) according to the manufacturer's instructions . Groups were compared by paired student's t - test . A value of p 0.05 was considered significant . To investigate differences in the lysosome compartment of pmacs compared to bmdms, we quantified lysosome volume in these two sets of primary macrophages using the lysosomotropic dye lysotracker red coupled with flow cytometric analysis . Using this approach, lysosome content was significantly increased in pmacs relative to bmdms (figures 1(a) and 1(b)). In line with this finding, pmacs also had significantly higher levels of the lysosomal membrane protein lamp1 compared to bmdm (figures 1(c) and 1(d)). To assess lysosomal protease function, we analyzed the activity of cathepsin b using the fluorogenic substrate magic red . As shown in figures 1(e) and 1(f), cathepsin b activity was increased in pmacs when compared to bmdms . Lysosomes have several distinct morphologic appearances that can vary based on cell type and activation status including spheroid, ovoid, or tubular . In addition, the size of lysosomes can vary dramatically . To visualize lysosomes in pmacs and bmdms, we utilized two complementary immunofluorescent approaches: (1) lysotracker red staining and (2) tetramethylrhodamine- (tmr-) dextran staining . Lysotracker red freely crosses cell membranes and is concentrated in acidic organelles whereas tmr - dextran is taken up via endocytosis and reaches lysosomes in ~30 min . In addition, the morphology was more frequently ovoid and tubular in pmacs and spherical in bmdms (figures 2(a)2(d)). Notably, pmac lysosome morphology is similar to that described from adipose tissue macrophages during high fat feeding . Stimulation of bmdms or pmacs with lps did not lead to a significant change in lysosome morphology (data not shown). Emerging data indicates that lysosome biogenesis and function are regulated in part at the transcriptional level . Therefore, we compared the expression of several lysosome and autophagy related genes in bmdms and pmacs . Consistent with the flow cytometry and imaging data, the expression of genes encoding cathepsin proteases, lysosomal membrane proteins, and the vacuolar atpase was increased in pmacs relative to bmdms (figure 3(a)). In addition, the autophagy related genes lc3 and p62 were also expressed at a higher level in pmacs (figure 3(a)). Thus, mrna expression of lysosome and autophagy related genes is increased in pmacs, which is likely related to the expanded lysosome compartment observed in these primary macrophages . At the protein level, pro - cathepsin d protein content was increased in pmacs relative to bmdms (figures 3(b) and 3(c)). Similar to the flow cytometry data, lamp1 protein content trended towards an increase in pmacs compared to bmdms (figures 3(d) and 3(e)). Interestingly, the molecular weight of lamp1 was significantly greater in bmdms suggesting a higher level of glycosylation, a finding which could further affect lysosome function and stability in these two types of primary macrophages (figure 3(d)). To investigate whether the observed differences in lysosome phenotype modulate macrophage inflammatory responses, we explored the lipotoxic inflammasome as a model system . We, and others, have shown that the combination of lps and the saturated fatty acid (sfa) palmitate triggers the release of il-1 through an nlrp3 and caspase 1-dependent mechanism [2022]. In prior studies using this system, it has been shown that il-1 release from pmacs is dependent on lysosome damage and cathepsin proteases, whereas the role of lysosomes in bmdms is less clear . To directly compare pmacs and bmdms using this system, we simultaneously stimulated both cell types with lps in the presence of increasing concentrations of palmitate and the release of il-1 was quantified . Both pmacs and bmdms secreted il-1 in a dose dependent fashion; however, pmacs consistently secreted 510 times more cytokines than bmdms despite using a similar number of cells (figures 4(a) and 4(b)). Importantly, fa uptake was similar between the pmacs and bmdms (data not shown). Consistent with prior studies, il-1 release from pmacs was almost completely prevented by the cathepsin b inhibitor, cao74, or lysosomal acidification inhibitor, bafilomycin (baf), in palmitate - lps treated pmacs (figure 4(c)). This was not the case in bmdms where il-1 release was unaffected by cao74 and actually increased with baf (figure 4(d)). Thus, lysosome inhibitors differentially influence lipotoxic inflammasome activation in pmacs compared to bmdms, an observation that may account for some of the discrepancies between prior publications [2022]. More importantly, these findings argue that the baseline differences in lysosome content and function between pmacs and bmdms can alter the response of these cells to inflammatory stimuli . Lysosomes are also important for the degradation of autophagic cargo, which can also modulate the inflammasome response . Therefore, using atg5 ko cells, we tested the impact of autophagy deficiency on activation of the lipotoxic inflammasome in pmacs versus bmdms . As can be seen in figures 4(e) and 4(f), the phenotypes observed using autophagy deficient cells were different in these primary macrophage systems . In pmacs, atg5 ko cells had a modest increase in il-1 release after lps treatment (figure 4(e) inset), but il-1 secretion following palmitate - lps stimulation was reduced compared to wt cells (figure 4(e)). In distinction, atg5 ko bmdms released more il-1 in response to both lps and palmitate - lps treatment (figure 4(f)). This was not related to differences in atg5 protein expression, which was similar between the two types of macrophages (figure 4(g)). Instead, the development of lysosome dysfunction in pmacs treated with palmitate - lps may explain why genetic autophagy deficiency does not further enhance inflammasome activation . In contrast, atg5 ko bmdms phenocopy cells treated with baf, which also inhibits autophagic flux . These findings argue that genetic or pharmacologic inhibition of autophagy in bmdms unmasks an important suppressive role for this degradative process on lipotoxic inflammasome activation . Further investigation will be necessary to evaluate the mechanisms that account for these differential phenotypes . Interestingly, recent evidence suggests that autophagy can increase lysosome activity, which could be particularly important for bmdms as they have less active lysosomes at baseline [25, 26]. Taken together, this data suggests that pmacs and bmdms respond to lipotoxic stimulation through fundamentally different mechanisms, and at least part of these differences is related to distinct lysosome phenotypes . The findings presented in this paper illustrate several significant differences between lysosomes in pmacs and bmdms . Specifically, the activated lysosome compartment in pmacs may reflect the signals they receive in the inflammatory setting of peritonitis . In contrast, bmdms are nave macrophages that have not encountered activation signals . Using the example of the lipotoxic inflammasome, we also provide evidence that the distinct lysosome phenotypes observed in bmdms and pmacs can influence macrophage inflammatory function . Thus, when conducting experiments of lysosome function with primary macrophages, it is critical to account for baseline lysosome phenotype to ensure appropriate interpretation of data . We would argue that the choice of bmdm versus pmac for experimentation should be tailored to the specific research question, and a low threshold should exist for comparing multiple macrophage populations . In addition, further comparative analysis of lysosomes between pmacs, bmdms, and other in vivo isolated macrophages will be necessary to shed additional light on the dynamic regulation of lysosomes in these important leukocytes.
As a national reference center for q fever, our center receives samples from france and other regions for serologic, molecular, histologic, and immunohistochemical analyses as described (57). In 2012, we received a cardiac valve sample from a patient in cayenne with q fever endocarditis who had undergone surgery in martinique . In the same year, we collected 4 heparinized blood samples from patients with acute q fever from cayenne that were collected before initiation of antimicrobial drug treatment . Serologic titers of igg1 increased to 51,200 in this patient, and results of quantitative pcr (qpcr) and immunohistochemical analyses of the valvular sample were positive for c. burnetii (figure 1). The other 4 patients (2 men and 2 women) had fever and acute pneumonia, and 2 of them also had increased transaminase levels . The heparinized blood samples were tested by using a specific qpcr; samples from only 1 patient were positive for c. burnetii . Immunohistochemical detection of coxiella burnetii in resected cardiac valve of a 60-year - old man with q fever endocarditis, cayenne, french guiana . We successfully cultured c. burnetii from the valvular sample after incubation for 16 days and from 3 blood samples after incubation for 25, 32, and 32 days . Paradoxically, the only blood sample that was positive by qpcr was negative by culture . Spacers cox 51 and cox 20 were the most discriminating spacers in identifying this genotype, and the 3 isolates from the blood cultures were also identified by these 2 spacers as genotype 17 (figure 2). This genotype had been identified in our laboratory only once, in 2000 in an aortic valve of a 40-year - old french man who had undergone surgery in france, had q fever endocarditis, and was co - infected with streptococcus oralis . Retrospectively, we found that patient had lived in cayenne for years before his diagnosis with q fever endocarditis . Therefore, all genotype 17 isolates were obtained from who lived or had lived in cayenne, making it unique to this area . We determined the antimicrobial drug susceptibilities of these isolates, and the mic of doxycycline was 0.25 g / ml for all isolates (10). Phylogenetic diversity of 34 genotypes of coxiella burnetii identified by using multispacer sequence typing (mst). Scale bar indicates nucleotide substitutions per site . For the past 10 years, routine cell culture for c. burnetii has been performed in our laboratory . We found that the proportion of isolates obtained from blood samples of patients with acute q fever was higher for patients from cayenne than for patients from metropolitan france . We obtained 3 isolates from 5 blood samples from untreated patients in cayenne and 3 isolates from 65 samples from patients in metropolitan france (p = 0.003, by fisher exact test). However, we did not find any difference with respect to the culture delay between patients from the 2 locations . Our work shows that genotype 17, a unique genotype, is circulating in cayenne . This genotype is related to genotypes that harbor the qph1 plasmid, which causes the most severe clinical forms of acute q fever in experimental animal models (11,12). Only bacteria from this group and bacteria containing the qpdv plasmid have been found in cases of human acute q fever (9,13). We believe that severity of q fever infection is related to the strain of c. burnetii circulating in cayenne . Because the population in french guiana is a large diversity of creole, amerindian, maroon, caucasian, and asian persons (14), we excluded the hypothesis that genetic susceptibility of patients from cayenne to c. burnetii infection might be related to severity of the disease . We observed a higher rate of strain isolation from the blood of patients from cayenne than from the blood of patients from metropolitan france . This difference may be related to a higher bacterial load in blood or a greater ability of the bacteria to grow on cell cultures . This second hypothesis seems more likely because even if the observed delay for the first culture was the same for genotype 17 and other genotypes, the strain from cayenne was isolated more frequently in the cell line that we used (hel cells) than other strains of c. burnetii from france . In addition, we did not find a higher number of dna copies in the blood of patients from cayenne, and qpcr results were not correlated with culture results . C. burnetii isolates from cayenne were susceptible to antimicrobial drugs, particularly doxycycline . These isolates cause acute pneumonia and endocarditis . Among the 34 genotypes identified in our study by multispacer a larger biodiversity of strains has been observed in samples from patients in metropolitan france (9), where 21 genotypes circulate (figure 2). In contrast, during the recent q fever outbreak in the netherlands, it appears that a single strain (genotype 33) was responsible for the epidemic (15). We believe that there has been an epidemic developing in cayenne since 1996 that is caused primarily, if not solely, by a single strain that has circulated since at least 2000, whose reservoir is unknown (1,2). In conclusion, c. burnetii genotype 17 is circulating in french guiana and causing acute infections and endocarditis . This strain is epidemic and most likely causes more acute infections with exacerbated immune responses than other known genotypes of c. burnetii.
Dr . Alexandre batista liver hemangiomas are the most common benign liver tumors . Diffuse hepatic hemangiomatosis (dhh) is a rare disorder, usually occurring in the neonate, characterized by extensive replacement of liver parenchyma with hemangiomatous lesions . It is frequently associated with a palpable abdominal mass and high - output heart failure, with high mortality rate in neonates . Dhh in the adult, especially without extra - hepatic involvement, is extremely rare . Hemangiomatosis differs from multiple or giant hemangiomas in that the boundary of the lesion is ill - defined, while in the latter, the liver parenchyma surrounding the lesion is compressed by a fibrous capsule forming the wall of the hemangioma . An asymptomatic 68-year - old man was referred to our hospital due to the presence of numerous nodular hepatic lesions accidentally detected on ultrasound . There was a history of heavy drinking (alcohol intake around 70 - 90 g / d) and no prior history of medication or steroid intake . Physical examination upon admission revealed diffuse hepatomegaly, with no associated cardiac murmur or leg edema . Ui / l). Abdominal ultrasound (us) showed multiple ill - defined sub - centimeter hyperechoic hepatic nodules, predominately in the right lobe, with no posterior acoustic shadowing or significant through transmission and absence of high vascularity on color doppler [figure 1]. 68-year - old male referred to our hospital due to multifocal hepatic lesions, subsequently diagnosed as diffuse hepatic hemangiomatosis . A) ultrasound examination demonstrates multiple ill - defined yperechoic nodules dispersed throughout the hepatic parenchyma, mostly sub - centimeter and with right hemi - liver predominance . Countless predominantly sub - centimeter nodular lesions with low t1-weighted signal intensity (si) and moderately high t2-weighted si were depicted . Liver lesions showed moderately restricted diffusion, and post - contrast fat - suppressed t1-weighted images showed predominantly hypovascular features with retention of contrast in later phases . A us - guided biopsy using an automated gun with an 18-gauge needle was performed collecting three representative fragments . 68-year - old male referred to our hospital due to multifocal hepatic lesions, subsequently diagnosed as diffuse hepatic hemangiomatosis . A) axial fatsaturated t2-weighted image shows countless small - sized moderately bright lesions (arrows). B) diffusion weighted image shows these lesions with high signal intensity (b = 600 sec / mm) (arrows), which may be seen in liver hemangiomas secondary to t2 shine - through effect . Axial c) pre - contrast and (d - f) post - contrast fat - saturated t1-weighted images show arterial homogeneous enhancement (arrows, d), some exhibiting portal phase peripheral nodular enhancement (arrow, e) and late centripetal filling (arrows, f). Pathology revealed endothelial - lined sinusoidal proliferation with erythrocyte content, consistent with hepatic hemangiomatosis [figure 3]. Definitive diagnosis of dhh was established based on a combination of imaging and pathological findings and clinical / imaging follow - up . Two years following the diagnosis, the patient's hepatic function and imaging findings remain stable, with no relevant associated symptoms . 68-year - old male referred to our hospital due to multifocal hepatic lesions, subsequently diagnosed as diffuse hepatic hemangiomatosis . Ultrasound guided biopsy samples stained with hematoxylin and eosin a) 4 10, b) 40 10; c) 40 10). Reveal numerous dilated nonanastomotic vascular spaces, which were lined by flat endothelial cells and supported by fibrous tissue endothelium . Chronic inflammatory infiltrates with peri - portal distribution are also present (black outlined asterisk, c). Imaging plays a vital role in the detection and characterization of multifocal liver lesions in adults . There are numerous causes including benign and malignant neoplasms, infectious lesions, and inflammatory conditions . The us evaluation showed multiple sub - centimeter, ill - defined, hyperechoic nodules dispersed throughout the liver parenchyma, predominantly in the right hemi - liver, with very little or no flow seen on color doppler . The differential diagnoses included metastatic disease, multifocal nodular fatty infiltration of the liver, bile duct hamartomas (bh), or other less - frequent entities such as dhh or angiosarcoma . Metastatic liver involvement usually occurs in a patient with a known extra - hepatic primary neoplasm . Small hypervascular liver metastases (e.g. From breast or neuroendocrine tumors) may mimic small early uniform enhancing hemangiomas; however, these lesions tend to fade or wash out, whereas hemangiomas are more likely to maintain the si or fade toward background isointensity . Also, the majority of liver metastases shows heterogeneous or ring enhancement on the arterial phase images, confirming their definite diagnosis . Multifocal nodular fatty deposition is an unusual pattern of liver steatosis that consists of multiple round or oval fat foci randomly distributed throughout the liver . Fat deposits appear isointense or hyperintense to the liver on in - phase images, dropping si on out - of - phase images . Fat accumulation does not show restriction or shine - through effect on diffusion - weighted images . Other clues indicative of multifocal fat deposition are the lack of mass effect, stability in size over time, and contrast enhancement similar to or less than that of the surrounding liver parenchyma . Bh, also called von meyenburg complexes, originate from embryonic bile ducts that fail to involute . On ultrasound, bh have been described as innumerable tiny hypoechoic or hyperechoic foci measuring less than 10 mm and distributed uniformly throughout the liver, with some displaying a comet tail artifact . Differences in echogenicity may be due to the size of the dilated bile duct component, which, at a certain size, would behave like other microcystic structures . On mri, bile duct hamartomas usually appear as multiple tiny cystic lesions, strongly hyperintense on t2-weighted images . After gadolinium administration, some authors described a thin rim of enhancement, which correlates with the compressed liver parenchyma surrounding the lesions . Internal progression or retention it is very aggressive and sometimes may mimic hemangioma on dynamic studies, showing high si on t2-weighted images, since both tumors contain abundant blood - filled vascular spaces; also, heterogeneous centripetal and progressive enhancement has been described . Dhh is a rare, although benign, condition that is characterized by diffuse replacement of liver parenchyma by hemangiomatous lesions . The natural history and prognosis of hepatic hemangiomatosis are poorly understood due to its rarity . Hemangiomatosis is different from multiple or giant hemangiomas in that the boundary of the lesion is ill - defined, while in the latter, the liver parenchyma surrounding the lesion is compressed by a fibrous capsule that forms the wall of the hemangioma . Dhh can occur in all age groups, but is most frequent in the neonate where it can result in high - output cardiac failure and significant mortality . It is usually asymptomatic in adulthood, being extremely rare when not associated with giant cavernous hemangiomas and even rarer when no other organ is affected besides the liver . Two patterns of dhh are described, the diffuse (non - nodular) and the nodular pattern, the former being the predominant pattern, representing approximately two - thirds of the cases . Sonographically, the liver affected by dhh appears as homogeneous hyperechoic areas with poorly defined margins (diffuse pattern) or a heterogeneous echo pattern with multiple discrete or coalescent small hyperechoic nodules (multinodular pattern). These nodular lesions are variable in size (usually <5 - 10 mm), appearing as countless oval or round hyperechoic nodular lesions . Very little or no flow, the areas affected by hemangiomatosis may also display a diffuse non - nodular pattern composed of tiny cystic spaces separated by numerous thin hypointense septa, creating a lattice - like appearance or a diffuse nodular pattern consisting of multiple small discrete and coalescent nodules on t2-weighted imaging . The extremely low velocity of blood flow within hemangioma relates to the absence of flow void seen in mri imaging . Dynamic contrast - enhanced mri shows heterogeneous enhancement in these areas during the arterial phase that becomes more homogeneous during portal and delayed phase imaging . The multinodular type, as was our case, exhibits small discrete and coalescent nodules with early homogeneous enhancement or the typical peripheral discontinuous enhancement during the arterial phase, showing uniform late retention of contrast . In conclusion, dhh with no associated cavernous hemangiomas and without extra - hepatic involvement is an extremely rare condition . Despite its rarity, it can be suggested in adults presenting with multiple, predominantly sub - centimeter hepatic lesions demonstrating mri features of hemangiomas . Mr imaging may be superior to us and computed tomography (ct) for the diagnosis and is the preferable cross - sectional imaging technique for long - term follow - up.
In the outpatient setting, actinic keratosis is one of the most commonly treated skin conditions, the prevalence of which increases with cumulative skin exposure and increasing age . Rates of 1125% have been reported in the northern hemisphere and of 4060% in australia . Actinic keratoses are strong predictors of squamous cell carcinomas and are believed to be precursors of squamous cell carcinomas of the skin . A number of studies have been published recently concerning modalities of photodynamic therapy (pdt) treatment for actinic keratosis in which the efficacy of several photosensitisers and/or light sources were compared,,, . The most recent of these is the study by dirschka and colleagues which appeared recently in the british journal of dermatology and in which a gel formulation of 5-aminolaevulinic acid (bf-200 ala) was found to be superior to methyl-5-aminolaevulinate (mal). In the same study, the narrow - band light sources were found to be more effective than the broad - band light sources, although the narrow - band sources were associated with a higher occurrence and severity of adverse effects, which included pain, burning, erythema and exfoliation, irrespective of the photosensitizer used . While such a multicenter, randomized study is to be commended, the authors in our opinion failed to appropriately consider their findings in the light of previous studies, since a number of pertinent and highly relevant articles were not mentioned . An important study in this context is the study by von felbert and colleagues with a longer follow - up time of 12 months (compared to 12 weeks in the study of dirschka et al .) And in which the use of narrow - band and broad - band sources were found to be equally effective . One reason given by von felbert and colleagues for the comparable efficiency of these sources was the use of a more advanced and efficient broad - band optical filter (in comparison to the broad - band sources used in previous studies). However, not only failed to discuss reasons for the differences between the narrow - band and broad - band types of sources used, but also did not comment on the differences found between the various types of broad - band sources employed . Additionally, despite the findings of von felbert et al . Who found the use of a broad - band source with water filter to be associated with less pain than that caused by a narrow - band source, and a study by apalla et al . Who found the use of a broad - band source without a water filter to be associated with pain, dirschka and colleagues did not consider the fact that broad - band sources would result in absorption over a broader spectral range (and possibly encompassing several maxima) of the photosensitizer as has been discussed previously additionally, previous studies in which no differences were found between narrow- and broad - band sources,, were not considered . Their study also demonstrated that the conditions under which pdt was applied had a pronounced impact on the efficiency of the treatment . For mal, the von felbert study also found considerably higher response rates (80% total clearance) as compared to the dirschka et al . Possible reasons for such a pronounced difference between the response rates in these two studies were however not discussed by dirschka and colleagues . While the extent of pain experienced during photodynamic therapy (which is deemed to be the main side - effect of pdt,) was documented by dirschka et al ., possible reasons for the greater incidence of pain during the use of the narrow - band sources were not considered . In the dirschka et al . Study, a comparison of the photosensitizers used showed bf-200 ala to be superior to mal, but again, a study with contradictory findings (gholam et al .) Is not discussed . While we do not wish to infer that the study performed by dirschka and colleagues is in any way erroneous, we would nevertheless like to point out that the discussion of the results and the authors selection of cited literature appears to be biased towards studies supporting the authors own findings . The problem of such a bias has recently been outlined, together with the need for an up - to - date selection of literature and the recommendation that a concealment of contradictory or critical literature should be avoided . As a result of the rather selective choice of literature provided and the lack of a thorough consideration of the relevant studies already published, the article by dirschka and colleagues leaves a number of questions unanswered . Furthermore, the corresponding editorial published in the same issue of the british journal of dermatology is unfortunately also based on the acceptance of the superiority of bf-200 ala in comparison to mal as well as the superiority of narrow - band in comparison to broad - band sources and fails to address the fact that several other studies have found contradictory results . Certainly, sweeping statements claiming e.g., narrow - band sources to be preferable to broad - band sources or bf-200 to be better than mal should not be made on the basis of a single study, especially since such statements may lead to rapid and ungrounded propagation of therapeutic protocols . In conclusion, it would appear that the last word concerning the optimal photosensitizer and radiation source for the treatment of actinic keratosis has not yet been spoken . Therapy for actinic keratosis should be a carefully chosen compromise between undesired side - effects and therapeutic efficacy and needs to be based on a consideration of all of the relevant clinical studies.
Whereas the management of localized renal cell carcinoma (rcc) has evolved toward minimally invasive and nephron - sparing surgery, the treatment of endophytic renal tumors has remained problematic . These tumors typically require precise intracorporeal suturing and complex reconstruction, with the added time constraints imposed by renal ischemia . Even open partial nephrectomy is acknowledged to be technically demanding in this situation, with increased complication rates compared with procedures for tumors in peripheral locations . In addition, serial studies of laparoscopic partial nephrectomy (lpn) report higher postoperative complication rates, which result in more ischemic time and longer hospital stays for endophytic tumors [2 - 4]. With the increasing application of minimally invasive surgery, several energy - based tissue ablation technologies including cryoablation and radiofrequency ablation are being investigated . In these procedures, tissue is destroyed in situ, thereby avoiding the complications induced by renal ischemia and surgical excision . Although energy - based ablation may injure the collecting system, clinical and preclinical studies on cryoablation have demonstrated the ability of this technique to manage tumors located almost within the collecting system without compromising the integrity of the underlying structure [5 - 7]. In addition, the laparoscopic approach allows the surgeon to position the cryoprobe by using intraoperative ultrasonography more precisely and to move adjacent organs away from the ablation site . If the surgical and oncological outcomes support the feasibility and efficacy of laparoscopic cryoablation (lrc) for endophytic renal tumors, and safety can be maintained during the surgical procedure, this method may provide an alternative nephron - sparing surgery for selected patients . To determine this, we evaluated the intermediate - term outcome of lrc in patients with endophytic rcc . From june 2005 to may 2009, lrc was performed on 45 renal tumors in 39 patients at our institution . Indications for lrc included the presence of a localized, solid enhancing renal mass smaller than 4 cm in a patient at high risk for partial nephrectomy or older than 70 years of age . High operative risk in our institution was defined as an american society of anesthesiology (asa) physical status score of over 3 . Among these inclusion criteria for lrc, absolute indications included bilateral tumors and a patient with a solitary kidney or renal insufficiency . A patient with normal contralateral renal function but poor operability was defined as an elective indication . All patients underwent a preoperative radiologic evaluation with contrast - enhanced computed tomography (ct) or magnetic resonance imaging (mri) in the case of renal insufficiency to delineate the parameters of the renal lesion, including location, size, depth, and position relative to hilar vessels and the collecting system . All patients who underwent lrc in our institution were registered prospectively in a specific database that included all important information about tumor and patient characteristics including operative time, blood loss, hospital stay, pathology findings, and occurrence of complications . This allowed us to collect and analyze the data of the patients retrospectively, depending on the location of the tumor and pathologic findings . Three attending urologists reviewed the preoperative imaging studies for each patient to categorize the renal mass depending on tumor position . An endophytic location of a tumor in this study was defined as less than 40% of the lesion extending off the surface of the kidney (fig . 1), as originally described by finley et al . Among all patients who underwent lrc, 15 tumors from 14 patients who were confirmed as having rcc by intraoperative needle biopsy and patients were initially evaluated at 1 month and 3 months, then every 3 months during the first year . Follow - up evaluations involved a medical history update, physical examination, blood pressure measurement, contrast - enhanced ct or mri, chest radiography, measurement of serum electrolytes, liver function tests, and renal function tests . A lack of enhancement on ct or mri along with stable or decreased tumor size were considered signs of successful treatment . Recurrence was defined as increasing tumor size or lack of tumor shrinkage, with image enhancement . All lrc procedures were conducted by a single surgeon with use of nearly identical technique . For the laparoscopic procedure, a standard technique using three ports was performed . In general, tumors anterior to a horizontal line within the coronal plane through the renal hilum were approached transperitoneally, and tumors posterior to this line were approached retroperitoneally . Real - time intraoperative ultrasonography (ious: aloka dynaview ii, americanlab, miami, fl, usa) was used in all cases to identify the lesion and to determine the degree of the ice ball extension . The kidney was mobilized and the gerota's fascia was opened to facilitate identification of the tumor . Up to two needle biopsies were taken from the tumor before the insertion of a cryoprobe . Then 1.47 mm cryoprobes (icerod, oncura, plymouth meeting, pa, usa) were inserted, each of which induced a -40 isothermal lesion 14.5x34 mm in diameter and radiating from the ablation probe . Before insertion into the kidney, the proper number and positions of the cryoprobes temperature probes were then inserted into the deep margins of the tumor . In all cases, a double - freeze cycle was applied, with an intervening thawing process . At the base of a lesion in proximity to the collecting system, the extent of the ice ball was identified by ious as a complete loss of echogenic lesion in the circumferential area of the base . Hemostasis was achieved by filling the probe tract with fibrin glue (baxter, deerfield, il) and surgicel (johnson & johnson, irvine, ca, usa) after the second thaw allowed the safe removal of the cryoprobe . All data were analyzed with the statistical package for the social sciences, version 13 (spss inc, usa). Statistical comparison of pre- and postoperative values was performed with paired t - tests, with differences considered statistically significant if p<0.05 . From june 2005 to may 2009, lrc was performed on 45 renal tumors in 39 patients at our institution . Indications for lrc included the presence of a localized, solid enhancing renal mass smaller than 4 cm in a patient at high risk for partial nephrectomy or older than 70 years of age . High operative risk in our institution was defined as an american society of anesthesiology (asa) physical status score of over 3 . Among these inclusion criteria for lrc, absolute indications included bilateral tumors and a patient with a solitary kidney or renal insufficiency . A patient with normal contralateral renal function but poor operability was defined as an elective indication . All patients underwent a preoperative radiologic evaluation with contrast - enhanced computed tomography (ct) or magnetic resonance imaging (mri) in the case of renal insufficiency to delineate the parameters of the renal lesion, including location, size, depth, and position relative to hilar vessels and the collecting system . All patients who underwent lrc in our institution were registered prospectively in a specific database that included all important information about tumor and patient characteristics including operative time, blood loss, hospital stay, pathology findings, and occurrence of complications . This allowed us to collect and analyze the data of the patients retrospectively, depending on the location of the tumor and pathologic findings . Three attending urologists reviewed the preoperative imaging studies for each patient to categorize the renal mass depending on tumor position . An endophytic location of a tumor in this study was defined as less than 40% of the lesion extending off the surface of the kidney (fig . 1), as originally described by finley et al . Among all patients who underwent lrc, 15 tumors from 14 patients who were confirmed as having rcc by intraoperative needle biopsy and patients were initially evaluated at 1 month and 3 months, then every 3 months during the first year . Follow - up evaluations involved a medical history update, physical examination, blood pressure measurement, contrast - enhanced ct or mri, chest radiography, measurement of serum electrolytes, liver function tests, and renal function tests . A lack of enhancement on ct or mri along with stable or decreased tumor size were considered signs of successful treatment . Recurrence was defined as increasing tumor size or lack of tumor shrinkage, with image enhancement . All lrc procedures were conducted by a single surgeon with use of nearly identical technique . For the laparoscopic procedure, a standard technique using three ports was performed . In general, tumors anterior to a horizontal line within the coronal plane through the renal hilum were approached transperitoneally, and tumors posterior to this line were approached retroperitoneally . Real - time intraoperative ultrasonography (ious: aloka dynaview ii, americanlab, miami, fl, usa) was used in all cases to identify the lesion and to determine the degree of the ice ball extension . The kidney was mobilized and the gerota's fascia was opened to facilitate identification of the tumor . The fat overlying the tumor was placed aside and later retrieved for pathology examination . Up to two needle biopsies were taken from the tumor before the insertion of a cryoprobe . Then 1.47 mm cryoprobes (icerod, oncura, plymouth meeting, pa, usa) were inserted, each of which induced a -40 isothermal lesion 14.5x34 mm in diameter and radiating from the ablation probe . Before insertion into the kidney, the proper number and positions of the cryoprobes temperature probes were then inserted into the deep margins of the tumor . In all cases, a double - freeze cycle was applied, with an intervening thawing process . At the base of a lesion in proximity to the collecting system, the extent of the ice ball was identified by ious as a complete loss of echogenic lesion in the circumferential area of the base . Hemostasis was achieved by filling the probe tract with fibrin glue (baxter, deerfield, il) and surgicel (johnson & johnson, irvine, ca, usa) after the second thaw allowed the safe removal of the cryoprobe . All data were analyzed with the statistical package for the social sciences, version 13 (spss inc, usa). Statistical comparison of pre- and postoperative values was performed with paired t - tests, with differences considered statistically significant if p<0.05 . Among 45 renal tumors treated with lrc in our institution, 17 (37.8%) were defined as endophytic tumors . Among them, 15 tumors from 14 patients were confirmed as rccs and were exclusively enrolled in this series . Five tumors were centrally located, which was defined as being completely surrounded by normal parenchyma, with no visual clue regarding the location of the tumor on laparoscopy . One patient had a hilar tumor, which was defined as a tumor positioned medially within 5 mm of the renal artery or vein . The mean (range) age of the 14 enrolled patients was 61.4 years (range, 43 - 75 years) old . Four patients (28.6%) were over 70 years old, and their mean age was 72.8 years (range, 70 - 75 years) old with a mean asa physical status score of 2.3 (range, 1 - 3). The mean asa physical status score of the whole patient group was 2.9 (range, 1 - 4), and 85.7% (12/14) of patients had an asa physical status score over 3 . The indications for nephron - sparing surgery included a bilateral tumor in 2 patients, solitary kidney tumors in 3 patients, and chronic renal failure in 1 patient . The mean tumor size was 2.8 cm (range, 1.7 - 3.7 cm) and the mean operating time was 169.7 minutes (range, 110 - 220 minutes). The mean time of the first freeze cycle was 7.9 minutes, and the mean of the second freeze cycle was 7.4 minutes . In the case of five central tumors, more fat tissue overlying the kidney was removed to achieve an adequate image for monitoring the position of the cryoprobe tip . In the case of a hilar tumor, the major renal vessels, ureter, and renal pelvis were displaced from the ice ball with vascular loops, and rolled gauze was placed into the renal hilum . In all patients, multiple cryoprobes were used, and the mean number of cryoprobes needed was 3.2 (range, 2 - 5). The mean blood loss, measured as the amount in the suction device, was 99.5 ml (range, 40 - 150 ml). No major complications, including open surgical conversion and nephrectomy due to bleeding, occurred in any patient . The serum creatinine and hemoglobin levels checked 7 days after surgery showed no significant differences compared with preoperative levels . The average preoperative and postoperative creatinine levels were 1.31 mg / dl and 1.43 mg / dl, respectively (p=0.13). The average preoperative and postoperative hemoglobin levels were 13.94 g / dl and 13.21 g / dl, respectively (p=0.57). One patient who had a previous subtotal gastrectomy for early gastric adenocarcinoma needed a postoperative transfusion due to a low preoperative baseline hemoglobin level (8.4 g / dl). The mean hospital stay was 4.1 days (range, 3 - 8 days), and during this stay, no one experienced clinical signs or symptoms of collecting system injuries, including flank pain, fever, and leukocytosis . In the pathologic examination, all of the patients showed common clear cell carcinoma, rcc, except for two patients with papillary type 1 . The mean follow - up was 32.6 months (range, 12 - 51 months). Follow - up imaging studies revealed no evidence of hydronephrosis, and all patients remained asymptomatic . A 2.2 cm sized central tumor was in direct contact with the collecting system, as shown by the enhancing portion in the deep peripheral area of the cryoablated site in an initial follow - up ct taken 1 month after lrc . Due to his high anesthesiologic risk from myocardial infarction, which required vascular stenting 3 times, and the anterior location of the tumor, which disturbs deployment of the probes for other minimally invasive ablative techniques including radiofrequency ablation, this patient is currently under active surveillance . Follow - up images showed no increase in the enhancing lesion or distant metastasis for 24 months after the initial lrc . Except for this patient, all other patients have remained free of recurrence or metastasis as shown by the periodic radiologic workups . The mean size of these properly treated lesions was 2.750.72 cm, 2.960.42 cm, 2.710.26 cm, 2.430.19 cm, 2.240.21 cm, 1.260.31 cm, and 1.080.34 cm before surgery, and at 1 month, 3 months, 6 months, 9 months, 12 months, and 24 months, respectively (fig . 2, 3). Because small renal tumors emerge incidentally during imaging for various indications, questions persist about the best way to treat endophytic tumors with a minimally invasive technique . Although 3-, 5-, and 10-year - old data encourage the use of renal cryoablation, especially for tumors in peripheral locations [14 - 16], extending these findings to endophytic tumors is still under debate . Because of the proximity to the renal vasculature and the collecting system, the cryoablation of an intraparenchymal endophytic tumor can potentially be disturbed by natural heat sinks, including the blood and urine . Actually, wright et al reported two cases of treatment failure, both in endophytic tumors (i.e., completely intrarenal lesions), among 35 lrc lesions during an 18-month follow - up . In the multivariate analysis in their study, in contrast to this, hurby et al reported the successful treatment by lrc in all 11 patients with tumors located within 5 mm of the renal vasculature during 11.3 months of follow - up . Considering the technical difficulty of lpn in endophytic and hilar lesions, nisbet et al formulated an algorithm on the basis of published series outcomes that recommended lrc for endophytic and lpn for exophytic and mesophytic lesions . Our results provide additional evidence for the efficacy of lrc for the treatment of endophytic tumors . With intermediate - term follow - up, whereas 1 among 15 rcc lesions was managed improperly with lrc, the other lesions have remained free of recurrence or metastasis . Although we studied a small number of patients, the treatment failure rate of 6.7% in our series agrees with the positive margin rate of 1 - 7.4% in partial nephrectomy . This result provides us with technical clues for successful ablation in cases of endophytic lesions . We usually planned the position of the cryoprobe tip on the basis of the preoperative imaging, including ct or mri . In our series, the skin insertion site of each probe, the depth of insertion of the cryoprobe into the tumor in the operative filed, and the number of cryoprobes were decided on preoperatively on the basis of the shape, location, and diameter of the tumor . The ious image is also valuable, especially in guiding the location of the cryoprobe for endophytic tumors, because laparoscopic visual cues alone do not permit precise localization of cryoprobe tips, leaving ious as the only mode of real - time imaging . However, the image during freezing is distorted by several factors, including artifacts at the leading edge of the ice ball and inability to monitor the periphery of the mass, as noted by badger et al . In our lrc experience with endophytic tumors, the capacity of ious to monitor ice ball expansion is limited, especially for endophytic intraparenchymal tumors, because the collecting system itself behaves as an anechoic lesion . To inflict lethal freezing injury throughout the tumor volume while sparing normal healthy tissues, the targeted lesion should be located within the lethal area of cryoablation, and a temperature below -40 is usually required to effectively destroy malignant renal tissue . Unfortunately, no tool currently available can precisely define the three - dimensional configuration of this critical isothermal surface during cryoablation . Thus, we surmise that the use of multiple cryoprobes is more practical than focusing on the accuracy of the cryoprobe itself for conducting lrc for an endophytic lesion . Hypothetically, this approach might increase freezing efficiency by extending the coldest isothermal line, as compared with a single probe, and the distribution of the probes across the tumor might also compensate for an asymmetric tumor shape . Particularly for an intraparenchymal tumor, the use of multiple cryoprobes may have the additional advantage of evading the natural heat sink effect, a barrier intrinsic in low - temperature - based ablation . One challenge a surgeon faces in a partial nephrectomy, whether open or laparoscopic, on a patient with an endophytic renal tumor is reconstruction of the collecting system . Cryoablation averts this challenge, but not the risk of injury to the collecting system, with resultant urine leakage, particularly for endophytic and deep lesions . However, recent reports on renal cryoablation support the tolerance of the collecting system to the radiographic ice ball formed in the procedure . Targeted renal pelvic cryoablation resulted in no urinary extravasations from a total of 15 lesions in a swine model . In a clinical setting, six patients with intraparenchymal tumors who were treated by percutaneous cryoablation revealed no clinical evidence of ureteral sequelae . In a comparison of 11 lrc and 12 lpn procedures for hilar tumors that were defined as located within 5 mm of the renal vasculature, the lrc group experienced no complications, whereas the complication rate of the lpn group was 50%, with urine leakage being the most significant complication . Similarly, in our series of 14 patients with endophytic tumors, none showed clinical or radiological evidence of collecting system injury during the minimum follow - up of 28 months . These studies together support the safety of the cryoablative ice ball for the underlying structure of the kidney . We still recognize several limitation of this series, including this being the experience of a single institution for a relatively small number of cases . The noncomparative, retrospective study design may also limit the application of our findings . Obviously, to establish the effectiveness of a new technique, prospective studies must be conducted to compare the new technique with conventional methods . However, it deserves notice that our patient criteria for lrc included high risk for anesthesia with severe co - morbidity or relatively old age, leaving lrc as the last surgical treatment option . Still, the application of cryoablation to renal tumors is currently in the investigational stage, despite the promising reported clinical outcomes . Therefore, although our data showed the intermediate - term oncologic efficacy of lrc, longer follow - up with larger scale series is still needed before ablative technologies can be established as a valid alternative option for the treatment of renal tumors and before their indication can be expanded to endophytic renal tumors . In this series with an intermediate - term follow - up, lrc for small endophytic tumors showed acceptable oncological and surgical outcomes without adverse effects in the collecting system . These results also emphasize that careful preoperative design of the layout of the cryoprobes and the use of multiple cryoprobes should be given special consideration, especially in endophytic cases . However, further studies with longer term follow - up and larger patient groups are needed to establish the position of ablative technologies in the treatment of renal tumors and to extend their application to renal tumors in endophytic locations.
Doxorubicin (dox) is an important component of multimodality therapy for several antineoplastic combined chemotherapy protocols . It is used to treat a variety of malignacies such as leukemias, hodgkin and non - hodgkin lymphoma, and solid tumors (1). However, despite high efficacy, the major side effect of dox is cardio - toxicity, which has dramatically hindered its clinical usage for a prolonged period of time . The mechanisms of dox - induced cardio toxicity are not completely understood, but most evidences indicate the generation of reactive oxygen species (ros) involvement (2). Interestingly, some natural foods have been reported to contain substantial amounts of antioxidants and free radical scavenging agents . These compounds diminish some side effects of chemotherapeutic agents on normal cells by reducing their genotoxicity (2). There are many evidences showing the protective effects for different herbs against oxidative injury - related cardio toxicity, and among which n. sativa, zingiber officinale and glycyrrhiza glabra showed the highest protective effect . Its seeds have long been used in traditional medicine for a wide range of disorders including bronchial asthma, headache, infections, obesity, back pain, hypertension and gastrointestinal problems (4). These effects are mediated via different mechanism such as antioxidant (5), anti - inflammatory (6), anticancer (7), and antihistaminic effects (8). It has been shown that n. sativa attenuates isoproterenol induced myocardial infarction (mi) (9). Also, n. sativa oil supplementation reduces lead - induced cardio - toxicity by mechanisms related to its ability to decrease the pro - inflammatory cytokines; oxidative stress and cardiac tissue damage, and preserve the activity of antioxidant enzymes (10). Ebru et al (2008) showed that pretreatment with n. sativa oil decreased the subsequent cyclosporine - a injury in rat heart (11), recent studies revealed a protective effect of thymoquinone against doxorubicin - induced cardio toxicity (12 - 14). It is consumed worldwide as a spice and flavoring agent, and is attributed to have many medicinal properties (15). It is used in traditional medicine as carminative, and antipyretic, and in the treatment of pain, rheumatism and bronchitis (15). It has different pharmacological activities such as hepatoprotective (16), antiparasitic (17), antimalarial (18), antimicrobial (19), antidiabetic (20), and radioprotective effects (21). Ginger also is a potential remedy for cardiovascular diseases (22), and can prevent the development of morphine analgesic tolerance and physical dependence in rats (23). It is also used for the treatment of gastrointestinal disorders including gastric ulcerogenesis (24). The plant contains high level of phenolic and flavonoid compounds, responsible for its high antioxidant activities (25). G. glabra (licorice) is used widely as a flavoring and sweetening agent in tobacco products, chewing gum, candy, toothpaste and beverages . The therapeutic potential of g. glabra as a hypoglycemic (27), hypocholesteremic (28), anti - ulcer (29), anti - inflammatory (30), renoprotective (31), and antiatherogenic (32) have been demonstrated . In traditional medicine, it is useful in the treatment of agent renovascular and cardiovascular diseases, and hence constitutes as the major ingredient of polyherbal formulations indicated for cardiovascular diseases (31). The investigations have shown that g. glabra has protective effects against ischemic damages of the several body organs due to its potent antioxidant and free radical scavenging activity (27, 28, 30 - 32). Also, a recent study has shown that pretreatment with g. glabra significantly attenuates ischemic reperfusion induced myocardial injury by improving antioxidant status in heart (33). In addition, other species of g. uralensis, suppressed doxorubicin - induced apoptosis in h9c2, rat cardiac myoblast cells (34). Treatment with licorice extract significantly protected the mice against dox - induced cardio toxicity (35), and improved cardiac performance (36). Based on these findings, we hypothesized that the combination of these medicinal plants may produce a higher protective effect against doxorubicin - induced cardiomyopathy . Therefore, the present study was designed to investigate the potential effects of nigella sativa with glycyrrhiza glabra and zingiber officinale (ngz) extracts on cell viability, lipid peroxidation level, ros content and apoptotic induction in h9c2 cardiomyocytes . 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (mtt), thiobarbituric acid (tba), 2,7-dichlorofluorescin diacetate (dcfh - da), propidium iodide (pi), sodium citrate and triton x-100 were purchased from sigma (st . Louis, mo, usa). High glucose dulbecco s modified eagles medium (dmem), penicillin - streptomycin and fetal bovine serum were purchased from gibco . Trichloroacetic acid (tca) and malondialdehyde bis-(dimethyl acetal) (mda) were obtained from merck (darmstadt, germany). N. sativa seeds were collected from gonabad region (northeast of iran) and authenticated by herbarium of the ferdowsi university of mashhad (fumh) (voucher specimen = 293 - 0303 - 1). G. glabra rhizomes was purchased from local market and authenticated by fumh (voucher specimen number = 25947). Whereas z. officinale is not native to iran, afforded from a local herb market in mashhad and confirmed by plant specialist of fumh . The seeds of n. sativa were washed, dried, and crushed to a powder with an electric micronizer . The g. glabra and z. officinale rhizomes were peeled chopped into tiny bits, air - dried and were ground with a mechanical grinder . Each herbal sample was extracted separately in a soxhlet extractor with ethanol (70%) and the resulting extract was dried and kept at -20c until use . The cells were cultured in dulbecco s modified eagles medium (dmem) supplemented with 10% fetal bovine serum, 100 units / ml penicillin and 100 g / ml streptomycin . For the experiments, they were seeded in 96-well and 24-well culture plates for mtt / ros and mda assays, respectively . For apoptosis assay, cells were seeded at 100,000 cell / well in a 24-well plate . The cells were pretreated with each herbal extract alone (6 to 200 g / ml) for 4 hr and then incubation was continued in the presence of the herbal extract with or without 5 m doxorubicin for 24 hr . For assaying the effect of three extract in combination, the minimum effective concentration of each extract was chosen (n=12.5, g=25 and z=25 g / ml sum=62.5 g / ml) as the basis and one (62.5 g / ml), two (125 g / ml), four (250 g / ml) and eight (500 g / ml) folds of this concentration were applied . Cell viability was determined using a modified mtt assay as described previously (37). Briefly, mtt solution in phosphate - buffered saline (5 mg / ml) was added to each well at final concentration of 0.05% . After 3 hr, the formazan precipitate was dissolved in dmso . The absorbance at 570 and 620 nm (background) was measured using a statfax303 plate reader . The level of lipid peroxidation was estimated by measuring mda, which is the end product of lipid peroxidation (38). At the end of incubation, then, 400 l of tca (15%) and 800 l of tba (0.7%) were added to 500 l of cell samples . The mixture was vortexed and heated for 40 min in a boiling water bath . Then, 200 l of the sample was transferred to 96-well plate and the fluorescence intensity was read with excitation / emission of 480/530 nm . Intracellular ros level was evaluated using a fluorescent probe, dcf - da . At the end of incubation, the cells were treated (30 min) with dcfh - da (10 m) at 4c in the dark . Apoptotic cells were detected using pi staining of small dna fragments followed by flow cytometry . It has been reported that a sub - g1 peak that is reflective of dna fragmentation can be observed following the incubation of cells in a hypotonic phosphate - citrate buffer containing a quantitative dna - binding dye such as pi . Apoptotic cells that have lost dna will take up less stain and appear on the left side of the g1 peak in the histogram . Briefly, h9c2 cells were seeded in wells of a 24-well plate and treated according to mentioned protocol . Floating and adherent cells were then harvested and incubated at 4 c overnight in the dark with 750 l of a hypotonic buffer (50 g / ml pi in 0.1% sodium citrate with 0.1% triton x-100). Next, flow cytometry was carried out using a facscan flow cytometer (becton dickinson). Statistical analysis was performed using one way analysis of variance (anova) followed by tamhane s t2 post - hoc test . 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (mtt), thiobarbituric acid (tba), 2,7-dichlorofluorescin diacetate (dcfh - da), propidium iodide (pi), sodium citrate and triton x-100 were purchased from sigma (st . Louis, mo, usa). High glucose dulbecco s modified eagles medium (dmem), penicillin - streptomycin and fetal bovine serum were purchased from gibco . Trichloroacetic acid (tca) and malondialdehyde bis-(dimethyl acetal) (mda) were obtained from merck (darmstadt, germany). N. sativa seeds were collected from gonabad region (northeast of iran) and authenticated by herbarium of the ferdowsi university of mashhad (fumh) (voucher specimen = 293 - 0303 - 1). G. glabra rhizomes was purchased from local market and authenticated by fumh (voucher specimen number = 25947). Whereas z. officinale is not native to iran, afforded from a local herb market in mashhad and confirmed by plant specialist of fumh . The seeds of n. sativa were washed, dried, and crushed to a powder with an electric micronizer . The g. glabra and z. officinale rhizomes were peeled chopped into tiny bits, air - dried and were ground with a mechanical grinder . Each herbal sample was extracted separately in a soxhlet extractor with ethanol (70%) and the resulting extract was dried and kept at -20c until use . The cells were cultured in dulbecco s modified eagles medium (dmem) supplemented with 10% fetal bovine serum, 100 units / ml penicillin and 100 g / ml streptomycin . For the experiments, they were seeded in 96-well and 24-well culture plates for mtt / ros and mda assays, respectively . For apoptosis assay, the cells were pretreated with each herbal extract alone (6 to 200 g / ml) for 4 hr and then incubation was continued in the presence of the herbal extract with or without 5 m doxorubicin for 24 hr . For assaying the effect of three extract in combination, the minimum effective concentration of each extract was chosen (n=12.5, g=25 and z=25 g / ml sum=62.5 g / ml) as the basis and one (62.5 g / ml), two (125 g / ml), four (250 g / ml) and eight (500 g / ml) folds of this concentration were applied . Cell viability was determined using a modified mtt assay as described previously (37). Briefly, mtt solution in phosphate - buffered saline (5 mg / ml) was added to each well at final concentration of 0.05% . After 3 hr, the formazan precipitate was dissolved in dmso . The absorbance at 570 and 620 nm (background) was measured using a statfax303 plate reader . The level of lipid peroxidation was estimated by measuring mda, which is the end product of lipid peroxidation (38). At the end of incubation, then, 400 l of tca (15%) and 800 l of tba (0.7%) were added to 500 l of cell samples . The mixture was vortexed and heated for 40 min in a boiling water bath . Then, 200 l of the sample was transferred to 96-well plate and the fluorescence intensity was read with excitation / emission of 480/530 nm . Intracellular ros level was evaluated using a fluorescent probe, dcf - da . At the end of incubation, the cells were treated (30 min) with dcfh - da (10 m) at 4c in the dark . Apoptotic cells were detected using pi staining of small dna fragments followed by flow cytometry . It has been reported that a sub - g1 peak that is reflective of dna fragmentation can be observed following the incubation of cells in a hypotonic phosphate - citrate buffer containing a quantitative dna - binding dye such as pi . Apoptotic cells that have lost dna will take up less stain and appear on the left side of the g1 peak in the histogram . Briefly, h9c2 cells were seeded in wells of a 24-well plate and treated according to mentioned protocol . Floating and adherent cells were then harvested and incubated at 4 c overnight in the dark with 750 l of a hypotonic buffer (50 g / ml pi in 0.1% sodium citrate with 0.1% triton x-100). Next, flow cytometry was carried out using a facscan flow cytometer (becton dickinson). Statistical analysis was performed using one way analysis of variance (anova) followed by tamhane s t2 post - hoc test . Incubation with doxorubicin significantly decreased cell viability to 622% compared to control of control (p<0.001). Cell viability was assayed to determine the optimum concentrations necessary for the three extracts (n, g and z) to protect h9c2 cells against dox - induced cytotoxicity . The results demonstrated that, in comparison with dox, n. sativa increased cell viability at doses of 25 to 200 g / ml dose dependently that maximum effect was produced at effect at concentration of 50 g / ml (75.40.84, p<0.01), and z. officinale increased cell viability at doses of 50 to 200 g / ml, its maximum effect was at dose of 100 g / ml (752.5, p<0.01), while g. glabra increased cell viability at dose of 100 g / ml (73.51.5, p<0.05) (figure 1a). Effect of extracts alone (figure 1a) or ngz (figure 1b) on percentage of protection against dox - induced cytotoxicity in h9c2 cells . Cells were pretreated with different concentrations of ngz for 4 hr before exposure to 5 m of dox for 24 hr . Dox decreased cell viability to 62.22 p<0.001 versus control. p<0.05, * p<0.01 and * * * p<0.001 versus dox results of experiment with combination extract (ngz) showed that all doses (62.5, 125, 250 and 500 g / ml) protected h9c2 cells against dox and increased cell viability significantly . The best protection was obtained at dose of 250 g / ml (90.291.2, p<0.001, 70% protection). The results showed that dox significantly increased mda (p<0.001) and ros (p<0.001) levels in h9c2 compared with control cells . In the presence of dox, treatment with 125 g / ml (p<0.05), 250 g / ml (p<0.001) and 500 g / ml (p<0.01) doses of ngz significantly reduced the level of mda . The most reduction of mda was observed at the dose of 250 g / ml (control 1001.4, dox 2357.9 and 250 g / ml ngz 1195.18). Also this combination diminished significantly ros production in comparison with dox, at dose of 125 g / ml (p<0.01), 250 g / ml (p<0.001) and 500 g / ml (p<0.01) (figures 2a and 2b, respectively). The lowest production of ros was at the dose of 250 g / ml (control 274, dox 1208 and 250 g / ml ngz 454). Effect of ngz on dox - induced mda production (figure 2a) and reactive oxygen species (ros) generation (figure 2b) in h9c2 cells . Cells were pretreated with different concentrations of ngz for 4 hr before exposure to 5 m of dox for 24 hr . Data are expressed as mean sem of three separate experiments p<0.001 versus control, * p<0.05, * * p<0.01 and * * * p<0.001 versus dox apoptosis in h9c2 cell line was detected with flow cytometry using pi staining . Cells were pretreated for 4 hr with various concentrations of the ngz and exposed to dox for 24 hr . Analysis of the subg1 peak in flow cytometry histograms revealed the induction of apoptosis in cells treated with dox (p<0.001). Ngz decreased apoptotic induction significantly at the doses of 62.5 g / ml (p<0.05), 125 g / ml (p<0.01), 250 g / ml (p<0.001) and 500 g / ml (p<0.01) (figure 3). The most reduction of apoptotic rate was at the dose of 250 g / ml (control 121.4, dox 714 and 250 g / ml ngz 37.52.5) the effects of the ngz on apoptosis in h9c2 cells using pi staining and flow cytometry . P<0.001 versus control, * p<0.05, * * p<0.01 and * * * p<0.001 versus dox incubation with doxorubicin significantly decreased cell viability to 622% compared to control of control (p<0.001). Cell viability was assayed to determine the optimum concentrations necessary for the three extracts (n, g and z) to protect h9c2 cells against dox - induced cytotoxicity . The results demonstrated that, in comparison with dox, n. sativa increased cell viability at doses of 25 to 200 g / ml dose dependently that maximum effect was produced at effect at concentration of 50 g / ml (75.40.84, p<0.01), and z. officinale increased cell viability at doses of 50 to 200 g / ml, its maximum effect was at dose of 100 g / ml (752.5, p<0.01), while g. glabra increased cell viability at dose of 100 g / ml (73.51.5, p<0.05) (figure 1a). Effect of extracts alone (figure 1a) or ngz (figure 1b) on percentage of protection against dox - induced cytotoxicity in h9c2 cells . Cells were pretreated with different concentrations of ngz for 4 hr before exposure to 5 m of dox for 24 hr . Dox decreased cell viability to 62.22 p<0.001 versus control. p<0.05, * p<0.01 and * * * p<0.001 versus dox results of experiment with combination extract (ngz) showed that all doses (62.5, 125, 250 and 500 g / ml) protected h9c2 cells against dox and increased cell viability significantly . The best protection was obtained at dose of 250 g / ml (90.291.2, p<0.001, 70% protection). The results showed that dox significantly increased mda (p<0.001) and ros (p<0.001) levels in h9c2 compared with control cells . In the presence of dox, treatment with 125 g / ml (p<0.05), 250 g / ml (p<0.001) and 500 g / ml (p<0.01) doses of ngz significantly reduced the level of mda . The most reduction of mda was observed at the dose of 250 g / ml (control 1001.4, dox 2357.9 and 250 g / ml ngz 1195.18). Also this combination diminished significantly ros production in comparison with dox, at dose of 125 g / ml (p<0.01), 250 g / ml (p<0.001) and 500 g / ml (p<0.01) (figures 2a and 2b, respectively). The lowest production of ros was at the dose of 250 g / ml (control 274, dox 1208 and 250 g / ml ngz 454). Effect of ngz on dox - induced mda production (figure 2a) and reactive oxygen species (ros) generation (figure 2b) in h9c2 cells . Cells were pretreated with different concentrations of ngz for 4 hr before exposure to 5 m of dox for 24 hr . Data are expressed as mean sem of three separate experiments p<0.001 versus control, * p<0.05, * * p<0.01 and * * * p<0.001 versus dox cells were pretreated for 4 hr with various concentrations of the ngz and exposed to dox for 24 hr . Analysis of the subg1 peak in flow cytometry histograms revealed the induction of apoptosis in cells treated with dox (p<0.001). Ngz decreased apoptotic induction significantly at the doses of 62.5 g / ml (p<0.05), 125 g / ml (p<0.01), 250 g / ml (p<0.001) and 500 g / ml (p<0.01) (figure 3). The most reduction of apoptotic rate was at the dose of 250 g / ml (control 121.4, dox 714 and 250 g / ml ngz 37.52.5) the effects of the ngz on apoptosis in h9c2 cells using pi staining and flow cytometry . P<0.001 versus control, * p<0.05, * * p<0.01 and * * * p<0.001 versus dox cardiovascular risk factors enhance the production of ros generated by mitochondrial electron - transport chain, xanthine oxidase, nadph oxidase and uncoupled nitric oxide synthases in cardiomyocytes (39). Oxidative stress occurs when production of ros exceeds the capacity of antioxidant defense systems (catalase, sod and glutathione peroxidase) (40). Because of its deleterious effects weak antioxidant capacity in cardiomyocytes may be a risk factor responsible for their high sensitivity to oxidative damage (42), and a promising approach to cardioprotection is the use of pharmacological tools to reduce oxidative stress in the heart (37). In the present study, we used h9c2 cells as a pharmacological model to evaluate the potential cardioprotective effects of three different prevalent medicinal plants, n. sativa, glycyrrhizin glabra and zingiber officinale alone and in combination with all three (ngz). The results showed that the combination of ngz improves their protective effects against dox - induced oxidative stress in h9c2 cells . H9c2 cells are morphologically similar to immature embryonic cardiomyocytes . Considering that these cells preserve electrical and hormonal signal pathways found in adult cardiac cells (43), they are a useful model for studying oxidative stress - induced cardiomyocyte damage (44). In this model, dox significantly increased the level of ros and lipid peroxidation and induced the rate of apoptosis . These changes are similar to the dox - induced deleterious effects on normal cardiac cells, which lead to the loss of cardiomyocytes viability (2). In this study, pretreatment with each of the three herb extracts alone produced some degree of protection in a concentration dependent manner . These findings are in accordance with previous studies which show the capability of cadioprotective effects of n. sativa, g. glabra and z. officinale (10 - 14). In mice, thymoquinone, the pharmacologically active component of n. sativa, reduces dox - induced cardio toxicity without reducing the serum concentration of dox (12). Also, studies have shown that thymoquinone potentiates cisplatin antitumor activity and protects against cisplatin - induced nephrotoxicity in mice and rats (45). A recent investigation has shown that n. sativa oil supplementation attenuates lead - induced cardio toxicity by mechanisms related to its ability to decrease the pro - inflammatory cytokines; oxidative stress and cardiac tissue damage, and preserve the activity of antioxidant enzymes (10). Ebru et al (2008) showed that pretreatment with n. sativa oil decreased cyclosporine - a injury in rat heart (11), and later studies revealed a protective effect of thymoquinone against doxorubicin - induced cardio toxicity (12 - 14). Recently, it was shown that gingerol protected cardiomyocytes against dox - induced toxicity through its antioxidative effect and modulation of nf-b as well as apoptosis (46). Glycyrrhizin, the chief sweet - tasting constituent of g. glabra (liquorice) root and its metabolites (glycyrrhizic acid or glycyrrhizinic acid) induce apoptosis and cell growth inhibition in various cancer cells, including human stomach cancer cells, promyelotic leukemia hl-60 cells and hepatoma cells (47, 48). Moreover, experimental results showed that the combination of antibiotic anti - cancer drug with 18-glycyrrhetinic acid (derivative of the beta - amyrin type obtained from the hydrolysis of glycyrrhizic acid) exhibits a synergistic toxic effect on cancer cell lines (49). The extract of glycyrrhiza uralensis suppresses doxorubicin - induced apoptosis in h9c2 rat cardiac myoblasts (34). Recent experimental observations reported that z. officinale is an effective anticancer agent (50). Also, in a polyherbal preparation, ginger was one of the components and reported to be effective against dox - induced cardio toxicity without interfering its antineoplastic activity (51). Thus, it is of great interest to investigate the effect of combination of these extracts on dox - induced disorders . In this study, the combination of extracts (ngz) augmented their protective effects, suggesting the presence of a positive interaction between components . In this experiment, however, the maximum protective effect was achieved with combination of the threshold concentrations of the extracts and the protection increased in concentration dependent manner to a maximum protection of more than 2.5 fold . These observations do not show an additive effect, but implicates the presence of a positive interaction between extracts against dox - induced apoptosis in h9c2 cells . In high concentrations, n. sativa did not produce any further protective effect, while the effects of the two other extracts were decreased in high concentrations (figure 1a). The decreasing effect of g. glabra or z. officinale at concentration of 200 g / ml could not be explained by a non - specific effect such as ionic disturbances . However, at higher concentration (500 g / ml) in mixed extracts, the effect was reduced . This may suggest the presence of some components in crude extracts, which may have toxic effect and reduce the protection . The molecular mechanisms for protective effect of each extract and augmenting effect of their combination (ngz) are not clear . However, it has been shown that glycyrrhizate could ameliorate rabbit myocardial ischemia - reperfusion injury through p38mapk pathway (52). Also, treatment with glycyrrhiza uralensis extract significantly protected mice from dox - induced cardio toxicity, by decreasing levels of serum ldh and creatin kinase - mb improving heart morphology and increasing gsh - p(x) activity and gsh level (35). Also, results revealed that glycyrrhizin and glycyrrhetinic acid directly affected cardiac performance (36). In this paper, we showed that the protective effects may be mediated by reducing oxidative stress, which could decrease the production of ros, lipid peroxidation and diminished apoptotic induction . It is interesting that in comparison with the effect of each extract alone, the combination produced a stronger protective effect . Therefore, it can be concluded that cardioprotective effect of ngz is mediated at least in part by its active ingredients . However, further investigations are needed to reveal molecular mechanisms to increase the possible positive interactions and diminish negative interactions in combination . These must be included in fractionation of the extracts to assess the effect of each fraction in combination . These may lead to find purified active ingredients, which could be used in a clinical trial . It was shown that all of three extract produced protective effect on h9c2 cell against dox - induced toxicity with different potencies, but similar efficacy with a maximum effect of about 30 percent . The effects of extracts in combination showed a positive interaction between components of different extracts, which augments their protective effect . These observations could suggest new experiments that can be performed to test the roles of each extract components in the combination . These may lead to the development of a new class of drugs to prevent cardio toxicity of dox and improve its anticancer effect.
Rapidly increasing rates of diabetes mellitus (dm) throughout the developed world represent an emerging epidemic with profound consequences . Approximately 30% of patients with dm develop chronic kidney disease (ckd), accounting for 45% of end - stage renal disease (esrd) cases in the u.s . Racial / ethnic minority populations are disproportionately affected by dm and its complications (2). The 2012 united states renal data system report showed the incidence of new esrd cases in african americans (aas) and hispanics was, respectively, 3.4 and 1.8 times higher than whites (1). Further, the incidence of esrd attributed to dm or hypertension was found to be 12 times higher among aas compared with whites in a 12-year follow - up cohort study of 9,082 aa and white adults between 30 and 74 years of age (3). However, in the general population, the prevalence of early ckd was not found to be elevated in minorities, suggesting a racial / ethnic difference in ckd progression (4). Although the etiologic causes for the increased rate of progression of ckd- and dm - related esrd in aas and hispanics compared with whites remains unclear, it appears to involve both sociocultural and biologic factors (5). Inflammation plays a central role in the pathogenesis of many renal diseases (6,7). Studies suggest that inflammatory mechanisms significantly contribute to the development and progression of ckd (8,9). Epidemiological studies have shown that c - reactive protein (crp), the most extensively studied inflammatory marker, is associated with microalbuminuria in diabetic patients and in the general population (1013). Inflammation has been reported in the early stages of ckd (estimated glomerular filtration rate [egfr]> 90 ml / min/1.73 m and microalbuminuria) in patients with type 1 dm (14,15). Despite the knowledge that aas and hispanics are disproportionately affected by ckd, and the potential role of inflammation in the pathophysiology of early ckd, little is known about the potential contribution that inflammation may play in racial / ethnic disparities when the egfr is still preserved . We postulated that in contrast to the general population, the prevalence of early ckd in patients with primarily type 2 dm would be higher in aas and hispanics than in whites . In the current study, we tested this hypothesis in the national health and nutrition examination survey (nhanes) 19992008 and speculated that elevated levels of systemic inflammatory markers in general, and crp in particular, may be associated with the posited increase in early ckd (egfr 60 ml / min/1.73 m and urinary albumin excretion [uae] 30 g / ml) in racial / ethnic minorities . The study included participants of the nhanes 19992008, conducted by the national center for health statistics (nchs) of the centers for disease control and prevention (cdc). Nhanes used a highly stratified multistage probability sampling (total n = 51,623) and used oversampling of the elderly (n = 7,094), non - hispanic blacks (n = 7,765), and hispanics (n = 8,688). Descriptions of the survey, sampling procedures, and details of the laboratory tests evaluated can be found on the cdc website (http://www.cdc.gov/nchs/nhanes/nh3rrm.htm#refman). Analysis was limited to adults 20 years of age, because those <20 years of age did not have plasma glucose tested . (n = 117) and those with missing laboratory data (n = 335) were excluded . The total analytic sample was 2,310 adults with dm (early ckd [n = 693] and those with no ckd [n = 1,459]). Subjects were classified as aa (n = 647), hispanic (n = 799), white (n = 864), and asian / other (n = 91). (n = 2,038) or had a fasting plasma glucose (fpg) level 126 mg / dl (n = 1,508). Of the 2,310 participants with dm, 2,292 participants answered the question regarding the use of insulin, and of these, 462 participants said they take insulin . Of those, one participant said he began using insulin before 20 years of age, the majority being likely to have type 2 dm, although a few may have type 1 dm . Thus, 0.03% participants with dm (unadjusted) have type 1 dm and because of this low number, all subjects with dm were analyzed together . There was no difference in any of our analyses if the one participant <20 years of age was excluded . Data were analyzed for age, sex, race / ethnicity, history of hypertension, cardiovascular disease (cvd), smoking, use of metformin, pioglitazone, statin, ace inhibitors (aceis), and angiotensin receptor blockers (arbs), systolic and diastolic blood pressure (sbp and dbp), bmi, duration of dm, fpg, hemoglobin a1c (hba1c), serum albumin, total cholesterol, hdl cholesterol (hdl - c), triglyceride, inflammatory markers (crp, uric acid, fibrinogen, and white blood cell counts [wbcs]), uae, serum creatinine, and egfr . For categorical analyses, subjects were classified as obese / nonobese according to the bmi level using a cutoff of 30 kg / m . Similarly, based on the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure guidelines, a cutoff for hypertension of 140/90 mmhg was used . Cutoffs for total cholesterol 240 mg / dl and triglyceride 200 mg / dl were used . Crp was categorized according to the distribution of the variables into tertiles (<0.2 mg / dl, 0.200.56 mg / dl, and 0.57 mg / dl). Subjects were classified according to the egfr level and uae level as follows: subjects with egfr 60 ml / min/1.73 m and uae <30 g / ml were considered as having no ckd (n = 1,459). Subjects with egfr 60 ml / min/1.73 m and uae 30 g / ml were considered as having early ckd (n = 693). Prior to the use of select continuous variables in multiple linear regression analyses, the following variables were log transformed because their distribution was skewed: duration of dm and laboratory variables (uae, crp, total cholesterol, hdl - c, egfr, fpg, hba1c, uric acid, and wbc). Prevalent cvd was defined as a stated history of physician - diagnosed myocardial infarction or stroke, electrocardiographic evidence of myocardial infarction, or history of a revascularization procedure . Smoking status was defined as any participant who had smoked at least 400 cigarettes in their lifetime and was currently smoking at their baseline examination . Uric acid was classified into two groups using the cutoff point 7 mg / dl . Descriptive statistics were used to characterize the subjects (mean sd for continuous variables and weighted percentages for categorical variables). To test the statistical differences between the groups, test for categorical variables and two - sided student t tests for continuous variables were used, and p <0.05 was considered significant . Linear regression analysis (for the continuous outcome, uae) was used to determine the relationship between crp and the level of uae . Multiple linear regression models were used to adjust for confounding variables, and the adjusted regression coefficient, standard error, and p value were reported . The -coefficient represented the rate of change in the outcome variable (uae) brought about by the main independent variable (crp) and each of the other independent variables (confounders). Two sets of multiple linear regression models were examined . In the first set, crp was log transformed and used as the continuous variable . In the second set, crp was divided into tertiles (normal, mid-, and highest crp tertiles) representing normal, moderate, and elevated crp levels . Variables considered as confounders in the multivariate analysis were age, sex, bmi, duration of dm, smoking status, sbp and dbp, use of medications (oral hypoglycemic, statin, and acei or arb), total cholesterol, hdl - c, triglyceride, hba1c, fpg, comorbidity (hypertension and cvd), and additional postulated inflammatory markers (uric acid and wbc). The effect of each of the confounding variables was tested on the change of the coefficient of the linear regression of uae and crp . Significant variables in the linear regression were included in the final multivariate model as follows: model 1, adjusted for social variables (age, sex, race / ethnicity, bmi, duration of dm, and smoking); model 2, adjusted for variables as in model 1 plus log - transformed laboratory variables (total cholesterol, hdl - c, fpg, hba1c, uric acid, and wbc); model 3, adjusted for variables as in model 2 plus medication use (statin and acei) and comorbidity (hypertension and cvd). Data for the unadjusted as well as the adjusted models were reported for the total sample as well as by race / ethnicity . Multiple logistic regression was used to determine the association between race / ethnicity and both crp (0.2 mg / dl) and uae (30 g / ml), adjusting for the previously mentioned confounding variables . Data were presented as adjusted odds ratio (aor) and 95% ci for crp (0.2 mg / dl) and uae (30 g / ml), comparing aas and hispanics to whites (as reference). The data were analyzed using sas and the survey module of stata (release 10, 19842007 statistics / data analysis; statacorp, college station, tx). Sample weights (a value assigned to each case in the data file so that the statistics computed from the data were more representative of the population) provided by the nchs were used to correct for differential selection probabilities (i.e., oversampling of elderly and minorities) and to adjust for noncoverage (i.e., inadequacies in the sampling frame resulting from omissions of some housing units in the listing of area segments and omissions of people with no fixed address) and nonresponse . For the subpopulation analysis (i.e., data for each race / ethnic group),, several replacement approaches were used where 1) the single unit contributed nothing to the standard error, 2) it was replaced with the average of the variance, or 3) it was centered at the grand population mean . The study included participants of the nhanes 19992008, conducted by the national center for health statistics (nchs) of the centers for disease control and prevention (cdc). Nhanes used a highly stratified multistage probability sampling (total n = 51,623) and used oversampling of the elderly (n = 7,094), non - hispanic blacks (n = 7,765), and hispanics (n = 8,688). Descriptions of the survey, sampling procedures, and details of the laboratory tests evaluated can be found on the cdc website (http://www.cdc.gov/nchs/nhanes/nh3rrm.htm#refman). Analysis was limited to adults 20 years of age, because those <20 years of age did not have plasma glucose tested . (n = 117) and those with missing laboratory data (n = 335) were excluded . The total analytic sample was 2,310 adults with dm (early ckd [n = 693] and those with no ckd [n = 1,459]). Subjects were classified as aa (n = 647), hispanic (n = 799), white (n = 864), and asian / other (n = 91). (n = 2,038) or had a fasting plasma glucose (fpg) level 126 mg / dl (n = 1,508). Of the 2,310 participants with dm, 2,292 participants answered the question regarding the use of insulin, and of these, 462 participants said they take insulin . Of those, one participant said he began using insulin before 20 years of age, the majority being likely to have type 2 dm, although a few may have type 1 dm . Thus, 0.03% participants with dm (unadjusted) have type 1 dm and because of this low number, all subjects with dm were analyzed together . There was no difference in any of our analyses if the one participant <20 years of age was excluded . Data were analyzed for age, sex, race / ethnicity, history of hypertension, cardiovascular disease (cvd), smoking, use of metformin, pioglitazone, statin, ace inhibitors (aceis), and angiotensin receptor blockers (arbs), systolic and diastolic blood pressure (sbp and dbp), bmi, duration of dm, fpg, hemoglobin a1c (hba1c), serum albumin, total cholesterol, hdl cholesterol (hdl - c), triglyceride, inflammatory markers (crp, uric acid, fibrinogen, and white blood cell counts [wbcs]), uae, serum creatinine, and egfr . For categorical analyses, subjects were classified as obese / nonobese according to the bmi level using a cutoff of 30 kg / m . Similarly, based on the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure guidelines, a cutoff for hypertension of 140/90 mmhg was used . Cutoffs for total cholesterol 240 mg / dl and triglyceride 200 mg / dl were used . Crp was categorized according to the distribution of the variables into tertiles (<0.2 mg / dl, 0.200.56 mg / dl, and 0.57 mg / dl). Subjects were classified according to the egfr level and uae level as follows: subjects with egfr 60 ml / min/1.73 m and uae <30 g / ml were considered as having no ckd (n = 1,459). Subjects with egfr 60 ml / min/1.73 m and uae 30 g / ml were considered as having early ckd (n = 693). Prior to the use of select continuous variables in multiple linear regression analyses, the following variables were log transformed because their distribution was skewed: duration of dm and laboratory variables (uae, crp, total cholesterol, hdl - c, egfr, fpg, hba1c, uric acid, and wbc). Prevalent cvd was defined as a stated history of physician - diagnosed myocardial infarction or stroke, electrocardiographic evidence of myocardial infarction, or history of a revascularization procedure . Smoking status was defined as any participant who had smoked at least 400 cigarettes in their lifetime and was currently smoking at their baseline examination . Uric acid was classified into two groups using the cutoff point 7 mg / dl . Descriptive statistics were used to characterize the subjects (mean sd for continuous variables and weighted percentages for categorical variables). To test the statistical differences between the groups, test for categorical variables and two - sided student t tests for continuous variables were used, and p <0.05 was considered significant . Linear regression analysis (for the continuous outcome, uae) multiple linear regression models were used to adjust for confounding variables, and the adjusted regression coefficient, standard error, and p value were reported . The -coefficient represented the rate of change in the outcome variable (uae) brought about by the main independent variable (crp) and each of the other independent variables (confounders). Two sets of multiple linear regression models were examined . In the first set, crp was log transformed and used as the continuous variable . In the second set, crp was divided into tertiles (normal, mid-, and highest crp tertiles) representing normal, moderate, and elevated crp levels . Variables considered as confounders in the multivariate analysis were age, sex, bmi, duration of dm, smoking status, sbp and dbp, use of medications (oral hypoglycemic, statin, and acei or arb), total cholesterol, hdl - c, triglyceride, hba1c, fpg, comorbidity (hypertension and cvd), and additional postulated inflammatory markers (uric acid and wbc). The effect of each of the confounding variables was tested on the change of the coefficient of the linear regression of uae and crp . Significant variables in the linear regression were included in the final multivariate model as follows: model 1, adjusted for social variables (age, sex, race / ethnicity, bmi, duration of dm, and smoking); model 2, adjusted for variables as in model 1 plus log - transformed laboratory variables (total cholesterol, hdl - c, fpg, hba1c, uric acid, and wbc); model 3, adjusted for variables as in model 2 plus medication use (statin and acei) and comorbidity (hypertension and cvd). Data for the unadjusted as well as the adjusted models were reported for the total sample as well as by race / ethnicity . Multiple logistic regression was used to determine the association between race / ethnicity and both crp (0.2 mg / dl) and uae (30 g / ml), adjusting for the previously mentioned confounding variables . Data were presented as adjusted odds ratio (aor) and 95% ci for crp (0.2 mg / dl) and uae (30 g / ml), comparing aas and hispanics to whites (as reference). The data were analyzed using sas and the survey module of stata (release 10, 19842007 statistics / data analysis; statacorp, college station, tx). Sample weights (a value assigned to each case in the data file so that the statistics computed from the data were more representative of the population) provided by the nchs were used to correct for differential selection probabilities (i.e., oversampling of elderly and minorities) and to adjust for noncoverage (i.e., inadequacies in the sampling frame resulting from omissions of some housing units in the listing of area segments and omissions of people with no fixed address) and nonresponse . For the subpopulation analysis (i.e., data for each race / ethnic group), there were multiple sampling units with a single observation . To accommodate for this, several replacement approaches were used where 1) the single unit contributed nothing to the standard error, 2) it was replaced with the average of the variance, or 3) it was centered at the grand population mean . Of the 2,310 subjects with primarily type 2 dm and complete data, 864 were white, 647 were aa, and 799 were hispanic (table 1). Overall, 65% had hypertension, 19% had prevalent cvd, and 27% were smokers . Collectively, 30% used metformin and 6% used pioglitazone as the hypoglycemic agent, and their use did not differ between racial / ethnic groups . Statin and acei therapies were used by 29 and 31% of the patients, respectively, and only hispanic patients had significantly less (p <0.01) use of these medications compared with whites . Arbs were used by 6% of patients, and their use among all ethnic groups did not differ . Aas had higher levels of sbp, dbp, bmi, hba1c, and hdl - c, but lower serum triglyceride levels, compared with whites and hispanics (p <0.01). Relative to white subjects, hispanics had higher levels of total cholesterol, fpg, triglycerides, and dm duration and lower bmi (p <0.05). Relative to whites and aas, hispanics had a lower prevalence of cvd, uric acid, and history of smoking (p <0.01). Aas had higher crp and fibrinogen levels (p <0.01 and p <0.05, respectively) and lower wbc levels (p <0.01) relative to whites and hispanics . Importantly, aas had higher serum creatinine compared with hispanics or whites, but both aas and hispanics had higher egfr (which is adjusted for age and sex) compared with whites (p <0.01). In addition, aas and hispanics had higher uae relative to whites (p <0.01). Of the study population with early ckd (n = 693), 59% were white, 21% were aa, and 20% were hispanic . Aas (36%) and hispanics (34%) had a higher prevalence of early ckd relative to whites (26%, p <0.001) and the total population (29%). Characteristics of study participants by race / ethnicity table 2 shows the population characteristics by absence or presence of albuminuria defined as uae <30 g / ml or uae 30 g / ml, respectively . Of the 2,152 subjects, 693 had uae 30 g / ml . Patients with uae 30 g / ml were further stratified into macro- (uae 300 g / ml) and microalbuminuria (uae = 30299 g / ml) in order to assess the relationship between these levels of albuminuria and the independent variables . There were statistically significant differences (p <0.05) between no ckd and early ckd groups in all the variables except smoking, use of dm medications, statin, and arb, hdl, triglycerides, and egfr . Diabetic participants with early ckd had a significantly higher history of hypertension, prevalent cvd, use of acei, sbp, dbp, bmi, duration of dm, fpg, hba1c, crp, uric acid, fibrinogen, wbc, and serum creatinine compared with those with no ckd (p <0.01). Serum albumin levels were slightly lower in diabetic patients with uae 30 g / ml, relative to those with uae <30 g / ml (p <0.04). Demographic and clinical characteristics of diabetic patients with and without early ckd in the group divided into macro- and microalbuminuria, those with macroalbuminuria had statistically significant higher levels of sbp, hba1c, and uric acid and higher reported history of hypertension, but lower serum albumin (p <0.05), reflecting the relationship of some parameters consistent with more advanced disease . In addition, in relation to the entire cohort, microalbuminuria was present in 24% of white subjects, 27% of aas, and 27% of hispanics, whereas macroalbuminuria was present in only 3% of white subjects, 10% of aas, and 7% of hispanics (data not shown). In the adjusted logistic regression analyses (controlling for age, sex, bmi, smoking, dm duration, total cholesterol, hdl - c, fpg, hba1c, uric acid, wbc, statin or acei use, cvd, and hypertension), relative to whites, aas had higher adjusted odds of crp 0.2 mg / dl (aor 1.81, p = 0.007) and hispanics had higher odds of having uae 30 g / ml (aor 1.65, p = 0.03) (fig . Aor for crp (0.2 mg / dl) and uae (30 g / ml) among diabetic patients comparing aas and hispanics to whites (as reference). Crp and uae are adjusted for age, sex, bmi, smoking, dm duration, total cholesterol, hdl - c, fpg, hba1c, uric acid, wbc, medication use (statin and acei), cvd, and hypertension . * p = 0.03; * * p = 0.007 ., aor;, lower ci;, upper ci . In the first set of the multiple linear regression analyses, the crp data were transformed into log crp to achieve normal distribution and used in unadjusted and adjusted analyses with log uae for the total sample (n = 693) and for each racial / ethnic group . In the unadjusted model for the overall sample population, . However, in the adjusted model, crp was a strong predictor for uae for the total sample as well as for whites and hispanics (table 3a). Multiple linear regression models for the association between log - transformed uae and crp in patients with early ckd, n = 693 in the second set of the multiple linear regression models, crp was divided into tertiles in order to provide more sensitivity between crp and uae (i.e., comparing uae in the presence of normal, moderate, and very elevated crp levels). The crp tertiles were as follows: first tertile, which was used as the reference, <0.2 mg / dl; mid- or second tertile, 0.200.56 mg / dl; and highest or third tertile, 57 mg / dl . In an unadjusted linear regression analysis, an association between uae and the second crp tertile was found among hispanics (p = 0.04). In model 1, which was adjusted for social variables, hispanics in the second crp tertile had significant associations with higher uae relative to those in the reference tertile . In model 2, adjusted for model 1 plus laboratory variables, hispanics had significant associations with uae in both the second and third crp tertiles . However, in model 3, adjusted for model 2 plus medication use and comorbidity, both aas and hispanics had a significant association with higher uae in the second crp tertile, but only hispanics had an association with higher uae in the third crp tertile (table 3b). Furthermore, in the adjusted model for the total sample, uric acid, but not wbc, was a significant predictor of uae in both the total sample (adjusted = 0.74 0.24, p = 0.003) and in aas (adjusted = 1.50 0.35, p = 0.001; data not shown). Of the 2,310 subjects with primarily type 2 dm and complete data, 864 were white, 647 were aa, and 799 were hispanic (table 1). Overall, 65% had hypertension, 19% had prevalent cvd, and 27% were smokers . Collectively, 30% used metformin and 6% used pioglitazone as the hypoglycemic agent, and their use did not differ between racial / ethnic groups . Statin and acei therapies were used by 29 and 31% of the patients, respectively, and only hispanic patients had significantly less (p <0.01) use of these medications compared with whites . Arbs were used by 6% of patients, and their use among all ethnic groups did not differ . Aas had higher levels of sbp, dbp, bmi, hba1c, and hdl - c, but lower serum triglyceride levels, compared with whites and hispanics (p <0.01). Relative to white subjects, hispanics had higher levels of total cholesterol, fpg, triglycerides, and dm duration and lower bmi (p <0.05). Relative to whites and aas, hispanics had a lower prevalence of cvd, uric acid, and history of smoking (p <0.01). Aas had higher crp and fibrinogen levels (p <0.01 and p <0.05, respectively) and lower wbc levels (p <0.01) relative to whites and hispanics . Importantly, aas had higher serum creatinine compared with hispanics or whites, but both aas and hispanics had higher egfr (which is adjusted for age and sex) compared with whites (p <0.01). In addition, aas and hispanics had higher uae relative to whites (p <0.01). Of the study population with early ckd (n = 693), 59% were white, 21% were aa, and 20% were hispanic . Aas (36%) and hispanics (34%) had a higher prevalence of early ckd relative to whites (26%, p <0.001) and the total population (29%). Table 2 shows the population characteristics by absence or presence of albuminuria defined as uae <30 g / ml or uae 30 g / ml, respectively . Of the 2,152 subjects, 693 had uae 30 g / ml . Patients with uae 30 g / ml were further stratified into macro- (uae 300 g / ml) and microalbuminuria (uae = 30299 g / ml) in order to assess the relationship between these levels of albuminuria and the independent variables . There were statistically significant differences (p <0.05) between no ckd and early ckd groups in all the variables except smoking, use of dm medications, statin, and arb, hdl, triglycerides, and egfr . Diabetic participants with early ckd had a significantly higher history of hypertension, prevalent cvd, use of acei, sbp, dbp, bmi, duration of dm, fpg, hba1c, crp, uric acid, fibrinogen, wbc, and serum creatinine compared with those with no ckd (p <0.01). Serum albumin levels were slightly lower in diabetic patients with uae 30 g / ml, relative to those with uae <30 g / ml (p <0.04). Demographic and clinical characteristics of diabetic patients with and without early ckd in the group divided into macro- and microalbuminuria, those with macroalbuminuria had statistically significant higher levels of sbp, hba1c, and uric acid and higher reported history of hypertension, but lower serum albumin (p <0.05), reflecting the relationship of some parameters consistent with more advanced disease . In addition, in relation to the entire cohort, microalbuminuria was present in 24% of white subjects, 27% of aas, and 27% of hispanics, whereas macroalbuminuria was present in only 3% of white subjects, 10% of aas, and 7% of hispanics (data not shown). In the adjusted logistic regression analyses (controlling for age, sex, bmi, smoking, dm duration, total cholesterol, hdl - c, fpg, hba1c, uric acid, wbc, statin or acei use, cvd, and hypertension), relative to whites, aas had higher adjusted odds of crp 0.2 mg / dl (aor 1.81, p = 0.007) and hispanics had higher odds of having uae 30 g / ml (aor 1.65, p = 0.03) (fig . Aor for crp (0.2 mg / dl) and uae (30 g / ml) among diabetic patients comparing aas and hispanics to whites (as reference). Crp and uae are adjusted for age, sex, bmi, smoking, dm duration, total cholesterol, hdl - c, fpg, hba1c, uric acid, wbc, medication use (statin and acei), cvd, and hypertension . In the first set of the multiple linear regression analyses, the crp data were transformed into log crp to achieve normal distribution and used in unadjusted and adjusted analyses with log uae for the total sample (n = 693) and for each racial / ethnic group . In the unadjusted model for the overall sample population, however, in the adjusted model, crp was a strong predictor for uae for the total sample as well as for whites and hispanics (table 3a). Multiple linear regression models for the association between log - transformed uae and crp in patients with early ckd, n = 693 in the second set of the multiple linear regression models, crp was divided into tertiles in order to provide more sensitivity between crp and uae (i.e., comparing uae in the presence of normal, moderate, and very elevated crp levels). The crp tertiles were as follows: first tertile, which was used as the reference, <0.2 mg / dl; mid- or second tertile, 0.200.56 mg / dl; and highest or third tertile, 57 mg / dl . In an unadjusted linear regression analysis, an association between uae and the second crp tertile was found among hispanics (p = 0.04). In model 1, which was adjusted for social variables, hispanics in the second crp tertile had significant associations with higher uae relative to those in the reference tertile . In model 2, adjusted for model 1 plus laboratory variables, hispanics had significant associations with uae in both the second and third crp tertiles . However, in model 3, adjusted for model 2 plus medication use and comorbidity, both aas and hispanics had a significant association with higher uae in the second crp tertile, but only hispanics had an association with higher uae in the third crp tertile (table 3b). Furthermore, in the adjusted model for the total sample, uric acid, but not wbc, was a significant predictor of uae in both the total sample (adjusted = 0.74 0.24, p = 0.003) and in aas (adjusted = 1.50 0.35, p = 0.001; data not shown). Population, we found an increased percentage of aas and hispanics with type 2 dm and early ckd associated with both crp and uae . Our analyses of the adjusted models indicate that uae among people with early ckd was significantly elevated in hispanics compared with whites, and crp was significantly elevated in aas compared with whites . Our findings of elevated crp in aas are consistent with previous reports, such as the midlife in the united states (midus) study (16). However, the midus study was limited to investigating the association between ethnicity and crp in a biomarker substudy group of pooled patients, a portion from a random national sample and a portion from an enriched group of aa participants from milwaukee (16), which may confound both the findings and the generalizability . Despite these limitations, the midus study found that early life adversities (e.g., school failure, relation with parents, parental unemployment, or alcohol problem) correlated with high concentrations of inflammatory markers (crp, interleukin-6 [il-6], fibrinogen, and e - selectin) at midlife for aas, but not for whites . This pattern may be associated with the development of an accelerated course of age - related chronic diseases, including dm and its complications in aas (16). In our study, hispanics had higher adjusted odds of uae 30 g / ml compared with whites, whereas the trend toward higher odds for uae 30 g / ml among aas did not reach statistical significance . This trend among aas compared with whites is consistent with the nhanes iii population where the prevalence of albuminuria (albumin - to - creatinine ratio 30 mg / g) was 12% among aas and 8% among whites (17). Further, in the kidney early evaluation program, albuminuria was prevalent in 11% of aas and 8% of whites (18). In the fully adjusted multiple linear regression model, we found a modest association between uae and crp for aas (second tertile only) and more robust for hispanics (both second and third tertile) in comparison with whites . Our finding of an association between crp and uae is also consistent with earlier reports supporting an association between crp and micro / macroalbuminuria in high - risk populations such as those with dm (10,19), although none of these studies had reported ethnic differences in the level of inflammation and uae in the setting of early ckd . Others have also demonstrated that crp is associated with microalbuminuria in the general population (12,13), and in the multi - ethnic study of atherosclerosis cohort, this association was found to be stronger in nonwhite men and aa women (20). Indeed, in our study, the relationship between crp and uae with early ckd was most robust in hispanics, less so in aas, and not significant in whites . The weaker association of crp and uae in aas is consistent with two recent studies of patients treated with maintenance hemodialysis (21,22) that found higher crp and il-6 levels were less predictive of mortality in aas than whites, suggesting that although crp levels are generally higher in aas, clinical outcomes in aas may be less dependent on the level of crp . We also considered wbc and uric acid, which were previously used as inflammatory markers in an nhanes iii analysis (23). The multiple linear regression models indicated that uric acid, and not wbc, was a significant predictor of uae . Studies suggest that uric acid may cause microvascular complications through endothelial dysfunction, increased activity of the renin angiotensin aldosterone system, and induction of the inflammatory cascade including increased crp levels (24). However, due to the paradoxical pro- and antioxidant properties of uric acid (25), we limited our discussion mainly to crp as a marker of inflammation and adjusted the data for both uric acid and wbc in multivariate analyses . Even after this adjustment, aas still had significantly higher aor for having elevated crp levels, suggesting that the presence of systemic inflammation in aas may occur at a very early stage in ckd, while egfr is still preserved . We speculate that this relationship may ultimately contribute to their accelerated and disparate disease progression, which is observed later in the course of ckd . Recent investigations support the mechanistic notion that type 2 dm includes an inflammatory component that significantly contributes to the genesis and progression of ckd (26). Several clinical and experimental studies have shown that a variety of inflammatory molecules, such as crp, il-6, and monocyte chemoattractant protein-1, are involved in the setting of diabetic nephropathy (27,28). In addition, studies show that crp is not only a prototypic marker of inflammation but may directly influence ckd . Using a mouse model of type 1 dm, liu et al . (29) showed that crp promotes ckd by enhanced activation of transforming growth factor- (tgf-)/smad and nuclear factor-b (nf-b) signaling pathways . Crp also induced il-6 and thrombospondin (tsp-1) mrna and protein expression in human renal tubular epithelial hk-2 cells via activation of the p38 mitogen - activated protein kinase and nf-b signaling pathways as well as tgf-1 expression, suggesting that crp plays an important role in the propagation and prolongation of inflammation in renal fibrosis (30). Most importantly, in the current context, crp also promotes proinflammatory cytokine production (31), leading to mesangial cell proliferation, matrix overproduction, and increased vascular permeability resulting in albuminuria (32). Human and murine macrophages exposed to high glucose concentrations show increased levels of crp mrna and protein biosynthesis and secretion, suggesting that crp - mediated proinflammatory effects could be triggered locally by macrophage - produced crp in addition to the effect of circulating and liver - derived crp (33). It has also been demonstrated that crp promotes the differentiation of human monocytes toward a proinflammatory phenotype (34) and induces macrophage colony stimulating factor release via upregulation of nf-b, resulting in increased macrophage recruitment and proliferation (35). It is well known that macrophages infiltrate the glomeruli and/or interstitium in the kidney tissue in diabetic patients with nephropathy, and the intensity of the interstitial infiltrate is proportional to the rate of subsequent decline in renal function (36). Our finding of an association between crp and uae at early ckd seems to be logical as vascular (and possibly microvascular) endothelial damage begins before it becomes clinically apparent even when gfr> 90 ml / min/1.73 m (37). Improving our understanding of the mechanism(s) by which inflammatory molecules such as crp may contribute to the development of albuminuria can help direct new interventions . For example, oxidative stress modulators that could potentially activate keap1-nrf2 and suppress inflammation in ckd (38) may be effective for counteracting crp activation of tgf-/smad and nf - kb signaling pathways (29,30). Unfortunately, the recent clinical trial using bardoxylone methyl, one such oxidative stress modulator, was stopped prematurely (39), possibly secondary to the detrimental effects of degradation products (40). However, modified analogs addressing this pathway and others outlined above may open doors for developing new therapeutic targets, especially in ethnic minorities . First, it is a cross - sectional study and can provide only associations and not causation . Despite the efforts of nhanes to enroll a random representative noninstitutionalized sample of the u.s . Population, people attending the study visits may differ from those not attending in subtle ways that may affect the results of this study . Although we tried to control for the potentially confounding variables further, data on risk factors were ascertained only at baseline; therefore, we could not systematically control for differences in the severity of risk factors or perform time - dependent analyses . Although crp is a widely used marker of inflammation, the test is not established to be highly sensitive or specific to ckd, and the lack of data on other inflammatory markers such as interleukins and tnf- may limit our ability to interpret a broader assignment of inflammation to the observed racial / ethnic differences in ckd . Balanced against these limitations, the strength of this study lies in the analysis for crp and covariates in a large, nationwide, population - based sample that includes a well - validated approach to interviews, laboratory, and physical examination . In summary, we showed here that patients with dm and early ckd had increased crp levels compared with those with dm and no ckd . We also showed that diabetic aas and hispanics had a higher prevalence of early ckd compared with whites, and we found higher levels of uae in aas and hispanics, higher absolute crp levels in aas, and an association between crp and uae in aas and hispanics (stronger in hispanics), providing additional evidence for a role of inflammation in the increased prevalence of early diabetic ckd in both ethnic groups . The association between uae and crp in aas and hispanics may impact ckd progression, accounting in part for the exceptionally high rates of dm - related esrd in minorities . Prospective studies are required to assess whether a targeted intervention to reduce the level of inflammation is an effective strategy to prevent or slow the progression of ckd in dm and whether there is particular efficacy for select interventions across racial / ethnic groups . First, it is a cross - sectional study and can provide only associations and not causation . Despite the efforts of nhanes to enroll a random representative noninstitutionalized sample of the u.s . Population, people attending the study visits may differ from those not attending in subtle ways that may affect the results of this study . Although we tried to control for the potentially confounding variables further, data on risk factors were ascertained only at baseline; therefore, we could not systematically control for differences in the severity of risk factors or perform time - dependent analyses . Although crp is a widely used marker of inflammation, the test is not established to be highly sensitive or specific to ckd, and the lack of data on other inflammatory markers such as interleukins and tnf- may limit our ability to interpret a broader assignment of inflammation to the observed racial / ethnic differences in ckd . Balanced against these limitations, the strength of this study lies in the analysis for crp and covariates in a large, nationwide, population - based sample that includes a well - validated approach to interviews, laboratory, and physical examination . In summary, we showed here that patients with dm and early ckd had increased crp levels compared with those with dm and no ckd . We also showed that diabetic aas and hispanics had a higher prevalence of early ckd compared with whites, and we found higher levels of uae in aas and hispanics, higher absolute crp levels in aas, and an association between crp and uae in aas and hispanics (stronger in hispanics), providing additional evidence for a role of inflammation in the increased prevalence of early diabetic ckd in both ethnic groups . The association between uae and crp in aas and hispanics may impact ckd progression, accounting in part for the exceptionally high rates of dm - related esrd in minorities . Prospective studies are required to assess whether a targeted intervention to reduce the level of inflammation is an effective strategy to prevent or slow the progression of ckd in dm and whether there is particular efficacy for select interventions across racial / ethnic groups.
. An earlier version of this paper was written as part of charlotte khlbrandt's msc dissertation project at the london school of hygiene and tropical medicine . Ck conducted the literature review, collected and analysed the data, interpreted the results and drafted the paper . We would like to thank dr alex mold, who co - supervised the msc dissertation and commented on a draft of this paper . We are also grateful for the wellcome trust's commitment to cover open access charges . . An earlier version of this paper was written as part of charlotte khlbrandt's msc dissertation project at the london school of hygiene and tropical medicine . Ck conducted the literature review, collected and analysed the data, interpreted the results and drafted the paper . We would like to thank dr alex mold, who co - supervised the msc dissertation and commented on a draft of this paper . We are also grateful for the wellcome trust's commitment to cover open access charges.
Obesity is an isolated risk factor for cardiovascular disease; it can lead to cardiac hypertrophy followed by dilated cardiomyopathy, predisposing to fatal arrhythmias . 1 obesity also causes organ - specific changes, which are due to a direct mechanical effect of the adipose tissue or occur systemically through humoral mediators and metabolic adjustments that change heart hemodynamics and geometry, as well as possibly lung function . 1 3 a recent epidemiological study 4 found a relationship between lung function and left ventricular mass (lvm), although the relationship differed between genders . Functional effects of obesity that are associated with being male or female are common in pulmonary function testing, since fat concentrated in the chest (android obesity) could lead to deeper changes in lung function than could fat concentrated in the hips (gynecoid obesity). In contrast, another study, which had a case - control design and involved children with and without metabolic syndrome, found no relationship between lung function and lvm . 5 recent evidence suggests that inflammatory mediators act independently of confounding variables on cardiac remodeling in obese individuals, 2 6 as well as on lung function, 7 8 primarily at the level of the small airways . In contrast, there is evidence of purely mechanical cardiopulmonary effects, with no effect of inflammatory mediators, 9 10 or an interaction of mechanical and inflammatory factors in cases of lung function and asthma associated with obesity . 11 therefore, the primary objective of this exploratory study was to look for correlations between lung function and cardiac dimension variables, in order to test the hypothesis that regardless of being a purely mechanical or gender - related factor, the relative size of the small airways (dysanapsis), as measured indirectly by the ratio of fef25 - 75% to fvc (fef25 - 75/fvc,%), is correlated with left ventricular hypertrophy (remodeling), since the small airways are especially susceptible to mechanical - inflammatory interactions and bronchial hyperreactivity . Similarly, we aimed to determine the degree of association between lung function and ventricular mass in the study population, with the goal of guiding future studies related to common mechanisms of cardiac and pulmonary impairment in morbid obesity . To the best of our knowledge, based on our review of currently available databases (bireme, scielo, pubmed, cochrane library, and google academic), this is the first study to investigate this relationship in morbidly obese individuals . We planned this study based on secondary data obtained from a bariatric surgery outpatient clinic, which is a state referral center for this type of surgery . In this retrospective study, we reviewed the medical records of all obese individuals who were candidates for bariatric surgery between january of 2006 and december of 2010, with the total number of individuals being 192 . Individuals underwent a standardized clinical assessment, which was based on an instrument designated " clinical assessment form for obese patients " (a protocol at the department of bariatric surgery), in which detailed information was obtained on anthropometric parameters, degree of obesity, comorbidities, and pressure levels, among other clinical data and relevant tests, assessed by physicians, nutritionists, and nurses working in that department . The aim of the medical record review was to systematically collect the following clinical data on and test results for morbidly obese individuals (bmi 40 kg / m 12: (i) anthropometry; (ii) simple spirometry; (iii) m - mode echocardiography; (iv) reporting of bronchial asthma (diagnosis and/or treatment); (v) reporting of current or former smoking; and (vi) reporting (diagnosis and/or treatment) of systemic arterial hypertension (sah). Current or former smoking refers to a longer than 1-year history of smoking, regardless of the number of pack - years . Sah was defined as a current drug treatment for sah or at least two arterial blood pressure measurements 140/90 mmhg . Data on the forms, as well as test results, were only accepted if they had been properly recorded within 1 year before surgery . Only 45 patients met all of the above inclusion criteria, of whom 3 were excluded because of an echocardiographic report of acoustic window impairment caused by obesity and 3 were excluded because they did not meet the spirometry quality criteria . The main reasons for exclusion of the remaining cases were not being diagnosed with morbid obesity (136 individuals) and being a morbidly obese patient with no spirometric data (4 individuals), no echocardiographic data (5 individuals), or neither (2 individuals). Anthropometric data were obtained by using a stadiometer and a scale for obese individuals, and bmi was calculated by the formula weight / height (in kg / m . Waist circumference values were not collected, since such values were lacking in many cases . The study was approved by the human research ethics committee of the federal university of mato grosso do sul and was in compliance with the declaration of helsinki . Spirometry with forced expiratory maneuver was performed in the pulmonary function section of the department of pulmonology of the university . All tests met the acceptability criteria established in the brazilian guidelines for pulmonary function testing, 13 and values were corrected to body temperature, pressure saturated ., rio de janeiro, brazil), with individuals in a sitting position and wearing a nose clip . The equipment was always calibrated in the morning, in accordance with the manufacturer instructions, and the tests were always conducted by one of two trained spirometry technicians belonging to the department of pulmonology . At least three acceptable maneuvers were always performed, and instantaneous flow was expressed as that obtained from the one maneuver with the highest sum of fvc (in l) and fev1 (in l / min)., 14 and the mean sd of the study population was recorded . Echocardiography was performed by two cardiologists, who used a nemio 17 - 2005 echocardiograph (toshiba, tokyo, japan). Those two cardiologists, one of whom is one of the present authors, work specifically in the echocardiography section of the hospital . For the purposes of this study, only data acquired in m - mode were collected; left ventricle end - diastolic volume (lvdv) and lv end - systolic volume (lvsv) were indirectly measured using the teicholz formula, and lvm was calculated using the formula by devereux et al . 15 the measurements were obtained from parasternal lv cross - sections at the level of the papillary muscles . Left atrial (la) dimensions, lv end - diastolic diameter (lvdd), and lv end - systolic diameter (lvsd) were also measured . We used as criteria for lvm indexation both height to the power of 1.7 (lvm / m, which is a criterion recommended for obese individuals, and height squared (lvm / m . Relative lv posterior wall thickness (rlvpwt) was obtained by dividing lv posterior wall thickness (lvpwt) by lvdd . Our sample size calculation (pass software, version 11; ncss llc, kaysville, ut, usa) showed that 40 individuals would be enough to provide a power of 80% for a significant correlation coefficient of 0.40 at = 0.05 (fef25 - 75/fvc,% vs. lvm / lvpwt / rlvpwt). For multiple linear regression analysis (based on lvm), 40 individuals would be necessary to provide a power of 98% and an r= 0.37, in a model with four independent variables (weight, fev1, fvc, and fef25 - 75/fvc,%). The categorical variables reporting of smoking, reporting of asthma, and reporting of sah were also computed, and gender and reporting of sah were coded as a binary variable for purposes of statistical adjustment . Correlations between various spirometric and echocardiographic variables were assessed by pearson's correlation test . Only lvm and lvpwt required log transformation to fit a normal distribution . The relationship between reporting of asthma (diagnosis and/or treatment) or current / former smoking and fef25 - 75/fvc,%, below or above the lower limit of the normal range, was assessed by fischer's exact test . The relationship between fef25 - 75/fvc,% and lvpwt / rlvpwt was tested by partial correlation analysis, adjusted for weight, gender, and sah, which are traditionally the most important determinants of lv hypertrophy in obesity . In order to determine which variable would be the best predictor of lvm (dependent variable), we tested a stepwise multivariate linear regression model, in which the independent variables were only the variables showing a significant correlation (p <0.05) with lvm, whether indexed or not . For all calculations and graphs, we used the ibm spss statistics software package, version 20.0 (ibm corp ., of the 45 patients for whom all data were originally available for inclusion, 6 were excluded because of reported failures in m - mode echocardiography or in pulmonary function testing, which did not meet quality criteria . The anthropometric and demographic data of the 39 patients included in the study are shown in table 1 . Females predominated in the study (74.3%), the mean age of the participants was 35.5 7.7 years, and 8 participants were considered super - obese (bmi> 55 kg / m . Sah (diagnosis and/or treatment) was reported to be present in 21 individuals (54%). Only 1 individual reported both asthma and smoking . The mean values of la diameter, lvm, and lvm / m were increased relative to normal mean values for the brazilian population . 16 table 1.characteristics of the individuals included in the study (n = 39).a characteristic result age, years35.5 7.7height, cm163.1 9.1weight, kg131.4 25.9bmi, kg / m249.2 7.6bs, m22.3 0.3gender (m / f), n / n10/29asthma (y / n), n / n7/32smoking (y / n), n / n6/33sah (y / n), n / n21/18bs: body surface; m: male; f: female; y: yes; n: no; and sah: systemic arterial hypertension . Bs: body surface; m: male; f: female; y: yes; n: no; and sah: systemic arterial hypertension . Parameter result spirometry fev1, l2.8 0.6fev1,% predicted87.9 11.8fvc, l3.4 0.8fvc,% predicted88.4 11.7fev1/fvc,% 83.1 6.4fef25 - 75/fvc,% 99.5 30.2echocardiography la, mm36.2 4.1st, mm11.6 4.4lvpwt, mm10.7 2.3rlvpwt0.2 0.1lvdd, mm48.2 4.1lvsd, mm29.6 3.7lvdv, ml108.9 21.7lvsv, ml34.9 11.2lvm, g248.3 84.9lvm, g / m 106.7 30.4lvm, g / m 92.1 26.1la: left atrium; st: septal thickness; lvpwt: left ventricular posterior wall thickness; rlvpwt: relative left ventricular posterior wall thickness; lvdd: left ventricular end - diastolic diameter; lvsd: left ventricular end - systolic diameter; lvdv: left ventricular end - diastolic volume; lvsv: left ventricular end - systolic volume; and lvm: left ventricular mass . La: left atrium; st: septal thickness; lvpwt: left ventricular posterior wall thickness; rlvpwt: relative left ventricular posterior wall thickness; lvdd: left ventricular end - diastolic diameter; lvsd: left ventricular end - systolic diameter; lvdv: left ventricular end - diastolic volume; lvsv: left ventricular end - systolic volume; and lvm: left ventricular mass . Chief among the significant correlations is the weak direct relationship between bmi and the variables lvdd(r = 0.359; p <0.05), lvsd (r = 0.387; p <0.05), lvdv (r = 0.387; p <0.05), and lvsv (r = 0.425; p <0.01). A moderate inverse correlation was found between fef25 - 75/fvc,% and the variables of lv remodeling (table 3) in the univariate analysis, and this correlation remained statistically significant for lvpwt (r = 0.355; p <0.05) and rlvpwt (r = 0.349; p <0.05) even after adjustment for weight, gender, and sah (figures 1a and 1b, respectively). The correlation between indexed lvm (in g / m and fef25 - 75/fvc,% was borderline for statistical significance (p = 0.05; figure 1c). Interestingly, fef25 - 75/fvc,% did not correlate with the variables of lv internal diameter or lv volume . Fischer's exact test showed no statistically significant association between fef25 - 75/fvc,%, below or above the lower limit of the normal range, and reporting of asthma or smoking (p> 0.05 for both). Table 3.univariate correlations of anthropometric and spirometric variables with transthoracic echocardiography variables (m - mode) in the sample as a whole (n = 39). Parameter lv hypertrophy lv diameter / volume lvm, g lvm, g / m lvm, g / m st, mm lvpwt, mm rlvpwt la, mm lvedd, mm lvesd, mm lvedv, ml lvesv, ml anthropometry bmi, kg / m 0.1750.1920.2270.0780.0920.0460.0690.359 * 0.387 * 0.387 * 0.425 weight, kg0.476 0.2930.3120.1600.360 * 0.1690.380 * 0.447 0.476 0.455 0.556 spirometry fev1, l0.590 0.388 * 0.401 * 0.1620.389 * 0.2120.473 0.406 0.388 * 0.386 * 0.346 * 0.380 * 0.1640.429 0.2640.513 0.365 * 0.3150.366 * 0.418 fvc,% predicted0.0870.1590.2560.0710.0170.0610.0190.1330.0480.1940.038fev1/fvc,% 0.0030.0770.0400.0020.1140.1450.0820.1180.1970.0470.167fef25 - 75/fvc,% 0.3970.2750.3180.0540.453 0.404 0.1680.0090.0410.0570.020lv: left ventricular; lvm: lv mass; st: septal thickness; lvpwt: lv posterior wall thickness; rlvpwt: relative lv posterior wall thickness; la: left atrium; lvdd: lv end - diastolic diameter; lvsd: lv end - systolic diameter; lvdv: lv end - diastolic volume; and lvsv: lv end - systolic volume . p <0.05; p = 0.05; p <0.01 . Lv: left ventricular; lvm: lv mass; st: septal thickness; lvpwt: lv posterior wall thickness; rlvpwt: relative lv posterior wall thickness; la: left atrium; lvdd: lv end - diastolic diameter; lvsd: lv end - systolic diameter; lvdv: lv end - diastolic volume; and lvsv: lv end - systolic volume . * p <0.05; p = 0.05; p <0.01 . Figure 1.correlation of fef25 - 75/fvc,%, adjusted for the variables weight, gender, and systemic arterial hypertension, with left ventricular posterior wall thickness, in a; with relative left ventricular posterior wall thickness, in b; and with left ventricular mass adjusted for body size in obese individuals, in c. stepwise multiple linear regression analysis (table 4) showed that the variation in lvm among morbidly obese individuals is better predicted by fvc (in l), which explained 36.9% (p <0.0001) of the variation in lvm in the study population . The best predictor of lvm indexed to height squared and lvm indexed to height to the power of 1.7 was fev1 (in l / min; p <0.05 for both; table 4). Table 4.stepwise multiple linear regression for the dependent variable left ventricular mass (in grams or indexed to body surface area; n = 39). Dependent variable predictor variable beta 95% ci adjusted r p lvm, gfvc, l0.600.43 - 1.130.369 <0.001lvm, g / m fev1, l / min0.380.29 - 2.80.126 <0.05lvm, g / m fev1, l / min0.400.2 - 1.30.161 <0.05lvm: left ventricular mass . This retrospective study shows there is an independent association between the relative size of the small airways (fef25 - 75/fvc,%) and echocardiographic parameters of ventricular hypertrophy in morbidly obese individuals . In addition, fvc (in l) and fev1 (in l / min) were found to be important predictors of lvm in grams or indexed to body surface area . Both lvm and lv internal diameters are increased in obesity, regardless of sah . 17 in our study, we found no relationship of bmi with lvm in grams or indexed to body surface area or with lvpwt, but we found a relationship of bmi with lv internal diameters and lv volumes, which is consistent with the literature . 2 17 this positive association is likely to be due to increased preload and increased cardiac output, causing ventricular dilatation, which could later progress to lv remodeling . 18 the literature shows inconsistent associations between bmi and spirometric results, with some studies showing an association 19 and others not showing any . 20 this is probably due to differences in gender ratio among studies, since the android type of obesity favors the correlation between bmi and lung function, unlike the gynecoid phenotype, which does not produce excessive accumulation of fat mass in the chest . In our study, females predominated (74%), which partly explains the lack of correlation between any spirometric variable and bmi . The relationships of fvc, fev1, and fef25 - 75% with lvm and lvpwt have been studied in (non - obese) elderly individuals with cardiovascular disease, and the results differ among studies in regard to the direction of the correlation, with correlations being positive 21 or negative, 22 which more strongly reflects loss of lung function associated with advanced age and with the effects of sah and pulmonary hypertension, as well as with the pulmonary restrictive effects of cardiomegaly . One study 4 showed an inverse relationship between lvm and fvc in nonsmoking females and a direct relationship between lvm and fvc in nonsmoking males under 60 years of age . The fact that most of our sample consists of females (74%) suggests that, in morbidly obese individuals, the direction of the correlation may be different for some lung function variables . Fef25 - 75/fvc,% is a measure that corrects to some extent for the large variability found in fef25 - 75% alone and reflects changes predominantly in the small airways, being adjusted for lung size on the basis of fvc . 17 the correlations of fef25 - 75/fvc,% with lvm indexed to body surface area (in g / m and with lvpwt / rlvpwt, correlations that remained after adjustment for weight, gender, and sah, possibly reflect the direct mechanical effects of obesity, but may also suggest that there are other independent (inflammatory or lipotoxic) mechanisms . Since research on the subject is scarce, there is limited evidence that the small airways are independently affected by obesity, as has been reported in nonsmoking males . 23 the hypotheses raised in that study 23 include an increase in blood volume in obese individuals, causing bronchial vessel congestion; the presence of increased levels of very - low - molecular - weight lipoproteins, which could trigger the release of histamine; and altered lipoprotein metabolism in obesity, which could elicit and amplify these effects . Recent data in the literature also indicate that obesity is characterized by hyperresponsiveness to methacholine, predominantly in the small airways, 24 and that this hyperresponsiveness correlates better with fef25 - 75/fvc,% . 25 on this point, a recent study suggests that groups of obese individuals with hyperresponsiveness are associated with greater lvm . 26 small airway hyperresponsiveness in obese individuals also could be partially explained by dysanapsis (which is assessed indirectly by fef25 - 75/fvc,%), a term coined by green et al . 27 to explain the large interindividual variability in airway size, regardless of lung parenchyma size . An important predictor of lvm, bmi correlated directly with cardiac size and mass in several studies, although lean body mass remains a better predictor of lvm . 2 28 29 this retrospective study found no correlations between bmi and lvm (in grams or indexed to body surface area), which is in agreement with another study, 30 and this is possibly due to differences in obesity phenotypes, prevalence of sah, and number of individuals studied . Of note in our study is the fact that fvc (in l) was the best predictor of variation in lvm (in g), explaining 37% of this variation in the study population, which suggests that reduced lung volume may be an important variable in establishing a predictive model for lvm in obese individuals in future studies . In turn is the fact that our sample was small, consisting of candidates for bariatric surgery, was limited by the criterion of including only morbidly obese and super - obese individuals, and was based on criteria that were unclear in the medical charts, such as the diagnosis of asthma or sah . In addition, data on diabetes were not collected, although the relationship between diabetes and lvm is inconsistent in the literature . Other important limitations were the limited acoustic window in the analysis of m - mode echocardiographic variables in obese individuals and the lack of a specific, standardized protocol for m - mode echocardiography, since we did not obtain data on inter - rater agreement for the two echocardiographers . In this regard, because this was a retrospective study, we sought not to use echocardiographic data for which accuracy is significantly decreased by the effects of obesity on the acoustic window, such as ejection fraction and doppler echocardiography data . We therefore conclude that the small airways in morbidly obese individuals have a correlation with cardiac hypertrophy, regardless of usual anthropometric variables, gender, or sah . This study reveals that factors other than mechanical and/or hemodynamic limitations imposed by increased body mass may be important in the joint changes seen in the small airways and in cardiac hypertrophy . In addition, further studies are needed to examine the impact that lung function parameters have on predictive equations for lvm in obese individuals . Verificar a existncia de correlaes entre variveis de funo pulmonar e de dimenses cardacas em obesos mrbidos, buscando testar a hiptese de que o tamanho relativo das pequenas vias areas correlaciona - se com hipertrofia cardaca esquerda de forma independente . Estudo retrospectivo de 192 pronturios mdicos que continham um protocolo clnico de candidatos cirurgia baritrica entre janeiro de 2006 a dezembro de 2010 . Dos 192 pacientes avaliados, 39 (29 mulheres) preencheram os critrios de incluso . A mdia do imc foi de 49,2 7,6 kg / m, e a mdia de idade foi de 35,5 7,7 anos . As correlaes da fef25 - 75/cvf,% com a espessura da parede posterior do ventrculo esquerdo e a espessura relativa da parede posterior do ventrculo esquerdo se mantiveram estatisticamente significativas aps ajuste para peso, sexo e histria de hipertenso arterial sistmica (r = 0,355 e r = 0,349, respectivamente). Aps anlise de regresso linear multivariada stepwise, a cvf e o vef1 foram os principais determinantes da massa ventricular esquerda (em gramas ou indexada para rea de superfcie corporal). Uma reduo no tamanho relativo das pequenas vias areas parece apresentar uma correlao independente com hipertrofia cardaca relacionada obesidade, independente de fatores que afetam a mecnica respiratria (imc e peso), sexo ou histria de hipertenso arterial sistmica . O vef1 e a cvf, por outro lado, podem ser importantes determinantes preditivos da massa ventricular esquerda em obesos mrbidos . A obesidade um fator de risco isolado para as doenas cardiovasculares, podendo levar a hipertrofia cardaca seguida de miocardiopatia dilatada, predispondo a arritmias fatais.a obesidade tambm provoca alteraes rgo - especficas, causadas por efeito mecnico direto do tecido adiposo, ou, sistemicamente, atravs de mediadores humorais e ajustes metablicos que alteram a hemodinmica e a geometria cardaca, assim como, possivelmente, a funo pulmonar . 1 3 um estudo epidemiolgico recente 4 encontrou uma relao entre funo pulmonar e massa ventricular esquerda (mve), ainda que diferente entre os sexos . Efeitos funcionais da obesidade associados ao sexo masculino ou feminino so comuns no estudo da funo pulmonar, pois uma obesidade concentrada no trax (obesidade androide) poderia levar a alteraes mais profundas na funo pulmonar do que um tipo de obesidade mais acentuada no quadril (obesidade ginecoide). Outro estudo, ao contrrio, com desenho caso - controle em crianas com e sem sndrome metablica, no encontrou relao entre funo pulmonar e mve . 5 evidncias recentes sugerem a ao de mediadores inflamatrios de forma independente de variveis de confuso no remodelamento cardaco em obesos, e tambm de forma independente sobre a funo pulmonar, principalmente ao nvel das pequenas vias areas . Ao contrrio, existem evidncias de efeitos puramente mecnicos cardiopulmonares, sem efeito de mediadores inflamatrios, ou uma interao de fatores mecnicos e inflamatrios no caso de funo pulmonar e asma associada obesidade . 11 assim, o objetivo principal do presente estudo exploratrio foi verificar a existncia de correlaes entre variveis de funo pulmonar e de dimenses cardacas, buscando testar a hiptese de que o tamanho relativo das pequenas vias areas (dysanapsis), mensurado indiretamente pela razo do fef25 - 75% com a cvf (fef25 75/cvf,%), correlaciona - se com hipertrofia cardaca esquerda (remodelamento) independente de ser um fator meramente mecnico ou de sexo, pois as pequenas vias areas so especialmente susceptveis a interaes mecnico - inflamatrias e hiper - reatividade brnquica . No mesmo sentido, objetivamos conhecer o grau de associao entre funo pulmonar e massa ventricular na populao estudada, orientando futuros estudos relacionados aos mecanismos comuns de comprometimento cardaco e pulmonar na obesidade mrbida . At o melhor que pudemos averiguar nas bases de dados atualmente disposio (bireme, scielo, pubmed, cochrane library e google acadmico), este o primeiro estudo a investigar essa relao em obesos mrbidos . Planejamos o presente estudo com base em dados secundrios de um ambulatrio de cirurgia baritrica, referncia estadual para esse tipo de cirurgia . No presente estudo retrospectivo, foram revisados os pronturios de todos os indivduos obesos candidatos cirurgia baritrica entre janeiro de 2006 e dezembro de 2010, totalizando 192 indivduos . Os indivduos se submetiam a uma avaliao clnica padronizada, baseada em um instrumento denominado " ficha clnica de avaliao de obesos " (protocolo do servio de cirurgia baritrica), no qual eram detalhados parmetros antropomtricos, grau de obesidade, comorbidades e nveis pressricos, entre outros dados clnicos e exames relevantes, avaliados por mdicos, nutricionistas e enfermeiras dedicados ao servio . A reviso dos pronturios visou coletar os seguintes dados clnicos e de exames, de maneira sistemtica, em indivduos com obesidade mrbida (imc 40 kg / m 12: (i) antropometria; (ii) espirometria simples; (iii) ecocardiografia modo m; (iv) relato de asma brnquica (diagnstico e / ou tratamento); (v) relato de tabagismo atual ou passado; e (vi) relato (diagnstico e / ou tratamento) de hipertenso arterial sistmica (has). A asma foi definida como o relato de diagnstico mdico atual ou prvio . O tabagismo atual ou prvio refere - se histria de tabagismo por mais de um ano, independente de carga tabgica . A has foi definida como o tratamento atual medicamentoso para has ou como pelo menos duas medidas de presso arterial 140/90 mmhg . Os dados da ficha e os resultados dos exames s eram aceitos se estivessem dentro do prazo de at um ano antes da cirurgia e estivessem adequadamente preenchidos . Somente 45 pacientes preencheram todos os critrios de incluso citados acima, sendo que foram excludos 3 casos com laudo ecocardiogrfico declarando janela acstica prejudicada pela obesidade e 3 casos que no preencheram os critrios adequados de qualidade para a espirometria . As principais causas de excluso dos demais casos foram no ser diagnosticado com obesidade mrbida (em 136 indivduos), assim como no haver dados sobre espirometria (em 4), ecocardiograma (em 5) ou dos dois exames (em 2) em pacientes com obesidade mrbida . Os dados antropomtricos foram coletados em estadimetro e balana apropriada para obesos, sendo que o imc foi obtido pela equao peso / altura (em kg / m . Os valores de circunferncia abdominal no foram coletados, pois estavam ausentes em muitos casos . O estudo foi aprovado pelo comit de tica em pesquisa em seres humanos da universidade federal de mato grosso do sul, estando de acordo com a declarao de helsinki . A espirometria com manobra expiratria forada foi realizada no setor de funo pulmonar da disciplina de pneumologia da universidade . Todos os testes preencheram os critrios de aceitabilidade das diretrizes brasileiras de funo pulmonare tiveram os valores corrigidos para body temperature, pressure saturated . Os exames foram realizados em um espirmetro vitatrace vt 130 (pr mdico ltda ., rio de janeiro, brasil), com os indivduos em posio sentada e utilizando um clip nasal, e o equipamento foi calibrado sempre pela manh, conforme as instrues do fabricante, e realizados sempre por uma de duas tcnicas treinadas em espirometria pertencentes ao servio de pneumologia . Eram sempre realizadas pelo menos trs manobras aceitveis, e o resultado de fluxos instantneos foi expresso como aquele obtido do exame com a maior soma da cvf (em l) e vef1 (em l / min). Os valores de referncia para a populao brasileira foram aqueles de pereira et al.,sendo registrado o valor mdio dp da populao estudada . O ecocardiograma foi realizado com ecocardigrafo nemio 17 - 2005 (toshiba, tquio, japo) por dois mdicos cardiologistas com rea de atuao especfica do setor de ecocardiografia do hospital, incluindo um dos autores . Para a finalidade do presente estudo, foram coletados somente os dados obtidos pelo modo unidimensional (modo m), sendo que tanto o volume diastlico final do ventrculo esquerdo (vdve) como o volume sistlico final do ve (vsve) foram medidos indiretamente pela frmula de teicholz, e a mve foi calculada usando a frmula de devereux et al . 15 as mensuraes foram obtidas por cortes paraesternais transversais do ve ao nvel dos msculos papilares . Tambm foram mensuradas as dimenses de trio esquerdo (ae), dimetro diastlico final de ve (ddve) e dimetro sistlico final de ve (dsve). Usamos como critrios para a indexao do valor da mve tanto a altura elevada potncia de 1,7 (mve / m, um critrio recomendado para obesos, como a indexao da mve ao quadrado da altura (mve / m . A espessura relativa da parede posterior do ve (erppve) foi obtida pela diviso da espessura da parede posterior do ve (eppve) pelo ddve . Utilizando um clculo amostral no programa pass, verso 11 (ncss llc, kaysville, ut, eua), demonstramos que 40 indivduos seriam suficientes para fornecem um poder de 80% para um coeficiente de correlao significativo de 0,40 a um nvel a = 0,05 (fef25 - 75/cvf,% vs. mve / eppve / erppve). Para a anlise de regresso linear mltipla (com base na mve), seriam necessrios 40 indivduos para um poder de 98% e um r= 0,37, assumindo quatro variveis independentes no modelo (peso, vef1, cvf e fef25 - 75/cvf,%). As variveis categricas de relato de tabagismo, asma e has tambm foram computadas, sendo que sexo e relato de has foram codificados como uma varivel binria para efeitos de ajuste estatstico . Foram realizados clculos de correlaes entre diversas variveis de espirometria simples e ecocardiogrficas pelo teste de correlao de pearson . Somente as variveis mve e eppve precisaram ser transformadas em log para assumir distribuio normal . A relao entre diagnstico e / ou tratamento relatado para asma ou tabagismo atual / passado e fef25 - 75/cvf,%, abaixo ou acima do limite inferior da normalidade, foi calculada pelo teste exato de fischer . A relao entre fef25 - 75/cvf,% e eppve / erppve foi testada por correlao parcial, ajustada para peso, sexo e has, que so tradicionalmente os determinantes mais importantes da hipertrofia de ve na obesidade . Para testar qual varivel seria a melhor preditora da mve (varivel dependente), testamos um modelo de regresso linear multivariada stepwise, tendo como variveis independentes somente as variveis cuja correlao foi significativa (p <0,05) em associao com a mve, indexada ou no . Para todos os clculos e grficos utilizamos o programa estatstico ibm spss statistics, verso 20.0 (ibm corp ., armonk, ny, eua). Dos 45 pacientes originais que continham todos os dados para incluso, 6 foram excludos porque apresentavam falhas relatadas no ecocardiograma modo m ou no teste de funo pulmonar, sem preencher critrios de qualidade . Os dados antropomtricos e demogrficos dos 39 pacientes includos no estudo so mostrados na tabela 1 . O sexo feminino foi predominante no estudo (74,3%), e a mdia de idade dos participantes foi de 35,5 7,7 anos, sendo 8 considerados superobesos (imc> 55 kg / m . Asma e tabagismo em algum momento na vida foram relatados por 7 e 6 indivduos, respectivamente . O relato de has (diagnstico e / ou tratamento) estava presente em 21 indivduos (54%). Os valores mdios de dimetro de ae, mve e mve / m estavam aumentados em relao a valores mdios normais para a populao brasileira . 16 tabela 1.caractersticas dos indivduos includos no estudo (n = 39).a caractersticas resultados idade, anos35,5 7,7altura, cm163,1 9,1peso, kg131,4 25,9imc, kg / m249,2 7,6sc, m22,3 0,3sexo (m / f), n / n10/29asma (s / n), n / n7/32tabagismo (s / n), n / n6/33has (s / n), n / n21/18sc: superfcie corporal; m: masculino; f: feminino; s: sim; n: no; e has: hipertenso arterial sistmica.avalores expressos em mdia dp, exceto onde indicado . Sc: superfcie corporal; m: masculino; f: feminino; s: sim; n: no; e has: hipertenso arterial sistmica.avalores expressos em mdia dp, exceto onde indicado . Parmetros resultados espiromtricos vef1, l2,8 0,6vef1,% previsto87,9 11,8cvf, l3,4 0,8cvf,% previsto88,4 11,7vef1/cvf,% 83,1 6,4fef25 - 75/cvf,% 99,5 30,2ecocardiogrficos ae, mm36,2 4,1es, mm11,6 4,4eppve, mm10,7 2,3erppve0,2 0,1ddve, mm48,2 4,1dsve, mm29,6 3,7vdve, ml108,9 21,7vsve, ml34,9 11,2mve, g248,3 84,9mve, g / m 106,7 30,4mve, g / m 92,1 26,1ae: trio esquerdo; es: espessura do septo; eppve: espessura da parede posterior do ventrculo esquerdo; erppve: espessura relativa da parede posterior do ventrculo esquerdo; ddve: dimetro diastlico final do ventrculo esquerdo; dsve: dimetro sistlico final do ventrculo esquerdo; vdve: volume diastlico final do ventrculo esquerdo; vsve: volume sistlico final do ventrculo esquerdo; e mve: massa ventricular esquerda . Ae: trio esquerdo; es: espessura do septo; eppve: espessura da parede posterior do ventrculo esquerdo; erppve: espessura relativa da parede posterior do ventrculo esquerdo; ddve: dimetro diastlico final do ventrculo esquerdo; dsve: dimetro sistlico final do ventrculo esquerdo; vdve: volume diastlico final do ventrculo esquerdo; vsve: volume sistlico final do ventrculo esquerdo; e mve: massa ventricular esquerda . As entre as correlaes significativas, destacamos a relao direta e fraca entre o imc e as variveis ddve (r = 0,359; p <0,05), dsve (r = 0,387; p <0,05), vdve (r = 0,387; p <0,05) e vsve (r = 0,425; p <0,01). A fef25 - 75/cvf,% apresentou correlao inversa e moderada com as variveis de remodelamento de ve (tabela 3) na anlise univariada, mantendo a significncia estatstica mesmo aps ajuste para peso, sexo e has, para eppve (r = 0,355; p <0,05) e erppve (r = 0,349; p <0,05; figuras 1a e 1b, respectivamente). A correlao entre mve indexada (em g / m e fef25 - 75/cvf,% foi limtrofe para significncia estatstica (p = 0,05; figura 1c). De modo interessante, a fef25 - 75/cvf,% no se correlacionou com as variveis de dimetro interno e de volume de ve . O fef25 - 75% no apresentou correlao com qualquer varivel ecocardiogrfica . O teste exato de fischer no mostrou associao estatisticamente significativa entre o valor de fef25 - 75/cvf,% abaixo ou acima do limite inferior de normalidade e relato de asma ou tabagismo (p> 0,05 para ambos). Tabela 3.correlaes univariadas das variveis antropomtricas e espiromtricas com as variveis de ecocardiografia transtorcica (modo m) na amostra total (n = 39). Parmetros hipertrofia de ve dimetro / volume de ve mve, g mve, g / m mve, g / m es, mm eppve, mm erppve ae, mm ddve, mm dsve, mm vdve, ml vsve, ml antropomtricos imc, kg / m 0,1750,1920,2270,0780,0920,0460,0690,359 * 0,387 * 0,387 * 0,425 peso, kg0,476 0,2930,3120,1600,360 * 0,1690,380 * 0,447 0,476 0,455 0,556 espiromtricos vef1, l0,590 0,388 * 0,401 * 0,1620,389 * 0,2120,473 0,406 0,388 * 0,386 * 0,488 vef1,% previsto0,1470,2320,1540,1100,0290,1020,0150,2170,1960,2380,162cvf, l0,584 0,346 * 0,380 * 0,1640,429 0,2640,513 0,365 * 0,3150,366 * 0,418 cvf,% previsto0,0870,1590,2560,0710,0170,0610,0190,1330,0480,1940,038vef1/cvf,% 0,0030,0770,0400,0020,1140,1450,0820,1180,1970,0470,167fef25 - 75/cvf,% 0,3970,2750,3180,0540,453 0,404 0,1680,0090,0410,0570,020ve: ventrculo esquerdo; mve: massa ventricular esquerda; es: espessura do septo; eppve: espessura da parede posterior do ve; erppve: espessura relativa da parede posterior do ve; ae: trio esquerdo; ddve: dimetro diastlico final de ve; dsve: dimetro sistlico final de ve; vdve: volume diastlico final de ve; e vsve: volume sistlico final de ve . ve: ventrculo esquerdo; mve: massa ventricular esquerda; es: espessura do septo; eppve: espessura da parede posterior do ve; erppve: espessura relativa da parede posterior do ve; ae: trio esquerdo; ddve: dimetro diastlico final de ve; dsve: dimetro sistlico final de ve; vdve: volume diastlico final de ve; e vsve: volume sistlico final de ve . * figura 1.correlao de fef25 - 75/cvf,%, ajustada para as variveis peso, sexo e hipertenso arterial sistmica, com a espessura da parede posterior do ventrculo esquerdo, em a; com a espessura relativa da parede posterior do ventrculo esquerdo, em b; e com a massa ventricular esquerda ajustada para o tamanho corporal em obesos, em c. o modelo de regresso linear mltipla stepwise estudado (tabela 4) mostrou que a variao na mve entre obesos mrbidos mais bem prevista pela cvf (em l), que explicou 36,9% (p <0,0001) de sua variao na populao estudada . A mve indexada para altura ao quadrado e a mve indexada para a potncia de 1,7 tiveram no vef1 (em l / min) o melhor preditor (p <0,05 para ambos; tabela 4). Tabela 4.regresso linear mltipla stepwise para a varivel dependente massa do ventrculo esquerdo (em gramas ou indexada para rea de superfcie corporal; n = 39). Varivel dependente varivel preditora beta ic95% r ajustado p mve, gcvf, l0,600,43 - 1,130,369 <0,001mve, g / m vef1, l / min0,380,29 - 2,80,126 <0,05mve, g / m vef1, l / min0,400,2 - 1,30,161 <0,05mve: massa ventricular esquerda . O presente estudo retrospectivo mostra uma associao independente entre o tamanho relativo das pequenas vias areas (fef25 - 75/cvf,%) e parmetros ecocardiogrficos de hipertrofia ventricular em obesos mrbidos . L) e o vef1 (em l / min) se mostraram importantes preditores da mve em gramas ou indexada para rea de superfcie corporal . A mve e os dimetros internos de ve esto aumentados na obesidade, independentemente de has . 17 nosso estudo no mostrou relaes do imc com a mve em gramas ou indexada para rea de superfcie corporal nem com a eppve, mas mostrou uma relao do imc com os dimetros internos e os volumes de ve, concordante com a literatura, sendo provvel que essa associao positiva ocorra por conta do aumento da pr - carga e do dbito cardaco, causando dilatao ventricular, que posteriormente poderia evoluir para o remodelamento do ve . 18 o imc e resultados espiromtricos mostram associaes inconsistentes na literatura, ora mostrando associao, 19 ora no demonstrando associao, 20 e isso ocorre provavelmente pelas diferenas na proporo entre sexos nos estudos, pois a obesidade do tipo androide favorece a correlao entre imc e a funo pulmonar, ao contrrio do fentipo ginecoide, que no acumula excessiva massa gordurosa no trax . Nosso estudo apresentou uma predominncia do sexo feminino (74%), o que explica em parte a ausncia de correlao entre qualquer varivel de espirometria e o imc . As relaes entre cvf, vef1 e fef25 - 75% com mve e eppve foram estudadas em cardiopatas idosos (no obesos) e so discordantes, em estudos realizados, no sentido da direo da correlao, sendo positivas 21 ou negativas, 22 o que reflete muito mais uma perda da funo pulmonar associada idade avanada e aos efeitos de has e de hipertenso pulmonar, assim como aos efeitos restritivos pulmonares da cardiomegalia . Um estudo 4 mostrou uma relao inversa entre mve e cvf em mulheres no fumantes e uma relao direta para as mesmas variveis em homens com menos de 60 anos e no fumantes . Considerando que a maioria de nossa amostra de mulheres (74%), isso sugere que, em obesos mrbidos, algumas variveis de funo pulmonar podem ter direo da correlao diferente . A fef25 - 75/cvf,% uma medida que corrige parcialmente a grande variabilidade encontrada no fef25 - 75% isolado e reflete alteraes predominantemente de pequenas vias areas, ajustada ao tamanho pulmonar relativo cvf . 17 as correlaes da fef25 - 75/cvf,% com a mve indexada para rea de superfcie corporal (em g / m e com eppve / erppve, mantidas aps ajuste para peso, sexo e has, possivelmente refletem efeitos mecnicos diretos da obesidade, mas tambm podem sugerir que existam outros mecanismos independentes (inflamatrios ou lipotxicos). Por ser pouco estudado, h limitadas evidncias de que as pequenas vias areas so independentemente afetadas pela obesidade, como relatado em homens no tabagistas.as hipteses levantadas naquele estudo 23 passam pelo aumento da volemia em obesos, causando congesto dos vasos brnquicos, pela a presena de lipoprotenas de muito baixo peso em nveis aumentados, o que poderia deflagrar liberao de histamina, e pelo metabolismo alterado lipoproteico na obesidade, que poderia instigar e amplificar esses efeitos . Dados recentes da literatura indicam tambm que a obesidade caracterizada por hiper - responsividade metacolina, predominantemente nas pequenas vias areas, sendo essa melhor correlacionada com a fef25 - 75/cvf,% .sobre esse aspecto, um estudo recente sugere que grupos de obesos com hiper - responsividade esto associados a maior mve.a hiper - responsividade das pequenas vias areas em obesos poderia ser parcialmente explicada tambm pela dysanapsis (avaliada indiretamente pela fef25 - 75/cvf,%), termo cunhado por green et al.para explicar a grande variabilidade interindividual no tamanho das vias areas, independente do tamanho do parnquima pulmonar . Importante fator preditor da mve, o imc correlacionou - se, em vrios estudos, diretamente com o tamanho e a massa cardaca, embora a massa magra ainda seja uma melhor preditora da mve.o presente estudo retrospectivo no mostrou correlaes entre imc e mve (em gramas ou indexada para rea de superfcie corporal), corroborando outro estudo, e isso possivelmente ocorra devido a diferenas de fentipos de obesidade, prevalncia de has e nmero de indivduos estudados . A destacar em nosso estudo foi o fato de que a cvf (em (em g), explicando 37% da variao da mesma na populao estudada, sugerindo que a reduo dos volumes pulmonares pode ser uma varivel importante para futuros estudos no estabelecimento de um modelo preditor de mve em obesos . A mve, por sua vez, se correlaciona com a morbidade e a mortalidade cardiovasculares . Entre os fatores limitantes de nosso estudo est o fato de que nossa amostra foi pequena, composta por pacientes candidatos cirurgia baritrica, limitada pelo critrio de incluir somente obesos mrbidos e superobesos e baseada em critrios no bem estabelecidos nos pronturios, como o diagnstico de asma ou has . Alm disso, dados referentes a diabetes no foram coletados, embora sua relao com mve seja inconsistente na literatura . Outras limitaes importantes foram a janela acstica limitada na anlise das variveis do ecocardiograma no modo m em obesos e a falta de um protocolo especfico e padronizado na realizao do mesmo, pois no obtivemos dados de concordncia interexaminadores para os dois ecocardiografistas . Nesse pormenor, por se tratar de estudo retrospectivo, buscamos no utilizar dados ecocardiogrficos que perdem acurcia mais intensamente pelos efeitos da obesidade sobre a janela acstica, como a frao de ejeo e dados de ecodopplercardiograma . Conclumos, portanto, que as pequenas vias areas em obesos mrbidos tm uma correlao com a hipertrofia cardaca, independente das variveis antropomtricas usuais, sexo e has . O presente estudo revela que outros fatores, alm de fatores mecnicos e / ou hemodinmicos impostos pela massa corporal aumentada, podem ser importantes nas alteraes conjuntas de pequenas vias areas e hipertrofia cardaca . Alm disso, outros estudos so necessrios para verificar qual o impacto dos parmetros de funo pulmonar nas equaes preditivas de mve em obesos.
This was a retrospective study of 18 patients with inflammatory myofibroblastic tumors, diagnosed in the department of general pathology during the period of january 2011december 2015 . 10200). The clinical details and follow - up information of each patient were noted from the clinical work station . All classical tumors fulfilled the morphologic criteria described in the who classification of tumours of soft tissue and bone . Atypical cases were defined by increased cellularity, cellular atypia with large ganglion - like round to polygonal cells, multinucleated or anaplastic giant cells, atypical mitosis and necrosis . The archival tissues obtained from institutional and consultation files were fixed in 10% buffered neutral formalin and paraffin - embedded tissue blocks were prepared . Ihc slides and h&e slides prepared from formalin fixed, paraffin embedded tissue blocks were reviewed . Ihcs had been done on ventana benchmark xt (ventana medical systems, tucson, az, usa). The stain was considered to be positive if the tumor cells showed specific cytoplasmic and/or nuclear membrane staining for the particular antibody . The immunohistochemical stains were evaluated semiquantitatively as follows: 0, negative; 1 +, <10% of cells positive; 2 +, 10%50% of cells positive; and 3 +,> 50% of cells positive . Intra - abdominal tumors with epithelioid morphology, nuclear or perinuclear accentuation of alk immunopositivity and aggressive clinical behavior were categorized as epithelioid inflammatory myofibroblastic sarcoma . Ihc slides and h&e slides prepared from formalin fixed, paraffin embedded tissue blocks were reviewed . Ihcs had been done on ventana benchmark xt (ventana medical systems, tucson, az, usa). The stain was considered to be positive if the tumor cells showed specific cytoplasmic and/or nuclear membrane staining for the particular antibody . The immunohistochemical stains were evaluated semiquantitatively as follows: 0, negative; 1 +, <10% of cells positive; 2 +, 10%50% of cells positive; and 3 +,> 50% of cells positive . Intra - abdominal tumors with epithelioid morphology, nuclear or perinuclear accentuation of alk immunopositivity and aggressive clinical behavior were categorized as epithelioid inflammatory myofibroblastic sarcoma . A total of 18 cases of inflammatory myofibroblastic tumors were found during the study period . Of the 18 cases, 17 were institutional cases and one was a consultation case (case 2) from outside hospital . There were nine females and nine males with female to male ratio of 1:1 and the mean age was 23.8 years (range, 3 to 44 years). The most common locations were lung (six cases) and intra - abdominal region (six cases) followed by suprarenal, mastoid antrum, dorsum of nose, mediastinum, sphenoid wing, and breast (one case each). All the patients underwent surgical excision and a few patients had combination treatments: surgery plus chemotherapy or chemoradiotherapy . Limited follow - up information was available, ranging from 3 to 65 months (mean, 16.1 months). The tumor size ranged from 1 to 15 cm (mean, 5.6 cm). 1) exhibiting soft to firm variegated appearance with grey white to tan fleshy and focal mucoid areas . One case showed variable hemorrhage and necrosis on the cut surface (case 1). Three cases (cases 13, 15, and 18) had multifocal appearance of tumor (multicentric in origin) in various anatomic locations (table 2). Histologically, among the 18 inflammatory myofibroblastic tumors (including recurrent and metastatic cases), 14 cases had classic histologic features (fig . 2) and four cases had atypical histologic features, revealing diffuse and focal cytoplasmic ihc expression of alk-1 (fig . 3). Classification of histological patterns was done for all the cases according to the dominant tumor area, which included cellular (77.8%), myxoid (11.1%), and fibrous (11.1%) types . All the classical inflammatory myofibroblastic tumors displayed a bland spindle cell proliferation admixed with an inflammatory infiltrate composed of lymphocytes, plasma cells, and eosinophils . The atypical histologic features included increased cellularity, cellular atypia with large ganglion - like round to polygonal cells, multinucleated or anaplastic giant cells, atypical mitosis, and necrosis . Among the atypical cases, one case (case 1) showed dominant epithelioid morphology with focally ganglion like cells, hypercellularity, atypia, mitosis, and prominent nucleoli (fig . 4); one case (case 13) showed dominant spindle cell morphology and focal epithelioid morphology (fig . 5); remaining two cases (cases 2 and 6) showed atypical spindle cell morphology (fig . 6). Among the atypical cases, three cases had recurrence (cases 1, 6, and 13) and two cases had metastasis to the lung, liver (case 1), and pelvic bone (case 15). Spindled tumor cells of inflammatory myofibroblastic tumors showed cytoplasmic or membrane positivity for smooth muscle actin (12/16 cases), muscle - specific actin (3/6 cases), vimentin (8/8 cases), desmin (5/11 cases), and the spindled tumor cells were negative for cytokeratin, myogenin, cd34, cd21, cd117, h - caldesmon, tle-1, cd30, and cd15 (table 3). Immunohistochemical stain for alk-1 expression was available in all the 18 cases, demonstrating cytoplasmic reactivity of spindled or polygonal tumor cells for alk-1 in 10 cases (55.6%) and no reactivity in eight cases (44.4%) (tables 3, 4). Diffuse (3 +) cytoplasmic staining was observed in six cases and focal (2 +) cytoplasmic staining in three cases . An intra - abdominal inflammatory myofibroblastic tumor with dominant epithelioid morphology (case 1) revealed perinuclear accentuation of alk-1 immunoexpression, suggestive of epithelioid inflammatory myofibroblastic sarcoma (fig . 4); the remaining atypical cases with spindle cell morphology (including case 13 with focal epithelioid morphology) were negative for alk-1, suggestive of atypical inflammatory myofibroblastic tumors only (figs . 5, 6). Alk-1 positive tumors (55.6%) included five females and five males and the mean age was 23.8 years (range, 3 to 44 years), compared with the mean age of (four females and four males) 27.1 years (range, 18 to 41 years) for alk-1 negative cases . Recurrence was noted in 3/10 alk-1 positive (30%) and 3/8 alk-1 negative (37.5%) cases, whereas metastasis to the lung, liver and pelvic bone was noted in the alk-1 positive group (tables 4, 5). Alk-1 immunopositivity in neither of the classical (p = .275), atypical (p = .275) nor multifocal tumor (p>.99) types was statistically significant at 5% level of significance with fisher exact test . There was no statistically significant correlation between alk-1 expression and either of the recurrence (p>.99) or metastasis (p = .485) of inflammatory myofibroblastic tumors . The tumor size ranged from 1 to 15 cm (mean, 5.6 cm). 1) exhibiting soft to firm variegated appearance with grey white to tan fleshy and focal mucoid areas . One case showed variable hemorrhage and necrosis on the cut surface (case 1). Three cases (cases 13, 15, and 18) had multifocal appearance of tumor (multicentric in origin) in various anatomic locations (table 2). Histologically, among the 18 inflammatory myofibroblastic tumors (including recurrent and metastatic cases), 14 cases had classic histologic features (fig . 2) and four cases had atypical histologic features, revealing diffuse and focal cytoplasmic ihc expression of alk-1 (fig . 3). Classification of histological patterns was done for all the cases according to the dominant tumor area, which included cellular (77.8%), myxoid (11.1%), and fibrous (11.1%) types . All the classical inflammatory myofibroblastic tumors displayed a bland spindle cell proliferation admixed with an inflammatory infiltrate composed of lymphocytes, plasma cells, and eosinophils . The atypical histologic features included increased cellularity, cellular atypia with large ganglion - like round to polygonal cells, multinucleated or anaplastic giant cells, atypical mitosis, and necrosis . Among the atypical cases, one case (case 1) showed dominant epithelioid morphology with focally ganglion like cells, hypercellularity, atypia, mitosis, and prominent nucleoli (fig . 4); one case (case 13) showed dominant spindle cell morphology and focal epithelioid morphology (fig . 5); remaining two cases (cases 2 and 6) showed atypical spindle cell morphology (fig . 6). Among the atypical cases, three cases had recurrence (cases 1, 6, and 13) and two cases had metastasis to the lung, liver (case 1), and pelvic bone (case 15). Spindled tumor cells of inflammatory myofibroblastic tumors showed cytoplasmic or membrane positivity for smooth muscle actin (12/16 cases), muscle - specific actin (3/6 cases), vimentin (8/8 cases), desmin (5/11 cases), and epithelial membrane antigen (2/4 cases). The spindled tumor cells were negative for cytokeratin, myogenin, cd34, cd21, cd117, h - caldesmon, tle-1, cd30, and cd15 (table 3). Immunohistochemical stain for alk-1 expression was available in all the 18 cases, demonstrating cytoplasmic reactivity of spindled or polygonal tumor cells for alk-1 in 10 cases (55.6%) and no reactivity in eight cases (44.4%) (tables 3, 4). Diffuse (3 +) cytoplasmic staining was observed in six cases and focal (2 +) cytoplasmic staining in three cases . An intra - abdominal inflammatory myofibroblastic tumor with dominant epithelioid morphology (case 1) revealed perinuclear accentuation of alk-1 immunoexpression, suggestive of epithelioid inflammatory myofibroblastic sarcoma (fig . 4); the remaining atypical cases with spindle cell morphology (including case 13 with focal epithelioid morphology) were negative for alk-1, suggestive of atypical inflammatory myofibroblastic tumors only (figs . 5, 6). Alk-1 positive tumors (55.6%) included five females and five males and the mean age was 23.8 years (range, 3 to 44 years), compared with the mean age of (four females and four males) 27.1 years (range, 18 to 41 years) for alk-1 negative cases . Recurrence was noted in 3/10 alk-1 positive (30%) and 3/8 alk-1 negative (37.5%) cases, whereas metastasis to the lung, liver and pelvic bone was noted in the alk-1 positive group (tables 4, 5). Alk-1 immunopositivity in neither of the classical (p = .275), atypical (p = .275) nor multifocal tumor (p>.99) types was statistically significant at 5% level of significance with fisher exact test . There was no statistically significant correlation between alk-1 expression and either of the recurrence (p>.99) or metastasis (p = .485) of inflammatory myofibroblastic tumors . Inflammatory myofibroblastic tumors formerly described as inflammatory pseudotumor, with an unpredictable behavior ranging from benign to low - grade malignant, are classified as intermediate grade tumors, with potential for recurrence and rare metastasis, in the current who classification of tumours of soft tissue and bone . These tumors are described in children and young adults in various anatomic sites with a tendency towards local recurrence in about 25% of abdominopelvic lesions and a few cases of inflammatory myofibroblastic tumors may appear morphologically benign but the biological nature may be aggressive even when the histologic appearance is bland . These tumors were considered as a neoplasm after the discovery of clonal rearrangement of the alk gene on the short arm of chromosome 2 at 2p23 in 50%75% of extrapulmonary inflammatory myofibroblastic tumors . In a study by wang et al ., the most common site of inflammatory myofibroblastic tumors was abdominopelvic region (73.9%) followed by other locations . The present study included 33.3% each of intrapulmonary and intra - abdominal locations followed by other locations . There was slight male predominance in the literature; however, there was an equal incidence in each gender in the present study . Hussong et al . In 1999 stated that the combination of cellular atypia, presence of ganglionlike cells, p53 expression and dna aneuploidy are all possible predictors to identify the malignant change and aggressive behavior of inflammatory myofibroblastic tumors . In a study by coffin et al . The atypical features in his study were increased cellularity, cellular and nuclear pleomorphism, distinct fascicular and focal herringbone pattern, cellular atypia with large ganglion - like round to polygonal cells, multinucleated or anaplastic giant cells, atypical mitosis, and necrosis . Alk immunopositivity was noted in 78% atypical, 54% recurrent and 60% classical inflammatory myofibroblastic tumors . Metastatic inflammatory myofibroblastic tumors (10.1%) were negative for alk expression . In another study, there were 60% classical and 40% atypical inflammatory myofibroblastic tumors . Twenty - five percent of alk - positive and 63.6% of alk - negative inflammatory myofibroblastic tumors revealed recurrence . Statistically significant correlation was found between atypia, recurrence and metastasis . There was a significant correlation between alk expression and recurrence . Overall alk-1 immunopositivity was noted in 55.6% and negativity in 44.4% of inflammatory myofibroblastic tumors . There was no statistically significant correlation between alk-1 expression, atypia, recurrence and metastasis in the present study compared to the other studies, probably due to a small sample size and limited follow up data; a larger study may be taken up in the future to evaluate the statistical significance . Marino - enriquez et al . Proposed a term epithelioid inflammatory myofibroblastic sarcoma for epithelioid variant of intraabdominal inflammatory myofibroblastic tumors with a nuclear membrane or perinuclear pattern of alk expression and aggressive clinical behavior . In the present study, one patient with an intra abdominal mass, which was categorized as inflammatory myofibroblastic sarcoma, with epithelioid morphology and perinuclear accentuation of alk-1 expression, developed liver and lung metastasis and expired within 8 months . Alk gene rearrangement was demonstrated in all the tested cases of epithelioid variant of inflammatory myofibroblastic tumors by fluorescence in situ hybridization (fish). Multifocality of pulmonary and extrapulmonary pseudotumors had a high chance of recurrence compared with tumors confined to a single organ . Intraabdominal pseudotumors were associated with> 75% of the recurrences when compared to other extrapulmonary pseudotumors . In the present study study by chaudhary pointed out that alk gene rearrangements were often seen in inflammatory myofibroblastic tumors of children and young adults compared to adults over 40 years of age . The search for alk gene rearrangement by fish is a useful tool for the diagnosis of inflammatory myofibroblastic tumors . Reported a case of inflammatory pseudotumor of the head and neck mimicking a metastatic disease in a human immunodeficiency virus positive 49-year - old female . In immunocompromised patients, alk protein immunoexpression was detected in various other tumors including lipomatous tumors, rhabdomyosarcomas, ewing s sarcoma / primitive neuroectodermal tumors, malignant fibrous histiocytomas, and leiomyosarcomas . Except inflammatory myofibroblastic tumors, almost all other tumors displayed low level of alk expression . The differential diagnosis of these tumors includes both benign and malignant spindle or epithelioid cell tumors . The spindle cell tumors include fibroblastic / myofibroblastic tumors, fibrous histiocytoma, and solitary fibrous tumor . The tumors with epithelioid or round cell morphology include anaplastic large cell lymphoma, epithelioid leiomyosarcoma, rhabdomyosarcoma, undifferentiated sarcoma, myxoid / round cell liposarcoma, myxofibrosarcoma, dedifferentiated liposarcoma, poorly differentiated carcinoma, malignant melanoma, and epithelioid gastrointestinal stromal tumor . Anaplastic large cell lymphoma can be differentiated from epithelioid variant of inflammatory myofibroblastic sarcoma by immunoreactivity for cd30, cd3, and cd43, diffuse membranous or cytoplasmic staining of alk-1 and absence of ranbp2-alk rearrangement . High - grade leiomyosarcoma usually reveals fascicles of spindle and epithelioid cells with increased cellularity, cellular atypia, pleomorphism, cigar - shaped nuclei and abundant brightly eosinophilic cytoplasm . Alveolar rhabdomyosarcoma shows uniform round cells with scant cytoplasm and usually lacks myxoid background and inflammatory infiltrates . Dedifferentiated liposarcoma can have features of inflammatory myofibroblastic tumor, but with expression of mdm2 and absence of alk-1 . Epithelioid malignant peripheral nerve sheath tumor will be strongly and diffusely positive for s100 and negative for alk-1 and also smarcb1 . Malignant melanoma shows polygonal cells with prominent nucleoli and the cells are positive for s100 and human melanoma black 45 . Epithelioid gastrointestinal stromal tumors reveal immunoexpression of cd117, discovered on gist-1 (dog1) and negative for alk-1 [3 - 5]. Nsaids, steroids, chemotherapy, and radiotherapy have been used as adjuvant therapy when feasible . In a study by jacob et al ., systemic (liver and lumbar spine) involvement of inflammatory myofibroblastic tumor in a 45-year - old female, expressing alk gene rearrangement, responded very well to alk inhibitor (crizotinib). In a series of cases by butrynski et al ., multifocal intra - abdominal inflammatory myofibroblastic tumors which expressed alk gene rearrangement, were treated successfully by alk inhibitor (crizotinib). Fragoso et al . In 2011 stated that, although surgical resection is the preferred treatment for inflammatory myofibroblastic tumors, cases which are not amenable to surgical excision may be treated conservatively . In conclusion, this study demonstrates that inflammatory myofibroblastic tumors exhibit a wide age group distribution with an equal incidence in males and females . Most of the tumors with histological atypia were associated with recurrence, irrespective of alk-1 expression . Epithelioid inflammatory myofibroblastic sarcoma with perinuclear accentuation of alk-1 showed aggressive clinical course with metastasis and mortality . Metastasis was seen in alk-1 positive tumors . However, there was no statistically significant correlation between alk-1 expression, tumor type, recurrence, and metastasis . Overall, alk-1 ihc is a useful diagnostic aid in the appropriate histomorphologic context, especially in identifying epithelioid inflammatory myofibroblastic sarcomas . There were no definite histopathologic criteria to predict malignant transformation, recurrence, or metastasis . However, this study is limited by the very small number of cases, limited follow - up data and also the lack of identification of alk gene rearrangement by fish . A search for newer molecular tools by larger studies needs to be performed to identify prognostic features to guide treatment and predict the outcome.
Von ilberg et al . (1999) showed that it is possible to combine the electrical stimulation of a cochlear implant (ci) device with the acoustic stimulation of a hearing aid (ha). Initially, this technique was used for patients with pure - tone thresholds of more than 60 db in the low frequencies, but it is currently possible for partially deaf patients with normal low - frequency hearing (lfh) to receive implants . Patients with residual hearing benefit considerably from cis alone compared with conventional has (cullen et al . The advantages of hybrid hearing compared with ci only have been demonstrated by von ilberg et al . 2006, 2007) and hearing in complex listening situations (gifford et al . 2013) seems to improve to a greater extent with hybrid hearing than with conventional ci . To date, only a few studies have investigated the quality of life and subjective ratings from hybrid hearing patients (lenarz et al . This procedure minimizes the trauma to the low frequency region in the apical portion the cochlea . 2007), whereas other studies have reported stable hearing in the operated ear (skarzynski et al . 2007; lenarz et al . 2009). Nevertheless, the follow - up periods after surgery of these studies have generally been short . The first category includes those with natural lfh who are not supposed to need amplification . The second category includes people with lfh requiring ha amplification . Both categories of people will, if their lfh is preserved, use ci hearing in the middle and high frequencies, which is called cut off frequency ci (the stimulation is in the range of approximately 5008500 hz). The devices used by these patients will be switched to include lfh and are called conventional cis or full frequency cis (1258500 hz). After surgery, depending on the extent of surgery - induced hearing loss, the patients who are intended for hybrid hearing can be divided into the following three groups: a natural lfh with cut off frequency ci group, a ha - amplified lfh with cut off frequency ci group, and a full frequency conventional ci group . Only the two first groups are hybrid - hearing patients . It can be assumed that hybrid - hearing patients with natural lfh have better quality hearing than hybrid - hearing patients with ha - amplified lfh . 2012) called the first group electrical complement patients and the second group electro - acoustic stimulation patients . The aims of this study were as follows: evaluate patient satisfaction among patients intended for hybrid hearing and the relationship of patient satisfaction with hybrid hearing; relate hearing results in quiet and noisy conditions to subjective experiences and residual hearing; evaluate whether ongoing deterioration of residual hearing occurred in the hybrid - hearing patients . The study group consisted of 19 patients who were intended for hybrid hearing (table 1). The ages at implantation ranged from 10 to 82 years and the study group consisted of 10 female and nine male patients . Age, sex, age of hearing loss, surgery ear, date of activation, mode of fitting, and usage of contralateral hearing aid from september 2008 to november 2011, the first 24 consecutive, partially deaf patients intended for hybrid hearing underwent hearing preservation surgery in our department . The preoperative candidacy criteria were an unaided pure - tone threshold 65 db hl at frequencies 500 hz and> 80 db hl at frequencies 2000 hz in the ear that was intended for surgery, nearly symmetrical hearing in the contralateral ear (i.e., the better ear), and aided monosyllabic word scores below 60% in each ear . In january 2013, after all patients had used their device for at least 1 year, the patients were asked to participate in the study . The study was approved by the regional ethical review board in uppsala (2012/473). The pure - tone audiograms of five of these patients have earlier been presented by erixon et al . The patient questionnaire survey consisted of the following three parts: the international outcome inventory for hearing aids - swedish (ioi - ha), which was translated into swedish in 2005 and provided by the international collegium of rehabilitative audiology . The ioi - ha is a questionnaire that targets seven different domains via questions about the following seven topics: (1) ha use, (2) ha benefit, (3) residual activity limitations, (4) satisfaction, (5) residual participation restriction, (6) effect on others, and (7) quality of life . Factor 1 is the sum of items 1, 2, 4, and 7 and represents ci satisfaction, and factor 2 is the sum of items 3, 5, and 6 and represents participation restriction (kramer et al . The questionnaire has been validated and was initially developed for the assessment of ha outcomes (cox et al . 2000, 2003). This instrument consists of two parts, and we used the part that involved a visual analog scale with endpoints that are labeled (100 points) and worst imaginable health state (0 points). This survey was added to scrutinize residual hearing and increase the relevance of the results across different listening situations, such as listening in noisy condition and listening to music . Interested readers can access the nine - question survey in the appendix, supplemental digital content (http://links.lww.com/eandh/a188). Speech discrimination tests involving phonemically balanced monosyllabic words (ms) were performed with and without the has uni- and bilaterally at a comfortable level (6585 db sound pressure level; svensk talaudiometri, c - a tegnr ab: 1998). The hearing in noise test (hint), a speech recognition test in which everyday sentences are presented with noise, was performed for patients with sufficient hearing (hllgren et al ., we evaluated the pure - tone audiograms and best - aided ms and hint scores . A round window approach was used, and the electrode was inserted slowly in steps that were interrupted by instillations of corticosteroids into the middle ear (kenacort - t or triamcinolone solution, 40 mg / ml). A 24-mm long flexible electrode (med - el flexeas) was used in all cases with the exception of one patient who received a custom - made electrode (20 mm) from med - el . One month after surgery, a postoperative audiogram was taken, and the patients were fitted with ci processors (opus 2), and if needed, most of the patients were fitted with an ipsilateral ha at the same time (duet 2). The patients visited our clinic on 4 days during the first 2 weeks for adjustment and training and then visited at 5 weeks and 3, 6, and 12 months after the fittings . Group means and alterations in pure - tone thresholds, ms, hint, and patient satisfaction scores were assessed . The parameters were plotted, and the correlations were evaluated using excel (microsoft). The levels of significance (i.e., p values) were calculated using wilcoxon s matched pairs signed rank test . Speech discrimination tests involving phonemically balanced monosyllabic words (ms) were performed with and without the has uni- and bilaterally at a comfortable level (6585 db sound pressure level; svensk talaudiometri, c - a tegnr ab: 1998). The hearing in noise test (hint), a speech recognition test in which everyday sentences are presented with noise, was performed for patients with sufficient hearing (hllgren et al ., we evaluated the pure - tone audiograms and best - aided ms and hint scores . A round window approach was used, and the electrode was inserted slowly in steps that were interrupted by instillations of corticosteroids into the middle ear (kenacort - t or triamcinolone solution, 40 mg / ml). A 24-mm long flexible electrode (med - el flexeas) was used in all cases with the exception of one patient who received a custom - made electrode (20 mm) from med - el . One month after surgery, a postoperative audiogram was taken, and the patients were fitted with ci processors (opus 2), and if needed, most of the patients were fitted with an ipsilateral ha at the same time (duet 2). The patients visited our clinic on 4 days during the first 2 weeks for adjustment and training and then visited at 5 weeks and 3, 6, and 12 months after the fittings . Group means and alterations in pure - tone thresholds, ms, hint, and patient satisfaction scores were assessed . The parameters were plotted, and the correlations were evaluated using excel (microsoft). The levels of significance (i.e., p values) were calculated using wilcoxon s matched pairs signed rank test . Before surgery, seven patients were intended to use unamplified lfh and a ci, and 12 patients were intended to use an ipsilateral ha and a ci . At the time of the patient survey, six patients were using unamplified lfh and a ci . Nine patients were using amplified lfh and a ci, and four patients were using a conventional full - frequency ci (table 1). There were no surgical complications and no persistent increases in tinnitus or dizziness; however, one patient suffered a persistent loss of chorda tympani function . The mean ioi - ha score was 29 out of the possible 35 (sd 3.4, range 2235). All items are presented in table 4 separately for the hybrid hearing and nonhybrid hearing groups . Low - frequency hearing losses in the operated and contralateral ears after surgery (125, 250, and 500 hz) patient satisfaction, experience, and speech discrimination separately for the patients with natural low - frequency hearing, the patients with hearing aids in the low frequencies and the patients with a conventional ci the groups exhibited nearly identical results on all questions with the exception of question number 5 (residual activity limitations) for which the nonhybrid patients indicated greater limitations . The eq-5d visual analog scale was not completed by two patients (table 3). The mean value across the 17 responders was 78 (sd = 18.9, range: 27100). The group with an ipsilateral ha scored 79 (sd = 20.2), and the nonhybrid group scored 67 (sd = 21.2). The difference between the hybrid and nonhybrid patients was not statistically significant (p = 0.51). According to the nine - question survey, 18 patients were very satisfied with their ci, and all of the patients responded that they would recommend a ci to a person in a situation similar to their own . One patient used the ci for 4 to 8 hr per day, and one patient used the ci for 1 to 4 hr per day . Table 3 illustrates the results of the hearing in silence, hearing in noisy conditions, and hearing music tests . . Comparison of the patients with useful lfh and the full - frequency stimulation group revealed a trend toward higher scores in the former group . Regarding hearing in noisy conditions, the hybrid - hearing patients scored 3.3 (sd = 0.9) compared with 2.5 (sd = 1.0) for the full - frequency stimulation group . A similar difference was found in the hearing in silence test (4 [sd = 0] compared with 3.5 [sd = 0.6], respectively). There were no differences in general satisfaction, time of use, contralateral ha use, or expected hearing . Eleven of the 15 hybrid - hearing patients claimed that music sounded good or very good . All the nonhybrid patients and four of the hybrid patients did not appreciate music . Two of four patients who used the full - frequency stimulation claimed that they experienced no benefit from their residual hearing . One patient did not respond, and one patient with a 50 db lf pta claimed a benefit . One child stated that a fog lifted from her ears and everything went clear when she heard the high frequencies for the first time in her life . All patients exhibited hearing levels within the limits of the audiometer (120 db) within the range of 125500 hz . We calculated the lf ptas at frequencies of 125, 250, and 500 hz . Preoperatively, the mean lf ptas were 31 db hl (sd = 17, min = -5, max = 67) in the ears that underwent surgery and 30 db hl (sd = 16, min = 0, max = 63) in the contralateral ears . The lf ptas of each patient before surgery and at the follow - up are presented in figure 1 . The mean changes of residual hearing at different frequencies after surgery are shown in figure 2 . There was significant (p <0.05) hearing loss in the operated ears 1 month after the surgeries . The contralateral ears displayed no significant loss during the first year but did exhibit a significant loss between the first and second year . International outcome inventory for hearing aids results the low - frequency pure - tone averages (125, 250, and 500 hz) in decibels are presented for each patient before the operation, 1 month postoperatively and 1 and 2 years after fitting . The mean changes in residual hearing for each frequency 1 month after surgery, 1 and 2 years after fitting, and after 3 years for eight patients . Eight of the patients attended a 3-year follow - up visit . Among these patients, the mean lf pta loss in the operated ear was -29 db (sd = 19, max = -8, min = -58), and the loss in the contralateral ear was -9 db (sd = 7, max = 2, min = -17) 3 years after fitting . Two years after fitting, the mean lf pta losses across these eight patients were -21 db (sd = 17, max = 3, min = -50) in the operated ear and -7 db (sd = 5, max = 2, min = -13) in the contralateral ear . Among these eight patients, there was a significant loss in the operated ear during the 3rd year after surgery . A similar deterioration in hearing did not occur in the contralateral ear . In two children lfh was normal (<20 db) up to 500 hz in one child and up to 750 hz in the other child . After 2 years, the lf pta losses in two children were 7 and 22 db . The child who experienced the 22 db loss also experienced a deterioration of 8 db in her contralateral ear . Before surgery, the patients displayed performances of 20% ms recognition (sd = 19, max = 58, min = 0) in the ear intended for surgery and 35% (sd = 17, max = 64, min = 6) in the other ear in their best - aided conditions . The mss were presented at a mean sound intensity of 72 db (sd = 5, max = 85, min = 65). Binaurally, the patients displayed 39% ms recognition (sd = 17, max = 70, min = 10) at the mean sound intensity of 70 db (sd = 4, max = 80, min = 65). At 1-year follow - up (data from two patients were missing), the patients displayed 52% ms recognition performance (sd = 19, max = 80, min = 12) in the implanted ear in their best - aided condition . The mss were presented at a mean sound intensity of 69 db (sd = 3, max = 75, min = 65). Binaurally, the patients (data from three patients were missing) displayed 63% ms recognition performance (sd = 16, max = 88, min = 34) at a mean sound intensity of 69 db (sd = 3, max = 75, min = 65). At the 2-year follow - up (the data from one patient was missing), the patients displayed 45% ms recognition performance (sd = 27, max = 82, min = 0) in the operated ear . The mss were presented at a mean sound intensity of 68 db (sd = 4.2, max = 80, min = 65). Binaurally, the patients (all patients) exhibited 58% ms recognition (sd = 17, max = 90, min = 22) at the mean sound intensity of 67 db (sd = 3, max = 75, min = 65). Nine patients were unable to perform the hint test before surgery due to poor hearing . Ten patients presented with a mean hint result of a 15 db signal to noise ratio (snr) in the binaural best - aided listening situation (sd = 4, max = 10, min = 22). In the ear that was intended for surgery (nine patients), the snr was 16 db (sd = 6, max = 21, min = 3). At 1-year follow - up (18 patients), the mean binaural snr was 4.2 db (sd = 4, max = -1.6, min = 10.6), and in the operated ear (14 patients), the snr was 6.0 db (sd = 5, max = 17, min = 0). The individual results after 1 year are illustrated in table 3 . In the nine patients with preoperative hint scores, the binaural gain in the snr was 11 db after 1 year . A mean gain of 11 db was observed in the operated ears of the five patients as measured monaurally . After 2 years, hint scores were assessed bilaterally in all of the patients, and the snr was 4.6 db (sd = 3.4, max = -1.7, min 12.= 5). For the operated ear alone (14 patients), the snr was 5.8 db (sd = 3, max = 11.5, min = 0). The two children did quite well in the silent condition before surgery (one performed an easier test designed for children). After surgery, the children s hearing was approximately the same as it was before surgery . On the hint test, the children exhibited a gain of 1112 db and presented with snrs of -1.6 and 0.1 db after 1 year . There were tendencies for the hybrid - hearing patients to experience better hearing and be more satisfied, but these trends were not statistically significant for any of the questions . Both the patients who scored the best and worst on the ms and hint were found in the hybrid - hearing group . The patients perceptions of improved hearing in silence and noise were not correlated with the postoperative ms or hint scores or with the ms or hint gains . All patients with a snr ratio below 1 db in the hint experienced better or much better hearing in noisy environments after implantation . The mean ioi - ha score was 29 out of the possible 35 (sd 3.4, range 2235). All items are presented in table 4 separately for the hybrid hearing and nonhybrid hearing groups . Low - frequency hearing losses in the operated and contralateral ears after surgery (125, 250, and 500 hz) patient satisfaction, experience, and speech discrimination separately for the patients with natural low - frequency hearing, the patients with hearing aids in the low frequencies and the patients with a conventional ci the groups exhibited nearly identical results on all questions with the exception of question number 5 (residual activity limitations) for which the nonhybrid patients indicated greater limitations . The eq-5d visual analog scale was not completed by two patients (table 3). The mean value across the 17 responders was 78 (sd = 18.9, range: 27100). The group with an ipsilateral ha scored 79 (sd = 20.2), and the nonhybrid group scored 67 (sd = 21.2). The difference between the hybrid and nonhybrid patients was not statistically significant (p = 0.51). According to the nine - question survey, 18 patients were very satisfied with their ci, and one patient was satisfied . All of the patients responded that they would recommend a ci to a person in a situation similar to their own . One patient used the ci for 4 to 8 hr per day, and one patient used the ci for 1 to 4 hr per day . Table 3 illustrates the results of the hearing in silence, hearing in noisy conditions, and hearing music tests . . Comparison of the patients with useful lfh and the full - frequency stimulation group revealed a trend toward higher scores in the former group . Regarding hearing in noisy conditions, the hybrid - hearing patients scored 3.3 (sd = 0.9) compared with 2.5 (sd = 1.0) for the full - frequency stimulation group . A similar difference was found in the hearing in silence test (4 [sd = 0] compared with 3.5 [sd = 0.6], respectively). There were no differences in general satisfaction, time of use, eleven of the 15 hybrid - hearing patients claimed that music sounded good or very good . All the nonhybrid patients and four of the hybrid patients did not appreciate music . Two of four patients who used the full - frequency stimulation claimed that they experienced no benefit from their residual hearing . One patient did not respond, and one patient with a 50 db lf pta claimed a benefit . One child stated that a fog lifted from her ears and everything went clear when she heard the high frequencies for the first time in her life . All patients exhibited hearing levels within the limits of the audiometer (120 db) within the range of 125500 hz . We calculated the lf ptas at frequencies of 125, 250, and 500 hz . Preoperatively, the mean lf ptas were 31 db hl (sd = 17, min = -5, max = 67) in the ears that underwent surgery and 30 db hl (sd = 16, min = 0, max = 63) in the contralateral ears . The lf ptas of each patient before surgery and at the follow - up are presented in figure 1 . The mean changes of residual hearing at different frequencies after surgery are shown in figure 2 . There was significant (p <0.05) hearing loss in the operated ears 1 month after the surgeries . The contralateral ears displayed no significant loss during the first year but did exhibit a significant loss between the first and second year . International outcome inventory for hearing aids results the low - frequency pure - tone averages (125, 250, and 500 hz) in decibels are presented for each patient before the operation, 1 month postoperatively and 1 and 2 years after fitting . The mean changes in residual hearing for each frequency 1 month after surgery, 1 and 2 years after fitting, and after 3 years for eight patients . Eight of the patients attended a 3-year follow - up visit . Among these patients, the mean lf pta loss in the operated ear was -29 db (sd = 19, max = -8, min = -58), and the loss in the contralateral ear was -9 db (sd = 7, max = 2, min = -17) 3 years after fitting . Two years after fitting, the mean lf pta losses across these eight patients were -21 db (sd = 17, max = 3, min = -50) in the operated ear and -7 db (sd = 5, max = 2, min = -13) in the contralateral ear . Among these eight patients, there was a significant loss in the operated ear during the 3rd year after surgery . A similar deterioration in hearing did not occur in the contralateral ear . In two children lfh was normal (<20 db) up to 500 hz in one child and up to 750 hz in the other child . After 2 years, the lf pta losses in two children were 7 and 22 db . The child who experienced the 22 db loss also experienced a deterioration of 8 db in her contralateral ear . Before surgery, the patients displayed performances of 20% ms recognition (sd = 19, max = 58, min = 0) in the ear intended for surgery and 35% (sd = 17, max = 64, min = 6) in the other ear in their best - aided conditions . The mss were presented at a mean sound intensity of 72 db (sd = 5, max = 85, min = 65). Binaurally, the patients displayed 39% ms recognition (sd = 17, max = 70, min = 10) at the mean sound intensity of 70 db (sd = 4, max = 80, min = 65). At 1-year follow - up (data from two patients were missing), the patients displayed 52% ms recognition performance (sd = 19, max = 80, min = 12) in the implanted ear in their best - aided condition . The mss were presented at a mean sound intensity of 69 db (sd = 3, max = 75, min = 65). Binaurally, the patients (data from three patients were missing) displayed 63% ms recognition performance (sd = 16, max = 88, min = 34) at a mean sound intensity of 69 db (sd = 3, max = 75, min = 65). At the 2-year follow - up (the data from one patient was missing), the patients displayed 45% ms recognition performance (sd = 27, max = 82, min = 0) in the operated ear . The mss were presented at a mean sound intensity of 68 db (sd = 4.2, max = 80, min = 65). Binaurally, the patients (all patients) exhibited 58% ms recognition (sd = 17, max = 90, min = 22) at the mean sound intensity of 67 db (sd = 3, max = 75, min = 65). Nine patients were unable to perform the hint test before surgery due to poor hearing . Ten patients presented with a mean hint result of a 15 db signal to noise ratio (snr) in the binaural best - aided listening situation (sd = 4, max = 10, min = 22). In the ear that was intended for surgery (nine patients), the snr was 16 db (sd = 6, max = 21, min = 3). At 1-year follow - up (18 patients), the mean binaural snr was 4.2 db (sd = 4, max = -1.6, min = 10.6), and in the operated ear (14 patients), the snr was 6.0 db (sd = 5, max = 17, min = 0). The individual results after 1 year are illustrated in table 3 . In the nine patients with preoperative hint scores, the binaural gain in the snr was 11 db after 1 year . A mean gain of 11 db was observed in the operated ears of the five patients as measured monaurally . After 2 years, hint scores were assessed bilaterally in all of the patients, and the snr was 4.6 db (sd = 3.4, max = -1.7, min 12.= 5). For the operated ear alone (14 patients), the snr was 5.8 db (sd = 3, max = 11.5, min = 0). The two children did quite well in the silent condition before surgery (one performed an easier test designed for children). After surgery, the children s hearing was approximately the same as it was before surgery . On the hint test, the children exhibited a gain of 1112 db and presented with snrs of -1.6 and 0.1 db after 1 year . There were tendencies for the hybrid - hearing patients to experience better hearing and be more satisfied, but these trends were not statistically significant for any of the questions . Both the patients who scored the best and worst on the ms and hint were found in the hybrid - hearing group . The patients perceptions of improved hearing in silence and noise were not correlated with the postoperative ms or hint scores or with the ms or hint gains . All patients with a snr ratio below 1 db in the hint experienced better or much better hearing in noisy environments after implantation . In this study, we evaluate patient satisfaction among patients intended for hybrid hearing and relate hearing results in quiet and noisy conditions to subjective experiences and residual hearing . Results showed that electric stimulation provides a major contribution to speech comprehension in partially deaf patients and that all of the patients were satisfied with their cis regardless of the variation in the extent of hearing preservation . All of our patients reported that they would recommend a ci to a person in a situation similar to theirs, and this is a major sign of patient satisfaction . The overall benefits in high - frequency hearing seemed to outweigh the possible benefits of preserved lfh . Because only four patients lost the possibility of hybrid hearing, the cohort was too limited to assess the possible advantages of hybrid hearing . A control group lacking usable lfh in the ipsilateral ear could not be obtained because the majority of conventional ci patients have profound bilateral hearing loss across all frequencies . Within - subject comparisons were not possible because earplugs and ear defenders are estimated to mask only approximately 30 db in the lower frequencies, which is insufficient when lfh is normal . Plugging of the operated ear was performed in a few patients to evaluate lfh when considering full - frequency ci stimulation or ha . (2013) performed within - subject comparisons and showed that hybrid hearing is superior to nonhybrid hearing . The patient satisfaction survey employed by these authors only compared hybrid hearing with preoperative ha hearing . Because it can be assumed that patients with natural unamplified lfh might hear better than those with ha - amplified lfh, we found it useful to separate the results based on three ci groups . A disadvantage is that patients with similar lfh might choose different fitting strategies, which may include ipsilateral has . Thus, we compared the levels of satisfaction related to the actual lfh and those related to the different groups . (2013) analyzed a fairly small patient sample and also found no differences in patient satisfaction between the hybrid and nonhybrid patients . The ioi - ha scores of our patients were nearly identical to those reported by redfors et al . These authors also claimed that the patients with mixed hearing loss scored better than did the patients with only sensorineural hearing loss using a ha . Our results indicate that music perception was better among the hybrid - hearing patients than the nonhybrid patients . Contralateral hearing might also have contributed to the musical experiences because the patients with minimal lfh predominantly had similar situations in their contralateral ears . Some of the patients had normal hearing since birth, and others had limited memories of high - frequency hearing . Some of the patients were very satisfied by relatively small improvements, particularly if they had experienced a long - lasting period of profound hearing loss with limited hearing experiences . There are several methods to for examining hearing loss related to hearing preservation surgeries (incerti et al . Because all of our patients exhibited ski - slope shaped audiograms and because all of our patients had measurable hearing in the 125500 hz range postoperatively, we found our selected method to be practical and easy to use every day in the clinic . (2013) characterized hearing preservation either as complete (10 db) or partial (30 db). In this study, among the 66 patients, 43% exhibited complete preservation, and 74% exhibited partial preservation after 1 year . The rest of the patients 26% have no preserved hearing according to the criteria). In this investigation, 32% of our patients exhibited complete preservation, and 95% exhibited partial preservation after 1 month . At 1 year, the corresponding values were 21% and 89%, respectively; at 2 years, the values were 26% and 63%, respectively . These results indicate that the number of patients with completely preserved hearing was less in our study than in the study by lenarz et al ., but the number of patients with partially preserved hearing was higher in our study . A loss of 30 db could be the difference between using and not using an acoustic complement . Despite the ubiquitous hearing preservation after round window surgery, deteriorations in the lfh of the operated ears were observed during the 1st years . The finding that the loss was more advanced in the operated ear suggests that this loss might have been induced by the electrode array . The explanation for the less severe hearing loss that was observed in the nonoperated ears might be related to the etiologies of the hearing impairments of our patients (usami et al . 2012). We did not find a way to predict hearing deterioration immediately after surgery or thereafter . A better understanding of the etiology of hearing loss might contribute to such predictions in the future . Because the loss of residual hearing could not be predicted, the progression of lfh loss should always be considered and discussed in patient consultations, but this should not be considered a contraindication for implantation because patients will benefit from cis . If the lf pta is 50 db before surgery, the patients will likely not use ipsilateral has . The patients who switched from the hybrid hearing strategy to the full frequency program due to greater lfh loss did not complain, likely because they gained full - frequency ci hearing and maintained lfh in the contralateral ear . These patients were pleased with the easier handling of the processor, which did not obstruct the ear canal . The patients with initial unaided lfh, who deteriorated after fitting abandoned the hybrid - hearing strategy and used full - frequency cis . One patient preferred the full - frequency stimulation despite having normal lfh, and another patient chose to use a full - frequency ci stimulation and a ha during 2 years . These results indicate the remarkable potential of the listening brain and demonstrate the complexity of identifying the best - aided condition . Because our patients had natural lfh in their contralateral ears, they likely suffered less from the ipsilateral lfh loss . Further studies are necessary before standard indications for bilateral hybrid hearing implantations can be established . One year after surgery, the patients displayed a mean performance of 52% in the ms test in the operated ear . The hint results after 1 year are in accordance with those of gifford et al . After 2 years, slight reductions in the ms and hint scores were observed . When asked, the majority of the patients responded that they experienced stable or improved hearing . In many patients, factors, such as preoperative hearing ability, individual expectations, and environmental needs might explain these inconsistencies . These findings illustrate the value of assessing both patient satisfaction scores and audiological to understand the actual hearing situation . The two children included in this report were described separately because they seemed to exhibit the possibility for successful ci rehabilitation despite profound high - frequency deafness since childhood . Such children may benefit from receiving high - frequency cis early and experiencing improved hearing at a critical time of their lives . Because both of the children in our study had normal preoperative lfh, they might benefit from hybrid hearing for many years despite the ongoing deteriorations of their residual hearing . Such benefits of hybrid hearing might be found to be important for the optimal education and social development of these children . All of the patients were satisfied with their cis regardless of the variation in the extent of hearing preservation . The preservation of hearing constitutes an additional benefit that might improve hearing in noisy situations and music perception . We observed ongoing deterioration of the residual hearing of the operated ear that surpassed the deterioration of the contralateral ear . Based on our findings, we propose that most people with partial deafness should be offered hybrid hearing . Evaluations of patient satisfaction should be used with conventional hearing tests to increase our knowledge of ci hearing.
Geographic tongue (gt) was first reported as a wandering rash of the tongue in 1831; however, its etiopathogenesis has remained unclear . Gt is a common condition characterized by an asymptomatic presentation of multiple variable sized, well - demarcated, erythematous areas usually surrounded by elevated, yellowish - white borders, which usually occurs on the anterior 2 - 3 of dorsal tongue . Different risk factors have been proposed for gt such as genetic factors, hormonal changes and oral contraceptive pills, pregnancy, psychological findings and diabetes mellitus . Pregnancy constitutes a special psychological state characterized by a series of temporary adaptive changes in the body structure, which results in an increased production of estrogen and progesterone (100 fold or more). Increased prevalence of gt and pyogenic granuloma (pg) have been postulated during pregnancy . However, no documented study has been carried out to investigate any possible relation between increased level of estrogen and progesterone and gt prevalence in pregnant women . Therefore, the aim of this study was to compare the level of salivary estrogen and progesterone in otherwise healthy pregnant women with gt and without gt . This analytical - descriptive study consisted of 26 pregnant women seeking dental treatment in community clinics in esfahan province, iran (13 with gt, 13 without gt) with the age range of 18 - 45 years . Patients who suffered from dermatological disease, systemic disease, allergic and atopic conditions, immune disorders and smokers were excluded from the study . Examination procedures were based on the world health organization's guide to epidemiology and diagnosis of the oral mucosa disease and conditions . A lesion was classified as gt when: there was localized absence of filiform papillae the affected area was irregularly shaped the location of the affected area changes over time . A total of 26 patients were assigned to the case (n = 13) and control (n = 13) groups according to the criteria, which is followed to diagnose gt . The measurement of estrogen and progesterone levels was performed during the 1, 2 and 3 trimester of their pregnancy ., saliva can be easily collected by the subjects at repeated intervals and requires no special collection or storage equipment, saliva sampling was conducted in the present study . After 5 min, un - stimulated saliva samples were collected while patients were sitting in a comfortable position and spitting into the plastic tubes five times per min for 5 min . The samples were stored at -80c and estrogen and progesterone levels tested using statistical analysis enzyme immune assay procedure . Data analysis was performed using t - test and repeated measure anova (= 0.05) (spss version 14.5). This analytical - descriptive study consisted of 26 pregnant women seeking dental treatment in community clinics in esfahan province, iran (13 with gt, 13 without gt) with the age range of 18 - 45 years . Patients who suffered from dermatological disease, systemic disease, allergic and atopic conditions, immune disorders and smokers were excluded from the study . Examination procedures were based on the world health organization's guide to epidemiology and diagnosis of the oral mucosa disease and conditions . A lesion was classified as gt when: there was localized absence of filiform papillae the affected area was irregularly shaped the location of the affected area changes over time . A total of 26 patients were assigned to the case (n = 13) and control (n = 13) groups according to the criteria, which is followed to diagnose gt . The measurement of estrogen and progesterone levels was performed during the 1, 2 and 3 trimester of their pregnancy ., saliva can be easily collected by the subjects at repeated intervals and requires no special collection or storage equipment, saliva sampling was conducted in the present study . After 5 min, un - stimulated saliva samples were collected while patients were sitting in a comfortable position and spitting into the plastic tubes five times per min for 5 min . The samples were stored at -80c and estrogen and progesterone levels tested using statistical analysis enzyme immune assay procedure . Data analysis was performed using t - test and repeated measure anova (= 0.05) (spss version 14.5). The mean level of estrogen was 49.4 1.2 ng / mi and 52.32 1.3 in the 1, 71.05 1.7 and 74.12 2.2 in the 2 and 109.1 0.7 and 112.16 1.2 in the 3 trimester in control and case groups respectively [figure 1]. The mean level of estrogen in saliva 3 periods of pregnancy for both groups: case and control the results of t - test analysis showed no significant difference between both groups in each trimester of pregnancy (p = 0.07). The mean level of progesterone was 0.72 0.09 ng / mi and 0.72 0.04 in the 1, 1.14 0.11 and 1.21 0.13 in the 2 and 1.3 0.14 and 1.28 0.24 in the 3 trimester of pregnancy in control and case groups respectively [figure 2]. The mean level of progestron in saliva 3 periods of pregnancy for both groups: case and control the mean levels of progesterone in both groups revealed no significant difference in each trimesters of pregnancy (p = 0.06). Even though, there was no significant difference between the case and control groups regarding the level of sex hormones, but there was a significant difference between each trimester of pregnancy regarding the level of measured hormones in both case and control groups (p = 0.001). The present study was designed to compare the salivary level of estrogen and progesterone in pregnant women with and without gt for the 1 time . Numerous studies which have examined the reliability of saliva assay have announced that salivary levels can be a reliable indicator of serum concentration . Worthman et al . Revealed significant positive correlations (0.82) between sex hormones in serum and saliva . Therefore, in this study, saliva samples were collected to measure the level of sex hormones . Different studies have been carried out to assess the prevalence of gt in pregnant women . Reported a more common occurrence of mucosal lesions including gt and pg in pregnant women . Their findings showed that the prevalence of gt in pregnant and non - pregnant women was 3.23% and 0.72% respectively . These studies demonstrated that the increased level of sex hormones during pregnancy may play an etiological role in the high incidence of this lesion during pregnancy . Study revealed no significant difference between the prevalence of gt in pregnant and non - pregnant women . Since there was no conclusive study to evaluate any possible relation between elevated levels of sex hormones and gt prevalence during pregnancy, the present study was designed to measure the level of estrogen and progesterone hormone in pregnant women with gt and without gt . The findings of this study showed that there was no significant difference between the level of these hormones in pregnant women with gt and without gt . However, the salivary level of sex hormones significantly increased from 1 to 3 trimester and reached to its maximum level during the 3 trimester, which is in accordance to the results of the previous studies . Compared the level of sex hormones during three trimesters of pregnancy and 6 weeks post - partum . The mean level of these hormones increased during pregnancy, but it showed a significant reduction in 6 weeks post - partum . According to the results of the present study it can be concluded that the increased level of sex hormones is not the only etiological factor of gt in pregnant women and other factors such as genetic potential, human leukocyte antigen marker and stress may aggravate the incidence of this lesion.
Nanoscale building blocks have attracted much interest due to their potential applications in replacement of traditional materials in the coming next few decades . However, most of the work in the synthesis of nanocrystals using solution - phase chemical method is focused on vi - ii semiconductor systems and noble metals . Transition metal phosphides exhibit unique catalytic, optical, electronic, and magnetic properties depending on their phases . For example, fe3p is ferromagnet that has a high transition temperature (tc = 716 k), while fep2 is a small - bandgap semiconductor with a tc of 15 k. some preparation methods have been developed for the interest of studying the size - property relations of nanoscale iron phosphides . For example, gu et al . Prepared 200 nm orthorhombic fep via solvothermal synthesis employing fecl3 and na3p at 180c . Hu et al . Obtained a mixture of iron oxide nanoshells and hollow iron phosphide nanoparticles using sonichemistry method . Prepared fep nanoparticles via a de - silylation route using a highly reactive phosphine source, p(sime3)3 . Later, brock found that trioctylphosphine (top), which is a less - reactive reagent compared with p(sime3)3, can be used as phosphine source as well as surfactant and solvent in the preparation of mnp and fep using metal carbonyls as metal source . Liu et al . Synthesized fep nanorods by injecting fe(co)5/top into hot solution of top and trioctylphosphine oxide (topo). Hyeon et al . Studied the relation between the injection rate and length of fe2p nanorods synthesized using a syringe pump . Fe2p nanorods can also be prepared without using a syringe pump as shown in the work of whitmire et al . Who developed a single - source organometallic method to control morphology by varying the ratio of tri - n - octylamine (toa) to oleic acid (oa) in a one - pot reaction . We recently reported the synthesis of ni2p nanowires in the system of toa / oa via an injection approach . In this work, we present a primary result on the synthesis of iron phosphide nanowires using a solution - phase chemical approach . Instead of using toxic and unstable iron carbonyl compounds that were usually employed in most of previous work on the synthesis of iron phosphide nanowires, we developed a approach that uses a metal precursor of iron (iii) acetylacetonate (fe(acac)3),which is low toxicity, inexpensive and rather stable at ambient condition . The structure and magnetic properties of as - synthesized nanowires are also presented in this work . In a typical synthesis, 0.5 mmol of fe(acac)3(96%, acros) was added in 10 ml of oleylamine (om, 8090%, acros) and heated to 130c . The resulting solution was continuously injected into a stock solution of 5 ml of om, 5 mmol of top (97%, acros), and 2.5 mmol of topo (90%, acros) at 340c using a syringe pump . The whole injection time varied from 3 to 6 h. a one - pot reaction was also attempted by reacting a mixture of 0.5 mmol of fe(acac)3, 4 mmol of top, 2 mmol of topo, and 6 ml of om directly at 350c for 1 h. the obtained product precipitates were washed by hexane and acetone, and separated from solution by centrifugation . Powder x - ray diffraction (xrd) data were collected on a panalytical xpert pro x - ray diffractometer using cu k radiation . Transmission electron microscopy (tem) was performed on a tecnai f-30 transmission electron microscope . Samples for tem analyses were prepared by sonicating the as - synthesized powders in hexane and dropping a small volume onto a carbon - coated copper tem grid followed by solvent evaporation . Magnetic analysis was performed using a superconducting quantum interference device (squid) magnetometer (mpms-5). In a typical synthesis, 0.5 mmol of fe(acac)3(96%, acros) was added in 10 ml of oleylamine (om, 8090%, acros) and heated to 130c . The resulting solution was continuously injected into a stock solution of 5 ml of om, 5 mmol of top (97%, acros), and 2.5 mmol of topo (90%, acros) at 340c using a syringe pump . The whole injection time varied from 3 to 6 h. a one - pot reaction was also attempted by reacting a mixture of 0.5 mmol of fe(acac)3, 4 mmol of top, 2 mmol of topo, and 6 ml of om directly at 350c for 1 h. the obtained product precipitates were washed by hexane and acetone, and separated from solution by centrifugation . Powder x - ray diffraction (xrd) data were collected on a panalytical xpert pro x - ray diffractometer using cu k radiation . Transmission electron microscopy (tem) was performed on a tecnai f-30 transmission electron microscope . Samples for tem analyses were prepared by sonicating the as - synthesized powders in hexane and dropping a small volume onto a carbon - coated copper tem grid followed by solvent evaporation . Magnetic analysis was performed using a superconducting quantum interference device (squid) magnetometer (mpms-5). Figure 1a shows the low - resolution tem image of the iron phosphide wires prepared using injection method . The nanowires typically have a width of ~16 nm and a length of several hundred nanometers . The selected area diffraction (saed) pattern recorded from bundles of nanowires high - resolution tem (hrtem) images were taken on individual nanowires to observe their microstructures . The measured lattice fringes (0.344 nm) that are perpendicular to the growth direction correspond well to the lattice spacing of (001) planes, indicating a growth direction of . Park et al . Also reported a similar result for the growth direction of fe2p nanowires . Typical xrd pattern of the as - prepared iron phosphide samples is shown in fig . The observed diffraction peaks also can be indexed with hexagonal fe2p structure, agreeing well with the saed analytic result . Energy - dispersive x - ray spectroscopy (eds) analysis on individual nanowires (fig . Further quantitative analysis reveals a fe: p ratio of ~1.93:1, which is close to the stoichiometric ratio of fe2p . Normal tem image along with saed pattern (a), hrtem image (b), xrd pattern (c) and eds spectrum (d) of the as - prepared iron phosphide nanowires it can be inferred from the above results that the anisotropic shape of nanowires comes mainly from the intrinsically anisotropic nature of hexagonal crystalline structure . The monomer concentration of the injection route is relatively low, which favors the growth of existing seeds formed in the early stage of continuous delivery . Another important prerequisite for the formation of anisotropic nanostructures is related to the surfactants used . Usually, a multi - surfactant system favors the formation of an anisotropic nanostructure . In our case, the employed top and topo both can act as surfactants to cover different crystallographic surfaces of newly formed nanocrystal seeds, and eventually lead to the anisotropic growth toward c axis, forming a one - dimensional nanostructure . Our studies found that if the experiments were conducted below 320c, the as - synthesized products were mainly iron oxide nanoparticles, which indicates that necessary thermal energy is needed to decompose top to form active phosphorus atoms to take part in the phosphide formation . For example, iron nanoparticles were obtained when top concentration was relatively low (less than 2 mmol). However, when high concentration of top (e.g. 12 mmol) was used, the synthesized nanowires would have a curved morphology (see fig . 2a), reflecting the insufficient capability of top for the protection of nanowires sidewalls . A small variation of top amount (from 3 to 5 mmol) did not make apparent difference in the products, showing the robustness of our synthetic approach . The employed surfactant topo also plays an important role in controlling the product s morphology . If no topo is added, the synthesized products mainly exhibit a nanoblock - like morphology resulting from the agglomeration of many soft - warped nanowires . This indicates that the existence of topo may function as a sidewall protector that can keep iron phosphide nanowires grow straightly over hundreds of nanometers . However, high concentration of topo on the contrary will reduce the quality of nanowires and lead to the formation of some big nanoblocks in the products . Tem images of iron phosphide nanowires with a curved morphology synthesized using high concentration of top (a), and products synthesized using one - pot reaction (b) the formation process of fe2p nanowires may be as follows . At first, the complex of fe(acac)3-om decomposes to iron clusters once added to hot solvent of om . Then, these iron clusters will be attached to top molecules, and the p c bonds located at the particle surface may break and cause phosphorus to react with iron, forming iron phosphide . Once fe2p seeds with an anisotropic structure forms, the growth along direction will be favored due to the cooperation of anisotropic nature and the side - protection effect of surfactant molecules . 2b, the obtained products via one - pot reaction consist of nanowires plus nanosheets or nanoblocks . The shape singularity of the products from the one - pot reaction is not superior to the continuous - injection approach . Considering the robustness of the reaction it is certain that continuous - injection route is crucial to keep the singularity of the products . A possible reason is that one - pot synthesis incurs a high concentration of nuclei, some of which are unable to develop into nanowires due to insufficient protections of surfactant molecules, whereas for the injection approach, the nuclei concentration at the beginning stage is restricted by the delivering rate, and the relatively low concentration of nuclei plus the continuous delivery of source materials will allow nuclei to fully grow into nanowires . The hysteresis loop of as - prepared fe2p nanowires measured at 5 k is shown in fig . It is clear that the magnetization curve is difficult to be saturated and the unsaturated magnetization moment is 45.5 emu / g (1.16 b), which is much lower than the saturation magnetization (ms) value of bulk fe2p (2.81 b). Meanwhile, the measured coercive force is about 5,000 oe . A large coercive force has also been noticed in fe2p nanorod (hc = 3.9 koe) and nanoparticle samples (~5 koe). From the curve of fig . In fact, it looks like that the curve is a result of combination of two different loops . Such a slightly twisted loop indicates that a small amount of impurities may exist in the synthesized products, although their amounts may beyond the detection limit of xrd . Since squid is a characterization technology that is very sensitive to a trace amount of magnetic impurities, it can detect these impurities more easily . It is very likely that a small amount of fe or fe oxides constitute the impurities, since they were often found in the experiments performed at lower temperatures . Such impurities were considered as the main reason accounting for the twisted hysteresis loops of fe2p nanorods . Similar results have also been observed in fept nanoparticle samples, which also have a large coercivity . Figure 3b shows the zero - field - cooled (zfc) and field - cooled (fc) curves of fe2p nanowires measured in a temperature range of 5300 k. a high blocking temperature (~275 k) is observed, which is consistent with the hysteresis test at 300 k (not included in this paper). A hysteresis loop measured at 5 k and b zfc / fc curves at an applied field of 100 oe for the as - prepared iron phosphide nanowires iron phosphide nanowires have been synthesized through a nonaqueous approach that utilizes fe(acac)3 as an iron source, top as a phosphorus source and oleylamine as solvent . The as - prepared nanowires have a hexagonal fe2p structure and grow along c axis . The dimension of nanowires can be tuned via reaction parameters such as injection rate, reaction temperature, and top / topo ratio . Both injection method and one - pot reaction can produce iron phosphide nanowires, although the former can generate products with much better uniformity and yield . The magnetic tests reveal ferromagnetic characteristics below the blocking temperature, and a large coercive force of ~5,000 oe has been observed . The reported synthetic approach does not use commonly used toxic iron carbonyl compounds as iron precursor, which provides a convenient access to further studies of their special physical and chemical properties . This work was partially supported by the national natural science foundation of china (grant nos . 50701036 and 50671087) and the national outstanding youth science foundation of china (grant no . 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 source are credited . 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 source are credited.
Laparoscopic ventral incisional hernia (vih) repair has gained wide acceptance and increased popularity over the past decade . Multiple studies have documented the safety, technical feasibility, and efficacy of this technique . The extremely low rate of wound infection and recurrence are among the most important advantages of the laparoscopic approach . Because the prosthetic mesh is placed intraabdominally, many surgeons use expanded polytetrafluoroethylene (eptfe) grafts because of the low adhesive potential .,, high cost, inferior handling characteristics, and poor incorporation into the tissues are among the drawbacks of eptfe mesh . Another product, polypropylene (pp) nevertheless, long - term complications, such as severe adhesions, obstruction, and fistula formation, have been reported with pp ., these complications seem to occur when the pp mesh is placed in direct contact with the serosal surface of the bowel . However, if omentum is interposed between the mesh and viscera, adhesions should not involve loops of bowel and should be restricted to omentum only . Using this technique, franklin et al reported no adhesion - related complications in over 170 patients undergoing laparoscopic vih repair during a 30-month follow - up . Between 1999 and 2001, patients undergoing laparoscopic vih repair were prospectively enrolled in a surgical database . In 30 patients, pp mesh (prolene, ethicon corporation, somerville, nj) was used for the hernia repair; omentum was interposed between the mesh and the underlying bowel . The mesh was secured in place with a hernia stapler along the edges and nonabsorbable transcutaneous sutures at the corners, taking care to ensure that the mesh overlapped the fascial defect by at least 3 cm in each direction . At a mean follow - up of 14 months twenty patients agreed and were studied using an acuson xp/10, 7 mhz linear transducer, supplemented by a curvilinear transducer when appropriate . A visceral slide technique was used specifically to detect adhesions . At the time of follow - up, all patients were also examined for hernia recurrence, wound infection, and seroma formation . Satisfaction with the procedure (completely / somewhat / not satisfied) and pain at the operative site (mild / moderate / severe) were assessed by using a standard questionnaire . This method is based on the demonstration of intestinal movement at the abdominal wall interface during real - time ultrasound imaging . Such movement may occur spontaneously as a result of respiratory excursions or may be induced by manual compression . During longitudinal surface scanning, a normal spontaneous visceral slide may range from 2 cm to more than 5 cm in distance . Detection of a visceral slide that measures less than 1 cm is considered abnormal due to adhesions . This method is based on the demonstration of intestinal movement at the abdominal wall interface during real - time ultrasound imaging . Such movement may occur spontaneously as a result of respiratory excursions or may be induced by manual compression . During longitudinal surface scanning, a normal spontaneous visceral slide may range from 2 cm to more than 5 cm in distance . Detection of a visceral slide that measures less than 1 cm is considered abnormal due to adhesions . No detectable omental or bowel adhesions were found in more than half of the patients (figure 1). In some cases, mesh appeared to be somewhat corrugated, but normal visceral slide was detected in each instance (figure 2). Five patients had omentum that appeared to be densely adherent to the pp mesh, as detected by the lack of a normal visceral slide . In one of these patients, the bowel was visibly tethered underneath the omentum but was not adherent to the mesh . One patient had tethered bowel at the edge of the hernia repair with thickened omentum underlying the mesh . In another patient, the falci - form ligament had been divided to complete the hernia repair in that case . The incidence of adhesions (%) detected by ultra - sonography in 20 patents after laparoscopic ventral incisional hernia repair with polypropylene mesh and omental interposition . Ultrasonogram of the patient after laparoscopic ventral incisional hernia repair: mesh (arrow) appears to be corrugated . Nine patients had no pain at the surgery site, while 10 patients stated they experienced mild to moderate pain with activity, usually near the edges of the repair (figure 3). Only 2 patients complained of severe pain at the site of repair . One of these patients described severe pain with activity even though she had been pain - free for 1 year after surgery . This patient was noted to have a small recurrence of the hernia just lateral to the edge of the mesh (figure 4). Her initial repair 3 years earlier was accomplished using the separation of components technique, which led to a subsequent recurrence . Ultrasonogram of the patient with the recurrent hernia: omental herniation (thick arrow) is seen just lateral to the mesh (thin arrow). The reasons for less than complete satisfaction include unexpected postoperative pain in 1 patient and moderate crampy postoperative pain in a second patient . One patient had problems with a trocar site, and another patient felt that an area of attenuated fascia with underlying mesh constituted a recurrence . The use of prosthetic materials for the repair of a ventral incisional hernia has significantly decreased the number of recurrences for this condition . Moreover, the laparoscopic approach has helped to reduce the number of infections, recurrences, and the length of hospital stay . The formation of adhesions has been studied in numerous experimental models . In 1 such study, increasing adhesion development was noted from day 1 to day 7 after placement of the prosthesis, but surfaces that were already covered with mesothelial cells did not show progression . Thereafter, the bowel had a diminished tendency to form adhesions even in the absence of omentum . Another animal model was used to compare adhesion formation after open and laparoscopic placement of mesh . The study demonstrated that the area of mesh covered with adhesions and the grade of the adhesions were significantly greater in both groups undergoing celiotomy compared with the adhesion formation in the laparoscopic group . On histologic examination, the vascularity of the animals that underwent incision through skin and fascia was increased and slower to regress than in the laparoscopically treated animals . In the clinical setting, pp mesh has been used previously for open vih repair with a very low rate of enterocutaneous fistula formation, but fistulas have been reported as late as 15 years after the procedure . In laparoscopic vih repair, pp has been combined with omental interposition as protection against bowel adhesions . Colocutaneous fistula after laparoscopic vih hernia repair with intraperitoneal ptfe has also been reported; in this case, the laparoscopic staples or external postoperative trauma may have played a role . Ultrasound was used to detect adhesion formation at a time when this process was complete . Visceral slide detection by ultrasound was described by kodama et al in 1992 and was again validated in a french study with intraoperative observation and was noted to have a sensitivity of 77%, specificity of 74%, and a negative predictive value of 84% . The data in our study were prospectively collected, but the overall number of patients was modest . Nonetheless, a low recurrence and infection rate was confirmed as noted in other laparoscopic herniorrhaphy studies, and no mesh - related complications have been noted to date . Only 1 case of bowel adhesions to the edge of the mesh was found using ultrasound scanning at 24-month follow - up . Sixty - five percent of our patients did not have adhesions of bowel or omentum detectable on ultra - sound examination . This result is similar to that reported by franklin et al where two thirds of the patients had few or no adhesions . A decreased inflammatory response in the abdominal wall tissue might limit the inflammatory tissue reaction caused by the pp mesh in regards to adhesion with underlying viscera . The results of this study are important but somewhat limited by the fact that the results of individual sonographic examinations could not be independently verified . The negative predictive value of the visceral slide method is approximately 80%; therefore, it is possible that some patients who were deemed free of adhesions might indeed have had mild or filmy adhesions not detectable by ultrasound . In any case, these adhesions, even if present are unlikely to have any clinical impact, because no visceral complications have been observed . Late postoperative pain seemed to be mostly related to the suture points during strenuous activity . Although it was noted by half of the patients, it usually did not require medication and did not interfere with work or recreational activities . We have now gone to the use of absorbable monofilament material for the transfascial sutures . Our early results indicate that laparoscopic ventral incisional hernia repair with pp mesh and omental coverage is not associated with sonographically detectable visceral adhesions in most patients . Moreover, visceral complications such as bowel obstruction or enterocutaneous fistula do not occur . Further accrual of patients and careful follow - up is needed to better understand the problem of ventral hernias and their repair with mesh.
Keratoelastoidosis marginalis of hands (kemh), is an acquired, marginal, acrokeratoderma that predominantly affects the radial side of the index finger, first web space, and ulnar side of the thumb . Prolonged uv exposure, heavy manual work, and repeated trauma to the hands seem to be the etiologic factors . A 75-year - old farmer presented with asymptomatic lesions over the web spaces of the thumb and index finger of both hands and raised lesions over his face since 15 years . He denied any history of preceding trauma, drug intake, or contact with any products causing irritation . Cutaneous examination revealed hyperkeratotic and scaly papules arranged in a linear fashion along the radial margins the index fingers [figure 1a and b]. Skin over the face was thick, leathery, and inelastic with deep wrinkles over the forehead and cheeks . Provisional diagnosis of kemh with colloid milium with cutis rhomboidalis nuchae and favre racouchot syndrome was made . (a) hyperkeratotic and scaly papules arranged in a linear fashion along the radial margins of right and left index fingers . (c and d) thick, leathery, and inelastic skin over the face with deep wrinkles over the forehead and cheeks . (e) deep furrows were seen over the nape of neck (cutis rhomboidalis nuchae) a 67-year - old farmer presented with asymptomatic rough, scaly lesions over the web space of the thumb and index fingers of both the hands since 10 years . He denied history of preceding trauma, drug intake, or contact with any products causing irritation . Cutaneous examination revealed keratotic, crateriform papules over the radial aspect of both index fingers arranged in a linear fashion extending on to the first web space . Similar lesions were also present on ulnar aspect of left little finger [figure 2a c]. Examination of the face revealed yellowish discoloration of the skin with leathery texture with multiple deep wrinkles over the face . Back of the neck showed deep wrinkling and furrowing of the skin associated with a leathery texture, suggesting the chronic actinic damage of the skin . (a) keratotic crateriform papules along the radial aspect of the right index fingers arranged in a linear fashion extending onto the first finger web space . (b) keratotic crateriform papules along the radial aspect of the left index fingers arranged in a linear fashion . (c) similar lesions were also present on ulnar aspect of left little finger skin biopsy from keratotic plaques on the fingers of both the patients revealed compact hyperkeratosis and marked acanthosis . There was evidence of thickened, haphazardly oriented collagen bundles with basophilic degeneration [figure 3a c], and haphazardly oriented elastic fibers throughout the dermis with dilated blood vessels in the papillary dermis . Van gieson stain showed thickened, degenerated, haphazardly arranged collagen bundles throughout the dermis and fragmented bundles of elastin [figure 3d]. On clinicopathological correlation, a final diagnosis of kemh was made . (a) h and e stain, 40: compact hyperkeratosis, marked acanthosis with thickened haphazardly oriented collagen bundles . (b) h and e stain, 100: marked basophilic degeneration of collagen and haphazardly oriented elastic fibers were throughout the dermis . (c) h and e stain, 400: degenerated collagen and haphazardly oriented elastic fibers in the upper throughout the dermis . (d) verhoeff van gieson stain, 40: thickened, degenerated haphazardly arranged collagen bundles throughout the dermis and fragmented bundles of elastin a 75-year - old farmer presented with asymptomatic lesions over the web spaces of the thumb and index finger of both hands and raised lesions over his face since 15 years . He denied any history of preceding trauma, drug intake, or contact with any products causing irritation . Cutaneous examination revealed hyperkeratotic and scaly papules arranged in a linear fashion along the radial margins the index fingers [figure 1a and b]. Skin over the face was thick, leathery, and inelastic with deep wrinkles over the forehead and cheeks . Provisional diagnosis of kemh with colloid milium with cutis rhomboidalis nuchae and favre racouchot syndrome was made . (a) hyperkeratotic and scaly papules arranged in a linear fashion along the radial margins of right and left index fingers . (c and d) thick, leathery, and inelastic skin over the face with deep wrinkles over the forehead and cheeks . (e) deep furrows were seen over the nape of neck (cutis rhomboidalis nuchae) a 67-year - old farmer presented with asymptomatic rough, scaly lesions over the web space of the thumb and index fingers of both the hands since 10 years . He denied history of preceding trauma, drug intake, or contact with any products causing irritation . Cutaneous examination revealed keratotic, crateriform papules over the radial aspect of both index fingers arranged in a linear fashion extending on to the first web space . Similar lesions were also present on ulnar aspect of left little finger [figure 2a c]. Examination of the face revealed yellowish discoloration of the skin with leathery texture with multiple deep wrinkles over the face . Back of the neck showed deep wrinkling and furrowing of the skin associated with a leathery texture, suggesting the chronic actinic damage of the skin . (a) keratotic crateriform papules along the radial aspect of the right index fingers arranged in a linear fashion extending onto the first finger web space . (b) keratotic crateriform papules along the radial aspect of the left index fingers arranged in a linear fashion . (c) similar lesions were also present on ulnar aspect of left little finger skin biopsy from keratotic plaques on the fingers of both the patients revealed compact hyperkeratosis and marked acanthosis . There was evidence of thickened, haphazardly oriented collagen bundles with basophilic degeneration [figure 3a c], and haphazardly oriented elastic fibers throughout the dermis with dilated blood vessels in the papillary dermis . Van gieson stain showed thickened, degenerated, haphazardly arranged collagen bundles throughout the dermis and fragmented bundles of elastin [figure 3d]. On clinicopathological correlation, a final diagnosis of kemh was made . (a) h and e stain, 40: compact hyperkeratosis, marked acanthosis with thickened haphazardly oriented collagen bundles . (b) h and e stain, 100: marked basophilic degeneration of collagen and haphazardly oriented elastic fibers were throughout the dermis . (c) h and e stain, 400: degenerated collagen and haphazardly oriented elastic fibers in the upper throughout the dermis . (d) verhoeff van gieson stain, 40: thickened, degenerated haphazardly arranged collagen bundles throughout the dermis and fragmented bundles of elastin marginal papular acrokeratoderma are classified by rongioletti et al . As hereditary and acquired forms . Hereditary form with elastorrhexis (fragmentation of elastic fibers) is termed acrokeratoelastoides, whereas that without elastorrhexis is termed focal acral hyperkeratosis (fah). Hereditary forms also include punctuate palmoplantar keratoderma (ppk - pt), hereditary papulotransluscent keratoderma and mosaic acral keratosis, whereas the acquired forms are kemh or degenerative collageneous plaques of the hand and digital papular calcinosis . It is also known as marginal papular acrokeratoderma, collagenous and elastotic marginal plaques of the hands, and digital papular calcific elastosis . Burks et al . In 1960 reported the first five cases of this acquired disorder and termed it as degenerative collagenous plaques of the hands . Chronic sun exposure and manual labor that involves chronic pressure or repeated trauma may be contributing factors . The hyperkeratosis may be due to repeated episodes of hypoxia of the affected areas resulting from papillary dermal capillary occlusion during periods of pressure, in conjunction with compression from the elastotic material that accumulates in the dermis, although this hypothesis has not yet been proved . It has been reported in people ranging in age from 42 to 78 years, but it remains a disorder mostly affecting the elderly population . Clinically it presents as asymptomatic, white to yellowish, waxy scaly crateriform keratotic papules along margins of the hands . The radial side of the index finger, first web space, and ulnar side of the thumb are characteristically involved . Solar elastosis in the dermis may be associated with thickened, fragmented, often calcified elastic fibers between distorted, degenerated collagen bundles . Differential diagnosis of kemh includes acrokeratoelastoidosis of costa, focal acral hyperkeratosis, and punctuate palmoplantar keratoderma . Differential diagnosis of keratoelastoidosis marginalis of the hands keratoelastoidosis marginalis is a progressive disease of a benign nature . These include laser therapy, high - potency topical corticosteroids, topical tazarotene and tretinoin, oral isotretinoin, and cryotherapy . Retinoic acid (50 mg / d) has reportedly shown some response, although the results are unsatisfactory . Topical keratolytics such as salicylic acid and tretinoin provide temporary relief with recurrences on stopping application . Apart from lesions of kemh, both our patients also had features of chronic actinic damage, in the form of waxy, yellowish, deeply wrinkled skin over the forehead, cheeks, nape of the neck (cutis rhomboidalis nuchae) along with senile comedones and nodular elastosis (favre racouchot syndrome). The occurrence of seborrheic keratosis, solar elastosis, favre racouchot syndrome and cutis rhomboidalis nuchae are commonly noted signs of chronic actinic damage . However, the occurrence of kemh in occupations involving chronic sun exposure, such as farming, has rarely been reported . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed . The authors certify that they have obtained all appropriate patient consent forms . In the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal . The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
The system al cu has been investigated intensively during the last decades, mainly due to the importance of al - based alloys, for example in aviation and transport industry . In view of this, the main focus of most studies in the system is the very al - rich part . Although a lot of work was done in the cu - rich part as well, there are still some uncertainties and inconsistencies in the phase diagram present . His extensive paper gives an equilibrium phase diagram as well as manifold information on metastable phase equilibria which are not part of the current investigation . According to, the equilibrium phase diagram contains 5 intermetallic compounds stable at ambient temperature and 7 additional compounds stable at elevated temperature (see fig . 1). The phase diagram given by murray does not represent the current level of knowledge about the system . A more recent phase diagram combining the assessment of murray with new data from liu et al . Thermodynamic calculations in the system were performed by several authors concentrating on transition- and ordering phenomena,,,, as well as on atomic mobility . An overview on the al cu phases described by different authors,,,,,,,,,,,, is given in table 1 . The structure of the -phase with the composition al2cu was originally revealed by friauf and found to be tetragonal . According to murray additional investigations in the region between 31 and 37.5 at.% cu by goedecke and sommer indicate a composition of 32.4 at.% cu for at its formation temperature of 592 c . The eutectic line of the reaction l = (al) + ends at 32.05 at.% cu . Structural investigations of the compound -alcu have already been performed by preston who found an orthorhombic structure in a sample quenched from 602 c . Investigated slowly cooled samples of the same composition and proposed an allotropic transformation 1 2 on basis of structural differences compared to the work of preston . Were able to solve the structure of the low temperature phase which was found to be monoclinic . Preston suggested the structure to be orthorhombic (op16 or oc16), lukas and lebrun mentioned in their assessment of the al cu si system an orthorhombic cell with lattice parameters a = 4.087, b = 12.00, c = 8.635 and 32 atoms per unit cell . Although the supposed type of transition reaction was not mentioned explicitly, the assessed phase diagram by murray and riani et al . Obviously suggest a transition of higher order between 2 and 1 . According to the assessment of murray, the introduction of the high temperature phase 1 and 2 goes back to 1920 . However, the structure of 2 was solved for the first time in 1972 by el - boragy et al . The structure of 2-al2+xcu3 is of the nias - type with partial occupation of the additional interstitial position . The compound with the proximate composition al3cu4 (1/2 region) was also described by preston and bradley and was found to show a high and a low temperature modification . The work of murray suggests a transition temperature between 530 and 570 c but mentions other reported thermal effects between 373 and 450 c as well ., investigated as - cast and annealed samples with the composition al3cu4 . In the as - cast samples the authors find a mixture of an orthorhombic face - centered and an orthorhombic body - centered structure as well as a small amount of -al4cu9 . After annealing at 500 c for 10 h the of structure became the major phase thus the authors suggested a transition al4cu9 + oi electron probe micro analysis (epma) measurements indicated compositions of al43.2cu56.8, al41.3cu58.7 and al39.6cu60.4 for of, oi and -al4cu9, respectively . The crystal structures of 1 (fmm2, structure type al3cu4) and 2 (imm2, structure type al3cu4-) were finally solved by gulay and harbrecht using powder xrd, . The composition of the samples for structure analysis of 1 (al42.5cu57.5) and 2 (al43.2cu56.8) contradicts the findings of dong et al ., who allocated the face - centered symmetry to the phase with lower cu - content . The assessment of murray shows a low temperature phase 2 and a high temperature phase 1 with a slightly higher cu - content; the transition temperature is supposed to be between 530 and 570 c . Gulay and harbrecht, however, found the cu - richer phase 1 (only phase in a sample with the composition al42.5cu57.5) to be stable at 400 c . The cu - poorer phase 2 (only phase in a sample with the composition al43.2cu56.8) was found to be stable at elevated temperatures (530 c) and did not resist thermal treatment at 400 c . The authors claim that entropy provides an essential contribution to the stabilization of the 2 phase . The range from 60 to 70 at% bradley claimed that three different phases are present in this region: a cubic (), a monoclinic and a rhombohedral compound . Westman found the latter to crystallize in space group r3 m and confirmed the existence of a third phase of unknown structure between the cubic and the rhombohedral compound . Seshadri and downie claimed that there are only five intermetallic phases stable in the temperature range between 25 and 500 c, namely (1), (described by a cubic structure), 2, 2 and . The separation of 1 and is supported by an abrupt change in expansion coefficient from 1 to . In the assessed phase diagram the third phase of unknown structure mentioned above is not included since there is no consensus about its existence . Murray pleads diffusion couple experiments of funamizu et al . Which do not show any other phase between 1 and . More importantly, very slow cooling experiments performed by van sande et al . These two experiments support the non - existence of a third equilibrium phase in the indicated region . The existence of the high temperature phase 0 was demonstrated by thermal analysis but the transition 0 to 1 could not be confirmed metallographically . Examined the cu - rich part of the phase diagram by diffusion coupling, differential scanning calorimetry and high temperature xrd . The authors state that 0 crystallizes in the cu5zn8-type and they do not find a two phase field between 0 and 1, thus proposing a higher order transition between the two phases, in contradiction to murray s assessment . According to murray, quoting dawson, the high temperature phase 0 is formed peritectically from and liquid at 1037 c . Dawson determined the composition and stability range of 0 by metallography and dilatometry but the findings have never been reconfirmed and the structure of 0 remains unknown . Nevertheless, 0 was included in the equilibrium phase diagram . In 1998 diffusion couple experiments performed by liu et al . Showed a two phase region between and 0 and no single phase 0 was found . Additionally, the authors found only one peak in dsc measurements at 1019 c which they interpreted to be the solidus of the phase rather than the reaction temperature of the eutectoid transformation 0 investigated the region between 85 and 89 at.% cu at temperatures from 450 to 850 c, finding a square - like shaped phase which has, however, not been confirmed by other authors . The -phase and the two - phase region between and (cu) was frequently investigated and the assessment of murray gives a broad overview about the results of this research . It shows that the eutectoid temperature was found between 560 and 575 c which can be explained by the sluggishness of the reaction . Reaction temperatures between 515 and 540 c can be considered due to metastable eutectoid and peritectoid reactions ., later studies confirmed the peritectoid decomposition temperature to be 363 c at 77.25 at.% cu . According to murray s assessment, 2 has an ordered fcc structure with a long - period superlattice based on cu3au and al3ti (strukturbericht designations: l12 and d022, respectively). A more detailed description about investigations of the low temperature phase 2, including thermal analysis experiments in this region is given by adorno et al . . The samples were prepared from aluminum slug (99.999%), and copper wire (99.95%), both supplied by alfa aesar, karlsruhe, germany . The cu wire was reduced in a h2-flow at 300 c for 3 h. the calculated amounts of al and cu were weighted to an accuracy of 0.05 mg; the sample weight usually was 1000 mg . Sample homogenization was done in an arc furnace mam-1 by edmund buehler with a water - cooled copper plate and zirconium as the getter material . For homogenization of the sample, the resulting bead was turned and re - melted two times . The occurring mass loss during this procedure was found to be below 1% and therefore considered not to affect the sample composition significantly . The resulting bead was wrapped in molybdenum foil (99.97%, plansee se, reutte, austria) and annealed at 500 c under vacuum in a quartz glass tube for 24 days . Representative sections of all annealed samples were investigated by means of optical microscopy using a zeiss axiotech 100 microscope . The quantitative chemical analyses were performed on a zeiss supra 55 vp in combination with energy dispersive spectroscopy (eds) using the pure elements for calibration . Measurements of the phase composition were performed with a minimum of three different spots and the results were averaged . X - ray powder diffraction analyses were performed using a bruker d8 advance diffractometer operating in reflection mode (cu k1 radiation, lynxeye silicon strip detector). For selected samples high temperature x - ray powder diffraction analysis was applied using an anton paar xrk900 reactor chamber with an automated alignment stage . The temperature resolved measurements were performed under evacuated conditions . For evaluation of the resulting diffractograms, both at ambient as well as at elevated temperature, the software topas was used . The dta measurements were performed on a setaram setsys evolution 2400 (setaram instrumentation, caluire, france) and a netzsch dta 404 pc (netzsch, selb, germany). The measurement devices used pt / pt-10%rh thermocouples (type s) which were calibrated using the melting points of pure sn, au and ni . The samples with a weight of approximately 20 mg were placed in open alumina crucibles and measured under an argon flow of 50 ml min for the netzsch and 20 ml min for the setaram device . Applying a heating / cooling rate of 5 kmin, the possible mass loss during the dta investigations was checked routinely and no relevant mass changes were observed . Combining results from both dta and sem measurements, it was possible to obtain the complete description of the al cu system which is plotted in fig . 2 . It is, for the most part, in good agreement to the phase diagram of liu et al . And riani et al . The occurring invariant reactions together with the composition of the reacting phases and the reaction temperature are given in table 2, selected sem images taken in back - scattered electron (bse) mode are shown in fig . The al - rich part of the phase diagram has not been investigated extensively in the current study . Concerning the reaction temperatures and the phase composition, the solubility of cu in al was found to be 2.2(1) at.% at 500 c . The lattice parameters of the phase (ti12, al2cu - type) vary from a = 6.0718(1) and c = 4.8802(1) at 32(1) at.% cu to a = 6.0613(1) and c = 4.8724(1) at 33.6(2) at.% cu . The phase boundaries of the binary phases in the al - rich part of the phase diagram have been determined by means of sem measurements and are indicated as black dots in fig the phases 1-alcu and 2-alcu are supposed to be stable around 50 at% cu with a solubility range of 12 at.% cu . The results in the current investigations, however, reveal a shift of the composition toward the cu - rich side . The solubility limits of the phase 2-alcu at 500 c were confirmed by sem measurements . The cu - poor and the cu - rich composition limit were found at 51.9(5) at.% cu and at 54.8(5) at.%, respectively . Xrd analysis of the samples with the nominal composition al49cu51 (showing and 2 in equilibrium), al47.5cu52.5 and al46.5cu53.5 (showing single phase 2) and al45cu55 (showing 2 plus traces of 1) indicate a cu - rich solubility limit between 54 and 55 at.% cu which supports the sem measurements . The lattice parameters for 2 (mc20, alcu - type) range from a = 12.0925(1), b = 4.1001(1), c = 6.9085(1), = 55.03(1) on the cu - poor side to a = 12.2012(1), b = 4.0997(2), c = 7.0047(3) and = 54.787(1) for the cu - rich side of this compound . The phase diagrams of riani and murry indicate a transition temperature from the high temperature 1-phase to the low temperature 2-phase at 563 c at the cu - poor side and 560 c at the cu - rich side, . According to our measurements the transition temperatures are 574(3) c for the cu - poor side and 580(1) c for the cu - rich side . Consequently, we propose also different solid state reactions in this area: the eutectoid decomposition 1 = 2 + at 574(3) c and the peritectoid reaction 1 the peritectic decomposition temperature of the phase 1 was found to be 625(2) c which is in agreement with the phase diagrams mentioned above . The region of al4cu9 with the supposed high temperature modification 1 and the low temperature phase 2 is also complex . Gulay and harbrecht find the cu - rich phase 1 stable at 530 and 400 c, but the cu - poor phase 2 stable at 530 c and not stable at 400 c, . They discovered that after a heat treatment at 400 c, 2 is decomposed into 1 and small amounts of 2 . Additionally, gulay and harbrecht state that at 450 c the phase 2 segregates in a mixture of 1 and 2 . Therefore, the authors conclude that the temperature of the eutectoid decomposition of 2 is between 400 and 450 c . The present work shows a sample with the nominal composition of al45cu55 exhibiting 2 as major phase with very small traces of a second phase . Comparison of systematic extinctions of 1 and 2 suggests that the second phase is 1 . Due to the very low amount of 1 in the respective sample, sem analysis of this sample shows only one suitable measurement point at 56.3 at.% cu which can be assigned to the cu - poor solubility limit of 1 . Xrd analysis of a sample with the nominal composition al42.5cu57.5 shows 1 and some unidentified peaks (see table 3). These xrd results narrow the solubility limits of 1 (of88, al3cu4-type) between approx . 56 and 57.5 at% cu . In the present investigation, dta analysis of the sample with the nominal composition of al45cu55 does not show any effects related to the transition of 1 to 2, caused by the fact that the amount of 1 is very small in the sample . Dta analysis of the sample with the nominal composition al42.5cu57.5 shows an invariant effect at 561(2) c which is considered to be related to the reaction 2 these results do confirm the previous authors, concerning the stability of the low temperature phase 1 which is considered to be stable between ambient temperature and 561(2) c . Since the xrd results of the sample al45cu55 show small traces of 1 we suggest a transition temperature 2 = 1 + 2 above 500 c . The solubility limit at the cu - rich side of 1/2 was not accessible due to low contrast and fine microstructure . However, was found as the only phase present in al40cu60, while al42.5cu57.5 showed 1 with traces of . Therefore, the situation of the two phase field can be specified quite accurately . It was not possible to determine the phase boundaries between the two phases and 1 by sem measurements due to the lack of contrast and possibly very fine microstructure . In xrd it was possible to distinguish the single phase region (r3 m, a = 12.285(1), c = 15.1486(1)) from the single phase region 1 (p-43 m, a = 8.7068(3)) by peak splitting and selective peak broadening even though the patterns look very similar . Samples with the nominal composition al40cu60 to al37cu63 show as the only present phase . The sample al36cu64 shows a main pattern corresponding to plus some small extra peaks that could not be explained by or 1 . They may be caused by super structure reflections, corresponding to the monoclinic structure proposed by bradley and westman, which was omitted in the assessment of murray . + 1 = in the sample al38cu62 and al37cu63 can be explained by a shift of the 1 phase field toward the cu - poorer region at elevated temperatures and, therefore, a smaller amount of taking part in the reaction . This leads to a smaller endothermic effect and since the respective endothermic peaks in the samples al40cu60 and al39cu61 are already small the resulting effect in the samples al38cu62 and al37cu63 might be insufficient to observe . The region between 1 and (cu) has been the subject of an intensive research in the past and the present work does not provide any contradictory information . The solubility of al in (cu) as well as the upper solubility limit of 1 was confirmed by sem measurements . Dta analysis of samples in the respective area show very small thermal effects close to the solubility limit of (cu) at 567 c and thermal effects related to the formation of at higher temperature, which are indicated as diamond shaped points in fig . 2 . In general, solubility ranges and thermal stability of the high temperature compounds 1 and 2 could be confirmed in the present work by dta observations . Slight changes concerning reaction temperatures and solubility ranges are shown in table 2 . Since there is no consensus in literature concerning the transition of the high temperature phase 0 to the low temperature form 1 this area is of special interest . Analysis of thermal effects of samples in the respective field show very weak effects varying continuously with the composition . We did not observe any pointer for an invariant decomposition of 1 in any of the investigated samples . Therefore we conclude that the transition 1 = 0 is of higher order, in agreement with the previous obtained results by liu et al . . Structural analysis of a sample with the nominal composition al32cu68 confirm the structure given for 0 by liu (i-43 m, cu5zn8-type), and reveals a lattice parameter of a = 8.8692(1) at 900 c . According to murray, the high temperature phase 0 was included in the equilibrium phase diagram although its existence could not be confirmed . Did not find any evidence of its existence and consequently was not incorporated in the assessed phase diagram of riani et al . . Results of the present work confirm that there is no evidence of the existence of 0 and all observed dta effects in this composition area can be explained by the formation of the phases and 0 . Preston suggested the space groups cmmm or pban and orthorhombic lattice parameters were given by lukas and lebrun . In the current study, we used high temperature powder xrd data to establish a structural model for 1 . Measurements were carried out in a temperature range from 500 to 750 c at intervals of 25 c using a sample with the nominal composition al50cu50 . Up to 550 c show the low - temperature 2-phase in equilibrium with traces of the -phase . At 575 c a third pattern, 1, appears which is the only phase present at 600 and 625 c . Above 650 c only 2 is present and significant peak broadening can be observed, indicating the partial melting of the powder . After cooling back the sample to 500 c one again observes 2 and, the diffraction lines, however, are significantly broadened . Some unidentified peaks of very low intensity may be attributed to oxide formation at the sample surface during the long stay in the non ambient device . Since the amount of possible oxide is very small, further investigation was not performed . The pattern measured at 600 c could be successfully indexed using the orthorhombic unit cell suggested by lukas and lebrun . Cell refinement in space group cmmm yielded the lattice parameters a = 4.1450(1), b = 12.3004(4) and c = 8.720(1). According to the phase diagram discussion at 600 c the measured sample with the nominal composition al50cu50 is in equilibrium with a small amount of liquid phase . This affects the background of the xrd measurement, which was compensated by modeling an additional broad peak at 43.9(1) 2. further details of measurement and structure refinement of the 1 phase including the calculated errors of the parameters are listed in table 3 . The refined pattern of 1 is shown in fig . 6 . The structural model for 1 was established by a twofold approach . Given the similarities of the lattice parameters between the monoclinic phase 2 and the orthorhombic 1, aortho bmono, bortho amono and vortho 1.5vmono, we tried to develop the structural model by rearranging the atomic positions of the low temperature phase in the orthorhombic high temperature cell using the space group c222 . The atomic coordinates of this structural model were finally transformed to cmmm and standardized by applying the program structure tidy, . During the consecutive rietveld refinement, unusually large differences at the individual isotropic displacement factors were observed, indicating a decrease of electron density at some atomic positions . The occupations of al2, al3 and cu2 were found to be significantly reduced while all other sites were found to be fully occupied within 3 esd s and were therefore fixed during the final refinements . The final structural model shows reasonable displacement factors and the refined overall composition al14.1cu14.8 (equivalent to 51.2 at.% cu) is in excellent agreement with the al - rich phase boundary of 1 (51.5(5) at.% cu). More details on the crystal structure investigation can be obtained from the fachinformationszentrum karlsruhe, 76344 eggenstein - leopoldshafen, germany, (fax: + 497247 808666; e - mail: crysdata@fiz.karlsruhe.de) on quoting the depository number csd 423053 . The coordination polyhedra for the 7 independent positions of 1 are shown in fig . 7 . Apart from the high - symmetry positions cu3 and cu4, the coordination figures are quite irregular with coordination numbers between cn = 10 and cn = 14 . A comparison of the atomic arrangements in the low temperature phase 2 and the high temperature phase 1 is shown in fig . The figure shows the layer in (001) of orthorhombic 1 in comparison to the layer in (010) of the monoclinic 2, i.e. Both structures are projected along their short axis . All atoms shown are situated within the mirror plane at z = 0 and y = 0, respectively . The corresponding second layer of each structure (situated at z = 1/2 and y = 1/2, respectively) shows the same atomic arrangement shifted by in for 1 and in for 2 according to space group symmetry . 8 shows that both structures have a common structural motif; i.e. A diamond shaped unit consisting of 5 cu- and 4 al - atoms . These motifs are arranged in a rectangular pattern and interconnected along their corners in case of the high temperature structure . In the monoclinic structure while only one atom, al3, is not part of the diamond - shaped motif of 2, three of the seven sites in 1 (al1, cu2 and cu3) are not part of this motif . Although the two structures are obviously related it should be pointed out that it is not possible to transform one structure into the other in a simple way and a second order transition between 1 and 2 can be definitely ruled out . This is consistent with our phase diagram investigation which clearly indicates an invariant reaction related to the transformation from 2 to 1 . As an example, the dta curve for the sample with the nominal composition al50cu50 (used also for the high temperature xrd) is shown in fig . This effect is followed by the peritectic decomposition of (590 c) and 1 (625 c) and finally the liquidus effect at 804 c . Combining results from both dta and sem measurements, it was possible to obtain the complete description of the al cu system which is plotted in fig . 2 . It is, for the most part, in good agreement to the phase diagram of liu et al . And riani et al . The occurring invariant reactions together with the composition of the reacting phases and the reaction temperature are given in table 2, selected sem images taken in back - scattered electron (bse) mode are shown in fig . The al - rich part of the phase diagram has not been investigated extensively in the current study . Concerning the reaction temperatures and the phase composition, the solubility of cu in al was found to be 2.2(1) at.% at 500 c . The lattice parameters of the phase (ti12, al2cu - type) vary from a = 6.0718(1) and c = 4.8802(1) at 32(1) at.% cu to a = 6.0613(1) and c = 4.8724(1) at 33.6(2) at.% cu . The phase boundaries of the binary phases in the al - rich part of the phase diagram have been determined by means of sem measurements and are indicated as black dots in fig the phases 1-alcu and 2-alcu are supposed to be stable around 50 at% cu with a solubility range of 12 at.% cu . The results in the current investigations, however, reveal a shift of the composition toward the cu - rich side . The solubility limits of the phase 2-alcu at 500 c were confirmed by sem measurements . The cu - poor and the cu - rich composition limit were found at 51.9(5) at.% cu and at 54.8(5) at.%, respectively . Xrd analysis of the samples with the nominal composition al49cu51 (showing and 2 in equilibrium), al47.5cu52.5 and al46.5cu53.5 (showing single phase 2) and al45cu55 (showing 2 plus traces of 1) indicate a cu - rich solubility limit between 54 and 55 at.% cu which supports the sem measurements . The lattice parameters for 2 (mc20, alcu - type) range from a = 12.0925(1), b = 4.1001(1), c = 6.9085(1), = 55.03(1) on the cu - poor side to a = 12.2012(1), b = 4.0997(2), c = 7.0047(3) and = 54.787(1) for the cu - rich side of this compound . The phase diagrams of riani and murry indicate a transition temperature from the high temperature 1-phase to the low temperature 2-phase at 563 c at the cu - poor side and 560 c at the cu - rich side, . According to our measurements the transition temperatures are 574(3) c for the cu - poor side and 580(1) c for the cu - rich side . Consequently, we propose also different solid state reactions in this area: the eutectoid decomposition 1 = 2 + at 574(3) c and the peritectoid reaction 1 the peritectic decomposition temperature of the phase 1 was found to be 625(2) c which is in agreement with the phase diagrams mentioned above . The region of al4cu9 with the supposed high temperature modification 1 and the low temperature phase 2 is also complex . Gulay and harbrecht find the cu - rich phase 1 stable at 530 and 400 c, but the cu - poor phase 2 stable at 530 c and not stable at 400 c, . They discovered that after a heat treatment at 400 c, 2 is decomposed into 1 and small amounts of 2 . Additionally, gulay and harbrecht state that at 450 c the phase 2 segregates in a mixture of 1 and 2 . Therefore, the authors conclude that the temperature of the eutectoid decomposition of 2 is between 400 and 450 c . The present work shows a sample with the nominal composition of al45cu55 exhibiting 2 as major phase with very small traces of a second phase . Comparison of systematic extinctions of 1 and 2 suggests that the second phase is 1 . Due to the very low amount of 1 in the respective sample, sem analysis of this sample shows only one suitable measurement point at 56.3 at.% cu which can be assigned to the cu - poor solubility limit of 1 . Xrd analysis of a sample with the nominal composition al42.5cu57.5 shows 1 and some unidentified peaks (see table 3). These xrd results narrow the solubility limits of 1 (of88, al3cu4-type) between approx . 56 and 57.5 at% cu . In the present investigation, dta analysis of the sample with the nominal composition of al45cu55 does not show any effects related to the transition of 1 to 2, caused by the fact that the amount of 1 is very small in the sample . Dta analysis of the sample with the nominal composition al42.5cu57.5 shows an invariant effect at 561(2) c which is considered to be related to the reaction 2 these results do confirm the previous authors, concerning the stability of the low temperature phase 1 which is considered to be stable between ambient temperature and 561(2) c . Since the xrd results of the sample al45cu55 show small traces of 1 we suggest a transition temperature 2 = 1 + 2 above 500 c . The solubility limit at the cu - rich side of 1/2 was not accessible due to low contrast and fine microstructure . However, was found as the only phase present in al40cu60, while al42.5cu57.5 showed 1 with traces of . Therefore, the situation of the two phase field can be specified quite accurately . It was not possible to determine the phase boundaries between the two phases and 1 by sem measurements due to the lack of contrast and possibly very fine microstructure . In xrd it was possible to distinguish the single phase region (r3 m, a = 12.285(1), c = 15.1486(1)) from the single phase region 1 (p-43 m, a = 8.7068(3)) by peak splitting and selective peak broadening even though the patterns look very similar . Samples with the nominal composition al40cu60 to al37cu63 show as the only present phase . The sample al36cu64 shows a main pattern corresponding to plus some small extra peaks that could not be explained by or 1 . They may be caused by super structure reflections, corresponding to the monoclinic structure proposed by bradley and westman, which was omitted in the assessment of murray . + 1 = in the sample al38cu62 and al37cu63 can be explained by a shift of the 1 phase field toward the cu - poorer region at elevated temperatures and, therefore, a smaller amount of taking part in the reaction . This leads to a smaller endothermic effect and since the respective endothermic peaks in the samples al40cu60 and al39cu61 are already small the resulting effect in the samples al38cu62 and al37cu63 might be insufficient to observe . The region between 1 and (cu) has been the subject of an intensive research in the past and the present work does not provide any contradictory information . The solubility of al in (cu) as well as the upper solubility limit of 1 was confirmed by sem measurements . Dta analysis of samples in the respective area show very small thermal effects close to the solubility limit of (cu) at 567 c and thermal effects related to the formation of at higher temperature, which are indicated as diamond shaped points in fig . 2 . In general, solubility ranges and thermal stability of the high temperature compounds 1 and 2 could be confirmed in the present work by dta observations . Slight changes concerning reaction temperatures and solubility ranges are shown in table 2 . Since there is no consensus in literature concerning the transition of the high temperature phase 0 to the low temperature form 1 this area is of special interest . Analysis of thermal effects of samples in the respective field show very weak effects varying continuously with the composition . We did not observe any pointer for an invariant decomposition of 1 in any of the investigated samples . Therefore we conclude that the transition 1 = 0 is of higher order, in agreement with the previous obtained results by liu et al . . Structural analysis of a sample with the nominal composition al32cu68 confirm the structure given for 0 by liu (i-43 m, cu5zn8-type), and reveals a lattice parameter of a = 8.8692(1) at 900 c . According to murray, the high temperature phase 0 was included in the equilibrium phase diagram although its existence could not be confirmed . Did not find any evidence of its existence and consequently was not incorporated in the assessed phase diagram of riani et al . . Results of the present work confirm that there is no evidence of the existence of 0 and all observed dta effects in this composition area can be explained by the formation of the phases and 0 . Preston suggested the space groups cmmm or pban and orthorhombic lattice parameters were given by lukas and lebrun . In the current study, we used high temperature powder xrd data to establish a structural model for 1 . Measurements were carried out in a temperature range from 500 to 750 c at intervals of 25 c using a sample with the nominal composition al50cu50 . Up to 550 c show the low - temperature 2-phase in equilibrium with traces of the -phase . At 575 c a third pattern, 1, appears which is the only phase present at 600 and 625 c . Above 650 c only 2 is present and significant peak broadening can be observed, indicating the partial melting of the powder . After cooling back the sample to 500 c one again observes 2 and, the diffraction lines, however, are significantly broadened . Some unidentified peaks of very low intensity may be attributed to oxide formation at the sample surface during the long stay in the non ambient device . Since the amount of possible oxide is very small, further investigation was not performed . The pattern measured at 600 c cell refinement in space group cmmm yielded the lattice parameters a = 4.1450(1), b = 12.3004(4) and c = 8.720(1). According to the phase diagram discussion at 600 c the measured sample with the nominal composition al50cu50 is in equilibrium with a small amount of liquid phase . This affects the background of the xrd measurement, which was compensated by modeling an additional broad peak at 43.9(1) 2. further details of measurement and structure refinement of the 1 phase including the calculated errors of the parameters are listed in table 3 . The refined pattern of 1 is shown in fig was established by a twofold approach . Given the similarities of the lattice parameters between the monoclinic phase 2 and the orthorhombic 1, aortho bmono, bortho amono and vortho 1.5vmono, we tried to develop the structural model by rearranging the atomic positions of the low temperature phase in the orthorhombic high temperature cell using the space group c222 . The atomic coordinates of this structural model were finally transformed to cmmm and standardized by applying the program structure tidy, . During the consecutive rietveld refinement, unusually large differences at the individual isotropic displacement factors were observed, indicating a decrease of electron density at some atomic positions . The occupations of al2, al3 and cu2 were found to be significantly reduced while all other sites were found to be fully occupied within 3 esd s and were therefore fixed during the final refinements . The final structural model shows reasonable displacement factors and the refined overall composition al14.1cu14.8 (equivalent to 51.2 at.% cu) is in excellent agreement with the al - rich phase boundary of 1 (51.5(5) at.% cu). More details on the crystal structure investigation can be obtained from the fachinformationszentrum karlsruhe, 76344 eggenstein - leopoldshafen, germany, (fax: + 497247 808666; e - mail: crysdata@fiz.karlsruhe.de) on quoting the depository number csd 423053 . The coordination polyhedra for the 7 independent positions of 1 are shown in fig . 7 . Apart from the high - symmetry positions cu3 and cu4, the coordination figures are quite irregular with coordination numbers between cn = 10 and cn = 14 . A comparison of the atomic arrangements in the low temperature phase 2 and the high temperature phase 1 is shown in fig . The figure shows the layer in (001) of orthorhombic 1 in comparison to the layer in (010) of the monoclinic 2, i.e. Both structures are projected along their short axis . All atoms shown are situated within the mirror plane at z = 0 and y = 0, respectively . The corresponding second layer of each structure (situated at z = 1/2 and y = 1/2, respectively) shows the same atomic arrangement shifted by in for 1 and in for 2 according to space group symmetry . 8 shows that both structures have a common structural motif; i.e. A diamond shaped unit consisting of 5 cu- and 4 al - atoms . These motifs are arranged in a rectangular pattern and interconnected along their corners in case of the high temperature structure . In the monoclinic structure while only one atom, al3, is not part of the diamond - shaped motif of 2, three of the seven sites in 1 (al1, cu2 and cu3) are not part of this motif . Although the two structures are obviously related it should be pointed out that it is not possible to transform one structure into the other in a simple way and a second order transition between 1 and 2 can be definitely ruled out . This is consistent with our phase diagram investigation which clearly indicates an invariant reaction related to the transformation from 2 to 1 . As an example, the dta curve for the sample with the nominal composition al50cu50 (used also for the high temperature xrd) is shown in fig . This effect is followed by the peritectic decomposition of (590 c) and 1 (625 c) and finally the liquidus effect at 804 c . The current work revealed significant improvements on the established phase diagram in the system al cu by solving several inconsistencies in literature . The al - rich part of the phase diagram could be confirmed . The phases 1 and 2 show a significant shift to the cu - rich side of the phase diagram and exhibit, contrary to previously published phase diagrams,,, a first order transition reaction . The 1/2 region was re - investigated and the recent findings of gulay and harbrecht, were widely confirmed . The transition between 0 and 1 does not show a two phase field thus indicating a higher order transition and confirming the results of liu et al . . The phase is orthorhombic (space group cmmm) and the lattice parameters are a = 4.1450(1), b = 12.3004(4) and c = 8.720(1).
Different factors, especially genetic, effect individual attitude to regular physical activity in iranian traditional medicine . Our aim was to conduct a comparative revision and evaluation of the effect of genetic factors on physical activity in classic medicine and iranian traditional medicine . In this study, we reviewed persian resources in the research center of traditional medicine at shiraz university of medical sciences . We also evaluated the texts on physical activity and genetics in pubmed and google scholar . The results of classical medicine revision showed the effect of especial genes on obesity and sedentary behavior . It is also derived from iranian traditional medicine resources that physical activity and sedentary behavior is affected by individual mizaj . The results showed that those with hot and cold mizaj have different genetic potentials in sedentary behavior and physical activity . Such categorization would be helpful in identifying illnesses due to sedentary life in special groups of people.
Many primary care physicians in nonacademic settings lack a collegial forum for engaging the clinical uncertainties inherent in their work . Practice inquiry is proposed as a set of small - group, practice - based learning and improvement (pbli) methods designed to help clinicians better manage case - based clinical uncertainty . Clinicians meet regularly at their offices / clinics to present dilemma cases, share clinical experience, review evidence for blending with experience, and draw implications for practice improvement . From 2001 through 2005, practice inquiry was introduced to sites in the san francisco bay area as a demonstration effort . Meeting rosters, case logs, a feedback survey, and meeting field notes documented implementation and provided data for a formative, qualitative evaluation . 2006, 98 clinicians in 11 sites continue to hold regularly scheduled group meetings . Of the 118 patient cases presented in the seven oldest groups, clinician clinician feedback and meeting transcript data provided insights into how busy practitioners shared cases, developed trust, and learned new knowledge / skills for moving forward with patients . Ongoing clinician involvement suggests that practice inquiry is a feasible, acceptable, and potentially useful set of pbli methods . Two of the practice inquiry s group learning tasks received comparatively less focus: integrating research evidence with clinical experience and tracking dilemma case outcomes . Future work should focus on reducing the methodological limitations of a demonstration effort and examining factors affecting clinician participation . Set - aside work time for clinicians, or other equally potent incentives, will be necessary for the further elaboration of these pbli methods aimed at managing uncertainty . Primary care physicians (pcps) in nonacademic settings have few safe and reliable forums where they can reflect and learn from the clinical dilemmas inherent in their work . With the collaborative learning of residency training no longer available, clinicians often adopt idiosyncratic approaches when they encounter patient - care situations that cause them to question the limits of their own knowledge, what is knowable, and how to distinguish between their own knowledge limits and that of the medical canon in short, clinical uncertainty.1 the methods pcps have traditionally relied upon to tackle uncertainty include: curbsiding colleagues, consulting print and electronic literature, making referrals to specialists, employing watchful waiting, and cme courses for generalized updates in knowledge and skills.24 in addition to questions about method effectiveness,57 the current era of exponential knowledge growth, expanding patient volume, competing evidence - based care agendas (e.g., acute, chronic, and preventive), and the public s expectations for health fixes have made dependence on these familiar methods more difficult.811 nonetheless, the need for managing case - based dilemmas remains critical as clinical uncertainty affects care outcomes, resource utilization, and patient and physician satisfaction.1215 although contexts for coping with uncertainty have changed, most physicians would support light s 1979 observation that regardless how technically developed a professional field is, it will define the treatment of problematic cases as its true work.16 social constructivist learning theorists,17,18 medical educators,1921 and primary care researchers22 identify the problematic patient case as a powerful professional learning opportunity . Whether and how one decides to take on these problems in the swampy lowlands23 of practice become, according to guest, decisions about deliberate practice.24 practitioners develop expertise when they move from their comfort zones to examine problems at the upper limit of the complexity they can handle; they learn, and iteratively gain mastery through cycles of reflecting on practice, obtaining feedback, and adjusting performance.24,25 recent emphasis on practice - based learning and improvement defined by moore and pennington as an approach for reflection on and appraisal of one s own delivery of clinical care that results in pursuing an opportunity for improvement,26 pbli encompasses multiple methods that range from individual learning portfolios containing practice question literature searches to virtual clinician groups using interactive methods to discuss real patient cases.2729 whereas the effectiveness of many pbli methods is unknown, social interaction, a key element in some pbli approaches, appears to increase physician satisfaction with learning and improve certain practice and patient outcomes.30,31 a real - time, face - to - face, pcp small group could be an attractive pbli venue for tackling case - based dilemmas . In addition, newer methods model the advantages of learning with and from colleagues (e.g., problem - based learning in medical school32 and case - based ebm skill training in resident clinics33). A pcp office could form the learning practice that rushmer, cervero, parboosingh, and frankford advocate for supporting clinicians over time as they create, take apart, and reassemble strategies for patients whose problems are not in the book21,3436 and build a skills repository for uncertainty management.3739 engaging the problems of work at work allows for an explicit integration of clinical judgment with the system dimensions of practice21, making primary care practice both more efficient and personalized . We wondered whether a pbli experience focused on case - based clinical uncertainty would generate interest and commitment from pcps in the bay area of san francisco and designed practice inquiry (pi) as a set of methods to pilot in the office / clinic setting . In this study practice inquiry practice inquiry (pi) is a set of methods that structure how pcps could collaborate over time in learning from their patients case - based clinical uncertainties . They have been derived from the workplace focus of uk gps practice meetings,40,41 the relationship - centered emphasis of balint groups,42 and the ebm - based / reality - based direction of the canadian pcps small learning groups.43 the pi group consists of clinician colleagues, each with a patient panel, who work at the same practice site . Group members engage one another through flexible application of a dilemma case guide or a practice panel reflection guide . These guides suggest steps for identifying dilemma cases, exploring causes and consequences of uncertainty, searching for evidence, speculating about intervention options, and articulating new steps for moving forward with patients (see fig . 1 online). Clinician group members or an invited, external member facilitates by guiding discussion, supporting searches for and appraisals of evidence, and coordinating meeting logistics . Practice recruitment between fall 2001 and spring 2005, 30 physician practice leaders, known to the authors, were approached about holding initial pi meetings at their sites . Seventeen expressed interest; 13 cited limited time and satisfaction with current cme as reasons for declining . Of the 17 interested practices, 10 held initial pi meetings to introduce methods, and 7 continue to meet regularly (phase-1 groups, 65 clinicians). The remaining 3 discontinued meeting in early 2002 . Telephone interviews with these groups physician contacts regarding reasons for discontinuation cited lack of committed leadership and need for other types of clinical support . Four of the seven remaining interested practices began meeting in 2006 (phase-2 groups, 38 clinicians), and three have yet to initiate meetings (see fig . 2 for recruitment detail). Data collection and analysis with phase-1 clinicians consent, data from four sources were collected from fall 2002 through summer 2005 . These included: (1) pi meeting rosters with the date, location, attendance, and meeting length for each pi meeting; (2) dilemma case logs containing descriptions of case - based clinical uncertainties (coded by facilitators as predominately a diagnostic, therapeutic, relationship, or negative outcome, with validation by presenters at subsequent meetings), pertinent case data (e.g., patient age and current medications), and plans for follow - up (e.g., review specific literature); (3) formative feedback surveys, mailed in fall 2003 to all clinicians participating in active groups (see table 1 . ), which asked: what have you liked about practice meetings? (survey responses, identifiable only by group, were transcribed and analyzed by each author, as well as a psychologist educator and a family physician educator, expert in qualitative data analysis, using a first - stage in vivo coding approach).44 the authors met, discussed their coding schemas, compared approaches, selected one schema, and recoded sufficiently to generate agreed - upon themes . Ls recoded all data, refined themes, and circulated them for final approval; (4) handwritten field notes describing group discussions transcribed for 64 of the 137 pi meetings: 42 were facilitated or observed and transcribed by ls and 22 by lm . Of those transcripts judged to be most complete, table 1description of phase-1 practice inquiry groups by key characteristicsgrouplocation / sitefirst meeting date; pre pi, case - based cme?clinicians membership; specialty, mean attendancemeeting time, frequency, lengthfacilitatorgroup 1san francisco, caoctober 20025 fpadmin time, 1 h, every other monthrotated among group memberscounty - funded community health centerno4group 2san francisco, cadecember 20023 gim, 3 fpadmin time 1, 1/2 h, every other monthls (author)county - funded community health centerno5group 3oakland, caapril 20036 fp, 7 gim, 3 med subspecialistslunch, 1 h, twice monthlylm (author, physician group member)hmo medical center (department of medicine)no7group 4oakland, cajuly 200411 fp, 8 gim, 4 ped, 1 np, 1 palunch, 1/2 h, weeklyphysician group memberfederally funded community health center1/2 h, weekly no case log10group 5richmond, cafebruary 20058fp, 2 npafter work, 1 1/2 h, every other monthlscounty - funded community health centerno7group 6san francisco, camarch 20059 fpbefore clinic + admin time, monthlylsuniversity faculty practiceno6group 7san francisco, cajune 20055 fp, 1 gim, 1 npadmin time, 1 h, every other monthlscounty - funded community health centerno5these groups stopped meeting for 18 and 9 months, respectively, because of budget cuts; each resumed meeting in 2005.fp = family practitioner, gim = general internal medicine, ped = pediatrician, np = nurse practitioner, pa = physician assistant description of phase-1 practice inquiry groups by key characteristics these groups stopped meeting for 18 and 9 months, respectively, because of budget cuts; each resumed meeting in 2005 . Fp = family practitioner, gim = general internal medicine, ped = pediatrician, np = nurse practitioner, pa = physician assistant meetings and attendance the seven phase-1 groups met for periods of 7 months to 3 years as a result of different start dates within the 20022005 data collection period (see table 1). Attendance and group size remained stable over 137 pi meetings despite one- to three - member turnover mostly from job change . For five- to six - member groups, all clinicians working that day attended meetings . For nine - plus - member groups, three to six clinicians attended regularly, with an additional three to six attending on a less regular basis . Beginning in 2005 authors ls and lm facilitated four groups and one group, respectively; clinician members facilitated two groups . Clinical dilemma cases across the seven phase-1 groups, we documented 118 dilemma cases discussed in the 64 meetings analyzed . Eighty - five of the cases were presented as individual case dilemmas, and 33 were generated in 10 practice panel reflections; six of the seven groups completed at least one reflection . Of the total dilemma cases, patient relationship, 26% as treatment, 25% as diagnosis, and 10% as negative outcome dilemmas (see table 2 for representative dilemmas). Recurring treatment dilemmas included decision - making about incidental findings, morbid obesity, developmental disabilities coupled with chronic illness, and nonmalignant chronic pain with / without substance abuse . Table 2representative case dilemmas presented at practice inquiry meetings, phase-1 groupscase dilemmapatient clinician relationship#1this patient, also a friend, wants me to continue being his pcp after being diagnosed with prostate ca (at earlier social gathering, he asked me about difference in testicular size, and i told him not to worry).#2i do nt know how to work with angry, defeated patient who has fired previous physician, expects same - hour return phone calls, and berates me for lack of improvement in symptoms lacking organic basis.treatment#1i am seeing a 53-year - old female with chronic hep b, htn, dm, elevated ldl, and slightly elevated lfts . Should i start her on statins?#2i do nt know how to proceed with a morbidly obese, developmentally delayed woman of 43 with sleep apnea and presumed right - sided heart failure with pulmonary htn, who now wants bariatric surgery.diagnosis#1this is a 68-year - old female with a history of chemotherapy for lymphoma . She has new symptoms of a hot feeling in her body; she is afebrile with normal labs . How should i approach her workup?#2does this 30 year - old asian female have pcos and how can i help her become pregnant?negative outcomes#1i am hurt and confused regarding what i found out accidentally about a patient . A physician friend in private practice is now seeing my patient; patient left me because i did not prescribe a statin; she blames not being on statin for her subsequent tia . How could i have worked differently with this patient?#2i have male patient in late 1960s with presumptive diagnosis of temporal arteritis; on low - dose steroids for several years; was admitted to the hospital with altered mental status, nausea, vomiting, high white count and sed rate; biopsied for temporal arteritis negative; treated with abx . My realization: missed increasing dementia over time since family members brought him in and answered my questions.a third of individual case discussions produced literature searches done by group members or the facilitator . Systematic reviews and metaanalyses were the most frequent literature formats provided . Approximately one in five cases was discussed in subsequent meetings as follow - up; follow - up consisted of 2- to 4-min reports of patient status, literature usefulness, new questions, and/or implications for other patients . Representative case dilemmas presented at practice inquiry meetings, phase-1 groups clinician feedback seventeen of the 23 physicians who returned questionnaires produced 92 comments . Three categories accounted for the majority of comments: the value of being with colleagues (55%), group process / meeting logistics suggestions (27%), and the role of time(16%). Being with colleagues was further analyzed to yield four subthemes: (1) gaining renewal through reflection, (2) obtaining others perspectives, (3) developing collegial trust, and (4) learning specific information / skills (see table 3 for representative responses). Over half of the respondents commented on time issues related to participation; a third saw time constraints as deterring attendance . Table 3phase-1 groups feedback survey responses by coding group (n = 92)coding groupsn (% comments)representative commentsbeing with colleagues51 (55%) gaining renewal through reflection15(g2, 2) * although it was hard to break away from all our work responsibilities, the meetings have forced me to take protected time away from the daily grind to be more thoughtful...(g3, 2) i like the idea of reflecting on what we do rather than the daily do, do, do . In addition, to reflect on how what we do affects the patient physician relationship . Obtaining colleagues perspectives14(g2, 4) fresh eyes and ideas on old cases means new ideas...(g3, 3) exposure to a variety of physician / patient encounters and expectations developing trust as a group12(g2, 3) i learned about my colleagues . I was surprised by some of what was shared in terms of questions they had i would have thought that i was the only one. (g3, 8) helps develop a feeling of connectedness to peers, which balances out the isolation of clinical practice learning new information / skills10(g1, 3) learning efficient ways to deal with complicated but frequently occurring issues(g2, 5) making sure that my knowledge level is not falling too far behind!group process / meeting logistics24 (27%) more research time12(g1, 2) set aside time to research clinical questions more case follow - up4(g3, 2) follow - up cases previously discussed in a more formal way misc8(g2, 5) cme would make this more worth my time. (g1, 1) case presentation format all over the maprole of time15 (16%) create set - aside time9(g2, 3) meetings come out of clinical time instead of paperwork times . Time is a problem.6(g3, 7) want to continue but time is always a problem not codable in above categories2 (2%)(g3, 2) i want to continue. g2, 2 = survey respondent #2 in group 2 phase-1 groups feedback survey responses by coding group (n = 92) g2, 2 = survey respondent #2 in group 2 clinician group discussion analysis of the 15 pi meeting transcripts yielded four themes: acknowledging uncertainty, receiving validation, generating speculations, and envisioning practice change . In describing dilemmas, clinicians seemed to be willing to reveal knowledge gaps, cognitive biases, and unrealistic expectations: what should have told me it was temporal arteritis? When my gut says cancer, i get a bone marrow biopsy . If i had been more aggressive with his cocaine addiction, would this have prevented his hospitalization? These disclosures appeared to elicit validation from colleagues: amazing you got as far as you did...you got the tox screen! Patients choose you but you ca nt choose them! Why in 15 min should we take on more risks? Such interchanges stimulated speculative thinking as well as metastrategies: seems like you could create small successes to build on...like saying, wow, you took your hiv meds for 4 days! When i have a patient like this, i schedule a special appointment to do a family tree...then, it all becomes obvious . Clues that case presenters were thinking aloud, reframing assumptions, and imagining opportunities for change came through statements such as: you re suggesting she s not as worried about herself as i am and, if that s denial, maybe that s okay . I may not be taking this patient s depression as seriously as i should . In this first pi demonstration, as of summer 2006, 11 groups, composed of 98 clinicians, continue to meet on a regular basis . In the seven phase-1 groups, participants willingly presented individual dilemmas, validated each other s concerns, shared uncertainty management strategies, and responded positively to new clinical perspectives . Despite pcps beleaguered status in turbulent times,45 these findings suggest the feasibility, acceptability, and potential usefulness of these pbli methods . To address pi s usefulness for practice improvement will require more focused modeling and assessment . Analyses of meeting logs and feedback suggest that participants lacked sufficient opportunities to collaborate in searching for and appraising research evidence, and then blending it with clinical experience to apply to case dilemmas . Ebm enthusiasts advocate these skills but provide little guidance for their development.46,47 additionally, limited case follow - up may have truncated the iterative process of integrating feedback and new evidence into changing practice . To better support pcps in managing uncertainty, more meeting time should be spent on the deliberate practice of blending evidence with experience (e.g., per - case, focused analysis of guideline / relevance48) and using case follow - up insights to reconstruct practice20 for the individual patient while appreciating implications for the clinic / office as a whole . Future work must address methodological limitations that threatened this demonstration effort s external and internal validity . These include a convenience sample from one geographic area lacking private practice pcps, moderate clinician response to a cross - sectional feedback survey sent to three groups meeting in 2003, and qualitative analyses performed on a small meeting transcript sample and an incomplete dilemma case sample . In addition, limited data collection for achieving theoretical saturation and overlapping datasets hampering data triangulation remain as challenges . Further pi development, undertaken with rigorously designed, coordinated qualitative and quantitative methods,49 should examine how contextual variables (e.g., group facilitation) impact case discussion, development of uncertainty management skills, and clinician work - life satisfaction . Future pi development should not be predicated on the assumption that phase 1 and two groups will continue to thrive, and recruitment of new, heterogeneous groups representing different geographic areas will proceed unabated . Feedback suggests that holding meetings during personal / administrative may be suboptimal and result in even longer workdays . Would the 13 clinician leaders who originally rejected pi have reacted positively if compensated, set - aside time or credits toward board recertification were linked to participation?5053 because compensating attendance without evidence of impact would be ill - advised, primary care leadership should advocate for studies that examine diverse incentive structures for involvement in pbli efforts focused on clinical uncertainty and how that involvement affects practice and patient care.
The last few years have seen a rapid expansion in the use and availability of ablation techniques with hundreds of papers published . Radiofrequency (rf) remains the frontrunner in terms of cost, ease of set - up, versatility and flexibility but other techniques are catching up . Ablation with cryotherapy and microwave, which were previously only available at open laparotomy due to the large size of the probes, are now readily performed percutaneously, with a predictable reduction in morbidity . Cryotherapy offers the opportunity to monitor the ice - ball with ultrasound (us), computed tomography (ct) or magnetic resonance imaging (mri). Cell death occurs around 40c and this isotherm lies approximately 5 mm inside the ice - ball . More powerful microwave generators have been developed and active research into better impedance matching with specific tissues should result in improved performance . Microwave has several theoretical advantages; it can be designed to share a spherical geometry with most tumours and has a more predictable dose response curve . Simultaneous use of multiple energy sources (probes / electrodes) is now available in all the different technologies . Improvements in guidance include the development of real - time co - registration techniques, most commonly ct and us . Five percent dextrose is instilled into the area adjacent to the ablation to displace vulnerable structures, usually bowel, away from the ablation zone . A displacement of as little as 1 cm is enough to protect the bowel . Ablation is now accepted as the first line of treatment in patients with limited volume hepatocellular carcinoma (hcc) who are not candidates for transplantation . There is continuing debate in most other areas but the evidence is increasing for an important role in liver metastases, renal carcinoma, inoperable lung tumours and some bone tumours . Nephron - sparing surgery presents a challenge to the surgeon . Yet, there is increasing evidence that even moderate degrees of renal failure can significantly affect survival following cardiovascular events . This will increase the focus of all physicians on the need to preserve renal function wherever and whenever possible . One study found that 95.2% of patients had a glomerular filtration rate (gfr)> 60 ml / min per 1.73 m at 3 years post rfa compared with 70.7% post partial nephrectomy and only 39.9% post radical nephrectomy . Therefore patients with a solitary kidney and others with borderline renal function will increasingly be treated with ablation . Renal tumours up to 3.5 cm in diameter can be destroyed in situ by laser, rf or cryotherapy with virtually no damage to the surrounding normal renal tissue . Multiple renal tumours are not rare and can be difficult to resect without complications but complications are rare after ablation, particularly if a percutaneous approach is used . Bowel injury can be prevented by dextrose isolation but it remains necessary to maintain a distance of> 1 cm from the proximal ureter or cool the ureter via a stent as ablation can cause stricturing . Several series have now been published including one retrospective comparison with partial nephrectomy which showed comparable oncologic efficacy albeit with a shorter mean follow - up in the radiofrequency group (30 months versus 47 months). An apparently promising meta - analysis was heavily skewed away from recent innovations by the inclusion of early technology, failure to analyse by tumour size and location, and the stipulation that success after a single procedure was the main endpoint . Percutaneous ablative techniques are relatively easy to repeat compared with either surgery or laparoscopic cryotherapy . The question as to whether cryotherapy or radiofrequency is better is yet to be resolved and would be better addressed with mature technology and in a specific tumour cohort, e.g. Tumours <3.5 cm . This is predicted to be the single largest growth area in ablation over the next few years . Good results can be achieved in small, peripheral tumours . Both inoperable primary and limited numbers of metastatic tumours computed tomography fluoroscopy facilitates electrode placement as small, scirrhous lung lesions can be difficult to penetrate with a larger calibre needle . Pneumothorax occurs in about 40%, a similar incidence to that seen with trucut biopsy, but only a small percentage (1015%) require drainage . The likelihood of a pneumothorax increases with the length of aerated lung that is traversed by the electrode and is more common when treating multiple tumours . Infection is an unusual complication and in our series only occurred in patients with underlying asthma or chronic obstructive airways disease . Cavitation does not usually indicate infection but cavitating lesions are more likely to become infected . During treatment a penumbra of ground glass opacification (ggo) develops around the tumour representing the ablation zone and a surrounding inflammatory reaction . Histological studies have shown that the zone of cell death lies 24 mm inside the outer margin of the ground glass shadowing . This has been corroborated in a clinical study, with> 6 months follow - up, where the width of ggo at treatment was correlated with the development and location of recurrence . A minimum ggo of 4.5 mm is recommended to ensure complete ablation . Over time the ablation zone becomes increasingly dense and then reduces in size . At 12 months, up to 33% of successfully treated small lesions will have shrunk to a linear scar . Recurrence is identified by enlargement of the ablation zone, or a change in the shape of the zone indicating enlargement in one area or the development of focal nodular enhancement . Tumours <3.0 cm can usually be ablated at a single session, larger tumours, 3.05.0 cm, may require more than one ablation or other additional therapy . Multivariate analysis has shown size to be the dominant feature determining complete ablation, but contact with> 3 mm blood vessels or bronchi also increases the chance of recurrence . Current indications include patients with small volume, but inoperable metastases and early primary lung cancer in medically inoperable patients . Early clinical studies report 3- and 5-year survival of between 46% and 57% in patients with colorectal metastases, . Combinations of radiotherapy and rfa have been used to good effect in primary lung cancer in inoperable patients . The rapture study showed that lung function recovered quickly after an initial immediate post - treatment reduction and was maintained at pre - treatment values 12 months later . One of the first accepted indications for ablation was the minimally invasive treatment of benign osteoid osteomas . Malignant primary bone tumours are usually treated by chemotherapy, radiotherapy and surgery . However, if aggressive therapy is delivered at an early stage, recurrence can be very difficult to treat . Treatment by rfa may be curative, but is more likely to form part of a palliative treatment regimen . Ct or mr radiofrequency ablation and cryoablation have been advocated in the symptomatic palliation of bone metastases following radiotherapy . Initial results suggest that ablation can produce significant reductions in pain levels and analgesic requirements . It is important to select patients with a clearly defined and understood dominant site of bone pain . Some authors promote the combination of ablation and cementoplasty, others argue that cementoplasty alone would be adequate . Nephron - sparing surgery presents a challenge to the surgeon . Yet, there is increasing evidence that even moderate degrees of renal failure can significantly affect survival following cardiovascular events . This will increase the focus of all physicians on the need to preserve renal function wherever and whenever possible . One study found that 95.2% of patients had a glomerular filtration rate (gfr)> 60 ml / min per 1.73 m at 3 years post rfa compared with 70.7% post partial nephrectomy and only 39.9% post radical nephrectomy . Therefore patients with a solitary kidney and others with borderline renal function will increasingly be treated with ablation . Renal tumours up to 3.5 cm in diameter can be destroyed in situ by laser, rf or cryotherapy with virtually no damage to the surrounding normal renal tissue . Multiple renal tumours are not rare and can be difficult to resect without complications but complications are rare after ablation, particularly if a percutaneous approach is used . Bowel injury can be prevented by dextrose isolation but it remains necessary to maintain a distance of> 1 cm from the proximal ureter or cool the ureter via a stent as ablation can cause stricturing . Several series have now been published including one retrospective comparison with partial nephrectomy which showed comparable oncologic efficacy albeit with a shorter mean follow - up in the radiofrequency group (30 months versus 47 months). An apparently promising meta - analysis was heavily skewed away from recent innovations by the inclusion of early technology, failure to analyse by tumour size and location, and the stipulation that success after a single procedure was the main endpoint . Percutaneous ablative techniques are relatively easy to repeat compared with either surgery or laparoscopic cryotherapy . The question as to whether cryotherapy or radiofrequency is better is yet to be resolved and would be better addressed with mature technology and in a specific tumour cohort, e.g. Tumours <3.5 cm . This is predicted to be the single largest growth area in ablation over the next few years . Good results can be achieved in small, peripheral tumours . Both inoperable primary and limited numbers of metastatic tumours computed tomography fluoroscopy facilitates electrode placement as small, scirrhous lung lesions can be difficult to penetrate with a larger calibre needle . Pneumothorax occurs in about 40%, a similar incidence to that seen with trucut biopsy, but only a small percentage (1015%) require drainage . The likelihood of a pneumothorax increases with the length of aerated lung that is traversed by the electrode and is more common when treating multiple tumours . Infection is an unusual complication and in our series only occurred in patients with underlying asthma or chronic obstructive airways disease . Cavitation does not usually indicate infection but cavitating lesions are more likely to become infected . During treatment a penumbra of ground glass opacification (ggo) develops around the tumour representing the ablation zone and a surrounding inflammatory reaction . Histological studies have shown that the zone of cell death lies 24 mm inside the outer margin of the ground glass shadowing . This has been corroborated in a clinical study, with> 6 months follow - up, where the width of ggo at treatment was correlated with the development and location of recurrence . A minimum ggo of 4.5 mm is recommended to ensure complete ablation . Over time the ablation zone becomes increasingly dense and then reduces in size . At 12 months, up to 33% of recurrence is identified by enlargement of the ablation zone, or a change in the shape of the zone indicating enlargement in one area or the development of focal nodular enhancement . Tumours <3.0 cm can usually be ablated at a single session, larger tumours, 3.05.0 cm, may require more than one ablation or other additional therapy . Multivariate analysis has shown size to be the dominant feature determining complete ablation, but contact with> 3 mm blood vessels or bronchi also increases the chance of recurrence . Current indications include patients with small volume, but inoperable metastases and early primary lung cancer in medically inoperable patients . Early clinical studies report 3- and 5-year survival of between 46% and 57% in patients with colorectal metastases, . Combinations of radiotherapy and rfa have been used to good effect in primary lung cancer in inoperable patients . The rapture study showed that lung function recovered quickly after an initial immediate post - treatment reduction and was maintained at pre - treatment values 12 months later . One of the first accepted indications for ablation was the minimally invasive treatment of benign osteoid osteomas . Malignant primary bone tumours are usually treated by chemotherapy, radiotherapy and surgery . However, if aggressive therapy is delivered at an early stage, recurrence can be very difficult to treat . Treatment by rfa may be curative, but is more likely to form part of a palliative treatment regimen . Ct or mr radiofrequency ablation and cryoablation have been advocated in the symptomatic palliation of bone metastases following radiotherapy . It is important to select patients with a clearly defined and understood dominant site of bone pain . Some authors promote the combination of ablation and cementoplasty, others argue that cementoplasty alone would be adequate . A trial to establish the relative merits of the two techniques the last few years have seen a sharp increase in our understanding of ablation, maturation of the technology, an improvement in the safety profile, ablation efficacy and monitoring techniques and the publication of results in larger patient cohorts . Ablation will soon be sufficiently established to allow trials comparing ablation with conventional therapies in specific patient groups.
Number of (a) insertion / deletions, (b) high - confidence substitutions and (c) chromosomal rearrangements are plotted across all the samples from the 10 patients that were whole - genome sequenced . The primary means of validation was a deep sequencing validation experiment that included selected substitutions and indels from each sample, as described in extended data table 2 and supplementary information, section 2b . In addition, indels and rearrangements identified in wgs represent datasets orthogonal to the substitution data from which the subclones were identified . The subsets of samples in which validated substitutions, indels and rearrangements are found correlate strongly with the subclonal clusters identified from the clustering of substitutions from wgs, providing support for the existence of these subclones . For each patient, hierarchical clustering of the variant allele fraction (vaf) was performed separately for substitutions (a) and indels (b). Vafs are represented as a heatmap with deeper shades of red indicating a higher proportion of reads reporting the mutant allele . Above each heatmap, mutations are colour - coded according to the subclone they were assigned to by dirichlet process clustering of wgs data in the case of substitutions or by vaf for indels . For a22, additional samples not subject to wgs were included in the validation experiment . For these patients the phylogenetic tree from figure 2 was modified to incorporate these additional samples (c). Number of substitutions assigned to each subclone (d) and numbers of indels (e) and rearrangements (f) present in different subsets of samples are plotted as bar charts . Vafs from wgs and validation data, plotted as scatter plots (g), are very highly correlated . Validation strategy as described in extended data figure 2 . For a31 and a32, hierarchical clustering of the vaf was performed separately for substitutions (a) and (j) and indels (b) and (k). Additional samples for a31 and a32 are incorporated into the phylogenetic trees (c) and (l). Subclones for a31 cd and a32 ce are annotated in the corresponding 2d - dp plots (d) and (m). Numbers of substitutions in wgs data assigned to each subclone are plotted in (e) and (n). Vafs from wgs and validation data, plotted as scatter plots (f) and (o), are very highly correlated . Number of indels (g) and (p) and rearrangements (h) and (q) present in different subsets of samples are plotted as bar charts . Subclone colour keys for a31 and a32 (i and r) respectively . Validation strategy as described in extended data figure 2 . For a24 and a34, hierarchical clustering of the vaf was performed separately for substitutions (a) and (i) and indels (b) and (j). Indels that could not be assigned to any cluster (if any) are annotated with black . Additional samples for a24 and a34 are incorporated into the phylogenetic tree (c) and (k). The additional cluster in a24, supported by rearrangements only, is indicated by a light green branch in the tree . Numbers of substitutions in wgs data assigned to each subclone are plotted in (d) and (l). Vafs from wgs and validation data, plotted as scatter plots (e) and (m), are very highly correlated . Number of indels (f) and (n) and rearrangements (g) and (o) present in different subsets of samples are plotted as bar charts . Subclone colour keys for a24 and a34 (h and p) respectively . Validation strategy as described in extended data figure 2 . For a10 and a29, hierarchical clustering of the vaf was performed separately for substitutions (a) and (h) and indels (b) and (i). Indels that could not be assigned to any cluster (if any) are annotated with black . The additional sample (d) for a29 is incorporated into the phylogenetic tree (j). Validation experiment for a10-e, the prostate sample, gave very low coverage (d). Subclones for a29-a and a29-c are annotated in the 2d - dp plot (k). Numbers of substitutions in wgs data assigned to each subclone are plotted in (c) and (l). Vafs from wgs and validation data, plotted as scatter plots (d) and (m), are very highly correlated . Number of indels (e) and (n) and rearrangements (f) and (o) present in different subsets of samples are plotted as bar charts . Subclone colour keys for a10 and a29 (g and p) respectively . Validation strategy as described in extended data figure 2 . For a17 and a12, hierarchical clustering of the vaf was performed separately for substitutions (a) and (i) and indels (b) and (j). For a12, the c - specific cluster that is not present in substitutions is shown in very light green . Subclones for a17 ad are annotated in the 2d - dp plot (c). Numbers of substitutions in wgs data assigned to each subclone are plotted in (d) and (l). Vafs from wgs and validation data, plotted as scatter plots (e) and (m), are very highly correlated . Number of indels (f) and (n) and rearrangements (g) and (o) present in different subsets of samples are plotted as bar charts . Subclone colour keys for a17 and a12 (h and p) respectively . Validation strategy as described in extended data figure 2 . Hierarchical clustering of the vaf was performed separately for substitutions (a) and indels (b). Numbers of substitutions in wgs data assigned to each subclone are plotted in (d). Number of indels (e) and rearrangements (f) present in different subsets of samples are plotted as bar charts . Vafs from wgs and validation data, plotted as scatter plots (g), are very highly correlated . Rearrangements and copy number segments in the vicinity of the ar locus are shown for a31, a21, a29 and a10 . (is a tandem duplication whose existence is supported by tumour reads in adef but not c. however pcr - gel validation confirms its existence in the prostate sample c - the faintness of the band suggesting that this rearrangement is present subclonally in a31-c - as well as the prostate sample i, which was not subject to wgs . One tandem duplication is common to both prostate samples (shown in green) while the other is specific to sample c (dark pink). (b) in a21, there are 4 different sets of complex rearrangements, one shared by acdegh and the remainder specific to f, i and j. (c) rearrangements in the vicinity of the ar locus and inter - mutation distances for a29 plotted on a log10 scale for lesions specific to the metastasis (left) and specific to the prostate (middle). Each sample has a different set of complex rearrangements, which are associated with distinct kataegis events . (d) in a10, one tandem duplication is shared by cd while four others are each specific to a single sample . To determine validation rate for mutation calling, a custom capture sureselect design was used to sequence selected coding / non - coding loci to an average depth of 360 - 2000x . For loci with sufficient depth (> = 20x), the validation rate (the proportion of somatic variants) was calculated as described in extended data table 2 and supplementary information, section 3c . On average 95% and 86% of the substitutions and indels, respectively, were somatic . Validation for rearrangement calls was performed by pcr - gel electrophoresis, as described in supplementary information, section 3d . Pcr - gel experiments yielded a high validation rate for three of the four patients included in the validation . For this sample, we therefore assessed the veracity of the breakpoints by visual inspection of the associated copy number segments and confirmed that 82% were high - confidence events resulting in visible copy number changes . To identify potentially oncogenic events within regions of copy number changes, we intersected the affected genomic segments with genes previously shown to be recurrently amplified / deleted . Source column indicates the literature source of the gene as follows: pan_cancer = the cancer genome atlas (tcga) pan - cancer data set (zach, 2013), prostate = reports of genes specifically amplified / deleted in prostate cancer (taylor, 2010 and barbieri, 2012), cancer_gene_census = cancer gene census (futreal, 2004), literature = widely reported in cancer literature.
Unicystic ameloblastoma (ua) was first described by robinson and martinez in 1977 as a special type of ameloblastoma . Ua usually appears very similar to a non - neoplastic odontogenic cyst and is frequently clinically misdiagnosed as dentigerous cyst and odontogenic keratocyst hence, histological confirmation is mandatory from the management point of view . This report highlights the importance of clinical and radiographic features in the diagnosis and management of ua in a 5-year - old boy . A 5-year - old boy presented to our unit center for dental education and research, all india institute of medical sciences, new delhi, india with a painless hard swelling in the lower chin region of 3 months duration . Swelling was found to be approximately 2 cm 2 cm in size, overlying skin was normal in color and texture with no evidence of sinus / fistula . Swelling was non - tender, non - pulsatile, bony hard, non - fluctuant, non - compressible on palpation with expansion of both, buccal as well as lingual cortical plates and aspiration from the swelling yielded cystic color fluid . Panoramic radiographic revealed that unilocular well - defined radiolucent lesion extending from lower deciduous first mandibular molar to right lower canine with evidence of roots resorption [figure 1]. Pre - operative panoramic radiograph showing radiolucent lesion in the right side of the mandible and resorption of the roots of the teeth on the basis of lingual cortical bone expansion, presence of cystic fluid on aspiration and evident of root resorption of the teeth in the panorex, we made the provisional diagnosis of ua . Enucleation of the cyst with extraction of the involved teeth followed by application of carnoy's solution for 3 min over the cavity was planned under general anesthesia (ga). Histopathology report of the specimen revealed that type 1 ua as per the histological criteria defined by ackerman et al . (a) discrete epithelial lines in a connective tissue stroma (h and e, 10), (b) discrete follicles with a similarity to the stellate reticulum of enamel with a varying quantity of connective tissue stroma. (h and e, 40) patient has been on regular follow - up since 2 years, patient is doing well with no signs of recurrence [figure 3]. Post - operative panoramic radiograph showing bone regeneration in the affected area and movement of tooth buds into the regenerated bone at 2 years follow - up period ameloblastoma is rare before the age of 10 years . According to statistical analysis of 1036 cases of ameloblastoma collected from the literature by small and waldron the ua is considered a variant of the solid or multicystic ameloblastoma, accounting for 6% to 15% of all intra osseous ameloblastomas . Presence of lingual cortical plate expansion, cystic fluid on aspiration and presence of root resorption of teeth in panoramic radiograph helped us in making the preoperative diagnosis of ua over the dentigerous cyst without incisional biopsy (which requires ga in the child). Histopathological features of the tumor showed type 1 (tumor confined to the luminal surface of the cyst) ua as per the histological criteria defined by ackerman et al . Treatment modality of ua is being divided into following types - enucleation alone yielded the highest recurrence rate among all treatments (30.5%). A more conservative approach is enucleation with application of carnoy's solution and the extraction of closely related adjacent teeth has resulted in a recurrence rate of 16% . The success of the application of carnoy's solution after enucleation was thought to be due to both its penetration and fixation action . The usual practice is to apply the solution with cotton applicators or ribbon gauze for 3 - 5 min, rinse the bony cavity . The recurrence rate could even lower than reported, if the closely related teeth with tumor are extracted . Because in an attempt to preserve the tooth without damage, tumor remnants may be left around the tooth apex or root and these may lead to recurrence we carried out the same procedure in the case i.e. Enucleation followed by application of carnoy's solution for 3 min, extraction of all involved teeth and close follow - up . Second surgery (i.e. Incisional biopsy followed by definitive treatment) under ga in the child was not required due to proper preoperative planning of the case . At 2 years
Advanced albuminuria, one of the signatures of diabetic nephropathy, causes progressive loss of renal function and leads to terminal renal impairment . Damage to podocytes, which is considered to be a possible early pathological marker for diabetic nephropathy, is important due to its role in causing albuminuria and glomerular damage [13]. In diabetic nephropathy, it is necessary to study the regulation of the actin cytoskeleton directly in podocytes because the dysregulation of the highly specialized podocyte actin cytoskeleton is closely associated with albuminuria [4, 5]. Fyn is a member of the src family of kinases, and it responds to many stimuli to facilitate downstream signaling that regulates cell growth, adhesion, and motility [6, 7]. Earlier reports have demonstrated that fyn associates with multiple intracellular substrates, such as focal adhesion kinase (fak), paxillin, and -adducin, to regulate cytoskeletal architecture and cell - cell interactions [8, 9]. It resides in podocytes and binds directly to and phosphorylates nephrin during podocyte differentiation and in response to injury, and this phosphorylation event results in polymerization of actin filaments [1012]. Rho - associated coiled - coil forming protein kinase (rock) is a downstream effector of the small g protein rho, which has been implicated in a variety of biological functions including cell contraction, migration, adhesion, and gene expression . The rock kinases phosphorylate a variety of substrates, which collectively lead to increased myosin atpase activity and inhibition of the depolymerisation of actin and the assembly of stress fibers and focal adhesions; these events contribute to the reorganization of the actin cytoskeleton [11, 13]. Furthermore, it has been shown that rock may be a pivotal downstream target of fyn for f - actin formation in fibroblasts exposed to lysophosphatidic acid . The activation of rock in the kidney has been confirmed in models of diabetes both in vitro and in vivo [1517], whereas inhibition of rock ameliorated the structural changes in the diabetic kidney together with a modest antiproteinuric effect . In this study, we aimed to reveal the role of the fyn / rock signaling pathway in podocyte injury that was induced by high glucose and its interactions with paxillin . We found that exposure of podocytes to high glucose led to a rearrangement of the filamentous actin (f - actin), accompanied by paxillin phosphorylation and increased cell motility . Both knockdown of fyn by sirna and inhibition of rock by y27632 suppressed high glucose - induced f - actin rearrangement and paxillin phosphorylation . These findings demonstrated that high glucose - induced podocyte f - actin rearrangement was associated with the activation of fyn / rock signaling pathway and the axis - mediated phosphorylation of paxillin . Conditionally immortalized mouse podocytes (a kind gift from professor peter mundel) were cultured on type i collagen - coated dishes in rpmi1640 (hyclone, logan, utah, usa) supplemented with 10% fetal bovine serum (hyclone, logan, utah, usa), 100 u / ml penicillin, and 100 mg / ml streptomycin (hyclone, logan, utah, usa) under permissive conditions 33c plus 10 u / ml mouse recombinant -interferon (pepro technology, london, uk). Podocyte differentiation was induced by maintaining podocytes on type i collagen - coated dishes at 37c without -interferon for at least 14 days . Differentiated podocytes between passages 15 and 20 were incubated in serum - free rpmi1640 medium for 24 hours before being subjected to different treatments . To investigate the effect of high glucose stimulation on podocytes, cells were exposed to low glucose medium (rpmi1640 medium containing 5.5 mm glucose and 10% fetal bovine serum) or high glucose medium (rpmi1640 medium containing 30 mm glucose and 10% fetal bovine serum) for 48 hours . Podocytes incubated in rpmi1640 with 5.5 mm glucose plus 24.5 mm mannitol and 10% fetal bovine serum for 48 hours were considered as the control for osmolarity . Podocytes were pretreated with 10 m y27632 (merck chemicals ltd ., nottingham, uk), an inhibitor reported to be specific for rock, for 30 minutes, and then incubated in high glucose medium for 48 hours to investigate whether rock is involved in high glucose - induced podocyte injury . Podocytes were prepared the day prior to transfection . Following incubation with fyn sirna or cont - sirna for 24 hours, podocytes were then either incubated in standard - glucose medium or high glucose medium for an additional 48 hours . Cells that were not transfected which were exposed to standard - glucose medium were considered to be controls . Sirna oligonucleotides with the following sequences were designed: sirna, 5-cctgtatggaaggttcacaat-3; negative cont - sirna, 5-ttctccgaacgtgtcacgt-3. Transfection was performed in 24-well plates using 40 nm sirnas in lipofectamine 2000 (invitrogen, carlsbad, ca, usa). Podocytes were lysed in radioimmunoprecipitation assay (ripa) buffer at 4c for 10 minutes . Equal amounts of podocyte lysate were incubated with anti - fyn antibody (santa cruz biotechnology, ca, usa) at room temperature for 2 hours . Immunocomplexes were isolated using protein a / g plus - agarose (santa cruz biotechnology, ca, usa) and recovered by boiling for 3 minutes in 1x electrophoresis buffer . Proteins were eluted with sds - page sample buffer and immunoblotted with anti - src [py418] (invitrogen, ca, usa). The activity of rock was assessed by determining the phosphorylation state of myosin phosphatase targeting subunit 1 (mypt1), a downstream target of rock, using anti - mypt1 [py853] antibody (santa cruz biotechnology, ca, usa) and an appropriate secondary antibody (jackson immunoresearch laboratories, west grove, pa) for immunoblotting . Next, 50 g of total protein was separated by sds - page and transferred to nitrocellulose membranes; the membranes were then blocked with 1% polyvinylalcohol in pbs containing 0.2% tween 20 for 10 minutes and incubated at 4c overnight with primary antibodies diluted to various concentrations in blocking buffer (5% or 1% skim milk in pbs - tween (0.2% tween 20)) targeted against the following target proteins: fyn (1: 100), src [py418] (1: 500), paxillin [py31] (1: 5000), desmin (1: 500), vimentin (1: 500), synaptopodin (1: 200), mypt1 [py853] (1: 200), or -actin (1: 200). The membranes were then washed three times with pbs - tween for 10 minutes and incubated with specific peroxidase - conjugated secondary antibodies diluted in blocking buffer (5% skim milk in pbs - tween) for 2 hours at room temperature . Specific bands were detected using the ecl system and the bio - rad electrophoresis image analyzer (bio - rad laboratories, hercules, ca). Total rna was extracted from cells using the tripure reagent (takara, dalian, china) according to the manufacturer's instructions . A260/a280 ratios ranged from 1.8 to 2.0, which indicated a high purity of the extracted rna . Aliquots of total rna (1.0 g each) from each sample were reverse - transcribed into cdna according to the instructions of the primescript rt reagent kit (takara, dalian, china). Quantitative real - time pcr was used to detect the specificity and knockdown efficiency of the fyn sirna and to determine the steady - state mrna levels of synaptopodin and desmin and vimentin . Briefly, after reverse transcription of total rna, cdna was used as a template for the pcr reactions using gene - specific primer pairs . Amplification was performed using sybr premix ex taq kit (takara, dalian, china) in the lightcycler 480 real - time pcr system (roche applied science, penzberg, germany). The designed sequences were as follows: synaptopodin: sense, 5-cggagaatcaaaaccctcag-3, antisense, 5-caggacactgccatcagact-3; desmin: sense, 5-gtgaagatggccttggatgt-3, antisense, 5-gctggtttctcggaagttga-3; vimentin: sense, 5-gatcagctcaccaacgacaa-3, antisense, 5-gctttcggcttcctctctct-3; fyn: sense, 5-aaggataaagaagcagcgaaac-3, antisense, 5-tgcgtggaagttgttgtagttc-3; -actin: sense, 5-gtgggccgctctaggcaccaa-3, antisense, 5-ctctttgatgtcacgcacgatttc-3. Cells growing on glass slides were pretreated with different conditions and then fixed in 4% paraformaldehyde for 20 minutes followed by permeabilization in 0.3% triton x-100 for 10 minutes . After preincubation with 5% bovine serum albumin to block nonspecific binding, cells were individually incubated with primary antibodies against synaptopodin (proteintech group, chicago, il, usa), desmin (immunoway, newark, usa), vimentin (proteintech, chicago, il, usa), or paxillin [py31] (abcam inc ., after washing, the slides were stained with dylight 649-conjugated secondary antibodies (zsgb - bio, beijing, china) and double - stained with dapi to visualize nuclei . Cells were observed and images captured at randomly selected fields using an inverted phase fluorescence microscope (leica, wetzlar, germany). F - actin staining was in accordance with the procedure recommended by the dye manufacturer, and images were taken at randomly selected fields using an lsm780 confocal microscope (carl zeiss, jena, germany). Transwell cell - culture inserts (pore size 8 m; corning costar corp ., cambridge, ma, usa) were rinsed once with pbs and placed in rpmi1640 with 10% fetal bovine serum in the lower compartment . The heights of the medium in the upper and lower compartments were maintained at similar levels; thus, bulk flow was not due to a hydrostatic pressure gradient . Podocytes pretreated with different conditions were harvested with trypsin and resuspended in serum - free rpmi1640 medium; the upper chambers were seeded with 1 10 ml cells, which were then allowed to attach for 6 hours . After incubation for 12 hours at 37c, nonmigratory cells were removed from the upper surface of the membrane using cotton - tipped applicators, and migrating cells were fixed with 4% paraformaldehyde and stained with hematoxylin . The number of migrating cells in the center of a membrane (one field) was counted using phase contrast microscopy (leica, wetzlar, germany). Confluent monolayers of podocytes seeded onto type i collagen - coated six - well plates were pretreated with different conditions and scraped with a 20 l pipette and then allowed to migrate for 12 hours . The percentage of wound closure was captured using an inverted phase contrast microscope (leica, wetzlar, germany) and calculated using nih imagej . Migratory rates were calculated as (a b)/a 100%, where a and b reflect the width of the wound at 0 or 12 hours, respectively . Statistical significance was assessed using lsd t - tests and anovas, and values of p <0.05 were considered statistically significant . Podocyte foot processes (pfps) are characterized by a podocome - like cortical network of branched actin filaments, which are linked to the glomerular basement membrane at focal contacts to modulate the permeability of the filtration barrier via changes in pfp morphology [19, 20]. In this study, podocytes under low glucose conditions demonstrated that f - actin is distributed as obvious homogenous bundles that traverse the cell along the axis of the podocyte (figure 1). Exposure to high osmotic pressure for 48 hours caused no significant effect compared with incubation in a low glucose concentration . Podocytes stimulated with high glucose for 48 hours showed an assembly of f - actin in cortical regions, agminated f - actin along the cell periphery, and a slightly diffuse cytoplasmic distribution (figure 1). To fully characterize the changes in podocytes stimulated by high glucose, we further examined the expression of synaptopodin, desmin, and vimentin . Synaptopodin is an important actin - associated protein, a decrease of which has been confirmed as a remarkable phenomenon during podocytopathy [2123]. Vimentin and desmin, which are known markers of podocyte injury [24, 25], were also assessed in this study . We observed that podocytes cultured for 48 hours in high glucose demonstrated a significant downregulation of synaptopodin compared with podocytes cultured in low glucose conditions, at both the protein and mrna levels (figure 2), whereas high osmotic pressure showed no obvious effects . The effects of high glucose on synaptopodin expression were further confirmed by immunofluorescence, where reduced fluorescence staining was observed (figure 2). Moreover, significant increases in desmin and vimentin expression were also detected, at both the protein and mrna levels following 48 hours of incubation in high glucose, compared with incubation with low glucose (figures 3 and 4). Increased fluorescent staining further confirmed the high glucose - induced overexpression of desmin and vimentin in podocytes (figures 3 and 4). As a member of the src family of kinases, fyn is known as a nonreceptor tyrosine kinase that responds to many stimuli to regulate the cytoskeleton and process formation . The activity of fyn is regulated by intramolecular interactions and depends upon the phosphorylation state of two tyrosine residues, y529 and y418 . Dephosphorylation of y529 results in autophosphorylation at y418 in the activation loop of the catalytic domain of fyn, thereby stabilizing it in the active conformation [9, 26]. To detect activated fyn, podocyte lysates were immunoprecipitated with anti - fyn antibody followed by western blotting with anti - src [py418] antibody . As demonstrated by the western blotting (figure 5), high glucose promoted fyn activation in podocytes compared with cells incubated in low glucose conditions, with no alterations in total fyn expression noted . To gain insight into the mechanism of the high glucose - induced podocyte f - actin cytoskeleton reorganization, we utilized a small interfering rna (sirna) to knock fyn expression down . To validate the efficiency of the fyn sirna, podocytes were transfected with fyn sirna for 24 hours and then incubated in low glucose medium for another 48 hours . A significant reduction of fyn at both the protein and mrna levels was detected (figure 6). Podocytes transfected with fyn sirna for 24 hours and then incubated under high glucose conditions for 48 hours were used to perform further experiments . The high glucose - induced f - actin cytoskeleton reorganization was markedly ameliorated by fyn gene knockdown (figure 1). Furthermore, after silencing fyn expression with sirna, the alterations to synaptopodin, desmin, and vimentin were also ameliorated (figures 2, 3, and 4). Rock is a critical rho effector in the regulation of f - actin, focal adhesion formation, cell morphology, and cell motility . The activation of rho / rock in the kidney has been confirmed in models of diabetes both in vitro and in vivo [1517]. Here, we demonstrated that podocytes cultured in high glucose for 48 hours showed increased rock activity compared with podocytes incubated in low glucose conditions, shown by the increase in phosphorylation of mypt1, a downstream target of rock (figure 7). Knocking down fyn with sirna reversed the upregulation of rock activity stimulated by high glucose . Pretreatment with 10 m y27632, an inhibitor specific to rock, for 30 minutes, inhibited effectively the high glucose - induced rock activation (figure 7), with no significant alteration to total fyn expression noted (figure 5). The f - actin reorganization induced by high glucose was remarkably ameliorated by pretreatment with y27632 (figure 1). Moreover, the high glucose - induced alterations to synaptopodin, desmin, and vimentin were also partially reversed in the presence of y27632, at both protein and mrna levels (figures 2, 3, and 4). Paxillin is a phosphorylation - dependent protein that links the intracellular actin cytoskeleton to integrin - rich adhesion sites . The phosphorylation of paxillin tyrosine residue 31 (y31) is particularly important and prevalent in regulating downstream signaling and causes profound effects on the cytoskeleton, cell adhesion, and movement [2830]. In this study, an increase in tyrosine phosphorylation of paxillin at y31, which has been shown to be an effector of high glucose, was detected . Incubation with high glucose medium for 48 hours enhanced tyrosine phosphorylation of paxillin at y31 compared with podocytes incubated in low glucose conditions or exposed to high osmotic pressure, without alterations of total paxillin expression; high glucose promoted paxillin phosphorylation at y31 was markedly decreased by both fyn knockdown and rock inhibition (figure 8). Moreover, the immunofluorescence data showed that the distribution of phosphorylated paxillin was predominantly along the cell periphery and was associated with reorganized f - actin (figure 9). These findings suggested that the fyn / rock signaling pathway potentially interacted with paxillin in podocytes under high glucose conditions . To examine coordinated sheet migration, we next performed a scrap - wound - healing assay on a monolayer of podocytes . Compared with podocytes in standard - glucose conditions, high glucose increased the speed at which the podocyte monolayer filled a uniform - width scratch wound in . Both fyn gene knockdown and inhibition of rock slowed high glucose podocyte wound healing (figures 10(a) and 10(c)). Next, we used the modified transwell migration assay and wound - healing assay to test the motility of cultured podocytes in different conditions . Podocytes subjected to high glucose showed significantly increased cell migration through the transwell filters compared with those incubated in low glucose or high osmotic pressure conditions . This effect was reversed partially by both fyn gene knockdown and inhibition of rock (figures 10(b) and 10(d)). Podocytes possess a contractile structure, composed of abundant microfilaments that modulate glomerular filtration, and their structure is based on an actin cytoskeleton [4, 5]. Interference with cytoskeleton interactions with the slit diaphragm or defects in cytoskeletal components will cause foot process effacement, alterations in cell motility, and disruption of kidney filtration . Accumulating evidence has indicated that podocyte damage is a prerequisite for diabetic nephropathy, which, characterized by advanced albuminuria, has become one of the leading cause of end - stage renal disease . It has been recently shown that effacement of podocyte foot processes occurs in diabetic nephropathy and that it is accompanied by a rearrangement of the actin cytoskeleton [3, 5, 32]. The podocyte cytoskeletal system includes actin regulatory proteins, such as -actinin-4, synaptopodin, and the most important component called f - actin . The actin cytoskeleton plays a crucial role in regulating the movement of cells via the highly dynamic and reversible polymerization of actin filaments, which provides the internal mechanical support that drives cell motility [4, 5]. In this study, under normal glucose conditions, podocytes showed obvious homogenous f - actin bundle distribution along the axis . Exposure to high glucose conditions for 48 hours led to the assembly of f - actin into cortical regions . And modified transwell migration assays and wound - healing assays revealed an increase in spreading and migration of podocytes cultured in high glucose conditions . Podocytes stimulated with high glucose showed significantly increased cell migration through transwell filters compared with cells subjected to normal glucose and high osmotic pressure conditions . Much work has demonstrated that increased podocyte motility may be an important aspect of podocytes depletion [4, 33, 34]. Therefore, it appeared that high glucose - induced dynamic rearrangement of the podocyte actin cytoskeleton, which subsequently promoted podocyte migration, which may initiate the depletion of podocytes and a vicious cycle of progressive glomerular damage . Synaptopodin is an important marker of podocyte foot processes necessary for the maintenance of appropriate process structure and function in podocytes . A decrease in synaptopodin has been confirmed as an important phenomenon in podocytopathy [21, 22]. The results of this study demonstrate a significant decrease in synaptopodin expression at both mrna and protein levels in podocytes cultured in high glucose conditions . Increased expression of desmin and vimentin, both observed in the present study, is associated with damage to podocytes in diabetic nephropathy [23, 24] and several other renal disease models [25, 35]. These observations revealed that high glucose - induced podocyte injury, characterized by a significant downregulation of synaptopodin together with an upregulation of desmin and vimentin at both mrna and protein levels . Fyn tyrosine kinase, a member of the src family protein tyrosine kinases, has been suggested to be involved in cytoskeletal regulation and cell - process branching in a variety of cell types [69, 12]. Since podocytes and neurons share many structural and molecular features, a similar mechanism may be involved in the modulation of podocyte processes . Previous studies have shown that, in podocytes, fyn directly binds to nephrin and phosphorylates the latter at tyrosine residues during podocyte differentiation . In our study, we found that the high glucose stimulation led to the activation of fyn, accompanied by a rearrangement of f - actin bundles . Gene knockdown of fyn by its specific sirna ameliorated this high glucose - induced fyn activation and f - actin remodeling, suggesting that fyn is a triggering factor for f - actin cytoskeletal rearrangement under high glucose conditions . Rock, the best studied downstream target of rhoa, is involved in a myriad of signaling cascades including the regulation of stress fibers, focal adhesion formation, and cell motility . It is also a pivotal controller of the actin cytoskeleton during cell morphogenesis and migration [11, 13, 14, 16]. In the kidney, rock activation was found in models of diabetes both in vitro and in vivo [1517]. And inhibition of rock helped ameliorate structural changes in the kidney together with a modest antiproteinuric effect . Our results presented that rock could be activated by high glucose stimulation, which occurred concomitantly with the rearrangement of f - actin . Pretreatment of y27632, the rock inhibitor, showed decreased activity of rock and partially reversed f - actin remodeling . Prior studies have suggested that, in fibroblasts, fyn kinase may act as an upstream factor of rock in the pathway governing the cytoskeletal changes and process remodeling [14, 38]. In our study, we found that gene knockdown of fyn remarkably inhibited rock activation, with reversed f - actin remodeling, whereas pretreatment of y27632 showed no significant effects on total fyn or phosphorylated fyn expression, suggesting that, under high glucose conditions, fyn promoted f - actin cytoskeletal remodeling via activation of rock . Paxillin serves as a phosphorylation - dependent signaling scaffold, functioning in integrating and disseminating signals that affect cell adhesion, motility, and actin cytoskeletal remodeling . A recent study suggested that lipopolysaccharide - induced podocyte injury was accompanied by increased paxillin phosphorylation and cell spreading . In this study, we observed significantly upregulated paxillin phosphorylation in podocytes after high glucose treatment, which occurred concomitantly with f - actin bundles redistribution . Extensive research on cancer has documented that fyn associates with intracellular substrates like fak, paxillin, and -adducin to regulate cytoskeletal architecture and cell - cell interactions [8, 40, 41]. In human umbilical vein endothelial cells, the activation of the rock signaling pathway was followed by tyrosine phosphorylation of paxillin, which further led to actin reorganization . Inhibition of rock, however, could attenuate bombesin - induced increase in tyrosine phosphorylation of paxillin in swiss 3t3 cells . Our findings provided credible data that the upregulation of paxillin activity by high glucose was markedly abolished by fyn gene knockdown or pretreatment of rock inhibitor, demonstrating that high glucose - stimulated phosphorylation of paxillin was in close association with fyn / rock signaling pathway in cultured podocytes . Found that lps - induced phosphorylation of paxillin was responsible for the actin reassembly in macrophage and reassembly - associated cell motility . It is hypothesized that reorganization of f - actin may be regulated by phosphorylated paxillin under high glucose conditions, which was triggered by the activation of fyn / rock signaling pathway . The dynamic regulation of the podocyte cytoskeleton is paramount for the appropriate function of kidney filtration barrier . In the current study, we sought further confirmation that the reorganization of the f - actin cytoskeleton, along with paxillin phosphorylation and increased cell motility, is a consequence of fyn / rock signaling pathway activation in high glucose - induced podocyte injury, represented by alterations to synaptopodin, desmin, and vimentin expression . These observations shed light on one possible mechanism responsible for podocyte dysfunction in diabetes mellitus and may facilitate the development of novel strategies for treating diabetic nephropathy by targeting cytoskeletal rearrangement in podocytes.
The ability to compare the relative effectiveness of different technologies and treatment modalities is now imperative in order to meet the growing demand for more rigorous outcomes research . Today, there are many data available to conduct comparative effectiveness research (cer): 1) data from surveys and 2) data from secondary databases; however, many lack important information . Both of these types of data sources have their own particular limitations . While prospectively collected observational cohorts lend themselves to longitudinal epidemiologic data for research, they are traditionally limited in external validity, specificity of measurement on key exposures and outcomes, and historially dated beyond application to current therapies . Secondary databases, including administrative claims data collected by the infrastructure of the healthcare system for the purposes of payment for services, also have limitations, as they are typically restricted to billing claims and provide limited clinical detail to confirm disease . Additionally, they often contain few patient characteristics (such as race and ethnicity) and have a low proportion of indigent and self - pay populations . These types of databases also lack in details on patients perception of disease or the impact of disease or treatment on humanistic outcomes . Electronic medical record (emr) systems, newly launched to replace paper - based chart systems in tracking clinical symptoms and disease, are still in the early stages of development . Moreover, they are frequently positioned in silos of healthcare practice traditionally physician practices with limited linkage to inpatient facilities, other specialists or other physician services, emergency and acute services, and pharmacy services . While each of these datasets provides unique information, they lack a significant amount of information about the patients themselves and their associated treatments . Given these challenges and data limitations, there is a need for new, advanced techniques to overcome the limitations of existing data sources, thereby enhancing their utility for cer . In the absence of the ideal data source, there is an alternative: a method whereby researchers can maximize the benefits of each individual type of data source, utilizing data linkage methods to fill in the gaps of one database with the strengths of another . Blanchette et al . Presented this approach at a workshop during the 13 annual european ispor congress in prague in 2010, and discussed preliminary results demonstrating the application linking the two administrative databases, at a workshop during the 16 annual international ispor meeting in baltimore, in 2011 . Deterministic linkage techniques can be applied when both datasets provide patient - identifying information that can be cross - matched between the two datasets . This is the preferred form of data linkage because the focus is on matching the same patient from both datasets . In the current climate of regulatory and legal restraints on the use of personal health information (phi), the utility of this approach is limited . In the absence of phi, a probabilistic method may be applied . However, the focus is not on identifying the same patient but rather on two matching patients with similar behavior and characteristics . The goal of both of these efforts is to identify the best method for pair - matching between datasets . Probabilistic linking methods incorporate varying strengths of identifiers, depending on information provided by the identifier . For example, matching on gender (male or female) is less likely to produce a matching of equivalent records than matching a record on the combination of birth month and year . Assigned weights are related to frequencies of occurrences of values in linked and unlinked pairs and also to the designated level of agreement . The determination of agreement can be further defined into subcategories: agreement (yes / no) or varying levels of partial agreement (e.g., matching of a date within 2 days before / after or matching within a week before / after). Using probabilistic linking methods generally increases the sensitivity and specificity of models over a deterministic method in linking records from disparate datasets . Propensity score methods are increasingly used to match similar individuals in observational studies, along with other approaches to control for channeling bias . Score matching, well - matched pairs are closely matched on the predicted probability of experiencing the dependent variable using relevant covariates . Matching techniques then employ a variety of approaches based on the predicted probability score (ranging from 0 to 1), including nearest neighbor matching heuristics, and often employing sampling without replacement . To demonstrate the utility and subsequent validity of probabilistic data linkage we employed a historical retrospective cohort design . Patients aged 40 and older with a principal or secondary diagnosis of copd (international classification of disease, 9 revision codes: 491.xx, 492.xx, and 496) and at least 3 years of continuous enrollment in a us health plan contributing to the database between january 1, 2004 and april 30, 2009 were selected from two administrative claims databases representing commercially insured us health plan enrollees . The index date was designated as the date of the first claim (defined by a 12-month wash - out pre - index period) for either study drug, treatment 1 or treatment 2 . The effectiveness measure was the incidence of copd - related exacerbation (hospitalization, emergency department visit or oral corticosteroid prescription) observed in the 12-month post - index period, with baseline characteristics being identified in the 12-month pre - index period (figure 1). Data linkage was performed by restricting the samples in each database to those with similar pre - index and post - index time periods (as described above). We then pooled data and developed a logistic regression model predicting the propensity to be in database a using overlapping pre - index variables including age, geographic region, comorbidities, and pre - index utilization of health services and respiratory medications (table 1). We then directly imputed the outcomes of each matched patient from database a to the matched patient from database b. finally, we conducted the comparative effectiveness study in each cohort (a, b, and a with imputed outcomes) (figure 2). Of 12,926 patients in database a and 21,334 patients in database b, we matched 11,040 patients in each database on the propensity score using a nearest neighbor matching algorithm which matches like pairs of patients from each database without replacement . Post - matching groups were balanced on all pre - index characteristics with the exception of older age (6574 and> 75 years) as well as representation in the southern region . The event rate of exacerbations in each cohort was nearly identical (treatment 1 [database a: 39.3%, database b: 39.7%, and database a with imputed outcomes: 40.1%] and treatment 2 [database a: 46.3%, database b: 47.1%, and database a with imputed outcomes: 46.8%]) as were the odds ratio and corresponding confidence intervals from the adjusted logistic regression models (or database a: 0.72, database b: 0.74, database a with imputed outcomes: 0.72) (table 2). This is not entirely surprising since the probability of being in each cohort was determined by the overlapping similarity in characteristics between the two cohorts, just as if the propensity score match was used in a traditional cohort analysis . The probabilistic linkage demonstrated that patients from different databases matched on similar pre - index characteristics may demonstrate similar outcomes in the post - index period . The implication of these findings and this approach is significant as it provides a case example for linkage of simulated cohorts from varying databases (e.g., emr and administrative datasets, administrative datasets and surveys, emr and surveys, etc . ). The creation of simulated cohorts could help explain a more complete picture of patient outcomes and allow researchers to explore all aspects of a patient experience for example, the use of administrative claims databases for comparative effectiveness research is often lacking key variables of interest such as income, race, lab results, clinical detail identified through notes, etc . Through this linking technique, one could impute these characteristics into a similar matched - patient cohort and assess the associations with the outcome . Similarly, a survey of patients could be augmented with key administrative and clinical detail . Even a clinical trial sample could be matched to administrative or electronic health record data to populate long - term outcomes . This technique has the ability to provide a view into the world of more complete data with further validation of the methods and use in diverse data sources . The divergence of the measures between the databases may also cause a divergence in like patients thereby introducing error in the outcomes assessment . Care should be taken to ensure that similar measures are used to build the logistic regression model to create the propensity score; the assumption is that probabilistic matching produces two individuals with similar attributes and not the same individual . However, in the absence of complete data, the approach offers a good start and paves the way for further examples of this type of linkage method to be explored with various databases . These data represent us - based administrative claims submitted from healthcare providers to health insurance plans and therefore there is a potential for miscoded claims . It is assumed that these patients fully utilize their health insurance benefits to avoid the full cost of the health service; however unlikely, there is a potential that the patient may be receiving health benefits from a provider who does not submit a claim for reimbursement to the patient s health insurance provider . The probabilistic linkage method for simulated cohorts demonstrated that patients from different us - based health plan administrative claims databases matched on similar pre - index characteristics may demonstrate similar outcomes in the post - index period . This is the first application of this method for comparative effectiveness research and further validation in diverse data - sets is required . Substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data (cmb, mr). Drafting the article or revising it critically for important intellectual content (cmb, md, apd, mr). Final approval of the version to be published (cmb, md, apd, mr). International committee of medical journal editors (icmje) potential conflicts of interests forms for each of the authors are summarized below . Original forms for each of the authors are available for download at: http://drugsincontext.com/download/dic.212258_coi.pdf cmb: payment or receipt of services for any aspect of the submitted work: astrazeneca funded the original study but did not fund the methodological process on which this paper focuses; financial activities outside the submitted work: consultancy for centecor (not related); employee of otsuka america pharmaceutical inc; grants / grants pending from ethicon (not related), bristol myers squib (not related), auxilium (not related), eli lilly (not related), glaxosmithkline (copd); stock / stock options from celgene, cubist, gilead; no other relationships / conditions / circumstances that present a potential conflict of interest . Mk: no payment or receipt of services for any aspect of the submitted work; financial activities outside the submitted work: employee of ims health, alexandria, va, usa; no other relationships / conditions / circumstances that present a potential conflict of interest . Apd: no payment or receipt of services for any aspect of the submitted work; financial activities outside the submitted work: employee of ims health, alexandria, va, usa; no other relationships / conditions / circumstances that present a potential conflict of interest . Mr: no payment or receipt of services for any aspect of the submitted work; financial activities outside the submitted work: grants / grants pending from astra zeneca, glaxosmithkline, boehringer ingelheim, pfizer; lovelace clinic foundation, albuquerque, nm, usa has received funding for respiratory related healthcare research studies; no other relationships / conditions / circumstances that present a potential conflict of interest.
The principal aim of this laboratory is the synthesis of conjugated unsaturated ketones as candidate antineoplastic agents . These compounds interact with thiols but in general, they have little or no affinity for amino and hydroxyl groups which are found in nucleic acids (1 - 3). Hence thiol alkylators may not have the genotoxic properties associated with a number of contemporary anticancer drugs (4). However after an initial chemical insult, certain neoplasms are more vulnerable to a subsequent cytotoxic effect than various non - malignant cells (5, 6). Hence by mounting the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore [ar - c = c - c(o)-c = c - ar] on heterocyclic and cycloaliphatic scaffolds, two sequential interactions with cellular thiols can take place which may be more detrimental to tumours than normal tissues . Such considerations led to the development of 3,5-bis(benzylidene)-4-piperidones 1a - d which demonstrated potent cytotoxic properties with the ic50 values in the low micromolar range against human molt 4/c8 and cem t - lymphocytes as well as murine l1210 lymphocytic leukemia cells (7, 8). The hypothesis of sequential cytotoxicity was advanced that the 1,5-diaryl-3-oxo-1,4-pentadienyl group interacts at a primary binding site and a side chain on the piperidine nitrogen may align at an auxiliary binding site which could enhance cytotoxic potencies . In order to evaluate this hypothesis, a novel series of n - aroyl-3,5-bis(benzylidene)-4-piperidone derivatives 2 - 7 were synthesized (8). In these compounds, the side chain contains atoms and groups capable of forming hydrogen and van der waals bonds and also an ionic bond in series 3 - 6 which could bind at an auxiliary binding site . When the potencies of the compounds in series 3 - 6 were compared with that of the analogs 1a - d which have the same aryl substituent, the ic50 values of the amides 3 - 6 were lower in 48% of the comparisons made while equipotency was noted in 35% of the cases (8). The proposed primary and auxiliary binding sites of cytotoxic n - aroyl-3,5-bis(benzylidene)-4-piperidone analogs the objectives of the present investigation are two folds . First, to gain some insight into the physicochemical properties of the 3,5-bis(benzylidene)-4-piperidone derivatives 1 - 7 that influence cytotoxic potencies, and second, to derive predictive 2d - qsar models that will be helpful to design new analogs with improved bioactivity . The in - vitro cytotoxic data used in this study were taken from the literature (8). The reported ic50 values were converted into logarithmic scale (pic50) and then were used for qsar analysis as dependent variables . The data set (n = 25) was divided randomly into two groups: calibration set (n = 19) and prediction set (n = 6). (table 1) (asterisk sign in the table demonstrate the prediction set). Descriptor generation the chemical structure of molecules was constructed using a hyperchem package (version 7; hypercube inc ., gainesville, fl, usa) and primarily were energetically minimized (100 steepest descent steps using mm force field with a gradient convergence value of 0.1 kcal / mol) using hyperchem software . The z - matrices of the structures were provided by the software and were then transferred to the gaussian 98 program . Gaussian 98 was used for semi - empirical molecular orbital calculations (am1) of the structures . A large number of molecular descriptors were calculated using hyperchem, gaussian 98 and dragon packages (10). Different functional descriptors such as topological, geometrical, and constitutional descriptors were calculated using dragon software . Moreover, some chemical parameters including molecular volume (mv), molecular surface area (sa), hydrophobicity (logp), heat of formation and hydration energy (he) were calculated using hyperchem software . Data processing and modeling the calculated descriptors were collected in a data matrix and generated descriptors were scaled using auto scaling procedure . Firstly, the descriptors were checked for constant or near - constant values and those detected were removed from the original data matrix . Then the correlation of descriptors with each other s and with the potency data was determined . Among the detected collinear descriptors (r> 0.8), one of them demonstrating the highest correlation with potency was retained, and the rest were omitted . In the model development step, leave - one - out cross - validation was used to optimize the models (e.g., selection of variables or no . Of principal components). The final models were validated using a set of 6 molecules in the test set, which did not contribute to the model development . Two different methods were used for the development of qsar equations (i) stepwise multiple linear regression (mlr), (iii) genetic algorithm partial least squares (ga - pls). These methods are well documented in the literature (11). In stepwise regression, the basic procedures involve (i) identifying an initial model, (ii) iteratively' stepping', that is, repeatedly altering the model at the previous step by adding or removing a predictor variable in accordance with the' stepping criteria' (in our case, probability of f = 0.05 for inclusion; probability of f = 0.1 for exclusion for the forward selection method), and (iii) terminating the search when stepping is no longer possible given the stepping criteria, or when a specified maximum number of steps have been reached . Specifically, at each step, all variables are reviewed and evaluated to determine which one will contribute most to the equation . This variable will then be included in the model, and the process starts again . A limitation of the stepwise regression search approach is that it presumes there is a single' best' subset of x variables and seeks to identify it . There is often no unique' best' subset, and all possible regression models with a similar number of x variables as in the stepwise regression solution should be fitted subsequently to study whether some other subsets of x variables might be better . In the present study, mlr with stepwise selection and elimination of variables the resultant models were validated by a leave - one - out cross - validation procedure (using matlab software) to check their predictive power and robustness . A genetic algorithm uses genetic rules such as reproduction, cross - over, and mutation to build pseudo organisms that are then selected, on the basis of a fitness criterion to survive and pass information on to the next generation . To select the most relevant set of descriptors, the evolution of population was simulated (12 - 15). Pls is a generalization of regression, which can handle data with strongly correlated and or numerous x variables (15). The linear pls model finds' new variables' (latent variables or x scores) that are a linear combination of the original variables . To avoid over fitting, a strict test for the significance of each consecutive pls component is necessary and then stopping when the components are non - significant . Cross - validation is a practical and reliable method of testing this significance (14). Application of pls thus allows the construction of larger qsar equations while avoiding over fitting and eliminating most variables . Pls is normally used in combination with cross - validation to obtain the optimum number of components (15)19]. In the ga - pls procedure, for each subset of descriptors (i.e., for each chromosome of the ga), a pls model was developed separately and consequently the number of latent variables was optimized . The pls regression method used was the nipals - based algorithm found in the chemometrics toolbox of matlab software (version 6.5; math work inc ., based on the haaland and thomas f - ratio criterion we performed the leave - one - out cross - validation procedure as it was applied in our previous article (16). The matlab pls toolbox developed by eigenvector company was used for pls and ga modeling.the predicted power of obtained equations was validated using cross - validated squared correlation coefficient (q2). In order to calculate q following equation was used . Q = 1 (ypred yact)2/ (yact ymean) where ypred, yact, and ymean are predicted, actual, and mean values of the pic50, respectively . (ypred - yact) is the predictive residual error sum of squares (press) (17). Moreover, the predictive squared correlation coefficients rpred was used for further evaluation of the predictive potential of obtained equations . To calculate ytest)/(ytest ytraining) where ypred(test) and ytest are predicted and observed activity values, respectively, of test set compounds, and ytraining is the mean activity value of training set (17). Variable importance in the projection in order to evaluate the relative importance of the variable appeared in the final ga - pls, variable importance in projection (vip) was employed (18). Vip is the sum over all model dimensions of the contributions variable influence (vin). For a given pls dimension, a (vin)ak is equal to the squared pls weight (wak)2 of that term, multiplied by the explained ss of that pls dimension (19). Y - randomization test y - randomization test is the technique ensures the robustness of a qsar model . In this procedure, for each created models, the dependent variable vector pic50 is randomly shuffled, and a new qsar model is developed using the original independent variable matrix . For acceptable specific qsar model, the new obtained qsar equation (after several repetitions) is expected to have low r and q values (16). The structural features and the experimental cytotoxic potencies (represented as pic50) of the 3,5-bis(benzylidene)-4-piperidone derivatives 1 - 7 used in this study are shown in table 1 (8). A sar study of these compounds demonstrated that the attachment of the n - aroyl group to the 3,5-bis(arylidene)-4-piperidones resulted in enhanced cytotoxic potencies toward murine l1210 lymphocyte leukemia cells and two human t - lymphocytes molt4/c8 and cem . It was suggested that in the case of various members of series 2 - 7, alignment of the n - aroyl groups with auxiliary binding sites takes place reinforcing the interaction of the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore with a primary binding site . Chemical structures of the n - aroyl-3,5-bis(arylidene)-4-piperidones 1 - 7 used in this study and their experimental and cross - validated predicted cytotoxic potencies (by ga - pls) toward murine l1210 cells and human molt 4/c8 and cem t - lymphocytes reprelative error of prediction . The compounds are included in the prediction set in order to find important structural features of the n - aroyl-3,5-bis(benzylidene)-4-piperidones which contribute to the cytotoxic potencies, a qsar study was performed using different molecular descriptors and chemometrics tools . In this study, the chemometrics methods such as stepwise multi - linear regression (mlr) and genetic algorithm - partial least squares (ga - pls) were used for modeling the relationship between the biological activity and molecular descriptors and the results were compared to find the best predictive cytotoxicity models . Separate stepwise selection - based mlr analyses were performed using different types of descriptors, and then a mlr equation was obtained using the pool of all calculated descriptors . As there are 19 molecules in the training set and according to the rule of thumb (the ratio of 5:1 for molecule variable; toplis ratio) (16), mlr models with a maximum number of 3 or 4 variables were selected . A small difference between the conventional and cross - validated correlation coefficients of the different mlr equations (table 2) reveals that none of the models are over fitted, which can be partially attributed to the absence of collinearity between the variables in one hand and using no extra variables on the other hand . The correlation coefficient (r) matrix for the descriptors used in different mlr equations shows that no significant correlation exists between pairs of descriptors (table 3). The results of multiple linear regression (mlr) analysis for the compound in series 1 - 7 with different type of descriptors squared correlation coefficient; standard error of regression; root mean square of cross validation; leave - one - out cross - validation correlation coefficient (rcv); f-statistics.squared correlation coefficient (r) was calculated using following equation: 1-((y experimental experimental))) equation for calculation of standard error of regression: [se= ((y experimental y calculated)/n - p') (n= number of samples, p'= number of model parameters)] correlation coefficient (r) matrix for descriptors used in multiple linear regression equations 1 - 3 . L1210 cell line . Stepwise selection and elimination of variables produced a three - parametric qsar equation 1, having moderate statistical quality (r = 0.70, se = 0.38, q = 0.50, rmscv = 0.45 and rpred = 0.69) (table 2 and 4). The selected variables demonstrate that chemical (heat of formation), topological and spatial parameters (molecular density and narumi harmonic topological index (hnar) mainly affect the cytotoxic potential of the 3,5-bis(arylidene)-4-piperidone derivatives against the l1210 cell line . The mlr equation 2 obtained from the pool of the calculated descriptors for the cem cell line possesses good statistical quality (r = 0.85, se = 0.25, q = 0.77, rmscv = 0.29 and rpred = 0.81) (table 2 and 4) and demonstrates that the anticancer property of the compounds is mainly dependent on chemical (heat of formation), topological and spatial (molecular density and average connectivity index chi-4:x4a) properties of the molecule . These results show that the parameters that influence anticancer properties of the compounds against the cem line are similar to those obtained for the l1210 cell line . The three - parametric qsar equation 3 (table 2) demonstrates the quantitative effects of the structural parameters on the cytotoxic potential of 3,5-bis(arylidene)-4-piperidone derivatives on the molt / c8 cell line . This model demonstrated high statistical quality (r = 0.81, se = 0.31, q = 0.72, rmscv = 0.34, and rpred = 0.64) (table 2 and 4) which reveals the significant effects of quantum (homo, dipole moment x (dmx)) and spatial parameter (molecular density) on the cytotoxic activity of the compounds . It is revealed that the molecular density plays a determinant effect on the cytotoxic potential of the compounds on this cell line . Therefore, the molecular density and topological parameter are important descriptors that should be taken into consideration in the designing of potent cytotoxic 3,5-bis (arylidene)-4-piperidones analogs . The results of qsar analysis by mlra and ga - plsb methods for the compounds in series 1 - 7 multiple linear regressions, genetic algorithm - partial least square, correlation coefficient of regression, leave - one - out cross - validation correlation coefficient (rcv), standard error of prediction regression, correlation coefficient of prediction regression in pls analyses, the descriptors data matrix is decomposed into orthogonal matrices, the scores of which are constrained to have inner relationships with the dependent variables . Therefore the multi - collinearity problem in the descriptors is omitted by pls analysis . To find a more convenient set of descriptors in pls modeling,, many different ga - pls runs were conducted using different initial sets of populations . Given 19 calibration samples, the leave - one - out cross - validation procedure was used to find the optimum number of latent variables for each pls model . For the l1210 cell line, the ga - pls model that resulted in the best fit contained 7 indices (two of these indices such as molecular density and heat of formation were also obtained by mlr in this cell line). Moreover, similar to the results of mlr analysis, ga - pls analysis also showed that topological parameters affect the cytotoxic potencies of the compounds in the l1210 cell line . As per ga - pls modeling, a combination of chemical (heat of formation, melting point), quantum chemical (dipole moment x), conformational (torsion energy) and spatial (molecular density) descriptors account for the cytotoxic potential of the compounds toward l1210 cells . The resulted ga - pls model possess very good statistical quality (i.e., r= 0.86, q= 0.66). The predictive ability of the model was measured by application to 6 external test set molecules (rpred = 0.81 and sepred = 0.37). The calculated values of pic50 obtained by the pls model in the l1210 assay (refined from cross - validation of external prediction set) are shown in table 1 . To measure the significance of the 7 selected pls descriptors, the variable importance in projection (vip) was calculated for each descriptor . According to the method described by eriksson et al . (20), x - variable (predictor variable) could be classified according to their relevance in explaining y - variable (predicted variable) i.e. Vip> 1.0 (highly influential), vip <0.8 (less influential), and 0.8 <vip <1.0 (moderately influential). The vip analysis of the descriptors of the input variables used in the pls equation (figure 2a) shows that the parameters such as topological index (chi3), molecular density and heat of formation are the most important indices in the qsar equation derived by pls analysis for l1210 cell line . The regression coefficients of the selected variables obtained by ga - pls model of qsar analysis for three different cell lines are illustrated in figure 3 . Variable importance for projection (vip) of the selected variables obtained by ga - pls model for the qsar analysis of three different cell lines: (a) l1210, (b) cem, and (c) molt / c8 regression coefficients of the selected variables derived by the ga - pls model for qsar analysis of three different cell lines: (a) l1210, (b) cem, and (c) molt / c8 the best ga - pls model to predict cytotoxic activities of the compounds in series 1 - 7 toward the cem cell line includes 10 indices (three of the indices are similar to that obtained by the mlr analysis). A combination of topological (x1a), chi-1(average connectivity index), pw3 (randic shape index), x3av (average valance connectivity), sic2 (structural information content: symmetry 2-order)), geometrical (sph (spherocity of molecule)), chemical (logp), spatial (molecular density), electrotopological state (sum of the e - state value for the ch group in the aromatic ring (s_aach) and sum of the e - state value for the methyl group (s_sch3)) indices account for the cytotoxic properties of the 3,5-bis(arylidene)-4-piperidones toward the cem cell line . The resulted ga - pls model showed high statistical quality (i.e., r = 0.87 and q = 0.71). The predictive ability of the model was measured by application to 6 external test set molecules (rpred = 0.83 and sepred = 0.35). The pic50 values used in the pls model (refined from cross - validation of external prediction set) are shown in table 1 . The vip analysis of pls equation presented in figure 2b suggests that among the selected descriptors, topological and electrotopological parameters such as x1a, pw3, x3av and estate keys (s_sch3 and s_aach) are important influential descriptors . Similar to the l1210 cell line, the molecular density of the compounds is also a determinant parameter that influence cytotoxicity of 3,5-bis(arylidene)-4-piperidones against the cem cell line . Molt 4/c8 cell line a combination of 8 selected variables such as chemical (hydration energy), topological (x2a), geometrical (pw3, j3d (3d - balaban index), sph), spatial (molecular density) and electrotopological state indices (sum of the e - state value for the ch group in the aromatic ring (saach) and sum of the e - state value for the methyl group (s_sch3)) influence cytotoxic potencies of the compounds in series 1 - 7 against the molt 4/c8 cell line as indicated by the best ga - pls model . Most of these variables contribute significantly to the ga - pls model as discussed previously for the cem cell line . The resultant ga - pls model demonstrated very high statistical quality [r = 0.91 and q = 0.77] [figure 3c]. The predictive ability of the model was measured by applying to 6 external test set molecules and the squared correlation coefficient for prediction (rpred) was found to 0.82 . The vip analysis of the ga - pls model presented in figure 2c showed that the molecular density, topological (x2a), geometrical (sph, j3d) and electrotopological state indices influence the cytotoxicity of the compounds in series 1 - 7 . The plots of the predicted pic50 values (cross - validated) against the experimental values for the l1210, cem and molt4/c8 cell lines by ga - pls analysis showed r values 0.86, 0.87 and 0.91, respectively (figure 4) which indicates that the qsar models are reliable to predict cytotoxicity of the 3,5-bis(benzylidene)-4-piperidones 1 - 7 . Plots of the cross - validated predicted cytotoxic potency values of activity by ga - pls against the experimental values for three different cell lines: (a) l1210, (b) cem, and (c) molt / c8 cell line all of the generated ga - pls models were further validated by applying the y - randomization test . Several random shuffles of the y vector were performed and the results are shown in table 5 . The low r and q values indicate that the good results in our original model are not because of a chance correlation or structural dependency of the training set . R and q values of ga - pls models after several y - randomization tests the statistical parameters of qsar analysis by mlr and ga - pls are demonstrated in table 4 . The results indicate that ga - pls analysis is more accurate for predicting the cytotoxic potential of 3,5-bis(arylidene)-4-piperidones in all three cell lines . The cross - validation statistics reported in table 4 suggest the higher prediction ability of the ga - pls model . This can be a result of the more number of descriptors used by ga - pls with respect to mlr analysis . As far as the less parametric model is obtained by mlr analysis, the results of mlr analysis are more descriptive for interpretation of the structure - cytotoxic relationship . However for the prediction of the cytotoxic potential of novel compounds of these series, the ga - pls model is more useful . It is clearly understood that some of the spatial, (electro) topological and chemical descriptors are important structural parameters that mainly affects the cytotoxic potencies of these compounds in all three cell lines . The molecular density of compounds is one of the most important spatial descriptors that should be considered in the design of novel compounds of these series . As the results of the sar study of various members of these series suggest, alignment of the n - aroyl groups with auxiliary binding sites takes place reinforcing the interaction of the 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore with a primary binding site (8). This result can be interpreted by the important structural descriptor molecular density . It is obvious that the orientation of n - aroyl group for better interaction with a corresponding binding site is affected by molecular density . Other topological descriptors also affect this desirable orientation of the ligand and therefore play a determinant role in the binding of the ligands with its target receptors . The other important electro - topological parameter that should be considered is the electro - topological state indices which encode the electronic state of each atom type in the molecule: atom - type e - state indices . The results demonstrate that the sum of the electro - topological state of aromatic ch (s_aach) and the sum of the electro - topological state indices of methyl groups (e - state keys: s_sch3) are among the important influential descriptors in the cytotoxic potential of 3,5-bis(arylidene)-4-piperidones especially toward cem and molt / c8 cell lines . The sum of the electro - topological state indices of the aromatic ch and methyl groups especially should be considered in the employment of different n - acyl substituents . The results demonstrate that similar structural descriptors affect the cytotoxic potential of n - aroyl-3,5-bis(arylidene)-4-piperidones in three different cell lines . This result indicates that similar molecular mechanisms are involved in the cytotoxic activity of the compounds in these cell lines . In addition, the previous quantitative - cytotoxic activity study of asymmetrical 2,6-bis(benzylidene)cyclohexanones against mcf-7, sk - n - mc and mda - mb-231 cell lines (20), reported the spatial and topological parameters as determinant structural descriptors in cytotoxic potential of these compounds against the cell lines . This finding also confirms that the same molecular mechanisms are involved in the cytotoxic activity of different analogues of this scaffold.
A 65-year - old female patient presented in january 2013 with unstable angina . Coronary angiography showed a severely calcified long lesion of the proximal and mid segments of the left anterior descending coronary artery [video s1 (please see enhanced content link on title page)]. In view of the angiographic findings, a decision was taken to do a planned rotational atherectomy (ra), which was performed with the rotablator (boston scientific) using 1.5 then 1.75 mm burr sizes, followed by implantation of two overlapping 3.0 28 mm bioresorbable scaffolds (brs) (absorb, abbott). Post - dilatation was done using a 3.0 mm non - compliant balloon at 20 atm . Intravascular ultrasound (ivus) showed a good result with a minimal overlapping zone (fig . 1). A coronary angiography done 10 months later due to recurrent angina revealed a significant restenosis of the implanted brs . Optical coherence tomography (oct) showed a diffuse homogeneous intra - scaffold neointimal tissue with a minimum luminal area of 1.59 mm at the distal brs and 1.98 mm at the proximal one (fig . 2). A scoring balloon angioplasty using a 3.0 10 mm angiosculpt scoring balloon (angioscore) was performed followed by 3.5 20 and 3.0 20 mm pantera lux (biotronik) paclitaxel eluting balloons (deb) in the proximal and distal scaffolds, respectively . Oct clearly showed the neointimal cuts achieved by the scoring elements (white arrowheads, fig . 3), neointimal disruption with some intraluminal material (yellow arrowheads, fig . 3) and lumen expansion . At 2 years after balloon angioplasty (almost 3 years after brs implantation), the patient remained asymptomatic and a scheduled angiographic control showed a patent vessel with no significant lesions . Oct showed favorable evolution of the neointimal damage caused by the scoring balloon and maintenance of the initial lumen expansion, with partial resorption of the scaffold struts for which footprints were still visible (fig . C shows final result after implantation of two 3 28 mm bioresorbable scaffolds, with a good result in the final intravascular ultrasound (d) fig . 2in - scaffold restenosis . In a, b, the angiogram shows diffuse in - scaffold restenosis in the proximal and distal scaffolds . B is the final angiographic appearance after scoring balloon / drug - eluting balloon treatment . Optical coherence tomography (oct) images (a) and (c) show homogeneous intra - scaffold tissue . 3final result after scoring balloon / drug - eluting balloon treatment . In a, b, the final angiographic appearance after balloon - only treatment of the restenosis . Intraluminal material caused by neointimal dissection is also observed (yellow arrowhead) fig . 4follow - up angiogram and optical coherence tomography (oct) 2 years after treatment of the in - scaffold restenosis . A is done in a zone of muscular bridging showing the absence of significant stenosis in that area . The favorable evolution with disappearance of the neointimal damages caused by the scoring balloon is clear in the oct images b f, which also show partial resorption of the scaffold struts baseline angiogram and procedure . A baseline angiogram showing a calcified stenosis of the proximal and mid lad . C shows final result after implantation of two 3 28 mm bioresorbable scaffolds, with a good result in the final intravascular ultrasound (d) in - scaffold restenosis . In a, b, the angiogram shows diffuse in - scaffold restenosis in the proximal and distal scaffolds . B is the final angiographic appearance after scoring balloon / drug - eluting balloon treatment . Optical coherence tomography (oct) images (a) and (c) show homogeneous intra - scaffold tissue . The b represents oct cross section of the overlapping zone final result after scoring balloon / drug - eluting balloon treatment . In a, b, the final angiographic appearance after balloon - only treatment of the restenosis . D f lumen expansion is clearly visible via optical coherence tomography (oct). Intraluminal material caused by neointimal dissection is also observed (yellow arrowhead) follow - up angiogram and optical coherence tomography (oct) 2 years after treatment of the in - scaffold restenosis . A is done in a zone of muscular bridging showing the absence of significant stenosis in that area . The favorable evolution with disappearance of the neointimal damages caused by the scoring balloon is clear in the oct images b f, which also show partial resorption of the scaffold struts informed consent was obtained from the patient for being included in the study . The most important proposed advantage of brs over the current metallic stent technology is to restore the natural physiologic vasomotor function and to hinder late thrombotic events . In our case, the initial treatment strategy was using ra in disrupting the continuity of intravascular calcium rings facilitating brs implantation and potentially achieving a stent - free vessel in the long - term . Ra is a safe and effective method for lesion preparation before stent implantation and its use could be extrapolated before brs implantation, where lesion preparation is of utmost importance . However, additional vessel injury with ra has been previously suggested to be associated with increased late lumen loss . In our case, this mechanism appears very likely because the in - scaffold restenosis was diffuse and affected both implanted brss . The rate of target - lesion revascularization ranges from 1% to 3.3% after brs implantation [3, 4]. The most often used method for the treatment of in - scaffold restenosis is a drug - eluting stent (des), but there is no sufficient data supporting this approach, and the optimal treatment strategy is still uncertain . Drug - eluting balloons have demonstrated their efficacy in treatment of restenosis following metallic stent implantation . Additionally, scoring balloon predilatation prevents balloon slippage and enhances local plaque disruption, and its use in combination with deb angioplasty improved angiographic results in the isar - desire 4 study . In this reported case, by adopting the scoring / paclitaxel - coated balloon strategy, we tried avoiding a metallic stent implantation targeting to maintain the long - term benefits of brs . However, between 3 and 6 months and as a consequence of hydrolysis of ester bonds, the device loses its structural continuity, and the radial strength decreases considerably until it is lost . This loss of radial force is suspected to be source of recoil when balloon dilatation is performed alone after 6 months . In this patient, the vessel wall 10 months after brs implantation opposed minimal recoil to the scoring / deb treatment, which achieved sufficient stable lumen size and remained effective until 2 years of follow - up . In our case we tried to highlight the possibility of a balloon - only treatment of a restenosed bioresorbable scaffold . With an adequate initial lesion preparation, this strategy can be even applied in severely calcified coronary lesions . Finally, given the limited data, there is a need for further examination of the place of ra before brs implantation and the optimal treatment strategy for in - scaffold restenosis from future rcts.
Access to water in the required quantity is needed to achieve good personal and domestic hygiene practices1; while good quality water ensures that ingested water does not constitute a health hazard, even in a life time of consumption2 . It is however estimated that as much as 1.1 billion people do not have access to safe drinking water3, while the drinking of contaminated water is responsible for 88% of the over four billion cases of diarrhoeal diseases that occur in the world every year, and the 1.8 million deaths that result from them . It is also indirectly responsible for the 50% of childhood malnutrition that is linked to diarrhoral diseases, and the 860, 000 deaths that result from them each year4 the who estimates that 94% of diarrhoeal diseases are preventable through modifications to the environment4, with improved access to safe drinking water alone able to reduce diarrhea episodes by between 20% and 35%, according to two systematic reviews56 . These health benefits, and the fact that a ready access to water saves the time of water drawers for more productive activities explain why access to adequate quantity of safe water was made one of the millennium development goals7, and why it was recognized as one of the foundations of nigeria's developmental efforts, by the national economic empowerment and development strategy (needs) document8 . Target 10, goal 7 of the millennium development goal sets a 2015 target to reduce the proportion of people without access to safe water by half7; while the 2005 national water and sanitation policy expects a 100% coverage by 20119 . These targets would however require extra effort to achieve in the rural riverine communities of the niger delta region, considering the enormous effort required to make the huge water resources in the communities safe for drinking . According to the 2008 national demographic and health survey, access to safe drinking water is still low in the rural communities of nigeria at 43.8%, which is likely to be worse in the rural riverine communities of the niger delta, because of the widespread use of overhung toilets in the communities, and the poor quality of groundwater, linked to saline intrusion and high concentration of iron, manganese and arsenic, as a result of the geology of the area10 . It was not known the extent target 10, goal 7 of the mdg has been met in the communities, especially with the much publicized efforts of government and the oil companies in addressing the basic needs of the people of the niger delta . This study assessed the situation of the community water supply in 22 rural and semi - urban riverine communities in bayelsa and rivers states, in the niger delta; as part of a baseline health impact assessment study conducted in the communities for a gas pipeline project . The report the findings and the recommendations of appropriate technologies that could be implemented to hasten the pace of meeting the mdg in the communities are hereby presented . This was a descriptive cross - sectional study using a structured interviewer - administered questionnaire, field observations and focus group discussions carried out in 21 communities, in four local government areas in south - south nigeria . A triangulation of the qualitative research techniques were used to help gain a deeper insight into the context of the water situation in the study communities . These communities had a population of about 150, 000 people (projected with the 2006 national census). The study set to detect a 5% difference in access to safe water, with an alpha error of 5%, acceptable beta error of 20%, and a statistical power of 80%; and using the national average of access to safe water in rural communities of 43.8%11 . Using the usual formula for sample size determination for descriptive studies12 the data were collected by the author and trained assistants and analysed for the type, operation, maintenance and functionality of water facilities, and the microbiological status of the water, using the membrane filtration technique, with escherichia coli as the indicator organism14 . The data were analyzed according to the standard method12, and the results were considered to be significantly contaminated if they were found to be beyond the world health organisation (who) minimum acceptable values15 . The respondents had an average age of 29.20 + / 5.2 years; most (89.69%) had a secondary school education or less, and had spouses that were mostly engaged in fishing and farming (40.13%) (table 1). The socio - demographic characteristics of study participants table 2 shows the water and sanitation facilities of the respondents . The most common source of drinking water was surface water (37.9%), with bottled / sachet water serving the needs of up to 19% of the households . Household water and sanitation facilities most (61.2%) of the drawers of water for the households spent less than 15 minutes to complete the round trip to the water sources, while most (60.8%) of the households stored their drinking water in jerry cans . A lot (48%) of the drinking water used by the households were not treated, even as only 12.72% of them used an improved sanitation facility, and the two - week period prevalence of diarrhea amongst them was 27% . The number and type of water supply facilities in the 22 study communities are shown in table 3 . There were a total of 374 community water supply facilities in the communities, an average of 17 per community, but only 89 (23.80%) were functional as at the time of the study; an average of 4.4 per community . Only 46 (12.3%) of the water facilities provided piped supply, though with very few household connections, with only 17 (36.96%) of them functional as at the time of the study . The number and types of community water supply facilities in the study communities table 4 shows how the water facilities in the communities were provided . Most (64.7%) of the facilities were provided by government and its agencies like the niger delta development commission, but this included the protected hand - dug well constructed during the colonial and immediate post - colonial periods, and the hand - pumped well provided by several ad - hoc government water supply programmes . The sources of the community water facilities in the study communities the water facilities provided by the oil companies were either provided as part of their social responsibility to the communities, or as part of the remediation for an oil spill . Those provided as part of the company's social responsibility were mostly functional, mainly because they had functional committee for the maintenance of the facilities, though with most of the maintenance costs borne by the oil company . Table 5 shows the results of the microbiological analysis of the water sample collected from various water sources in the communities . Members of four of the communities had during the field study complained of the quality of water from their facilities, and some members had refused to drink from the facilities . The results of the microbiological analysis of the water sample collected from the various facilities in the communities more than two third (67.9%) of the samples tested were found to contain significant numbers of escherichia coli; especially the samples collected from surface water from which members of the communities routinely drank from . The study showed that the study communities were served by an average of 4.4 functional community water supply facilities, and that most of the inhabitants spent less than fifteen minutes to draw water from the facilities . This is consistent with the who recommendation of less than 15 minutes to and fro journey to the drinking water source, that ensures the provision of adequate quantity of water required to satisfy the drinking water and sanitation needs1316 . The situation in the study communities was also better than the figures obtained during the 2008 national demographic and health survey11 . According to the survey, only 71.9% of nigerians residing in the rural areas had access to water within 30 minutes, compared to the 91% obtained in the study . The situation in the study communities was helped by the efforts of the oil companies operating in the communities who provided more than 30% of the community water facilities in the communities . However, as much as 76.2% of the community water facilities in the study communities were not functional as at the time of the study (table iii). This has also been noted in other communities in nigeria17; and blamed on factors that include amongst others, the absence of a responsible body for the operation and maintenance of the facilities, and poor workmanship by dubious contractors17 . These factors were also noted in the study communities as most of the non - functional facilities were those provided by government and its agencies, without any arrangement for their maintenance and operation . On the other hand, facilities provided as part of an oil company's social responsibility were found to be mostly functional, because they had functional committees constituted and funded for the operation and maintenance of the facilities . The non - functional water facilities forced 66% of the households into drawing their water from non - improved sources . This is worse than the national average for rural areas of 53.4%11 and very unhealthy considering that as high as 67.9% of the water facilities were found to have e.coli count higher than the who recommended level15 . It is also not surprising that the two week period prevalence in the communities was 26.97%, much higher than the 8.9% average for rural areas in nigeria . The quality of water in the communities can be improved not only by ensuring the functionality of the water facilities, but most importantly by encouraging the use of point - of - use water purification systems . The use of point - of - use water purification systems would fully tap the huge surface water resources in the communities, and particularly discourage the use of the expensive, but dubious bottle / sachet water that was used by19.08% of the households to satisfy their drinking water needs . Point - of - use water purification systems have been found to deliver as much health benefits as an improved water source56, and among the most cost - effective approaches in preventing diarrhoeal diseases18 . Promoting the use of the point - of - use purification systems require a deliberate effort, especially because 48.03% of the households did not see the need to purify water of suspicious quality before drinking; while up to 29% use alum and cloth filtration that are not particularly effective in disinfecting the water in the communities . Even boiling that is often recommended has been found to fail under conditions of heavy faecal contamination as found in the study communities; because of the ease with which household utensils are recontaminated19 . More social marketing activities would therefore be needed in the communities to specifically promote the use of the coagulant / chlorine combination that was used by just 5.92% of the households, but have been found to be very effective520 . Members of four of the study communities had complained of the quality of water from their facilities . This is probably related to the high iron and manganese content of the water, as indicated by a previous study in the niger delta10 . Also, the fact that most of the water facilities in the communities had elaborate water treatment facilities points to the enormity of the problem posed by the high inorganic content of the ground water . The type of technology used to deal with the poor quality ground water is probably inappropriate, and responsible for the high level of non - functionality of the water facilities . The sustainable use of the water facilities therefore lies in adopting an appropriate technology that can address the need, yet simple enough to be operated and repaired by readily available expertise21 . There are already several low - cost and rugged technologies that can be applied at household and community levels in the communities for the treatment of water with high inorganic content2223; such technologies should as a matter of urgency be adopted and promoted in the communities . The communities had easy access to water supply, but most of the facilities were either contaminated or nonfunctional . The operation and management of the facilities by members of the communities, and the promotion of point - of - use purification systems are hereby advocated.
Transportation planning and public health have important historical connections . In the early 20th century transportation planning and public health held similar objectives . In 1909 marsh wrote, city planning is the adaptation of a city to its proper function . This conception can be indefinitely expanded but its significance will be appreciated if we admit that no city is more healthy than the highest death rate in any ward or block and that no city is more beautiful than its most unsightly tenement (p.27). Since the early 20th century, the relationship between transportation planning and public health has waxed and waned [1, 3]. There is growing recognition that integration of theories and methods from each discipline is beneficial for advancing research and practice . Of particular relevance to both transportation planning and public health are injuries due to traffic collisions . Worldwide, traffic collisions are one of the leading causes of death among youth and young adults . A number of intervention strategies including black spot analyses are used in transportation planning to reduce road fatalities . There is ongoing debate in the transportation planning literature that this type of intervention may have limited effectiveness in reducing road fatalities [68]. New approaches are needed to reduce the burden of road traffic fatalities . Given the need to reconnect transportation planning and public health, this paper will present rose's strategy of preventive medicine [9, 10], a promising intervention approach from public health . The prevention paradox states that a preventive measure which brings much benefit to the population offers little to each participating individual (p.38). The prevention paradox is based on an important distinction between an individual case and the incidence of cases in a population . A case is defined as an episode of disorder, illness, or injury affecting an individual, while incidence is defined as the number of cases of a disease that come into being during a specific time period (p.44). The basic principle of geoffrey rose's strategy of preventive medicine is that the causes of cases (i.e., why a specific individual is involved in a collision) are different from the causes of population incidence (i.e., why there are over 3 million collisions per year in the united states). Building from the prevention paradox, the primary concept of rose's strategy is that the majority of cases do not occur in individuals at high risk . Thus, a large number of people exposed to a small risk may generate many more cases than a small number exposed to a high risk (p.59). The recent study by beck provides an example of this principle related to road traffic fatalities . Show that pedestrians (13.7 deaths per 100,000 person trips), cyclists (21.0 deaths per 100,000 person trips), and motorcyclists (536.6 deaths per 100,000 person trips) are at higher risk of a fatal collision than motor vehicle users . Despite their higher risk, pedestrians, cyclists, and motorcyclists represent only 20.5% of those killed in traffic collisions . Figure 1 shows that in the usa the majority of trips (86%) and therefore exposure to causes of collisions are greater for motor vehicle users . Thus, the majority of those killed (76.6%) are motor vehicle users who are, on average, at a lower risk (9.2 deaths per 100,000 person trips) than other road users except public transit users . For interventions to be effective in reducing the total number of road deaths and injury, they must target fundamental causes (i.e., causes of incidence), which would result in a global reduction in exposure to a fundamental cause for the entire population . According to the strategy of preventive medicine road traffic fatalities can be neither understood nor properly controlled if high - risk road users are thought to constitute the entire problem . Policies and practices that attempt to prevent traffic fatalities must consider all modes of transportation because they all contribute to the total number of deaths (figure 1) [14, 15]. In transportation research, the volume and speed of motor vehicle traffic could be considered two of the fundamental causes of collisions, for all road users [16, 17]. Throughout the paper, we consider exposure to moving vehicles as a fundamental cause of road traffic deaths and injuries . In this example, a global reduction in the amount of kilometers driven would result in a reduction of the likelihood of collisions for all road users, a left shift in the risk of road death and injury (figure 2) [18, 19]. For all road users, including pedestrians and cyclists, a reduction in traffic volume contributes to a lower risk of injury and death . Significant public health benefits could be achieved through macrolevel interventions that influence the level of exposure to traffic volumes for all road users, for example, land use and transportation policies that encourage using safer transportation modes (e.g., public transit) and area - wide traffic calming schemes covering whole metropolitan areas, among others . In the usa, recent reductions of traffic volume were associated with a reduction in the total number of road fatalities . Rose's perspective is one way to critically appraise the challenge of reducing the overall burden of collisions while maintaining the benefits of effective mobility . The implementation of interventions supported by rose's strategy requires a dialogue between researchers, planners, policy makers, and the public . Throughout this discussion we must recall that the primary function of roadways is not to cause collisions but rather the mobility and productivity of the population . Because roadways have multiple functions, policy makers, planners, health officials, and the public may disagree on how to balance road safety with other competing functions . As rose states, if a problem is common and has been around for a long time, then people come to accept it even if it is large: it is the exceptional or new which causes alarm . The toll of deaths from road traffic accidents vastly exceeds that from crashing aero planes, but only the latter lead to public health inquiries (p.57). As the previous quote suggests, researchers must critically appraise their work and dialogue with policy makers and the public about complex transportation challenges in order to achieve an acceptable balance between the need for mobility and its consequences [2224]. Ideally, interventions will both improve mobility and reduce the public health burden of road traffic injuries . In particular researchers must be conscious of the potential influence of structural global economic forces lead by major car and oil companies in influencing road collisions research and discourse [21, 2527]. Beck et al . State, our findings suggest that a shift from passenger vehicle travel (lower risk) to nonmotorized travel (higher risk) could result in an overall increase in the numbers of people killed in traffic measures that prevent crashes and injuries for pedestrians and bicyclists are needed, especially given the recent focus on increasing physical activity through active travel (p.216). Suggests that the high - risk approach is a more acceptable intervention than a population approach, which would reduce exposure to fundamental causes, volume of motor vehicles in our example, for all road users . However, in general, interventions targeting high - risk groups (e.g., walkers and cyclists) have limited population wide benefit for injury prevention . Targeting the causes of incidence to reduce exposure to motor vehicle volume for all road users, the population approach, has the potential for a greater reduction in the total number of transportation fatalities in a population than the high - risk approach . Compares a population approach and a high - risk approach targeting pedestrians and cyclists . In the usa, between 1999 and 2003, a 25% reduction in the number of pedestrian and cyclist fatalities would have resulted in 1386 fewer deaths (1212 for pedestrians, 174 for cyclists). A greater number of lives would have been saved by a 4% reduction in the fatal risk for all road users (table 1). Table 1 estimates the hypothesized effect of decreasing fatal risk over the entire population of road users . It shows that a 10% and 15% reduction in the fatality risk for all road users would result in a reduction of 4213 and 6320 cases, respectively . These scenarios would benefit motor vehicle occupants but also pedestrians and cyclists . In urban settings, a risk reduction of 15% could be achieved through area - wide traffic calming interventions . Reducing the risk of road death and injury by intervening to reduce the level of exposure to traffic volumes, or other fundamental causes, first, the potential reduction in road injuries and deaths is much larger than with high - risk approaches . Second, a population - wide prevention strategy is of benefit to high - risk road users, whatever the criteria used to define high risk (e.g., transportation mode, alcohol consumption, age group). Third, it may also reduce other externalities including pollution, noise, physical inactivity of fundamental causes such as traffic volume [21, 29]. For decades, the strong relationship between traffic volume and road traffic injuries or death has been reported, at the street, city, region, or country levels [30, 31]. For all intersections from a road network, safety performance functions show that the expected number of collisions at intersections increases as traffic volume increases, though this relationship is not linear (figure 3). The traditional intervention strategy, recommended by the us department of transportation is to return deviant intersections to within an acceptable range of the average collisions rate at comparable intersections . As seen in figure 3(a), this targeted intervention approach aims to move the deviant intersections closer to the best fit regression line . This is an example of a high - risk approach that does not address the entire population of intersections or injured individuals . In montreal, for example, only 4% of pedestrian injuries occurred at 22 identified black spot intersections, whereas the remaining 96% of pedestrians were injured at more than 3500 different crash sites, none of which were black spots . The population strategy acknowledges that interventions should address the fundamental causes of road deaths and injuries, in our example, the level of exposure to moving vehicles over the entire population, and for all intersections . The population approach would ask whether it was possible to globally reduce the exposure to traffic volume (figure 3(b)) and subsequently reduce the risk of death and injury at any given intersection for all road users . As the arrows show in figure 3(b), the traffic volume and subsequent risk are reduced at every intersection . In theory, it would result in a greater reduction of the number of injured road users than targeting deviant streets and intersections . Rather we present a promising approach to think about road traffic fatalities . A detailed reading of rose's work [9, 10] will provide more precision and help clarify interested readers understanding . It should be noted that rose's ideas are not without considerable debate in public health [3335]. For example, modeling studies suggest that rose's approach is generally more cost effective; however, in certain cases a high risk approach can have better cost / benefit ratios . For example, recent studies suggest that when compared with other roads, streets with cycle tracks have a lower risk of injury for cyclists [36, 37]. As well, the potential of rose's strategy for reducing social inequities in health in general [34, 35] and in particular road fatalities for those residing in low income areas is still debated . A vulnerable population approach may be the most acceptable when attempting to act on social inequities; however, a recent study has shown that reducing traffic volume at all intersections of a canadian city would be most beneficial in poorer neighborhoods . Transportation planning and public health have important historical roots . To address common challenges, including road traffic fatalities, integration of theories and methods from both disciplines is required . Geoffrey rose's strategy of preventive medicine made an important theoretical contribution to public health and has applications to transportation . This paper allowed for both public health and transportation research to critically appraise their practice and engage in informed dialogue with the objective of improving mobility and productivity while simultaneously reducing the public health burden of road deaths and injuries.
A presentation to hospital with vertigo or dizziness is common [1, 2]. The usual reason is disabling dizziness leading to nausea, vomiting, and often prostration or inability to mobilise . Patients with these symptoms are challenging with difficulty determining which specialty to refer to . Otolaryngologists have expertise in this area; however, patients may be more appropriately referred to neurologists or general physicians in the first instance . We discuss the close interplay of pathophysiology relating to vertigo, nystagmus, oscillopsia, and the vestibulo - ocular reflex and its modulation by head titubation . It may be induced by stimulation of the intact sensorimotor system or by dysfunction of any of the stabilising sensory systems . General sensations of dizziness, in contrast, are often of nonvestibular aetiology . Whether this be an impending faint (type ii), dysequilibrium (type iii), or vague lightheadedness (type iv), it may be due to nonvestibular causes . These can include cerebrovascular disease, multiple sclerosis, cns tumours, migraine, or epilepsy . A sixty - four - year - old lady presented to her general practitioner (gp) with acute onset of unsteadiness and dizziness associated with movement . She visited her doctor with a complaint of feeling dizzy and faint but had a blood pressure of 113/70 mmhg . She had no abnormalities on general, vestibular, neurological, and otological examinations, including no nystagmus . The dizziness was described as repeated attacks of a sensation of internal moving; that is the patient was whirling, but the room was still . Her symptoms worsened over the following week, disturbing her daily activity and leading to nausea, vomiting, and prostration . The gp had noted that she had irregular and erratic eye movement and had developed a postural drop in her blood pressure, from 113/70 mmhg to 97/63 mmhg on standing . A week later she was permanently dizzy and nauseous and had noticed some difficulty in pronunciation of words . The dizziness was now described as a constant sensation that she was turning and tilting over, when she knew she was upright, and the room was still . There had also been the occasional sensation that the room was spinning around, although these were the only fleeting . She had no visual disturbance, hearing problems, weakness or numbness, and her neurological examination was normal . She was treated with antiemetics including prochlorperazine; with a presumed diagnosis of vestibular failure, and her gp referred her to the otolaryngology services for further treatment and to investigate for an organic cause . On admission on examination, she was noted to have a subtle head nodding tremor or titubation . This was associated with a low - frequency tremor of the upper limbs, which appeared to increase on intention . She had erratic eye movements and it was very difficult to elicit any nystagmus, even with frenzel glasses, whether spontaneous or positional . This was due to the difficulty in keeping her eyes open secondary to overwhelming nausea . The patient found that it very difficult to keep her eyes still and to focus on a stationary object and she was unable to smoothly pursue a moving target . There was clearly a fixation instability and it appeared to be in the form of conjugate involuntary saccadic oscillations . There was, however, a fleeting vertical nystagmus on upgaze, which may have indicated a gaze - evoked cause, but with no obvious horizontal element . A halmagyi - curthoys head thrust test was attempted, but proved difficult to interpret due to the stimulation of vomiting . It was noted, however, that the patient was unable to fix on a stationary object during head rotation indicating a probable suppression of the vestibulo - ocular reflex (vor). Due to the significant nausea and vomiting, a caloric canal test was not performed . She had subtle cerebellar signs on subsequent examination, including dysmetria and dysdiadocokinesis . Dix - halpike test was symptomatically positive, with vomiting but with no rotatory nystagmus and no positional dependence . She was given intravenous fluids and steroids and treated symptomatically for the nausea and vomiting . It was postulated that there was a central cause for her symptoms, due to the combination of cerebellar signs, fixation instability / opsoclonus, possible upbeat nystagmus, and vor suppression . Therefore, a magnetic resonance scan (mri) of the brain was ordered . This revealed a large space - occupying lesion in the right lobe of the cerebellum, with a smaller lesion in the left lobe (figure 1). The patient was referred directly to the neurosurgeons who performed a suboccipital craniectomy and excision of the cerebellar tumour . As a presentation to otolaryngology, the history of this case is not unusual ., it accounts for 13% of patients, but only 50% of those referred to otolaryngology services have an otological cause for their symptoms . It can be argued, therefore, that patients presenting with these symptoms should be referred to general physicians in the first instance, to rule out serious nonotological causes . The anamnesis of dizzy symptoms is especially important to elicit in detail in order to make informed decisions on referral route . Vertigo indicates a sensation of false movement (generally described like a rotation), but rarely, the patient can describe it like a sensation of tilt . In our case, the patient described both rotational and tilting sensations, whilst stationary and on movement . Most importantly was the constant sensation that she was turning but that the room was still . This specific symptom, and its duration, indicates an increased risk of central origin . With peripheral vestibular causes of vertigo this may be a result of oscillopsia, an optical illusion of oscillation sometimes as a direct result of nystagmus . Oscillopsia is common in bilateral labyrinthine abnormality, but the presence of vertical oscillopsia indicates a vertical nystagmus, which is indicative of a central lesion . In this case, spontaneous nystagmus, albeit fleeting, was vertical nystagmus and this is particularly specific to central lesions rather than peripheral vestibular lesions, and is a reliable indicator . Damage to the ventral tegmental tract, originating in the superior vestibular nucleus, leads to upward vestibular signals to the third nerve nucleus and so upbeat nystagmus (ubn). Therefore, a vertical nystagmus should give high index of suspicion for a central cause . The general inability to stabilise the eye, resulting in the erratic eye movement and opsoclonus seen may also indicate central pathology . Particular regions involved are the vermis, flocculus, and paraflocculus, which are responsible for coordinating smooth pursuit and adaptation of the vestibulo - ocular reflex (vor). These control mechanisms are essential to holding the eye steady for fixation, both immediately after saccades and in eccentric positions of gaze, particularly important when attempting to track a moving target . The lesions in the cerebellum may well have impaired these important mechanisms in this patient, leading to opsoclonus and an inability to fixate . It is also known that an impairment of adaptation of the vor, to compensate for movement, may lead to oscillopsia . Although the lesions seen on mri in this case are not in the vermis, surrounding oedema and paraneoplastic effects may be responsible for an impairment of vermis function . The general difficulty in diagnosis, detailed neurotologic examination, and testing in this case may have been influenced by the use of prochlorperazine . This is a phenothiazine antipsychotic agent which is very commonly used as a vestibular sedative for nausea due to vertigo . It is widely known, however, although affording symptomatic relief for the patient, this drug can result in a delay to vestibular compensation and influence diagnostic testing . This is defined as nodding head tremor with a frequency of 3 to 4 hz and may be seen in midline cerebellar disease [12, 13]. Titubation can occur in isolation or combined with a postural tremor elsewhere, especially in the arms . This can be seen in patients with essential tremor or dystonic tremor, associated with posterior fossa disorders . Essential tremor, such as this, is closely related to the oculomotor deficits described above . These include deficits of pursuit initiation and suppression of the vor . It has been shown that there is a strong correlation with the intensity of intention tremor and the oculomotor impairment and may indicate an impairment of the caudal cerebellar vermis . There is the potential, however, that the tremor and head titubation may have been an extrapyramidal akathisia as a result of prochlorperazine use . The closely related interplay of the mechanisms involved in this patient's symptoms are difficult to sequence . The cerebellar lesions are the only true evidence of a causal element to this case . However, it remains to be understood how these lesions had brought about the specific symptom complex identified . Was the vertigo a direct result of the lesion or was it secondary to an oscillopsia generated by nystagmus, or due to failure of the vor? Was ubn or gaze - evoked nystagmus present, or was it just a brief recognisable pattern in the otherwise erratic oculomotor impairment or opsoclonus, or secondary to loss of adaptation of the vor? Was the head titubation the key? Oscillopsia occurs when there is failure of the vor to adapt and compensate for head movements . Cerebellar lesions may impair the normal cerebellar adaptive mechanism for the vor and so impair its ability to compensate for movement of the head . Simultaneously, a cerebellar lesion may also lead to an essential tremor, manifesting as head titubation and upper limb tremor . Many of the signs seen in this case, such as the impaired vor, the titubation, opsoclonus, and dysarthria, are classically caused by cerebellar vermal lesions . However, the lesions seen on mri are in the deeper hemispheres . One may, therefore, suggest that surrounding oedema and local compression have led to vermal damage . It can be seen in figure 1 that there is a degree of cerebellar midline shift . One may conclude that the lesions themselves may have simultaneously caused a tremor and an inability for the vor to compensate for it . Oscillopsia due to an impaired vor has been shown to be more likely than oscillopsia due to a titubation . Also, the two cerebellar lesions may have had the two separate effects of tremor and impaired vor . The differentiation between central and peripheral causes of vertigo can be perplexing and often require the expertise of a specialist neurotologist . Many signs and tests, such as saccadic pursuit, gaze - evoked nystagmus, and the halmagyi - curthoys test, have recently been shown to be unreliable in distinguishing peripheral from central causes; therefore, an expert opinion may be required . However, whether the vertigo in this case was a result of an oscillopsia, nystagmus, or central cause, the referral route should initially be via a general physician to rule out such life threatening causes as central tumours . Suspicion of a central cause for vertigo on examination should prompt urgent imaging of the central nervous system.a vertical nystagmus should give high index of suspicion for a central cause.central vestibular lesions may present with a wide variety of symptoms and signs which may include impaired vor, head titubation and tremor, opsoclonus, and oscillopsia.initial referral of patients with disabling vertigo should be via general physicians . Suspicion of a central cause for vertigo on examination should prompt urgent imaging of the central nervous system . Central vestibular lesions may present with a wide variety of symptoms and signs which may include impaired vor, head titubation and tremor, opsoclonus, and oscillopsia.
The ability to navigate through unpredictable environmental changes such as cycles of feast and famine is critical for the survival of an organism . In order to adapt to fluctuations in nutrient availability, communication between multiple metabolic organs must be integrated to properly metabolise carbohydrates, proteins and lipids . Dysregulation of these processes can have an impact on survival during a fast or lead to obesity and related pathologies, such as type 2 diabetes, in response to overnutrition . However, the underlying mechanisms that maintain metabolic balance during extremes of nutrient challenge remain poorly understood . Insight into these mechanisms can be gained by studying the highly conserved nuclear hormone receptor (nhr) superfamily . The 48 human nhrs are genetic switches that control networks of genes in response to various physiological cues and orchestrate a diverse range of biological functions, including nutrient homeostasis [13]. Typical nuclear receptors are comprised of distinct functional domains, including an n - terminal transactivation domain, a highly conserved dna - binding domain (dbd) and a c - terminal ligand - binding domain containing a ligand - dependent transactivation function [1, 2]. Ligand binding induces a conformational change in the receptor that allows for differential recruitment of cofactors and subsequent modulation of nhr activity [36]. A unique feature of many of these receptors is their ability to be activated by lipophilic ligands derived from dietary nutrients such as long chain fatty acids and sterols . By controlling networks of genes in key metabolic organs, nhrs play critical roles in regulating the metabolism of nutrients in a tissue - specific manner . Nhrs also regulate the expression of core clock genes both centrally and peripherally to coordinate and establish daily rhythmicity . Recent studies show that certain members of the fibroblast growth factor (fgf) family are direct targets of nhrs and responsible for mediating many of their metabolic effects . Their importance in metabolism, combined with the ability to manipulate their ligand - dependent activity, make nhrs attractive drug targets for the treatment of metabolic disease . Here, we discuss the roles of nhrs, during feast and famine, in adipose tissue, liver and muscle, as well as their roles in establishing circadian rhythms and regulating fgfs . The primary function of wat is to synthesise and store triacylglycerol during periods of energy excess and to hydrolyse triacylglycerol to generate fatty acids and glycerol for use by other organs during periods of energy deprivation . Wat also secretes adipokines such as leptin and adiponectin that regulate energy intake, metabolism and insulin sensitivity . Excess storage of triacylglycerol in wat results in obesity and related disorders, including type 2 diabetes . Paradoxically, the metabolic abnormalities found in obesity are also found in lipodystrophies, which are characterised by the inability to properly store fat in adipose tissue . Therefore, an appropriate capacity to store triacylglycerol in wat in response to changes in nutrient availability is critical for metabolic homeostasis . While multiple nhrs have been shown to affect wat metabolism, peroxisome proliferator - activated receptor (ppar) is the most critical for proper wat function [9, 10]. Ppar is most highly expressed in adipose tissue and is best known for its role in regulating adipogenic and lipogenic pathways . Generation of whole - body and adipose - specific ppar knockout mice revealed that ppar is not only required for adipocyte differentiation but also for mature adipocyte function [1116]. Ppar also plays a critical role in glucose homeostasis by increasing the expression of glut4 and c - cbl - associated protein, as well as numerous secreted factors that affect insulin sensitivity, such as adiponectin, resistin, leptin and tnf in wat [1720] (fig . Activation of ppar specifically in adipocytes is sufficient to cause whole - body insulin sensitisation and, conversely, a dominant - negative mutation in a single allele of pparg in humans leads to partial lipodystrophy and insulin resistance, supporting its role in adipogenesis and insulin sensitivity [2124]. The thiazolidinediones (tzds), which are known agonists of ppar, exhibit potent adipogenic and glucose - lowering effects, and, despite their unwanted side effects, remain highly effective for the treatment of metabolic disease .fig . White adipose tissue (wat) synthesises and stores energy in the form of triacylglycerol (tag). During the fasted state, wat hydrolyses tag to generate nefa that can be taken up by other organs and used for energy production . Peroxisome proliferator - activated receptor (ppar) plays a major role in controlling the expression of genes involved in adipogenesis and lipogenesis . Ppar also controls the expression of secreted factors (transducers) involved in glucose and energy homeostasis such as leptin, adiponectin, resistin and tnf as well as fibroblast growth factor (fgf)1 and fgf21, which act locally in adipose tissue during the fed state to promote adipose remodelling and differentiation, respectively . (b) liver during feast and famine . During the fed state, excess glucose is taken up by the liver and stored as glycogen via glycogenesis or converted to nefa through de novo lipogenesis for synthesis and storage as tag . During the fasted state, prolonged fasting leads to de novo glucose synthesis in the liver through gluconeogenesis (gng). During starvation, when glycogen stores are depleted, the liver uses acetyl - coa to produce ketones by a process called ketogenesis, where ppar plays an important role . The glucocorticoid receptor (gr), oestrogen - related receptor (err) and hepatocyte nuclear factor 4 (hnf4) promote gluconeogenesis, while liver x receptor (lxr) and farnesoid x receptor (fxr) suppress gluconeogenesis . During the fed state high bile acid levels are then sensed by fxr, which inhibits their synthesis to prevent an accumulation of bile acids in the liver . Type i fibres preferentially oxidise fatty acids, while type ii fibres preferentially metabolise glucose . Ppar and err promote a type i fibre type in skeletal muscle and ppar promotes fatty acid utilisation, while nur77 promotes glucose utilisation (a) adipose tissue during feast and famine . During the fed state, white adipose tissue (wat) synthesises and stores energy in the form of triacylglycerol (tag). During the fasted state, wat hydrolyses tag to generate nefa that can be taken up by other organs and used for energy production . Peroxisome proliferator - activated receptor (ppar) plays a major role in controlling the expression of genes involved in adipogenesis and lipogenesis . Ppar also controls the expression of secreted factors (transducers) involved in glucose and energy homeostasis such as leptin, adiponectin, resistin and tnf as well as fibroblast growth factor (fgf)1 and fgf21, which act locally in adipose tissue during the fed state to promote adipose remodelling and differentiation, respectively . (b) liver during feast and famine . During the fed state, excess glucose is taken up by the liver and stored as glycogen via glycogenesis or converted to nefa through de novo lipogenesis for synthesis and storage as tag . During the fasted state, glycogen prolonged fasting leads to de novo glucose synthesis in the liver through gluconeogenesis (gng). During starvation, when glycogen stores are depleted, the liver uses acetyl - coa to produce ketones by a process called ketogenesis, where ppar plays an important role . The glucocorticoid receptor (gr), oestrogen - related receptor (err) and hepatocyte nuclear factor 4 (hnf4) promote gluconeogenesis, while liver x receptor (lxr) and farnesoid x receptor (fxr) suppress gluconeogenesis . During the fed state high bile acid levels are then sensed by fxr, which inhibits their synthesis to prevent an accumulation of bile acids in the liver . Type i fibres preferentially oxidise fatty acids, while type ii fibres preferentially metabolise glucose . Ppar and err promote a type i fibre type in skeletal muscle and ppar promotes fatty acid utilisation, while nur77 promotes glucose utilisation although multiple organs are involved in glucose homeostasis, the liver is the major player . During the fed state, excess glucose is taken up by the liver and stored as glycogen via glycogenesis or converted into fatty acids (de novo lipogenesis) for synthesis / storage as triacylglycerol . During the fasted state, stored glycogen is broken down by the liver to generate glucose by a process called glycogenolysis . Prolonged fasting or starvation induces de novo glucose synthesis in the liver through hepatic gluconeogenesis . During starvation, when glycogen stores are depleted and there is a reliance on fatty acids, the liver also uses acetyl - coa to produce ketones, by a process termed ketogenesis . Several nhrs have been implicated in the control of glucose homeostasis in the liver (fig . The first nhr to be linked with hepatic gluconeogenesis was the glucocorticoid receptor, which is activated by cortisol . During fasting, increased secretion of cortisol from the adrenal gland increases the activity of the glucocorticoid receptor in the liver, leading to transcriptional activation of hepatic gluconeogenic genes such as glucose-6-phosphatase (g6pc) and phosphoenolpyruvate carboxykinase (pepck, also known as pck1). Other nhrs, such as hepatocyte nuclear factor 4 (hnf4) and oestrogen - related receptor (err) have also been shown to promote hepatic gluconeogenesis during fasting [26, 27]. However, when fasted, they exhibit severe hypoglycaemia and impaired ketogenesis [2830]. Interestingly, the fatty acid synthase - dependent phospholipid 1-palmitoyl-2-oleoyl - sn - glycerol-3-phosphocholine (16:0/18:1-gpc) has been shown to be an endogenous ligand for ppar . During the fed state, liver x receptor (lxr) and lxr, which are activated by oxysterols, have been shown to suppress gluconeogenic enzymes and upregulate the expression of glucokinase to promote hepatic glucose utilisation, while the bile acid responsive farnesoid x receptor (fxr) suppresses gluconeogenic genes and increases glycogen synthesis . In addition to promoting glucose uptake and storage, the liver plays an important role in cholesterol, bile acid, fatty acid and triacylglycerol homeostasis . When cholesterol levels are high, oxysterols generated through cholesterol degradation activate lxr, which in the liver leads to the formation of bile acids, to avoid excess accumulation of cholesterol . High levels of bile acids are sensed by fxr, which inhibits their synthesis in order to prevent an accumulation of bile acids in the liver . During the fed state, lxr activation also leads to increased triacylglycerol accumulation in the liver by increasing the transcription of genes involved in hepatic de novo lipogenesis . While ppar has also been shown to increase the expression of some genes involved in lipogenesis, it also promotes the uptake and oxidation of fatty acids in mitochondria, peroxisomes and microsomes . Ppar activation leads to lower serum triacylglycerol levels through induction of lipoprotein lipase (lpl) activity via inhibition of apociii (lpl inhibitor) expression . In addition to the hypolipidaemic fibrate drugs that target ppar, it is possible that targeting of other nhrs in the liver may lead to promising therapeutics for the metabolic syndrome . Skeletal muscle is composed of multiple myofibres that differ in their metabolic properties, including oxidative slow - twitch (type i), mixed oxidative - glycolytic fast - twitch (type iia) and glycolytic fast - twitch (type iib) myofibres [36, 37]. Type i fibres preferentially oxidise fatty acids, while type ii fibres preferentially metabolise glucose . Adaptation to fasting in skeletal muscle is aimed at sparing glucose by switching to fatty acid oxidation . Suppression of glucose utilisation is accomplished via activation of pyruvate dehydrogenase kinase 4 (pdk4), which phosphorylates and thus inactivates the pyruvate dehydrogenase complex to prevent pyruvate oxidation and conserve lactate and alanine for gluconeogenesis . During the fed state, when insulin levels are high, skeletal muscle handles the majority of glucose disposal in the body . Nhrs play a critical role in controlling glucose and fatty acid metabolism in skeletal muscle (fig . Growth factor - inducible immediate early gene nur/77-like receptor (nur77), a nhr preferentially expressed in glycolytic vs oxidative muscle, promotes the expression of genes involved in glucose metabolism in skeletal muscle . On the other hand, ppar, which activates pdk4, promotes lipid utilisation and inhibits glucose uptake in skeletal muscle [38, 40]. Mice overexpressing ppar in skeletal muscle exhibit glucose intolerance but are protected from high fat diet (hfd)-induced obesity, while ppar null mice exhibit enhanced glucose tolerance, despite increased triacylglycerol accumulation in muscle and hfd - induced obesity . Ppar also promotes fatty acid utilisation in muscle but, in contrast to ppar, activation of ppar leads to enhanced glucose uptake and improved skeletal muscle insulin sensitivity, as evidenced by studies in mice with genetic ablation and ectopic expression of ppar [28, 4144]. Ppar has also been shown to activate a genetic programme that induces a type i endurance muscle fibre type switch, leading to an enhanced capacity for endurance running and the ability to oxidise both lipids and carbohydrates at higher rates . In this regard, err, which is expressed at much higher levels in type i vs type ii muscle fibres, has been shown to be critical for maintaining the highly vascularised and oxidative capacity of type i this ability to manipulate oxidative / glycolytic metabolism in skeletal muscle through nhrs represents a potential therapeutic avenue for treating metabolic disease . Mammals experience natural sleep / wake and fed / fasted cycles and most physiological processes are regulated in a circadian manner . These rhythms are generated by a master clock located in the suprachiasmatic nucleus (scn) of the hypothalamus, which synchronises physiology to day / night cycles . While the master clock, which is entrained by light onto the retina, can synchronise clocks in peripheral tissues, feeding / fasting rhythms also serve as strong entrainment factors for metabolic organs . At the genomic level, nhr signalling is intertwined with the core clock machinery, which consists of the transcription factors circadian locomotor output cycles kaput (clock) and brain and muscle arnt - like 1 (bmal1) and their co - repressors period (per) and cryptochrome (cry). The nhrs reverse c - erba (rev - erb) and rev - erb along with three retinoid orphan receptors (ror,,) regulate many metabolic pathways in a circadian fashion by binding to target genes with an ror response element . These receptors reciprocally regulate the expression of target genes, with rors serving as constitutive activators and rev - erbs acting as repressors (fig . 2). The circadian expression pattern of bmal1 is generated by the oscillating expression of both ror and rev - erb in the scn . Rev - erb and rev - erb are also involved in peripheral circadian regulation of liver metabolism by regulating the clock interestingly, haem has been identified as an endogenous ligand for rev - erb/ linking metabolism and circadian clock [50, 51]. Furthermore, many genes involved in both clock and metabolic functions harbour bmal1 and rev - erb/ binding sites, indicating an integrated mechanism of circadian gene expression by these transcription factors [49, 52].fig . Circadian rhythms are generated by a master clock located in the scn of the hypothalamus, which is entrained by light on the retina and can synchronise the clocks in peripheral tissues . Rev - erbs and rors reciprocally regulate the expression of target genes, including bmal1 . Rev - erbs act as transcriptional repressors, while rors act as activators circadian metabolism . Circadian rhythms are generated by a master clock located in the scn of the hypothalamus, which is entrained by light on the retina and can synchronise the clocks in peripheral tissues . Rev - erbs and rors reciprocally regulate the expression of target genes, including bmal1 . Rev - erbs act as transcriptional repressors, while rors act as activators the glucocorticoid receptor also plays a critical role in establishing circadian rhythmicity . Circulating levels of glucocorticoids the glucocorticoid receptor is also critical for resetting the clock in peripheral tissues, such as the liver, and there are glucocorticoid response elements (gres) in core clock genes such as per1 and per2 . It has also been shown that cry directly binds to the glucocorticoid receptor to regulate its activity . In addition to the glucocorticoid receptor, rev - erb/ and rors, many other nhrs have been shown to be expressed in a circadian fashion and play a role in circadian metabolism [54, 55]. In fact, in key metabolic organs such liver, muscle and adipose tissue, over half of the nhrs detected exhibit circadian expression, suggesting that changes in the expression of nhrs and their downstream target genes are likely to explain the cyclical behaviour of glucose and lipid metabolism . Recently, nhrs have been shown to exert control on nutritional homeostasis by regulating the expression of four different members of the fgf family [25, 56]. Fgfs are secreted heparan sulphate - binding proteins that act locally by signalling through fgf receptors and are typically involved in cell growth, angiogenesis and wound healing [57, 58]. In contrast, the endocrine fgfs (fgf 19, 21 and 23) have poor affinity for heparan sulphate and therefore circulate rather than being immobilised in the extracellular matrix . While the classical, paracrine fgfs require heparan sulphate for proper activation of fgf receptors, the endocrine fgfs require the or klotho co - receptors for signalling [56, 59]. The endocrine fgfs have received much attention for their role as metabolic regulators and are controlled by nhrs (fig . While fgf23, through its interaction with the vitamin d receptor, has been shown to be critical for controlling vitamin d metabolism, we will focus on the fxr fgf21 signalling pathways because of their relevance during feast and famine (fig . 3).fig . During the fed state ppar, which is activated by fatty acids, increases the expression of both fgf1 and fgf21 to promote adipose remodelling and adipocyte differentiation, respectively . During the fed state, bile acids activate fxr, which increases the expression of fgf15/19 to promote bile acid homeostasis . During the fasted state the fed state ppar, which is activated by fatty acids, increases the expression of both fgf1 and fgf21 to promote adipose remodelling and adipocyte differentiation, respectively . During the fed state, bile acids activate fxr, which increases the expression of fgf15/19 to promote bile acid homeostasis . During the fasted state, fatty acids activate ppar to control energy homeostasis fgf19 is a postprandial hormone, and the gene encoding this fgf is a direct bile acid - dependent target of fxr . The importance of fgf19 (fgf15 in rodents) in maintaining bile homeostasis has been demonstrated in both rodent studies and human clinical studies . In addition to its importance in bile acid metabolism, fgf19 has also been shown to lower blood glucose, hepatic triacylglycerol and cholesterol levels [60, 61] and induce protein and glycogen synthesis in the liver independently of insulin . However, unlike insulin, fgf19 does not activate the phosphoinositide 3-kinase (pi3k)akt signalling pathway but, rather, signals through the ras extracellular signal - related kinase (erk) pathway to promote protein and glycogen synthesis [62, 63]. Fgf19 also reduces hepatic gluconeogenesis by repressing the expression of the transcription cofactor ppar co - activator 1 (pgc1), and the pgc1 target genes, g6pc and pepck, also through a mechanism distinct from that of insulin . Unlike insulin, fgf19 does not increase lipogenesis and actually, as mentioned above, reduces hepatic triacylglycerol and cholesterol levels [60, 61]. While there are concerns regarding potential mitogenic effects [65, 66], its overlapping but unique functions with insulin make fgf19 an attractive target for the treatment of both type 1 and 2 diabetes . In addition to fgf19, fgf21 has emerged as a member of the endocrine fgfs that is regulated by an nhr to control metabolism . Initially identified as a factor induced by ppar in the liver in response to fasting, fgf21 is now recognised as a key player in the adaptive starvation response, helping to shift hepatic metabolism from carbohydrates to fatty acids and ketone bodies [67, 68]. Interestingly, fgf21 also has an important function in the fed state, where it is induced by ppar and works in a paracrine manner in adipose tissue to enhance adipogenesis [68, 69]. Fgf21 knockout mice exhibit impaired ppar signalling in adipose tissue, reduced adiposity and are resistant to the insulin - sensitising effects, as well as the undesirable side effects, of the tzds, which, as mentioned previously, are ppar agonists . Pharmacological administration of fgf21 reverses hepatic steatosis and improves glucose homeostasis in rodents and non - human primates [7072]. Recent studies have shown that fgf21 exerts its effects on glucose homeostasis through adiponectin, an adipokine known to enhance insulin sensitivity [73, 74]. It remains to be determined whether other metabolic effects of fgf21 are also mediated through adiponectin . In addition to its effects on peripheral metabolism, fgf21 was recently shown to act centrally to promote adaptations to fasting by increasing glucocorticoid levels, suppressing physical activity and altering circadian behaviour . Prevention of female reproduction is a critical adaptation to starvation, and fgf21 has been shown to exert an inhibitory effect on this process by acting on the scn in the hypothalamus . Future studies further addressing how the central and peripheral actions of fgf21 are coordinated to control metabolism will be an exciting new avenue of research [75, 76]. While the endocrine family of fgfs has received the most attention for its role in metabolism, a new role for the non - endocrine fgf1 in nutrient homeostasis has emerged . As part of a screen to identify genes that respond to feast and famine cues, fgf1 was found to be induced in wat in response to hfd and repressed during a fast, pointing to an unexpected metabolic function in both fed - state and fasted - state responses . Fgf1 was also shown to be induced in visceral adipose tissue in response to hfd or tzd treatment . Despite its implication in multiple physiological processes, fgf1 knockout mice exhibit no phenotype under standard laboratory conditions, which led to the long - held assumption that it was dispensable . However, when placed on an hfd, fgf1 knockout mice develop marked fibrosis in adipose tissue, structurally restricting adipose tissue expansion and resulting in a severe diabetic phenotype . Even more striking, upon withdrawal of the hfd the adipose tissue in these mice fails to properly contract, leading to severe necrosis of the adipose tissue . The induction of fgf1 expression in adipose tissue in the fed state is regulated at the genomic level by ppar, identifying a pparfgf1 signalling axis crucial for handling cycles of feast and famine . As global regulators of metabolism, nhrs play critical roles in key metabolic organs during feast and famine . Their importance in metabolism, combined with their ability to be modulated by small lipophilic ligands, make nhrs one of the most highly therapeutically targeted protein families, serving as targets for drugs including glucocorticoids, thyroid hormone, tamoxifen, fibrates and tzds . Although these nhr - targeted drugs are highly effective, there is the potential to improve on some of these therapies . For example, while highly effective for treating type 2 diabetes, tzds are accompanied by unwanted side effects, including fluid retention, weight gain, bone loss and congestive heart failure . New strategies are being developed for improving tzd - based therapies, one of which involves targeting downstream effectors of ppar, such as fgf21 or, potentially, fgf1 . In this regard, it will be important to dissect the relative roles of these two fgfs in adipose tissue, since they are both regulated in the fed state by ppar. Recently, a fgf21 analogue has been shown to improve dyslipidaemia in obese people, however only a trend toward glucose lowering was observed [78, 79]. Also, bone loss in these individuals was not examined, which will be important since fgf21 causes bone loss in rodents . While drugs are being developed for fgf21, it will be interesting to see what effect, if any, fgf1 has systemically . Also, the insulin - like properties of fgf19, combined with its ability to lower triacylglycerol and cholesterol levels and its nonlipogenic properties, make it an attractive therapeutic target of the treatment of both type 1 and 2 diabetes . While there are concerns about potential mitogenic effects of fgf19 [65, 66], therapies designed to dissociate its mitogenic effects from its metabolic ones might be possible . Additionally, targeting nhrs that affect circadian metabolism might be an interesting therapeutic avenue . In this regard, compounds that act as agonists for rev - erbs have been suggested to modulate the circadian clock and improve metabolic defects in mice [82, 83]. As our knowledge of nhr action in nutrient homeostasis continues to advance, these critical integrators of metabolism remain promising therapeutic targets for the treatment of metabolic disease.
Articular cartilage plays an integral role in the load - bearing ability of knee joints . In its healthy state, it provides excellent friction and lubrication to help sustain the rigorous dynamic loads that are placed on the knee joint during daily activities.1 typical articular cartilage consists of four major constituents: water, cells, proteoglycans, and collagen networks . Both proteoglycans and collagen are responsible for the load - bearing properties of the cartilage . The primary constituent of proteoglycans is aggrecan, while the majority of the collagen fibers are formed from type - ii collagen.2,3 cartilage extracellular matrix (ecm) metabolism is regulated by chondrocytes, which constitute only 5% of the total content of cartilage . Due to its avascular and aneural characteristics, the healing ability of cartilage is limited to such an extent that partial articular cartilage defects rarely or never heal spontaneously.4 this finding has prompted extensive worldwide research on cartilage transplantation with the goal of finding a sustainable solution . Most researchers opt to study cartilage in vitro to better understand the response of cartilage to various stimuli and to eliminate unrelated disturbances that may occur in vivo . There are various models for cartilage culture, including monolayer culture,5 - 8 micromass / pellet culture,9,10 scaffold culture,11,12 and explant culture.7,10, monolayer culture is a two - dimensional culture of chondrocytes on the flat surface of a plastic dish or culture flask . Micromass / pellet and scaffold cultures are three - dimensional (3d) culture systems that provide the cells with a suitable 3d environment to secrete and build a new ecm from scratch . Although 3d culture is clearly a better model than monolayer culture, it has been argued that chondrocytes in the newly - built matrix may act differently from those in naturally occurring ecm;8 therefore, on this basis, explant culture is advantageous . Through the direct culture of a small piece of tissue harvested directly from the host, explant culture provides a more controlled and physiologically relevant model, with both the morphological and biosynthetic characteristics of the chondrocytes remaining intact additionally, explant culture minimizes the complicated systemic and environmental parameters found in vivo.14,16 thus, explant culture is generally regarded as the superior model for the study of cartilage metabolism . Regardless of the various advantages provided by cartilage explant culture, the dynamics of cartilage metabolism after extraction from the host are still poorly understood . Admittedly, the vigorous extraction that is necessary to obtain a regularly sized cartilage explant places the adjacent cartilage under immense mechanical trauma . Thus, the primary focus of this study was to investigate the response of cartilage to the trauma sustained following explantation and to determine the time needed for stabilization . The latter consideration is particularly imperative, as the effects of the various stimuli applied to the cartilage explant can be accurately assessed only if the explant is stable . Previous research has shed some light on the dynamics of cartilage explant culture; however, our understanding of culture dynamics is incomplete and has been obstructed by differences in the anatomical location of the tissue harvests, the extraction method, the culture conditions, and the age of the tissue used in various studies.15 the aim of this study was to investigate the response of cartilage to the trauma sustained during explant extraction by observing biochemical changes (i.e., cell number, proteoglycan content, and collagen content following excision and culture of full - thickness explants with an intact subchondral bone layer). Furthermore, we aimed to determine whether chondrocytes can withstand the vigorous extraction process that is necessary for preparing an explant culture . Bovine metacarpal - phalangeal joints with intact synovial membranes from skeletally mature adult cows were obtained from a local abattoir . The joints were opened aseptically and sawn laterally at the joint end to extract two large bone - cartilage samples . These bone - cartilage samples were further processed in the laminar flow hood to remove most of the bone, leaving only 2 mm of the bone with the cartilage on top . The sawing process was accompanied by constantly irrigating the exposed cartilage surface with sterile phosphate - buffered saline (pbs) solution at 4c . Approximately twenty 6-mm - diameter cartilage disks were then extracted from the processed bone - cartilage samples using an in - house - built hollow punch . All of the explants were washed once with 70% ethanol for 30 seconds, once with sterile pbs containing antibiotics and three times with sterile pbs, with the exception of one group, which was washed with sterile pbs to assess the effect of washing with ethanol on cell viability . All of the disks were transferred to six - well techno - plastic - product (tpp) plates (trans - techno enterprise, malaysia), with four explants in each well . The explants were immobilized using 2% agar (fisher scientific, malaysia) and cultured in 5 ml / well of dulbecco's modified eagle's medium (d5921, sigma aldrich, malaysia) supplemented with 20% (v / v) fetal bovine serum (f9665, sigma aldrich, malaysia), 16 mm hepes (h0887, sigma aldrich, malaysia), 1.6 mm l - glutamine (g7513, sigma aldrich malaysia), 160 u / ml penicillin-160 g / ml streptomycin (p4333, sigma aldrich, malaysia) and 0.68 mm l - ascorbate (ducheta biochemie, malaysia).17 the explants were cultured at 37c under an environment of 95% air and 5% co2 for up to 17 days, and the medium was changed every two days . The explants in the first group were washed three times with sterile pbs, while those in second group were washed once with 70% ethanol, once with pbs containing antibiotics and three times with sterile pbs . The cell viability of the explants in each group were tested using trypan blue exclusion . The explants were harvested at days 0, 2, 10, and 17 of culture and washed with pbs . The samples were then digested with papain (p3125, sigma aldrich, malaysia) for 18 - 24 hours at 60c.18 next, the total dna content (cell number) of the digests was analyzed using a fluorometric hoechst 33258 assay.19 the remaining digests were stored at -80c for future analysis and analyzed colorimetrically to measure chondroitin-6-sulfate (c6s) and hydroxyproline content . C6s content was assayed using the dimethylmethylene blue (dmb) assay.20 the digests were acid - hydrolyzed for 18 hours at 110c before having their hydroxyproline content analyzed.21,22 all of the analytical data were normalized to the cartilage wet weight . The results obtained were expressed as the mean the standard error of the mean . The means of two or more different groups were compared using one - way between - groups analysis of variance (anova) and tukey's honestly significant difference (hsd) test, which were calculated using statistical package for social sciences (spss). The effect of the washing method on the cell viability of the explants was revealed by the significantly higher (90.331.61%) mean cell viability of the explants in the first group (washed three times with sterile pbs; n = 8) compared to those in the second group (washed once with 70% ethanol, once with sterile pbs containing antibiotics, and three times with sterile pbs; n = 8), which had a cell viability of 83.561.62% (p0.01). The mean wet weight of the cartilage explants (figure 1a) remained stable at 200.7 mg throughout the culture period . The mean cell density (figure 1b) was significantly lower at day 0 than during the remainder of the experiment (p0.05), with a value of 36.701.57 (* 10 cells / mg wet weight of cartilage). However, the cell density stabilized after day 2, with an average of 54.362.35 (10 cells / mg wet weight of cartilage). The mean c6s weight (figure 2a), which was normalized to the wet weight of cartilage, gradually decreased between day 0 (0.0360.002 g c6s / mg wet weight of cartilage tissue, hereafter abbreviated as w / w) and day 17 (0.0280.001 [w / w]) (p0.05). The mean c6s weight was 0.0330.002 (w / w) on day 2; on day 10, the weight was 0.0310.001 (w / w), which was also significantly lower than the value on day 0 . Similarly, the mean c6s weight normalized to the weight of dna (figure 2b) was found to be significantly higher (p0.05) on day 0 (0.1280.009 w / w) when compared to the values at the later time points . The values were relatively stable for these groups, with an average of 0.0760.004 (w / w). In contrast, the mean hydroxyproline weight, which was normalized to the cartilage wet weight (figure 3a), remained stable from day 0 to day 10, with an average value of 0.0260.001 (w / w). However, this value dropped significantly (p0.05) to 0.0180.0004 (w / w) on day 17 . The mean hydroxyproline weight normalized to the dna weight (figure 3b) was significantly higher (0.0960.005 w / w) on day 0 (p0.05) than at the later time points . The value stabilized at 0.0630.003 (w / w) between days 2 and 10 but significantly decreased to 0.0460.002 (w / w) on day 17 (p0.05). A cell viability test was performed to verify that washing the explants with 70% ethanol would not have a negative effect on cell viability . We found that, although washing with 70% ethanol did significantly decrease cell viability, the viability remained at a high level of 80% . The suitability of this treatment was further demonstrated by the fact that the chondrocytes responded with an increase in cell density after two days of culture . These observations suggest that only the chondrocytes in the superficial layers were killed by the ethanol, while the others remained unaffected . This tradeoff is worthwhile, as most of the explants that were not washed with 70% ethanol became contaminated after two days of culture and had to be discarded . The cartilage tissues were co - cultured with the underlying subchondral bone to avoid further damage to the cartilage tissue (especially the deep - zone tissue), which could occur while cutting the cartilage tissue out of the bone layer . Furthermore, previous studies23 - 25 have shown that co - culturing cartilage with osteochondral explants does not significantly affect the metabolism of the cartilage, despite the potential for the release of chemical factors, such as cytokines and/or free radicals . The fluorometric assay measured the dna content of only the live cells, as most of the dna from the dead cells was lost during dna extraction and discarded along with the cell debris pellet.26 we found that the cell density was the lowest on day 0, with a value that was 32% lower than the densities observed at the later time points . Due to the density of the extracellular matrix, it is unlikely that the increase in cell density after day 0 was due to cell proliferation . Instead, it is more likely that the explants studied at day 0 experienced more cell death than the explants that were given sufficient time to equilibrate . These cartilage explants were all washed once with 70% ethanol, which might have affected the chondrocytes in the superficial zone; if these cells had been given time to recover, they may have been able to survive . However, the explants on day 0 were digested immediately after the 70% ethanol wash . Therefore, those weakened cells from the superficial zone might have been unable to withstand the vigorous digestion and died, resulting in much lower cell viability . Consistent with this hypothesis, the cell density remained relatively stable after day 2, and there was no dna accumulation throughout the culturing process, indicating that no cell outgrowth occurred.27 the chondroitin-6-sulfate (c6s) content of the explants was found to be equivalent to the proteoglycan content of healthy cartilage . It was noted that, when the c6s content was normalized to the cartilage wet weight, the value exhibited a gradual decrease from day 0 . This observation is sensible if one considers that the collagen networks at the periphery of the explants were largely distorted during explant extraction . Therefore, it was inevitable that some of the proteoglycan would leak into the bathing culture medium . Although significant differences in the proteoglycan content were observed between day 0 and day 10 and between day 0 and day 17, the proteoglycan content become relatively stable after day 2, with no significant differences recorded after that time point . Additionally, when the c6s content was normalized to the dna weight, we found that the proteoglycan content remained relatively stable after day 2 . It can be argued that the high value at day 0 was due to the low cell density; furthermore, the proteoglycans bound for leakage were not given sufficient time to diffuse out of the cartilage by this assay point . Regarding the hydroxyproline weight / cartilage wet weight (w / w), we noted that this value remained very stable from day 0 to day 10 in full - thickness cartilage . This finding agrees with previous findings that the turnover rate for collagen is the slowest of all of the cartilage components.16 however, we observed an abrupt 30% drop in collagen content at day 17, suggesting that the collagens were slowly degraded after 10 days of culture . Because a partial or total loss of proteoglycan always precedes collagen degradation in diseased cartilage,28 - 30 the cartilage studied here was not developing into diseased cartilage, as the proteoglycan content remained relatively stable through day 17 . However, because the osteochondral explants were cultured for a prolonged period without any stimulation, it is possible that the unstimulated chondrocytes failed to maintain their normal metabolism, which led to gradual collagen degradation.16 consistent with this hypothesis, it is generally accepted that cartilage metabolism changes according to the direct mechanical loading exerted by an individual's daily activities . It would be interesting to determine if the cartilage explants would eventually develop into diseased cartilage after a culture period of more than one month and with no stimulation . According to a study by hoemann,18 the glycosaminoglycan (gag), collagen and dna contents in 1 mg of wet mass of typical articular cartilage samples are approximately 50 g, 150 g and 0.2 - 0.6 g, respectively . These values are comparable to the results obtained in this study, in which contents of 31 g of gag, 198 g of type - ii collagen (a conversion factor of 7.6 for extrapolating hydroxyproline content to type - ii collagen type ii31), and 0.42 g of dna were observed during the stable culture period . However, it should be noted that this study was performed in vitro, and the in vivo reproducibility of the biochemical content of cartilage tissue has not been verified . The results of this study were further compared to those of previous studies by dumont et al.14 and brighton et al.13 the explants used in the study by dumont et al . Were similar to the explants used in this study in the sense that both were cartilage - bone explants . Cultured eight small pieces of full - thickness cartilage derived from 8-mm - diameter samples, and those eight pieces were taken as equivalent to one explant . The results of the biochemical analyses performed in these previous studies differ slightly from those of the current study . Part of this difference could be attributed to the various anatomical harvest positions (the shoulder joint for dumont et al . And reported that 4 days were required for the gag content of their cartilage explants to stabilize at 22.5 g / ml after dropping from an initial value of 31 g / ml, while the cartilage explants in this study only needed two days to stabilize . The higher gag value found in this study (31 g / ml) may be due to the addition of serum.7 the average number of chondrocytes in 1 mg of cartilage after equilibration was found to be 38,300 cells / mg of cartilage, which is about 70% of the value found in this study (54,360 cells / mg of cartilage). Again, this difference could be due to the addition of serum in our study . However, the type - ii collagen content was similar in both studies (187 g / ml in the study by dumont et al . And found the type - ii collagen content to be constant for durations of up to 21 days, while, in this study, it decreased significantly on day 17 . Although brighton et al . Also studied the effect of electrical stimulation on cartilage explants, only the results of their control set were used for comparison . The authors found that their cartilage explants were relatively stable from day 0 of culture, and washing the explants with 70% ethanol did not appear to affect the cell number (reflected by the dna weight). They reported a proteoglycan content normalized to dna weight (w / w) of 65 on day 0, which was followed by an insignificant increase to 70 on day 3 and to 75 on day 10 before dropping back to 65 on day 17 . In this study, we observed that the gag content remained relatively stable, reaching an average of 76 after two days of equilibration . The results that brighton et al . Found for the hydroxyproline weight normalized to the dna weight (w / w) also showed a stable value, with an average of 46 from day 0 to day 2 . However, their collagen content increased to 65 on day 10 and decreased back to 51 on day 17 . A decrease in collagen content on day 17 was also observed in this study . Aside from the high collagen content normalized to the dna weight (w / w) found on day 0 (caused by the low cell density on day 0), the collagen content was quite stable in this study, remaining at a value of 63 prior to its abrupt decrease to 46 on day 17 . Based on the results of this study, we conclude that healthy cartilage responds well to the blunt trauma (exerted during fixation by the clamp) and the abrasive, thermal, and lacerative trauma (exerted during cutting with the saw and hollow punch) sustained by the adjacent cartilage during explantation . After the two - day equilibration period, the cartilage remained stable for up to 10 days of culture . Serum - supplemented culture medium, despite its potential for adverse effects (cell outgrowth, which did not occur in this study),27 did help the cartilage respond positively to the sustained trauma . The degradation of collagen on day 17, albeit not to diseased levels, showed that explanted cartilage could potentially develop into degenerated cartilage . Healthy cartilage metabolism was assessed by studying changes in gag, collagen, and dna content over 17 days of culture . In general, cartilage metabolism was found to be relatively stable for a culture period of up to 10 days if the explant was given two days to equilibrate . However, the collagen fibers in the cartilage begin to degrade if the culture period is longer than ten days, which means that the cartilage could potentially develop into degenerated cartilage . It is apparent that tissue stability and remodeling dynamics are complex and influenced by many parameters . In this study, we sought to report the dynamics of cartilage metabolism in the context of a generalized culture process; future studies should aim to elucidate the factors that affect the long - term culture characteristics of bovine articular cartilage . We would like to thank vincent murphy for his invaluable comments and adhli iskandar putera bin hamzah for his efforts in procuring the bovine feet . This research was funded by the institute of research management and monitoring, university of malaya, malaysia.
This is a retrospective cross - sectional study . There were 4,377 participants, all of whom underwent cardiac ct in a health promotion center at gangnam severance hospital (seoul, korea) between january 2008 and february 2013 . From 4,377 participants, we excluded men (n = 2,584) and premenopausal women (n = 748). Postmenopausal women 50 years or older with no menstrual periods for more than 12 consecutive months and women with elevated follicle - stimulating hormone levels (> 30 iu / l) were included in this study (n = 1,045). Women with diabetes mellitus (n = 59), excessive alcohol consumption (n = 7), viral hepatitis (positive results for hepatitis b surface antigen or anti hepatitis c virus; n = 19), liver cirrhosis or malignancy on ultrasonography (n = 6), and self - reported or medically verified history of cvd (n = 35) were excluded from this study . Height and weight were measured, and body mass index (bmi) was calculated by dividing weight (kg) by the square of height (m). Lifestyle, personal medical history of acute and chronic diseases, and medication history were assessed using a standard questionnaire . Systolic blood pressure (sbp) and diastolic blood pressure (dbp) were measured after a 5-minute rest . Fasting plasma glucose (fpg), aspartate aminotransferase, alanine aminotransferase, total cholesterol (tc), high - density lipoprotein cholesterol (hdl - c), triglycerides (tg), and c - reactive protein (crp) levels were determined by enzymatic methods using a hitachi 7600 - 120 automated chemistry analyzer (hitachi, tokyo, japan). Low - density lipoprotein cholesterol (ldl - c) was calculated according to the friedewald equation . Hepatitis b surface antigen levels and antibodies to hepatitis c virus were measured with a roche e-170 device (roche diagnostics, mannheim, germany). Fasting serum insulin was determined by chemiluminescence (radioimmunoassay kit; daiichi, kyoto, japan), and insulin resistance was estimated using the homeostasis model assessment of insulin resistance (homa - ir) index calculated from the following formula: homa - ir = fasting insulin (u / ml) fpg (mmol / l) / 22.5 . Diagnosis of fatty liver disease was based on an abdominal ultrasonography scan obtained with a 3.5-mhz transducer (hdi 5000; philips, bothell, wa). Abdominal ultrasonographic examination was performed by one of three experienced radiologists, all of whom were blinded to the laboratory and clinical details of participants at the time of the procedure . In the present study, liver with any degree of fat accumulation the number of participants with severe fatty liver disease was too small (n = 29); thus, the presence and severity of nafld were classified into three groups according to hyperechogenicity of liver tissue, degree of discrepancy between the liver and the right kidney, and visibility of vascular structures . The three groups were as follows: group i, no fatty liver disease; group ii, mild fatty liver disease; group iii, moderate to severe fatty liver disease . Cac was determined with a multidetector ct scanner (philips brilliance 64; philips medical system, best, the netherlands). We used a standard prospective electrocardiogram gating protocol with a step - and - shoot technique . The scanning protocol included a slice section collimation of 64 0.625 mm, rotation time of 420 milliseconds, tube voltage of 120 kv, and tube current of 210 mas . Quantitative cac score (cacs) was calculated with dedicated software and expressed as agatston score . Continuous variables with normal distribution were expressed as mean (sd), whereas continuous variables with skewed distribution were presented as median (interquartile range) and log - transformed for analysis . Logistic regression analysis was used to analyze the association between nafld and presence of cac while controlling for potential confounders . Covariates in the multivariable model, such as age, bmi, sbp, dbp, fpg, tc, ldl - c, tg, hdl - c, crp, and homa - ir, were chosen for clinical importance and statistical significance . Spss statistical package version 20.0 (spss inc, chicago, il) was used for all statistical analyses . This is a retrospective cross - sectional study . There were 4,377 participants, all of whom underwent cardiac ct in a health promotion center at gangnam severance hospital (seoul, korea) between january 2008 and february 2013 . From 4,377 participants, we excluded men (n = 2,584) and premenopausal women (n = 748). Postmenopausal women 50 years or older with no menstrual periods for more than 12 consecutive months and women with elevated follicle - stimulating hormone levels (> 30 iu / l) were included in this study (n = 1,045). Women with diabetes mellitus (n = 59), excessive alcohol consumption (n = 7), viral hepatitis (positive results for hepatitis b surface antigen or anti hepatitis c virus; n = 19), liver cirrhosis or malignancy on ultrasonography (n = 6), and self - reported or medically verified history of cvd (n = 35) were excluded from this study . Height and weight were measured, and body mass index (bmi) was calculated by dividing weight (kg) by the square of height (m). Lifestyle, personal medical history of acute and chronic diseases, and medication history were assessed using a standard questionnaire . Systolic blood pressure (sbp) and diastolic blood pressure (dbp) were measured after a 5-minute rest . Fasting plasma glucose (fpg), aspartate aminotransferase, alanine aminotransferase, total cholesterol (tc), high - density lipoprotein cholesterol (hdl - c), triglycerides (tg), and c - reactive protein (crp) levels were determined by enzymatic methods using a hitachi 7600 - 120 automated chemistry analyzer (hitachi, tokyo, japan). Low - density lipoprotein cholesterol (ldl - c) hepatitis b surface antigen levels and antibodies to hepatitis c virus were measured with a roche e-170 device (roche diagnostics, mannheim, germany). Fasting serum insulin was determined by chemiluminescence (radioimmunoassay kit; daiichi, kyoto, japan), and insulin resistance was estimated using the homeostasis model assessment of insulin resistance (homa - ir) index calculated from the following formula: homa - ir = fasting insulin (u / ml) fpg (mmol / l) / 22.5 . Diagnosis of fatty liver disease was based on an abdominal ultrasonography scan obtained with a 3.5-mhz transducer (hdi 5000; philips, bothell, wa). Abdominal ultrasonographic examination was performed by one of three experienced radiologists, all of whom were blinded to the laboratory and clinical details of participants at the time of the procedure . In the present study, liver with any degree of fat accumulation was considered nafld . The number of participants with severe fatty liver disease was too small (n = 29); thus, the presence and severity of nafld were classified into three groups according to hyperechogenicity of liver tissue, degree of discrepancy between the liver and the right kidney, and visibility of vascular structures . The three groups were as follows: group i, no fatty liver disease; group ii, mild fatty liver disease; group iii, moderate to severe fatty liver disease . Cac was determined with a multidetector ct scanner (philips brilliance 64; philips medical system, best, the netherlands). We used a standard prospective electrocardiogram gating protocol with a step - and - shoot technique . The scanning protocol included a slice section collimation of 64 0.625 mm, rotation time of 420 milliseconds, tube voltage of 120 kv, and tube current of 210 mas . Quantitative cac score (cacs) was calculated with dedicated software and expressed as agatston score . Continuous variables with normal distribution were expressed as mean (sd), whereas continuous variables with skewed distribution were presented as median (interquartile range) and log - transformed for analysis . Logistic regression analysis was used to analyze the association between nafld and presence of cac while controlling for potential confounders . Covariates in the multivariable model, such as age, bmi, sbp, dbp, fpg, tc, ldl - c, tg, hdl - c, crp, and homa - ir, were chosen for clinical importance and statistical significance . Spss statistical package version 20.0 (spss inc, chicago, il) was used for all statistical analyses . A total of 919 postmenopausal women were included in this study . Of these, 738 women had no cac (cacs = 0), whereas the remaining 181 women exhibited some evidence of cac (cacs> 0; 19.7%). Among women in this study, 294 women (31.9%) women were stratified into three groups by the presence and severity of fatty liver disease . Age, bmi, sbp, dbp, fpg, tg, aspartate aminotransferase, alanine aminotransferase, crp, and homa - ir increased according to the severity of fatty liver disease, whereas hdl - c decreased . The prevalence of cac and the mean cac score significantly increased with the severity of fatty liver disease (table 2). The association between fatty liver disease and presence of cac (cacs> 0) was further explored by categorizing the severity of nafld into three groups and by using the first group as reference (table 3). These relationships remained significant even after adjusting for well - established cardiovascular risk factors, including age, bmi, sbp, dbp, fpg, tc, ldl - c, tg, hdl - c, and crp (model 2). However, this association was attenuated after additional adjustment for insulin resistance (model 3). In this study, we investigated the relationship between nafld and prevalence of cac in postmenopausal women . We found that the prevalence of cac was significantly associated with nafld after adjustment for conventional cardiovascular risk factors, but this association was attenuated after adjustment for insulin resistance . To the best of our knowledge, this is the first study to investigate the correlation between nafld and presence of early coronary artery atherosclerosis in postmenopausal women . In this study, this prevalence was higher than that in premenopausal women (19.7%), which was determined by analyzing the subgroup of premenopausal women among 4,377 participants . Hamaguchi et al reported that the prevalence of nafld in japanese postmenopausal women was higher than that in premenopausal women and concluded that postmenopause was a risk factor for nafld . Although it remains unclear how estrogen deficiency increases the prevalence of nafld after menopause, several reports have suggested that estrogen deficiency affects body fat distribution, which increases accumulation of gluteofemoral fat and central fat . Hepatic estrogen receptors mediate estrogen action in the liver, and estradiol has been shown to play a favorable role in chronic liver disease . Thus, estrogen seems to exert a protective effect against nafld in women . In this study, we showed that most metabolic risk factors increased or decreased with the severity of nafld . Cac has been shown to be an independent predictor of future cardiovascular events . Moreover, several recent studies have reported the association between nafld and cac, but results have been inconsistent . As part of the diabetes heart study, mckimmie et al reported that coronary arterial calcium did not correlate with hepatic steatosis in women with type 2 diabetes . Another study of 398 white and black participants by ding et al did not find associations of liver fat attenuation with calcified coronary plaque . Recently, vanwagner et al showed that obesity attenuates the relationship between nafld and cac . In contrast, some studies have suggested that nafld might be an independent risk factor for cac, after accounting for conventional risk factors . Only a few studies have investigated the association between nafld and carotid intima - media thickness, which is another marker for subclinical atherosclerosis, after adjusting for insulin resistance and other traditional risk factors . However, whether nafld is an independent risk factor for increased intima - media thickness remains controversial . In the present study, similar to a previous study of healthy women, we found that the presence of cac increased with the severity of nafld in postmenopausal women . However, this association was attenuated after adjusting for insulin resistance, and insulin resistance was independently associated with cac in these adjusted models . This suggests that insulin resistance may act as a mediator of the relationship between nafld and cac . Although the pathogenesis of nafld and cac has not been fully elucidated, there are several explanations for the relationship between nafld and cac . One of the accepted hypotheses implicates insulin resistance as a major factor leading to nafld and cac . Menopause affects body fat distribution, and the resultant increases in body weight and body fat lead to insulin resistance, which might play an important role in the pathogenesis of clinical nafld and cac . Recently, rodrigues et al reported that insulin resistance, metabolic syndrome, and abdominal obesity were risk markers for the development of nafld in postmenopausal women . Therefore, although the mechanism underlying the relationship between nafld and cac in postmenopausal women is not completely understood, insulin resistance might provide a link between them . First, this is a cross - sectional study that cannot definitively establish causality . Second, this study lacked direct measures of body fat, which affects insulin resistance . Fatty liver disease was assessed by liver ultrasonography a technique that cannot detect fatty infiltration below 30% . However, ultrasonography is nonetheless a very useful noninvasive technique that is often used as the first - line imaging technique in clinical practice and epidemiological studies . Finally, because participants in the current study were enrolled in the same health promotion center, generalizability may be limited . Increased severity of nafld is significantly associated with cac, a marker of subclinical atherosclerosis . However, this association is attenuated after additional adjustment for insulin resistance; thus, nafld is not an independent factor for coronary atherosclerosis in postmenopausal women . Further prospective large - scale studies are required to explore these findings and to elucidate the mechanism for this association.
Macrolide, lincosamide and streptogramin b (mlsb) antibiotics are structurally different with a same mechanism of action . These drugs, especially clindamycin, are alternative drugs for some staphylococcus aureus infections such as skin and soft tissue infections particularly in penicillin - allergic patients (1, 2). Clindamycin resistance in s. aureus strains may be either constitutive (cmlsb) or inducible (imlsb). Relapse of infection in a patient with s. aureus endocarditis was reported in 1976 (3). Detection of isolates with inducible clindamycin resistance is a problematic issue for the routine diagnosis, because they are erythromycin resistant and clindamycin susceptible in vitro and in the condition that they are not place adjacent to each other . In these cases, in vivo treatment with clindamycin may lead to clinical therapeutic failures (4, 5). The aim of this study was to determine the incidence of inducible clindamycin resistance in s. aureus isolates collected from clinical specimens in this geographic area . Totally 162 s. aureus isolates were collected from clinical specimens of patients admitted to three university hospitals with 462, 420 and 300 bed capacities in kerman, south - east of iran, from march 2011 to february 2012 . Confirmation of s. aureus isolates and detection of methicillin resistant s. aureus (mrsa) were performed by phenotypic and genotypic methods as previously described (6). Only one isolate per patient this test was performed by placing an erythromycin (15 g) disk (mast, group ltd ., merseyside, uk) at a distance of 15 to 20 mm from clindamycin (2 g) disk (mast, group ltd ., merseyside, uk) on a mueller - hinton agar plate inoculated with 0.5 mcfarland standard bacterial suspensions . Following overnight incubation at 37c, flattening of the clindamycin zone adjacent to the erythromycin disk (d shaped) indicated inducible clindamycin resistance (7). From 162 s. aureus isolates tested for determination of inducible clindamycin resistance, 70 (43.2%) were mssa and 92 (56.8%) isolates were mrsa (table 1). Sensitivity to both erythromycin and clindamycin was significantly higher in mrsa compared to mssa isolates . Resistance to methicillin, erythromycin and clindamycin was observed in 92 (56.8%), 75 (46.29%) and 46 (28.4%) of the isolates, respectively . Inducible resistance to clindamycin was determined in 14 (8.64%) isolates (d - test positive, figure 1). Table 2 shows the distribution of d - test positive s. aureus isolates in clinical specimens . Among those, 11 (78.57%) abbreviations: mssa, methicillin susceptible s. aureus; mrsa, methicillin resistant s. aureus; ery, erythromycin; cli, clindamycin; s, sensitive, r, resistant; cmlsb, constitutive macrolide lincosamide and streptogramin b; imlsb, inducible macrolide lincosamide and streptogramin b. data are presented as no . (%). Flattening of the clindamycin zone adjacent to the erythromycin disk produces a d shape . (%). Including: blood, skin and soft tissue, cerebrospinal fluid . Clindamycin is an effective antibiotic agent used in the treatment of skin and soft - tissue infections caused by s. aureus isolates . This antibiotic has good tissue penetration, potential antitoxin effects, and also accumulates in abscesses (8). However, resistance to clindamycin may develop in s. aureus isolates with inducible phenotype and these isolates have a high rate of spontaneous mutation which would enable these isolates to develop constitutive resistance to clindamycin during therapeutic process (4). A positive d - test indicates the existence of inducible resistance to clindamycin . In this study 8.64% of 162 s. aureus isolates were d - test positive that is comparable with the findings of the studies by rahbar and hajia (9.7%) (9) and sedighi et al . The prevalence of inducible and constitutive clindamycin resistance differs in different geographic areas (12). In our study high number (46.29%) of s. aureus isolates were resistant to erythromycin . Among those, 14 (18.66%) isolates had positive and 15 (20%) had negative d - test results and 46 (61.33%) were constitutive clindamycin resistance . In this study the prevalence of inducible and constitutive resistance to clindamycin in mrsa (11.95% and 47.8% respectively) is higher than those in mssa (4.28% and 2.85%, respectively). The results of this study revealed that inducible resistance to clindamycin in s. aureus isolates is relatively high in this region . Therefore, d - test should be performed to prevent treatment failures of s. aureus infections caused by the agents that are resistant to erythromycin and sensitive to clindamycin.
Inflammation is a crucial physiological response for the maintenance of tissue homeostasis, protecting the host against invading microorganisms, foreign substances, or host self - disturbers, such as the molecules derived from damaged cells . After the host has been incited, important microcirculatory events occur in response to local release of proinflammatory mediators, such as histamine, prostaglandins, leukotrienes, cytokines, and chemokines, leading to higher vascular permeability and increased leukocyte recruitment . Leukocytes, such as neutrophils and macrophages, play a key role in inflammatory response, by releasing further inflammatory mediators and acting as effector cells and phagocytes to remove the inflammatory agent / stimuli . Despite the important roles of neutrophils for effective host defense, these cells can also cause tissue damage requiring appropriate regulation [3, 4]. Continuous inflammatory stimuli can lead to aggressive and/or prolonged inflammatory responses, which may be detrimental to the host, leading to chronic inflammation . The efficient removal of the inciting agent by phagocytes is the first signal for triggering proper resolution, through inhibition of proinflammatory mediators production and activation of their catabolism, resulting in the ceasing of further leukocyte recruitment . After that, proresolving pathways are activated in order to restore tissue structure, function, and homeostasis [7, 8]. In this context, anti - inflammatory and proresolving molecules such as specialized lipid mediators (lipoxin a4, resolvins, maresins, and protectins), peptides / proteins (melanocortins, galectins, and annexin a1), and several other substances of different natures are released at the site of inflammation [7, 9, 10]. These endogenous mediators are known for their ability to decrease endothelial activation, reduce leukocyte infiltration, and activate neutrophil apoptosis, which ensures their secure removal by scavenger macrophages through a process called efferocytosis (phagocytosis of apoptotic cells). Annexin a1 (anxa1) is an important glucocorticoid- (gc-) regulated protein, which contributes to the resolution of inflammation through various ways (figure 1). Moreover, anxa1 acts by inducing neutrophil apoptosis, modulating monocyte recruitment, and enhancing the clearance of apoptotic cells by macrophages . Emerging evidence suggests that anxa1 also induces macrophage reprogramming toward a resolving phenotype, another key event to restore tissue homeostasis . In this review, we summarize several physiological and potential therapeutic actions of anxa1 on inflammation resolution . In particular, this review highlights recent advances on the actions of this endogenous mediator and its potential clinical utility . Endogenous mediators of inflammation, such as anxa1, are potential therapeutic tools to control inflammatory diseases . Although whether clinical use of proresolving strategies will be useful for treating inflammatory maladies or will show significant undesirable effects remains to be elucidated, it is believed these will be effective and have fewer side effects due to their ability to mimic or induce natural pathways of the resolution phase of inflammation [8, 12]. Annexin superfamily is composed of 13 members, grouped in view of their unique ca - binding - site architecture, which enables them to peripherally attach to negatively charged membrane surfaces [1315]. Anxa1, also known as annexin i or lipocortin i, was originally identified as a gc - induced protein active on phospholipase- (pl-) a2 inhibition and prevention of eicosanoid synthesis [1618]. It was subsequently recognized as an endogenous modulator of the inflammatory response, through several studies, mainly those led by dr . This 37 kda protein consists in a homologous core region of 310 amino acid residues, representing almost 90% of the structure, attached to a unique n - terminal region . In addition to mediating membrane binding, ca ions can also induce a conformational change that leads to the exposure of the bioactive n - terminal domain [15, 21, 22]. In fact, studies on the anti - inflammatory activity of anxa1 revealed not only that the different functions of the protein lie within the unique n - terminus, but also that synthetic peptides from the n - terminal domain may mimic the pharmacological property of the whole protein, specifically binding to formyl peptide receptors (fprs). In inflammatory conditions intact anxa1 (37 kda) can be cleaved by proteinase-3 and neutrophil elastase generating the 33 kda cleaved isoform, which is believed to be inactive, and peptides derived from the anxa1 n - terminus [2325]. The main cleavage sites on anxa1 are located at a, v, and v, as identified by cleavage assays coupled to mass spectrometric analyses . Investigation of the anxa1250 peptide revealed a novel cleavage site at position 25, probably unmasked due to the simpler conformation of the peptide, compared with the full - length anxa1 . In fact, the substitution of the mentioned cleavage sites allowed the generation of metabolically stable forms of anxa1 and its peptide, respectively, named superanxa1 (sanxa1) and cleavage - resistant anxa1250 (cr - anxa1250). The proinflammatory nature of anxa1 cleavage products is supported by reports of increased levels of the 33 kda fragment in human and animal inflammatory samples, including bronchoalveolar lavage fluids [2830] and exudates [11, 25, 31, 32]. For instance, using a model of acute pleurisy, our research group has shown increased levels of the 33 kda breakdown product of anxa1 during the time points of high neutrophil infiltration into the pleural cavity followed by regain of the intact form during the resolving phase of the pleurisy . However, what the biological functions of this and other anxa1-generated peptides are is still unclear, and this matter deserves further investigation . Evidence for physiological function of anxa1 in modulating inflammation emerged from studies involving anxa1-null mice and anxa1 neutralization strategies . Anxa1-null mice are viable and have a normal phenotype until they are challenged with inflammatory stimuli when they show stronger and more prolonged inflammatory reaction when compared to the wild - type (wt) [3340]. Resistance to glucocorticoid treatment and aberrant inflammation in anxa1-deficient mice provided initial evidence for the physiological relevance of the protein . In the absence of anxa1, the inflammatory response is exacerbated as demonstrated by increased neutrophil extravasation following zymosan - induced peritonitis and endotoxin - induced uveitis . In addition, animals lacking this protein exhibited exacerbated arthritis severity and allergic response in ovalbumin - induced conjunctivitis . Anxa1 ko mice also showed increased atherosclerotic lesion size with an overall increase in lesional macrophages and neutrophils . Moreover, our research group has shown the prevention of spontaneous and dexamethasone - driven resolution of inflammation by using an anxa1 neutralizing strategy . Aside from the physiological role of the endogenous protein, pharmacological treatment with both human recombinant anxa1 and its n - terminal peptides exerts anti - inflammatory and proresolving effects in a variety of experimental models, highlighting their therapeutic potential for inflammation resolution [11, 26, 27, 41] and wound repair . Anxa1 exerts many of its anti - inflammatory and proresolving actions through the formyl peptide receptor type 2/lipoxin a4 receptor (fpr2/alx). This receptor, along with fpr1 and fpr3, composes a family of seven - transmembrane domain g protein - coupled receptors which share significant sequence homology . Fpr2/alx receptor is shared by a variety of other peptide / protein and lipid ligands, mediating diverse biological functions of relevance for host defence and inflammation . Interestingly, fpr2/alx agonists are associated with both proinflammatory (e.g., serum amyloid a and cathelicidin) and proresolving (e.g., anxa1 and lxa4) signalling pathways [43, 44]. However, how fpr2/alx can promote both inflammatory response and limit its duration and intensity still remains to be fully elucidated . It is noteworthy that distinct fpr2/alx domains are required for signalling by different agonists . Using fpr2/alx transfected cells and chimeric fpr1 and fpr2 clones, bena and col . (2012) identified that while anxa1-mediated signalling involves the n - terminal region and extracellular loop ii of fpr2/alx, saa interacts with the extracellular loops i and ii of the same receptor . Otherwise, lxa4 has been shown to activate fpr2/alx by interacting with extracellular loop iii and the associated transmembrane domain . The versatility of fpr2/alx receptors also seems to rely on the activation of receptor dimmers in a biased fashion . In contrast to the full - length anxa1, the short anxa1 derived peptide ac226 is able to activate all members of the human fpr family and induce fpr2/alx - fpr1 heterodimerization . These observations suggest that short anxa1 mimetic peptides might fulfill other functions at variance to those reported for the parental protein . However, a good degree of selectivity was retained by longer anxa1 derived anti - inflammatory sequences such as anxa1250 . Interestingly, the promiscuity of fpr2/alx seems to be linked to a network of resolution mediators as discussed by brancaleone and col . In fact, the authors provide strong evidence that the engagement of fpr2/alx by selective agonists (such as lxa4 and antiflammin 2) would induce anxa1 phosphorylation and mobilization in human pmn . In a similar vein, the proresolving mediator resolvin e1 (rve1) stimulates endogenous lxa4 production [51, 52]. Moreover, it has been shown that proresolving mediators such as resolvins and lxa4 induce further anti - inflammatory molecules in vivo, such as interleukin- (il-) 10 . Taken together, these data suggest that a proresolving cascade may be operating during resolution with fpr2/alx playing a central role in this process . During inflammation neutrophils are rapidly recruited to the infected or injured tissue . However, due to the potential tissue - damaging effects of pmn, their fine - tuned regulation at the inflammatory site is required . Indeed, exacerbated or overshooting inflammatory response with high neutrophil influx may account for chronic inflammatory diseases . Thus, restricting leukocyte infiltration to the tissue is an essential process for spontaneous or pharmacological - induced resolution of inflammation [4, 8]. Neutrophil trafficking to the site of inflammation requires adhesion and transmigration through blood vessels, which is orchestrated by molecules on leukocytes (e.g., 1, 2 integrins, and l - selectin) and on endothelial cells (e.g., vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and e - selectin). The leukocyte adhesion cascade is a tightly regulated process, subjected to both positive and negative regulators . For example, anti - inflammatory and proresolving mediators, such as anxa1, are well documented to counterregulate excessive neutrophil accumulation (an anti - inflammatory action). Human pmn interaction with endothelial cells during the early stage of inflammation promotes modulation of anxa1 in several ways, such as induction of gene expression and mobilization and cell surface externalization of intracellular anxa1 [72, 73]. In turn, the externalized protein acts as a brake for pmn adhesion to the microvascular wall, preventing overexuberant cell transmigration to the inflammatory site [4, 27, 72, 74]. (2008) reinforced the anti - inflammatory properties of pmn - derived microparticles containing functionally active anxa1 . Released upon adhesion to endothelial cells, these microparticles inhibit neutrophil / endothelium interaction under flow, in vitro, and pmn recruitment to an air pouch inflamed with il-1, in vivo . Moreover, microparticles derived from wt but not from anxa1-deficient neutrophils were able to inhibit il-1-induced leukocyte trafficking . Several studies using exogenously administrated anxa1 have provided further evidence for the modulating role of anxa1 on neutrophil trafficking . In vivo observations produced through intravital microscopy techniques indicated that anxa1 and ac226 administration to mice during zymosan - induced peritonitis produced detachment of adherent neutrophils from the vascular wall with consequent inhibition of neutrophil extravasation across mouse mesenteric postcapillary venules (table 1). Supporting these first findings, in vitro studies showed that recombinant anxa1 and its mimetic peptides display inhibitory effects on neutrophil rolling [26, 27, 54, 55] adhesion to endothelial monolayer [26, 27, 40, 48, 54, 55] and transmigration . Shedding of l - selectin appears to be one of the molecular mechanisms that mediate the effects of anxa1 and its n - terminal peptides on neutrophil recruitment . (2000) have described the ability of the anxa1 peptide ac925 to cause transient calcium fluxes and l - selectin shedding in human neutrophils . After that, the same mechanism was linked to the inhibitory effects of ac226 on pmn capture and rolling in a flow chamber assay . Similarly, promotion of l - selectin shedding was demonstrated for human recombinant anxa1 [57, 58], an effect mediated by cell surface metalloprotease (sheddase). (2015) brought further insights into the mechanisms behind the antimigratory effects of ac226 . According to the authors, the peptide dose dependently reduces the affinity of activated neutrophils for vascular cell adhesion molecule-1 (vcam-1) and intercellular adhesion molecule-1 (icam-1), a response abrogated in cells harvested from fpr2 knockout mice . They demonstrated that ac226 inhibits the adhesiveness of 1 and 2 integrins by downmodulating their affinity and valency, but without changing their cell surface expression . It was also demonstrated that ac226 interferes with the chemokine - driven activation of rap1, an essential step in integrin activation [76, 77]. (2010) combined in vitro and in vivo experimental strategies to show that anxa1 and its mutant cleavage - resistant form, sanxa1, are able to augment rolling velocity and reduce adhesion of pmn to endothelial cells through fpr2 receptors . (2013) demonstrated the anti - inflammatory actions of the longer acetylated anxa1 peptide anxa1250 and its cleavage - resistant form, cr - anxa1250 . Both displayed antimigratory effects in vivo, reducing leukocyte adhesion to inflamed cremaster venule, neutrophil migration into dermal air pouches in response to il-1, and neutrophil migration into peritoneum in response to zymosan . In vivo anti - inflammatory and antimigratory properties of the short anxa1 peptide ac226 have also been extensively demonstrated, as exemplified by its ability to inhibit carrageenan - induced pmn adhesion to the vasculature and extravasation into the peritoneal fluid . The peptide was also able to prevent neutrophil recruitment in myotoxin - induced peritonitis and during lung inflammation induced by intestinal ischemia / reperfusion . Moreover, ac226 showed potential benefits in an ocular model by inhibiting neutrophil influx, protein leak, chemical mediator release, and cox-2 expression during endotoxin - induced uveitis . The ac226 peptide also demonstrated antimigratory effects in a model of ovalbumin - induced allergic conjunctivitis, significantly reducing the clinical signs of conjunctivitis through the inhibition of leukocyte influx and cytokines and chemokines release, effects correlated with inhibition of the erk pathway . Interestingly, increased levels of erk phosphorylation were associated with exacerbated allergic response observed in anxa1-deficient mice in comparison to wt animals . Reinforcing the involvement of anxa1 pathway in neutrophil recruitment, anxa1-null mice demonstrated a higher extent of neutrophil extravasation in animal models of peritonitis [35, 74], allergic conjunctivitis, and uveitis . Anxa1 may also be tightly coupled to the anti - inflammatory properties of other fpr2/alx agonists such as lxa4 and antiflammin 2 (af-2). The nonapeptide af-2, which corresponds to region 246254 of anxa1, is known to interfere with pmn activation, chemotaxis, and adhesion to endothelial cells [60, 61], via fpr2/alx receptor . Also, lxa4 is a potent regulator of pmn trafficking in experimental inflammation [9, 82]. Interestingly, recent data indicated a crucial role for endogenous anxa1 in the detachment phenomenon promoted by both compounds . For instance, lxa4 and af-2 lost their antimigratory effects in anxa1 ko mice suggesting anxa1 as a downstream mediator of other proresolving and anti - inflammatory molecules . Neutrophils are produced in the bone marrow from myeloid stem cells, which in turn proliferate, differentiate into mature neutrophils, and are delivered into circulation . Although the circulatory half - life of neutrophils is now thought to be longer than previously estimated (days instead of hours), at inflammatory sites the constitutive apoptotic pathway is delayed by the action of local inflammatory mediators, resulting in increased neutrophil half - life, an effect that can be opposed by proresolving mediators including anxa1 and lipoxins . In addition to affecting the migration of leukocytes through fpr activation, strong evidence of the involvement of anxa1 on neutrophil apoptosis has emerged . Proapoptotic effect of anxa1 on neutrophils was first described in vitro associated with transient calcium fluxes and dephosphorylation of bad, an intracellular protein whose proapoptotic function is lost upon phosphorylation . Our group demonstrated the in vivo proapoptotic functions of endogenous anxa1 during self - resolving inflammation . In an acute pleurisy model, blockage of the anxa1 pathway by using a specific anti - anxa1 antiserum prevented dexamethasone- (dexa-) induced resolution of neutrophilic inflammation, abolishing morphological and biochemical apoptotic events in the pleural cavity . Anxa1 neutralization also hampered dexa - induced decrease of erk1/2 and ib- phosphorylation and bax accumulation . In addition, anti - anxa1 treatment prevented spontaneous resolution of neutrophilic inflammation, suggesting an important role of endogenously produced anxa1 in the proresolutive program . Furthermore, pharmacological administration of ac226 peptide promoted active resolution and augmented the extent of neutrophil apoptosis . These effects were prevented by the pan - caspase inhibitor zvad - fmk and linked to activation of the cell death pathways bax and caspase-3 and inhibition of the survival - controlling pathways mcl-1, erk1/2, and nf-b (figure 2). In a skin allograft model, pharmacological treatment with ac226 increased transplantation survival related to inhibition of neutrophil transmigration and induction of apoptosis, thereby reducing the tissue damage compared with control animals . In vitro, ac226 counteracted the survival signal in saa - treated neutrophils, an effect associated with caspase-3 cleavage and prevented by the jnk inhibitor . (2013) also demonstrated that anxa1250 and cr - anxa1250 peptides can override the antiapoptotic effect of saa in human neutrophils in vitro . This proapoptotic effect may have contributed to the in vivo anti - inflammatory and proresolving actions of the peptides characterized by reduced granulocyte counts and enhanced efferocytosis in peptide - treated mice during peritonitis . Anxa1 has also been described as a mediator of drug - induced apoptosis, supporting its involvement in the induction of cell death . The proapoptotic effect described for the histone deacetylase inhibitor (hdcai) fk228, in leukemia cells, was linked to the induction of anxa1 expression, externalization, and cleavage . Neutralization with anti - anxa1 antibody or gene silencing with anxa1 sirna inhibited fk228-induced apoptosis, suggesting the involvement of anxa1 in apoptotic cell death in response to hdcai . Recently, the in vitro ability of hdacis to promote apoptosis was also demonstrated in bone - marrow neutrophils from wt but not from anxa1 knockout mice . In vivo, hdacis significantly reduced neutrophil numbers and induced neutrophil apoptosis in a zymosan - induced peritonitis model . It is important to keep in mind that the proapoptotic effect of anxa1 can be underestimated in dynamic in vivo models of inflammation . Regarding other anti - inflammatory drugs, it is documented in a number of diverse experimental and clinical settings that small changes in apoptosis rates can promote dramatic changes in total neutrophil numbers over time . This observation is most likely due to rapid recognition and phagocytosis of apoptotic cells [8991]. Macrophage phagocytic clearance of apoptotic neutrophils plays an important role in the resolution of inflammation since this process prevents excessive neutrophil activation and the exposure of tissues to noxious neutrophil intracellular contents [92, 93]. For this reason, appropriate (nonphlogistic) monocyte recruitment from the bloodstream to inflammatory sites is a critical step in acute inflammation, enabling the clearance of apoptotic neutrophils and orderly progression towards resolution . It has long been established that extravasation of pmn to the site of inflammation contributes to the launch of monocyte recruitment, with pmn granule proteins being important monocyte attractors . Recent research from perretti's group indicates apoptotic neutrophils as the principal reservoir of anxa1, which acts as important recruiting agent for monocytes to orchestrate the second resolving phase of acute inflammation . Associating in vitro and in vivo experiments, professor mauro perretti's group filled an important gap in our knowledge by demonstrating the central role of the anxa1alx / fpr2 pathway in modulating monocyte recruitment . The authors demonstrated that intraperitoneal administration of anxa1 induced monocyte migration, an effect absent in fpr2 null mice . Supporting these findings, both anxa1 and fpr2/alx null mice challenged with intraperitoneal zymosan exhibited diminished recruitment of monocytes as compared to wt mice, despite the higher levels of chemoattractants . After initial steps of apoptosis, neutrophils lose their functional properties, such as the ability to move by chemotaxis, generate a respiratory burst, or degranulate . Furthermore, they exhibit alterations on their intracellular pathways and cell surface molecules while some externalized molecules, such as phosphatidylserines (ps), facilitate the recognition and removal of apoptotic neutrophils by macrophages [92, 96]. Recent studies have reported that anxa1 from apoptotic cells is involved in their phagocytic clearance . The first observation that anxa1 participates in the engulfment of apoptotic cells, they showed that anxa1 is exported to the outer plasma membrane of apoptotic lymphocytes, colocalizes with ps, and is required for efficient clearance of apoptotic cells, suggesting a role for anxa1 as bridging ps molecules on apoptotic cells to phagocytes . (2007) subsequently demonstrated that apoptotic neutrophils release anxa1, which acts on macrophages, promoting the removal of effete cells . Noteworthily, not only the intact form of anxa1 released by apoptotic cells but also the cleavage fragments, under 10 kda, were effective in stimulating efferocytosis . Studies have also documented macrophages as a source of endogenous anxa1, which in turn facilitates phagocytic uptake of apoptotic cells . (2005) showed that human macrophages release anxa1 upon treatment with gc and that this protein acts in autocrine or paracrine manners to increase the engulfment of apoptotic neutrophils . Additional experiments with anxa1-null mice provided further evidence for a functional role of anxa1 in efferocytosis, as macrophages derived from their bone marrow were defective in clearance of apoptotic cells . In fact, the authors demonstrated, in vitro, the ability of the anxa1 mimetic peptide ac226 to promote phagocytosis of apoptotic pmn by human macrophages, an effect associated with actin rearrangement in the phagocytic cells and abrogated in the presence of fpr antagonist . Subsequently, it was clearly demonstrated the nonredundant function of fpr2/alx receptor in ac226 induced efferocytosis since the peptide failed to exert its proefferocytic action on fpr2/alx deficient macrophages . Furthermore, yona and coworkers (2006) associated in vitro and in vivo strategies that indicated reduced phagocytosis of zymosan particles by anxa1 knockout macrophages . It has been proposed that anxa1 released by macrophages can opsonize apoptotic cells, probably by interacting with surface - exposed ps, enhancing their uptake by phagocytes . Interestingly, mcarthur's group demonstrated that the binding of microglial - derived anxa1 to ps on the surface of apoptotic neuronal cells is critically required for phagocytosis . Moreover, dalli and colleagues (2012) reported that anxa1 expressed by resident macrophages is a critical determinant for the clearance of senescent neutrophils in the bone marrow . Proefferocytic effects were also observed for anxa1250 and its cleavage - resistant form (cr- anxa1250), which stimulated efferocytosis in vitro by human and mice bone - marrow derived macrophages . This effect was confirmed in vivo in a zymosan - induced peritonitis model, when the peptides significantly reduced exudate neutrophil counts and increased the number of macrophages containing ingested pmn . Once phagocytic removal of apoptotic cells has failed, neutrophils undergo secondary postapoptotic necrosis, probably leading to the leakage of cytotoxic and antigenic intracellular contents into the surrounding tissue . Blume and col . (2012) revealed, in two complementary studies, the role of externalized anxa1 as a fail - safe mechanism after neutrophil transition from apoptosis to secondary necrosis . First, they described anxa1 externalization during secondary necrosis, which in turn promotes the removal of dying cells and prevents proinflammatory cytokine production . In the second study, they demonstrated that in vitro anxa1 proteolysis during secondary necrosis generates a monocytic find - me signal, contributing to the recruitment of monocytes and consequently preventing inflammation . The removal of apoptotic cells has dual importance: prevention of potentially toxic content release and induction of macrophage reprogramming toward a resolving phenotype [101103], another key event to restore tissue homeostasis . Accordingly, anxa1-induced efferocytosis is coupled with increased release of transforming growth factor- (tgf-) and lower levels of the proinflammatory cytokine il-6 [65, 67]. In agreement with this observation, impaired phagocytosis in anxa1-deficient macrophages is mirrored by increased release of tumor necrosis factor- (tnf-) and il-6 . Supporting an immunomodulatory effect of anxa1 on cytokine production, anxa1-null mice showed increased mortality in a model of lps - induced endotoxic shock which was correlated with increased activation of inflammatory cells . The authors detected delayed and more prolonged increase in the levels of tnf-, il-1, and il-6 in the blood of anxa1-null mice, as well as increased production of these cytokines by anxa1 ko macrophages . This data is consistent with the increased production of il-6 and tnf by stimulated anxa1 ko peritoneal macrophages in comparison to wt cells . Moreover, in vitro studies linked anxa1 to brain homeostasis, demonstrating that exogenous anxa1 can suppress microglial activation, limiting indiscriminate phagocytosis of healthy neurones and nitric oxide (no) production during the phagocytic reaction . Recently, the functional role of macrophage - derived anxa1 in modulating hepatic inflammation and fibrogenesis during nonalcoholic steatohepatitis (nash) progression was documented . Nash in anxa1 ko mice was characterized by enhanced lobular inflammation resulting from increased macrophage recruitment and exacerbation of the proinflammatory m1 phenotype . In line with these results, anxa1 administration to liver macrophages suppressed m1 activation, characterized by reduced expression of inos and il-12p40, and increased il-10 expression . Interestingly, activation of fpr2 by anxa1 skewed m1 macrophages to anti - inflammatory m2-like cells, attenuating the expression of il-6, il-1, and tnf- . Furthermore, cooray and col . (2013) revealed an anxa1-specific fpr2/alx proresolving signal pathway centered in p38, leading to the production of il-10 by human monocytes, an effect replicated in vivo after intraperitoneal anxa1 injection . Although uptake of secondary necrotic leukocytes was shown to be anxa1 independent, the protein has an anti - inflammatory action on macrophages, since phagocytosis of anxa1 knock - down necrotic cells induced increased release of proinflammatory cytokines tnf, il-6, and il-1 by phagocytic cells . (2011) added knowledge to the immunosuppressive actions of anxa1 derived from apoptotic pmn . According to the authors, the treatment of human monocytes with anxa1-containing supernatant of apoptotic granulocytes or ac226 peptide results in a significantly diminished release of proinflammatory cytokines when the monocytes are subsequently challenged with endotoxin . Taken together, these findings indicate that anxa1-induced efferocytosis collaborates with the resolution of inflammation by promoting the elimination of effete neutrophils allied to an alternative macrophage activation that downregulates the production of proinflammatory mediators . Anxa1 is a gc - regulated protein that modulates a wide range of cellular and molecular steps of the inflammatory response and is deeply involved in the endogenous mechanisms that are activated to bring about proper resolution . So, it is reasonable to suppose that anxa1-based pharmacologic strategies could be as effective as steroids, without their metabolic side effects . We have discussed here the ability of anxa1 and its mimetic peptides to limit neutrophil accumulation in the tissue . Besides limiting neutrophil recruitment and increasing neutrophil apoptosis indeed, anxa1 contributes to tissue homeostasis by inducing macrophage reprogramming toward a resolving phenotype . The combination of these mechanisms results in an effective resolution of inflammation, pointing to anxa1 and its mimetic peptides as promising therapeutic agents for treating inflammatory diseases . The promising findings on the potential therapeutic use of anxa1 in inflammatory diseases have stimulated the development of pharmaceutical formulations containing anxa1 mimetic peptides, such as the controlled - release hydrogels for dermal wound repair application and targeted polymeric nanoparticles . The latter demonstrated ability to enhance resolution in zymosan - induced peritonitis, promote colonic wounds healing, and protect hypercholesterolemic mice against advanced atherosclerosis . These pharmaceutical strategies offer further benefits, overcoming the critical pharmacokinetics of short peptides in vivo, protecting them from proteolysis during pharmacological treatment, and facilitating the delivery to injury sites.
A 50-year - old, 168 cm, 68 kg man without significant past medical history, was admitted to emergency room following a slip - down accident . On admission, neurologic examination confirmed a glasgow coma scale (gcs) of 15, and the pupils were symmetrical and reactive . Emergent skull x - ray series, facial computerized tomography (ct) and forearm x - ray revealed fracture of the frontal sinus, skull base, and right distal radius fracture . Computed tomography (ct) of the brain showed multiple small subdural pneumocephalus (fig . Emergent lumbar drainage was performed to manage csf rhinorrhea and empirical prophylactic antibiotic treatment was given . The patient's post - traumatic symptoms improved gradually in the surgical intensive care unit . At post - trauma 8 days, following trauma, the patient underwent orthopedic surgery for distal radius fracture under general anesthesia . Anesthesia was induced with intravenous thiopental 300 mg and 0.5 g / kg / min remifentanil infusion . Endotracheal intubation was performed after administering 50 mg rocuronium injection with 100% oxygen mask ventilation for 3 min without any difficulties . Anesthesia was maintained with oxygen (2 l / min)-air (2 l / min)-sevoflurane (1 - 1.5 vol%), continuous infusion of 0.125 - 0.5 g / kg / min remifentanil and 10 - 15 mg / h of rocuronium . Tidal volume and respiratory rate were adjusted to maintain end - tidal carbon dioxide tension at 30 - 35 mmhg . Following surgery, the patient was extubated in the operation room after gaining consciousness and spontaneous ventilation . He was then transferred to the post - anesthesia care unit where he recovered fully without complications . Ten hours later, the patient complained of severe, increasing, headache and exhibited abrupt deterioration in mental state . In view of his clinical aggravation, neurologic examination demonstrated a gcs 11 and emergent brain ct showed large quantity of both frontal and right occipital pneumocephalus (fig . High flow (8 l / min) oxygen via a partial non - rebreathing mask was administered in conjunction with supportive care for 2 days . No infectious neuromeningeal complications occurred during hospitalization, and the patient was discharged at post - trauma day 32 . It develops mostly from acute and subacute complications of head trauma, or as late complications during the recovery phase of head injury . Subarachnoid or subdural air accumulation is the most common form of pneumocephalus, and intracerebral pneumocephalus can occur albeit rarely . Intracranial air is accumulated as a result of communications between paranasal sinuses, mastoid air cells and the cranial fossa, associated with dural tear . The other mechanism of air penetration is a ball - valve effect like sudden increase of nasopharyngeal pressure, such as during coughing and sneezing, forcing air into the cranial cavity which subsequently remained trapped . Injuries of the paranasal sinuses, which are closely contiguous to bony structures, the dura mater and the brain frequently accompany head traumas . 75 - 90% of pneumocephalus are posttraumatic and the incidence of pneumocephalus secondary to head injury range from 0.5% to 13.2% . Other causes include facial tumors that eroded through the skull, gas - forming bacterial infections, paranasal sinusitis and neurosurgical and facial procedures . Pneumocephalus is mostly asymptomatic and self - limiting, because gas is not irritating to the surrounding brain . Intracerebral pneumocephalus have usually become sizable when it is confirmed by simple skull x - ray or brain ct . Tension pneumocephalus caused by large amounts air can evoke sudden or insidious mass effects capable of shifting midline structures, causing major neurological deficits . Representable iatrogenic pneumocephalus are related to positive pressure ventilation in the upper airway, such as nasal continuous positive airway pressure, face mask ventilation and even nasal oxygen cannulae . It is well known that positive airway pressure can result in pneumothorax, pneumomediastinum and subcutaneous emphysema . It is difficult to predict which head trauma patients who do not initially develop pneumocephalus will experience iatrogenic pnemocephalus during positive pressure oxygen therapies . The mechanism of air delivered via manually operated mask tracking into the cranial cavities appears to be identical to that in traumatic pneumocephalus . Because csf leakage is usually a result of osteomeningeal fistula, csf rhinorrhea is commonly associated with pneumocephaus, as demonstrated in this patient . His initial primary pneumocephalus accompanied by rhinorrhea was completely subsided following lumbar drainage and general supportive care, but post - anesthetic secondary pneumocephalus probably developed during anesthesia induction using ppmv . Therefore, avoidance of excessive positive pressure during mask ventilation is recommended to prevent pneumocephalus . Because most mild pneumocephalus usually recover spontaneously within one week, its management includes strict bed rest in the semi - fowler position, analgesia and antibiotics . Prevention of intracranial pressure elevation by avoiding the valsalva maneuvers, coughing, sneezing, nose blowing, and excessive physical activities is important to avoid pneumocephalus aggravation . Because severe headache, personality change, disorientation, and mass effect associated with midline structure shift are the symptoms and signs of tension pneumocephalus, emergent surgical procedures, such as subdural drainage to decrease intracranial pressure, should be considered immediately after confirming the diagnosis with x - rays or brain ct . When the patient experienced severe headache and became rapidly lethargic, analgesia, antibiotic and high flow, high fio2 oxygen via a partial non - rebreathing reservoired mask were administered under a presumed diagnosis of impending tension pneumocephalus . The management principle is as follows: high fio2 oxygen reduces blood and brain tissue nitrogen concentration, thereby increases the nitrogen concentration gradient that promotes pneumocephalus gas absorption . To avoid tension pneumocephalus in head trauma patients, lma or tracheostomy tube intubation in addition, anesthesiologists should be aware of potential complications caused by low intracranial pressure during anesthesia in head trauma patients . In theory, hypotension, hypovolemia, excessive hypercapnia and head - up position during anesthesia can reduce intracranial pressure that facilitates air influx through open osteomeningeal fistulae secondary to head trauma, causing pneumocephalus . In conclusion, anesthesiologists and surgeons should be aware of pneumocephalus as a potential complication during positive pressure mask ventilation for facial trauma, secondary to air invasion through osteomeningeal fistulae . Close monitoring for pneumocephalus development should be mandatory following general anesthesia or during respiratory care with continuous positive airway pressure in head trauma patients.
The rna content of ev has been studied extensively; however, only a handful of reports have characterized the dna content of ev . The reason for this could be attributed to gaps in the scientific literature in understanding how dna could be packaged inside ev . Nevertheless, several groups have reported the presence of mtdna, ssdna, and dsdna from ev from various sources (table 1). The presence of mtdna, but not of genomic dna, was detected from ev from glioblastoma cells and astrocytes.4 however, upon dnase treatment of ev, it was found that a significant portion (~95%) of mtdna detected was shown to be free dna attached to the surface of ev.4 in another study, ev from malignant tumor cells (glioblastoma, medulloblastoma, atypical teratoid rhabdoid tumor, and melanoma cells) were found to contain ssdna, including genomic dna and retrotransposon elements.5 the ssdna detected from cancer cellderived ev was found to contain high levels of the cmyc oncogene . Others have reported the presence of dsdna in ev from various cancer cell lines (melanoma, breast, lung, prostate, pancreatic cancers), demonstrating that dsdna in ev could be a common feature of cancer cellderived ev that may be representative of the whole genomic dna.6 summary of dna detected from various ev sources ev, extracellular vesicle . The presence of dna in ev has sparked considerable interest in ev as novel mediators of horizontal gene transfer (hgt). However, very little is known about the origin and biological significance of dna in ev . The mechanisms of dna packaging in ev are poorly understood and whether dna is packaged within the membranebound space of ev remains unclear . Recent studies have revealed that dna is not contained within the membraneenclosed space of ev, but is largely attached to the outer surface of ev.7, 8 indeed, treatment of ev with nucleases such as dnase i and exonuclease iii before dna extraction significantly reduces the overall dna detected from cancer cellderived ev (yumi kawamura and takahiro ochiya, 2017, personal observation), demonstrating that dna is mainly associated with the outer ev membrane (fig . It has been suggested that the outer surface of ev are capable of interacting with various proteins, nucleic acids, and other surface membranes regulating their aggregation, motility in the circulation, and functionality in targeted sites.9, 10, 11 therefore, it is likely that dna associated with ev (termed evdna) may have some physiological significance in recipient cells and may play an important role, particularly in the cancer microenvironment . (a) hct116 ev were untreated or pretreated with dnase i, exonuclease iii, or rnase a, and extracted for dna and/or rna . (b) concentration of dna from hct116 ev was significantly reduced after treatment with exonuclease iii and dnase i. (c) rna concentration was unchanged after rnase a treatment of ev . The size of dna and rna extracted was measured with bioanalyzer (agilent technologies, santa clara, ca, usa). (d) dna size was reduced after treatment with exonuclease iii and dnase i. (e) detection of small rna species in ev was unaffected by rnase a treatment, as indicated by red arrows . Possible origins for evdna include cellfree dna (cfdna) found in apoptotic bodies,12, 13 vesicles released by apoptotic cells,14 or cfdna attached to the surface of ev.7, 15 circulating cfdna is mostly doublestranded, ranging from frequently observed size fragments of 180 bp to fragments larger than 10 000 bp.16 it is generally regarded that cfdna is released from dying cells during the process of apoptosis or necrosis, from both tumor and nontumor cells . Studies have shown that the concentration of cfdna is increased in patients with cancer, in comparison to healthy individuals.17 interestingly, reduced dnase activity was observed from plasma samples of cancer patients, which may indicate impaired degradation and clearance of cfdna in cancer.18 taken together, reduced dnase activity in cancer could also be related to higher dna concentrations detected from cancer cellderived ev . Given that cfdna from cancer patients can induce the oncogenic transformation of normal cells,19 cfdna on ev may carry substantial biological relevance in the tumor setting . Previous reports have shown that exogenous dna associated with ev is taken up more efficiently than free dna, thereby suggesting that dna associated with ev may be involved in their uptake at recipient cells.20 in addition to this, it has been shown that cancer cellderived ev take part in premetastatic niche formation21 and show organotropism towards potential metastatic sites.22 these findings lead to an intriguing hypothesis that cancer cellderived evdna including oncogenes could be transported to potential sites of metastasis by ev, inducing tumorfavorable effects (prometastatic effects) such as genetic instability in recipient cells, eventually leading to the modulation of the cancer microenvironment and premetastatic niche formation . In fact, we have seen that dna associated with cancer cellderived ev contains the oncogenic kras mutation, which is reflective of the parental cell line (fig . Although it remains unclear whether dna associated with cancer cellderived ev is functional in evrecipient cells, under certain conditions, these oncogenic mutations may have the potential to transform normal cells . Kras mutations are present in extracellular vesicle (ev)dna from the hct116 cell line . Dna was extracted from dnase i and exonuclease iiiuntreated hct116 ev and sequenced . Both hct116 cell dna and evdna have the heterozygous mutation in the kras gene, a substitution of the g nucleotide to a at c.38 . However, the kras gene was not detected in evdna when hct116 ev were pretreated with dnase i before dna extraction . Have assessed the ability of hgt by ev containing oncogenic sequences . In 2014, they showed that dsdna fragments including fulllength hras could be incorporated in ev from hras transformed cell lines.23 however, a followup study by the same group demonstrated that ev containing oncogenic hras failed to permanently transform immortalized fibroblasts.24 these data indicate cellular defense mechanisms that prevent the delivery and integration of evdna into the genome of normal cells . As previous studies have shown that apoptotic bodies can horizontally transfer oncogenes such as hras and cmyc to neighboring cells deficient of p53,13, 25 the integration of oncogenic sequences carried by ev could also be regulated by similar cellular defense mechanisms . Deciphering the cellular conditions that would allow the integration of exogenous dna into the host genome would be important in understanding the extent to which ev can mediate the hgt of oncogenes and other exogenous dna to evrecipient cells . The disparities in dna detected from ev reflect the complexity and heterogeneity of ev populations . However, given the importance of ev in intercellular communication and their ability to transport genetic material to neighboring and distant cells, determining the origin and physiological significance of evdna could be relevant in understanding their functions in living systems . Finally, the detection of dna from cancer cellderived ev may have potential uses as circulating biomarkers for detecting tumor dna from body fluids such as blood, urine, and saliva . Another important feature of cancer cellderived ev is the discovery of retrotransposon elements . In 2011, it was reported that cancer cellderived ev are highly enriched in retrotransposon elements including line1 (l1), alu and human endogenous retroviruses (herv).5 this is an intriguing discovery as it indicates a new modality for retrotransposons to spread horizontally from one cell to another . Retrotransposon elements, also known as jumping genes, are mobile genetic elements that replicate through rna intermediates that are reverse transcribed and inserted into new genomic locations . Following the complete sequencing of the human genome, it has been shown that more than 40% of the human genome is derived from retrotransposon elements.26 retrotransposons are capable of altering the genome by causing mutations, deletions and rearrangements, and their effects range from local genetic instability to largescale genomic variation . In somatic cells, however, active retrotransposition has been implicated in various diseases, and more than 70 diseases have been documented to be associated with heritable and somatic retrotransposition events.27, 28 although the ability of retrotransposons to replicate and integrate into the host genome is well studied, the ability to transfer from one cell to another remains largely unknown . As these elements themselves are not infectious, it has been suggested that horizontal transmission would be unlikely without the involvement of a vector . In addition to this, others have pointed out the instability and rapid degradation of rna intermediates in the extracellular environment.29 the discovery of retrotransposon rna in ev may indicate an alternative mechanism for the intercellular transfer of retrotransposon elements . Considering the protective features of ev and their ability to carry rna to neighboring and distant cells, this finding has important implications in understanding the mechanisms of somatic retrotransposition and the involvement of ev containing active retrotransposon elements in the development and spread of genetically influenced diseases . Somatic l1 insertions have been found in the apc tumor suppressor locus in colorectal cancer,30 and within the myc locus in breast cancer.31 since then, wholegenome and exome sequencing studies have identified somatic retrotransposon insertions from colorectal, lung squamous, head and neck, and endometrial carcinomas.32, 33 as indicated by these studies, somatic retrotransposon insertions are believed to play an important role in the tumorigenesis of various cancers . Showed that ev can transfer retrotransposon rna to normal cells.5 however, the functional impact of retrotransposon transfer in evrecipient cells has not been reported . Delivery of retrotransposons to distant sites, thereby avoiding immunerecognition in a tumor setting.34 given that active retrotransposon elements can yield extensive genomic insertions, further analysis will be important in observing the effects and activities of evmediated transfer of active retrotransposon elements in recipient cells . Understanding this new biological concept will elucidate the spread of retrotransposons by ev and their potential involvement in the formation of genetic instability in target cells (fig . 4). Proposed model of the horizontal transfer of genetic material by extracellular vesicles (ev) in the cancer microenvironment . Genetic material such as oncogenic sequences and retrotransposon elements can be propagated by cancer cellderived ev . Upon uptake at recipient cells, dna may be integrated into the genome of the recipient cell and cause phenotypic changes leading to niche expansion and tumor growth in neighboring cells, or the generation of the premetastatic niche at distant sites . Retrotransposition events in neural progenitor cells have been shown to be important in the generation of genetic mosaicism that contributes to neuronal diversity and brain plasticity.35 however, accumulating evidence shows that the deregulation of retrotransposons may cause deleterious effects in neuronal genomes, which may lead to the development of neurodegenerative diseases . Altered levels of retrotransposon expression and increased retrotransposition insertions have been observed in neurodegenerative and neuropsychiatric diseases . For instance, hyperactive retrotransposition and increased l1 copies have been reported in schizophrenia, alzheimer's disease (ad) and parkinson's disease (pd). In addition to this, active herv have been identified in neurons of patients with amyotrophic lateral sclerosis (als), and have been associated with neuronal injury and death of motor neurons.36, 37 however, it is still unclear how the deregulation of retrotransposon elements can cause neurodegenerative diseases . Neurons are particularly vulnerable to the accumulation of dna damage as a result of their limited capacity for cell replacement in adulthood . The accumulation of dna lesions is considered to be a major contributor to ageassociated neurodegenerative diseases such as ad, as well as pd.38, 39, 40 in addition to this, impaired dna damage repair has been documented in many neurodegenerative diseases and is considered to be an important indication preceding neurodegeneration . 39, 41 multiple dna repair molecules have been found to be mutated or deregulated in ad patients.41 dna lesions and double strand breaks (dsb) are particularly vulnerable to retrotransposon insertions,42 and are thought to be associated with increased retrotransposition events in neurodegenerative diseases.43, 44 ev have been suggested to play a significant role in the dissemination of protein aggregates in the brain, such as amyloid and tau in ad, and synuclein in pd.45, 46 the accumulation of pathogenic proteins has been associated with the development of neurodegenerative diseases (reviewed in47). Given that increased retrotransposition insertions in neurons are prominent in various neurodegenerative diseases, ev may also be responsible for the dissemination of not only deleterious proteins but also of genetic material such as deregulated retrotransposon elements . Furthermore, accumulating evidence suggests that distinct mechanisms might be involved in the onset of disease, spread, and neuronal death.48, 49 therefore, the development of neurological diseases could be modulated by various factors not limited to pathogenic protein aggregation in the brain . Clinical studies have shown that the spread of neurodegenerative diseases follows stereotypical patterns, and may derive from a focus of genetically altered cells.50 the idea that the disease is initiated in a focal area suggests that somatic mosaicism may be responsible for the genetic changes in initiating cells . Indeed, it has been suggested that somatic retrotransposition in neurons can cause mutations that may be involved in the onset of neurodegenerative diseases.44 however, the mechanisms that underlie the spread of the disease from the initial focus to other regions of the brain remain to be fully determined . The role of ev in the spread of deleterious cargo may explain the anatomical spread of neurodegenerative diseases from one brain region to an adjacent region . Considering that retrotransposons can act as insertional mutagens that can target dna dsb, it is plausible to hypothesize that the effects of ev containing active retrotransposon elements can exacerbate the spread of insertional mutagenesis in affected neurons, which may play a role in the potential spread of neurodegeneration . Although the involvement of retrotransposons in ev and their activity in the progression of neurodegenerative disease has not been demonstrated so far, this finding would be important in understanding the spread of somatic retrotransposition in the brain . The analysis of evmediated transfer of active retrotransposons will add to the current understanding of the role of ev not only in the degeneration of neurons but also in the development and spread of various human diseases associated with deregulated retrotransposition events . The discovery of genetic elements in ev, in particular retrotransposon elements, signifies notable importance in understanding the mechanisms of genome evolution by exogenous genetic elements carried by ev . The horizontal transfer of retrotransposons has been critical in generating genome diversity that has led to the evolution of various organisms . Genomewide comparisons of humans and other primate genomes have revealed the magnitude and significance of retrotransposon elements in human evolution.27 an example of horizontal transfer of retrotransposons is the case of bovb, an l1 retrotransposon that has been found to be widespread among mammalian species.51 however, the precise mechanisms regarding the transfer of retrotransposons between species remain largely unknown . The successful horizontal transfer of a retrotransposon requires the delivery of the rna intermediate, reverse transcription and integration into the host genome . It has been regarded that retrotransposons do not have the ability to infect other cells, and therefore would not be able to disperse horizontally across species without a vector involved . Retroviruses, which have the ability to assemble virus particles that can exit the cell, have been proposed as potential vectors for the horizontal transfer of retrotransposons . Recently, it was shown that ev are able to transfer hervk hml2 rna to normal cells, suggesting that a functional envelope gene may not be required for herv to infect other cells.5 this discovery raises the possibility of an evmediated mechanism for the dispersal of retrotransposons, and opens a new door into understanding their basic nature and scope in interspecies transfer . A recent review by nolte't hoen et al . Discusses the many aspects in which ev resemble retroviruses . And retroviruses share similar structural aspects including size and buoyancy, but can also be similar in functional aspects, such as virus propagation . Ev generated from virusinfected cells (termed virusinduced ev) have been shown to incorporate viral proteins and fragments of the viral genome, which can be transferred to target cells.52 the authors argue that virusinduced ev and viruslike particles are intermediate entities that are indistinguishable from one another . These intriguing similarities with retroviruses raise the possibility of ev as potential transporters of retrotransposon elements . Moreover, intercellular communication by ev is not unique to eukaryotic cells . The release of membrane vesicles is an evolutionarily conserved mechanism in eukaryotes, bacteria, and archaea, and is a basic feature in many organisms.53 in addition to this, the mechanistic strategies of ev release are similar, typically involving the highly conserved endosomal sorting complex required for transport (escrt) machinery, which are key components in the excision of membrane necks in the biogenesis of mvb, cytokinesis, and budding of enveloped viruses.54, 55, 56 the conserved feature of ev production and transmission may indicate their potential ability for interspecies transfer of genetic material . Several reports have demonstrated the transfer of ev across species and their involvement in interspecies communication . The delivery of infectious agents by ev has been reported in host pathogen interactions by intracellular and extracellular eukaryotic parasites57 and in human fungal interactions.58 ev have also been implicated in plant fungal interactions, in the secretion of virulence and defense molecules by both fungi and plants.59 furthermore, preliminary studies have shown that ev from food sources are taken up by intestinal macrophages and stem cells in humans, inducing the expression of target genes in recipient cells.60 the ability of ev to carry genetic material across species suggests their potential role in the transfer of exogenous genetic material into the human genome . Further studies examining the distribution of genetic elements such as retrotransposons by evmediated mechanisms may consequently open up new ways of considering the functional impacts of ev in genome modulation, and will provide additional evidence for external influence on the evolution of the human genome . The presence of genetic material in ev has heightened considerable interest in their potential as mediators of hgt not only between cells in a single living organism, but also across different biological species . Further studies assessing the functionality of genetic material in evrecipient cells will help to better define their roles as transgenomic agents and their involvement in modulating various cellular responses . Ev are packaged with abundant cellular information; however, the mechanisms that regulate ev cargo selection is still poorly understood . A deeper understanding of the biogenesis and cargo selection of ev may elucidate many of the biological processes that are regulated by ev, permitting an understanding of inter and intracellular communication by ev.
The paradigm of intercellular cell signaling mechanisms has until recently been assumed to be limited to direct cell cell contact and via local soluble factor release . However, there has been increasing interest in the role of plasma membrane - derived small lipid / protein vesicles in cell cell communication in the last decade . Microparticles (mp) are one such type of vesicles gaining more recognition as their biological roles are being explored . Although there is no standardized terminology for these small extracellular vesicles, the term, microparticle, usually refers to a subset of extracellular, cell membrane - derived vesicles ranging from 0.1 to 1 m in diameter, shed directly from the plasma membrane through the process of ectocytosis (gasser and schifferli, 2004; cocucci et al ., 2009 although mp are shed from steady - state cells, elevated levels are observed in actively proliferating cells and cells activated by a wide range of stimuli, from mitogens and inflammatory mediators, to antigenically foreign compounds, such as pathogen components, as well as various other stress - inducing challenges in both in vitro and in vivo models (ratajczak et al ., 2006; in vivo, mp are most commonly found in blood, however, they are also observed in other bodily fluids, such as synovial fluid (berckmans et al ., 2005; distler et al ., 2005b) and urine (smalley et al ., 2008). Microparticles are often compared with exosomes and apoptotic bodies (table 1), however, while superficial structural similarities exist between them, both the mechanism of mp formation and their subsequent composition are fundamentally different (thry et al ., 2009; mause and weber, 2010; gyrgy et al ., 2011). Mp shedding is a multi - enzyme - regulated process . Under normal conditions, the asymmetric composition of the plasma membrane is maintained by three principal translocase enzyme families of flippases, floppases, and scramblases, which actively maintain phosphatidylcholine (pc) and sphingomyelin (sm) on the external leaf, while phosphatidylserine (ps) and phosphatidylethanolamine (pe) are kept on the internal leaf . On cellular activation, these enzymes are inhibited following an influx of ca into the cytoplasm, and consequently the normal phospholipid asymmetry is disrupted (coltel et al ., 2006; piccin et al ., 2007; pap et al ., this is accompanied by the loss of actin spectrin anchorages and the degradation of actin filaments by calpain . These protrusions are pinched off as individual mp in a rho - associated kinase i (rock - i)-dependent manner from the cell membrane, with ps and pe exposed on the outer surfaces . In contrast, exosomes fall into the 50100 nm range and are formed by inward budding into the endosomal lumen of small intra - luminal vesicles (ilv) that aggregate to become multivesicular bodies (mvb). Mvb subsequently fuse with the plasma membrane to release the ilv into the extracellular space as exosomes (thry et al ., exosomes are enriched in cd63, and major histocompatibility complex (mhc) i and ii molecules, but tend to have low levels of ps and pro - coagulation factors on the surface (denzer et al ., 2000; thry et al ., 2009; mause and weber, 2010; gyrgy et al ., 2011). Apoptotic bodies, yet another type of subcellular vesicle, are much larger (14 m) and are essentially fragments of cells produced in late stage apoptosis (table 1). The process by which mp are produced results in their possessing many of the characteristics of the cell from which they originated . This includes many of the surface molecules, such as integrins, receptor ligands, co - stimulatory molecules, as well as cytoplasmic contents, such as activated signaling molecules and even genetic materials e.g., mrna (baj - krzyworzeka et al ., 2006; aliotta et al ., 2010) and mirna (yuan et al ., 2009). Mp either attach and fuse to their target cell (del conde et al ., 2005), or are internalized and processed by pinocytic or phagocytic mechanisms (dasgupta et al . Since mp carry biological molecules from their cells of origin, they may induce associated responses in their target cells, depending on the source of mp, the cell type on which they act and the route by which they are incorporated . Given that mp are produced in response to many stress conditions and are involved in several immunological processes, it seems likely that mp are involved in the regulation of the immune response against invading pathogens, however, their potential role in infectious diseases has not been extensively studied . This review aims to consolidate current evidence on their biological role and venture opinion on their potential impact in infectious disease . One of the hallmarks of bacterial, viral, fungal, or parasitic infection is the formation of an inflammatory milieu around infected or affected cells . Pathogen - associated molecular pattern (pamp) recognition receptors (prr), such as toll - like receptors, certain classes of rna helicases, like the retinoic acid inducible gene - i (rig - i), and nod - like receptors (nlr), specifically detect components of the invading pathogen and trigger a cascade of intracellular signaling events that lead to the activation of transcription to initiate the first wave of innate immune responses (creagh and oneill, 2006; seth et al ., 2006). However, endotoxins, produced by bacteria during infection, or the sheer load of replicating intracellular virus would undoubtedly cause stress in affected cells . Indeed, generation of mp may be closely associated with the initiation of apoptosis and necrosis (ardoin et al ., 2007). Whether stress - inducible proteins, such as hypoxia - inducible factor 1 (hif-1), heat - shock factor (hsf-1), heat - shock protein (hsp) family, nuclear factor b (nf-b), jun nh2-terminal kinase (jnk), and p53 (ohiro et al ., 2003; razorenova et al ., 2005; thompson and locarnini, 2007; zinkernagel et al ., 2007; de maio, 2011; li et al ., 2011; rawat and mitra, 2011; zhang et al ., 2011) and mhc class i polypeptide - related sequence a and b (mica, micb; stern - ginossar and mandelboim, 2009) are involved, remains to be determined . Nevertheless, a broad array of pro - inflammatory cytokines, such as tumor necrosis factor (tnf), interleukin-1 (il-1), il-6, and type 1 interferons (ifn - i) are produced in response to infection (medzhitov and horng, 2009), and many of these induce mp shedding in the absence of infection (combes et al ., 1999; sheremata et al . 2008). Not surprisingly, therefore, both stimulation in vitro with lipopolysaccharide (lps; aharon et al ., 2008; bernimoulin et al ., 2009) and intravenous injection of bacterial endotoxin in vivo (zubairova et al ., 2006; wang et al ., furthermore, elevated blood mp levels are also observed in septic patients (nieuwland et al ., 2000; mostefai et al ., 2008), in human immunodeficiency virus (hiv) patients (aupeix et al ., 1997) and ebola virus - infected macaques (geisbert et al ., 2003), as well as malaria patients and plasmodium - inoculated laboratory animals (combes et al ., 2004, 2005; penet et al ., 2008; couper et al ., 2010; pankoui mfonkeu et al ., many activated immune cells not necessarily infected by pathogens, such as monocytes, macrophages, dendritic cells (dc), and lymphocytes, produce mp when activated by relevant stimuli . Mp have been shown to be produced by monocytes and macrophages stimulated with a23187 (cerri et al .,, 2006; aharon et al ., 2008), and starvation (aharon et al ., 2008), by dc stimulated with lps (obregon et al ., 2006, 2009), by t cells stimulated with agonistic cd3 antibodies, fas ligand, heat, actinomycin - d, staurosporine, pha, pma (scanu et al ., 2008), and by neutrophils stimulated with fmlp and c5a (gasser and schifferli, 2004). In addition, mp are produced by cells isolated from patients with autoimmune disease, such as diabetes (martin et al ., 2004), as well as vascular disease (tesse et al ., 2005). The wide variety of possible stimuli producing mp in the absence of infection makes it difficult to predict pathways leading to mp production in infectious disease, or predict their function in this context and few experimental models utilize infectious pathogens . Nevertheless, these studies provide valuable clues to what possible responses might occur in infection . As mentioned above, elevated blood mp levels have been reported in models of sepsis, viral infection, and cerebral malaria . Because of the generalized inflammatory conditions that such events generally create, it seems reasonable to propose this would be the case for most, if not all infections accompanied by significant hematogenous spread, i.e., during parasitemia, bacteremia, viremia or fungemia . Furthermore, due to the propensity of mp to carry contents derived from their cells of origin, it is likely that they contain antigens, either present or induced in infected cells . Plasmodium parasite antigens were detected in the mp purified from the plasma of infected animals (couper et al ., 2010). This is unlikely to be unique to parasitic infections, as infecting viruses inevitably become an integral part of the cell, inducing a range of virus - specified proteins crucial for their replication and these could become incorporated into the mp contents, either specifically or non - specifically . Thus, mp could remotely convey viral antigens via the blood stream to professional antigen - presenting cells (apc), such as macrophages and dc residing centrally in the spleen and liver without the necessity for their direct interaction with infected cells . Both organs normally have high throughputs from the systemic circulation, increasing the likelihood that mp will interact with apc . From a host point of view, this maintains a desirable separation between the peripheral site of infection and the site of immune induction . Additionally, tnf - stimulated, endothelial cell - derived mp have the capacity to induce activation and maturation of plasmacytoid dc, although not conventional dc (angelot et al ., 2009). Together, such remote interactions could increase the probability of detecting infection early, enabling prompt initiation of innate and adaptive responses by apc responding to mp in the blood . Indeed, it has been shown that mp produced by mycobacterium - infected macrophages were able to activate and present antigen to naive t cells (ramachandra et al ., 2010), thus obviating the necessity for direct apc and t cell contact to generate a t cell response . In these experiments, mp were better at inducing t cell proliferation than exosomes . (2010) also demonstrated the activation of bone marrow - derived macrophages, with upregulation of cd40 and tnf expression . This raises the further possibility that mp may facilitate the mobilization of subsets recruited from the bone marrow during infection (king et al ., 2007; getts et al ., 2008). As well as accepting mp by fusion, dc also produce mp following activation . Lps - induced mp from dc can transfer antigen to recipient dc, which can present this to t cells (obregon et al . . Other cellular membrane function can be transferred by mp from activated to nave t cells via the tetraspanin, cd81, important for adhesion and lymphocyte maturation (fritzsching et al ., 2002; quast et al ., 2011). Furthermore, cd154 platelet - derived mp introduced into cd154 mice triggers cd4 t cell - dependent production of antigen - specific igg and germinal center formation, suggesting that with the right surface molecules mp can fully activate the adaptive immune response (sprague et al ., 2008). Indeed, mp transfer could explain the rapid exchange of t cell receptors (tcr) observed in the early stages of t cell activation to expand antigen - specific t cell clones (chaudhri et al ., 2009). Irrespective, it seems likely that mp containing pathogen antigens along with the necessary co - stimulatory molecules can activate t cells, bypassing cell cell contact, thus enabling a wider, more efficient initiation, amplification and/or modulation of early adaptive immune responses . Microparticles may also contain pro - inflammatory cytokines, such as il-1, providing an alternative or additional pathway for cytokine secretion into the extracellular space to modulate local cytokine / chemokine signaling (mackenzie et al . Depending on their mode of manufacture and cellular release, it seems highly likely that other cytokines / chemokines, may make use of this pathway (garcia et al ., 2005; dean et al ., 2009). Certainly, t cell - derived mp have been shown to activate monocytes (distler et al ., 2005a; huber et al ., 2007; scanu et al ., 2008) and monocytes activated with mp from pha- or pma - stimulated blood t cells produce tnf, il-1, and soluble interleukin-1 receptor antagonist (sil-1ra; scanu et al ., 2008). Mp derived from t cells and monocytes also induce nf-b - dependent expression of matrix metalloproteinases (mmp; distler et al ., 2005b), as well as some pro - inflammatory cytokines and chemokines such as il-6, il-8, macrophage chemoattractant protein (mcp)-1, mcp-2, and regulated upon activation, normal t cell expressed and secreted (rantes) in synovial fibroblasts (berckmans et al ., 2005; distler et al ., 2005b). Monocyte / macrophage - derived mp also upregulate inflammatory mediator synthesis by epithelial cells (cerri et al ., 2006). Not surprisingly, under certain conditions mp can also induce anti - inflammatory il-10 expression in monocytes (kppler et al ., 2006) and transforming growth factor 1 (tgf-1) in macrophages (sadallah et al ., mp can even be pro - apoptotic, especially in macrophages, which can undergo apoptosis upon uptake (distler et al . For example, neutrophil - derived mp specifically upregulate the expression of tgf-1, which helps to suppress the inflammatory response (distler et al . The mp used in these models induce macrophage apoptosis, possibly because the mp were generated from apoptotic cells, which might have transferred the apoptotic signals to the macrophages (bing et al ., 2008). This is similar to the behavior of macrophages to dampen inflammation after ingesting apoptotic bodies, to prevent over - reaction (savill et al ., 2002). However, these studies did not use pathogens and macrophages activated by plasmodium were in contrast highly pro - inflammatory (couper et al .,, mp might contain factors induced or expressed by the infecting organism that could impede or abrogate the early induction of an effective immune response . For example, influenza a ns1 protein expressed intracellularly has the capacity to suppress ifn - i production and inhibit apoptosis to enable better viral replication (pauli et al ., 2008; wang et al ., several flavivirus subunits interfere with ifn - signaling at various levels and in different ways (reviewed in daffis et al . Such molecules, carried into cells yet to be infected could arguably give the infecting organism a survival advantage, enabling greater levels of replication in the host for longer periods of time, thus increasing the probability of transmission to vectors and/or to the next host . In the case of arboviruses, considering the relatively short times that organisms spend in the blood stream, even small increases in concentration and time spent in the blood stream could potentially subtend a significant survival advantage . The inflammatory conditions created by mp could in principle play important roles in controlling infection and pathogen clearance . How crucial these roles are in the immune system remain unclear, but as one of many factors that regulate the immune response against invading pathogens, mp could significantly influence the outcome for the host, both during the initial knockdown phase of controlling pathogen and in the ongoing adaptive response to it . Pro - inflammatory cytokines induced by mp would act to manage and contain the infection, while chemoattractant proteins would send signals to recruit leukocytes to the site of infection to eliminate the pathogen . Indeed, a further interesting characteristic of mp is their ability to upregulate adhesion molecules such as intercellular adhesion molecule-1 (icam - i; berckmans et al ., 2005) in target cells and promote monocyte recruitment (mause et al ., 2005). Along with ability of mp to induce mmp expression and tnf release, the endothelium becomes more permeable for immune active proteins and leukocytes to access the site of infection . Although this is an important step to enable pathogen clearance, it can be a double - edged sword in certain infectious diseases . In cases of lethal flavivirus encephalitis, in which immunopathology is one of the prominent features of pathogenesis, excessive monocyte infiltration of the central nervous system (cns), and the accompanying pro - inflammatory cytokine spike are a major cause of mortality (king et al ., 2007; getts et al ., 2008). We hypothesize that during viremia preceding encephalitis, flavivirus infecting endothelium, and blood monocytes induce mp, both as a consequence of infection and via tnf released by infected cells (cheng et al ., brain barrier, potentially enabling virus access to the cns (wang et al ., 2004). Mp from tnf - activated endothelium could further activate uninfected monocytes and endothelium, while mp from infected cells could carry viral antigen, as well as activation signals to the liver and spleen . Early antigen presentation would likely advantage the host in generating anti - viral immune responses . However, wnv - infected cns neurons recruit activated (ccr2) monocytes from the bone marrow to the brain via ccl2 (getts et al ., 2008). Here, tnf- and mp - mediated activation of cns endothelium would facilitate macrophage margination, adhesion, and diapedesis in response to the chemokine gradient, thus potentially increasing numbers of monocytes recruited to the brain and exacerbating immunopathology (figure 1). Putative role of mp in immunopathogenesis of flavivirus encephalitis . After local replication of virus at the site of infection, the ensuing viremia results in endothelial and monocyte infection . Mp produced as a result of infection and via the consequent tnf secretion, are transported in the blood stream . On the one hand, mp from tnf - activated endothelium could further activate uninfected monocytes and endothelium at remote sites, such as in the brain, while mp from infected cells could carry viral antigen and activation signals to liver and spleen . Early antigen presentation would likely advantage the host by generating anti - viral immune responses . On the other hand, endothelial activation may enable increased migration of monocytes into the brain in response to ccl2 secreted from infected neurons . While this immigration may initially enable early viral clearance, over - exuberant immigration of activated monocytes that differentiate into macrophages may be immunopathological for neurons via toxic soluble factors produced . This does not exclude a possible trojan horse scenario, in which virus may also further gain access to the brain via the cerebral endothelium and/or via infected immigrant monocytes during encephalitis . Brain barrier in cerebral malaria via monocyte, tnf, and mp activities (combes et al ., moreover, in mice deficient in the floppase, atp - binding cassette transporter a1, which modulates ps distribution on the outer plasma membrane leaflet, mp production is reduced in cerebral malaria, abrogating neurological signs in this model, and presenting the possibility for pharmacological modulation of mp to ameliorate immunopathology (combes et al ., 2005; penet et al ., 2008). Immunological aspects aside, mp could potentially also be used directly as transport by viruses, including flavivirus, influenza, and herpes simplex virus (hsv) that propagate via a process of ectocytosis from the plasma membrane of an infected cell . In a model of hiv-1, the glycome profile and the budding signal of the virion were similar to that of the mp secreted in conjunction, suggesting that the mechanism for viral propagation was similar to that of the formation of mp (krishnamoorthy et al . Even if mp formation selectively excludes budding virions, the mp may still potentially contain unpackaged viral genome and other viral proteins necessary for replication in the target cell . In either scenario, mp may be used by viruses as trojan horses to spread to other cells . Indeed, hiv-1 induces mp that express cd45, cd80, cd86, as well as mhc - i and ii (esser et al ., 2001), thus, potentially masking themselves as being of host origin . In addition, hiv also induces ccr5-containing mp and can transfer this receptor to other peripheral blood mononuclear cells (pbmc) deficient of endogenous ccr5, enabling infection of these cells (mack et al ., 2000). However, the success of allogeneic transplantation of 32/32 ccr5 stem cells in keeping the patient hiv free, indicates that much more remains to be understood about these processes (htter et al ., 2009). As with all emerging areas, a number of technical issues remain to be fully resolved . For example, the accurate determination of mp size and phenotype, their clear distinction from and relationship to, other membrane vesicles, and their pure separation for functional experimental purposes, remain a significant challenge (gyrgy et al ., 2011). While mp clearly have likely roles in homeostasis and inflammation, few studies have to date directly addressed their involvement in infectious disease . Thus, many questions remain to be answered, perhaps most important among which are, whether the production of mp as a programmatic response to infection confers an overall species survival advantage . At the mechanistic level, also, how cellular decision - making occurs to produce mp, what they should contain, and whether production of the full range of membranous vesicles is required and/or produced kinetically to accommodate host requirements from initial infection to disease resolution, remains to be elucidated . As with most defenses against invading pathogens, on the one hand, the transfer of phenotype and function by mp may be of considerable benefit to the host, in the early warning and initiating of immune defenses at both innate and adaptive levels . On the other, however, this may exacerbate immune responses leading to immune pathology, or be commandeered by the invading pathogen for its own survival . 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.
Malaria is one of the world's leading killers, having a greater morbidity and mortality than any other infectious diseases, with greater mortality in children and pregnant women . Malaria is caused by a parasite of the genus plasmodium and transmitted by female anopheles mosquito . Renal damage and hemolysis induced by malaria, especially p. falciparum and p. vivax, occur between 2 and 6% of hospitalized patients with a mortality that can reach up to 50% . The pathogenesis of renal damage and hemolysis during malaria infection is multifactorial and not well characterized, but several studies suggest involvement of cytoadherence of parasitized erythrocytes and proinflammatory response as well as oxidative stress [35]. This has prompted research towards the discovery and development of new, safe, and affordable antihemolysis drugs during malaria infection . In this plant extracts are commonly investigated in several parts of the world for their possible activities for using in malaria [710]. Moreover, many studies recently endorsed the use of traditional medicinal plants to treat diseases [1116]. This plant is widely found in asia and southeast asia including thailand and used for general weakness, malaria fevers, and chronic rheumatism . This result is confirmed by the existence of phenolic and flavonoids such as catechin, luteolin, and borapetoside . These are responsible for high antioxidant activity and other activities including anti - inflammation, antimicrobials, and anticancer [1921]. The antioxidant properties of t. crispa stem extract make it useful for the treatment of diseases resulting from oxidative stress such as cardiovascular disease, cancer, liver damage, and renal disease . It also has been used as an antimalarial agent in vivo [22, 23]. However, it has not been reported yet to use t. crispa stem extract to treat renal damage and hemolysis induced malaria infection . Hence, the present study was aimed to investigate the protective effects of t. crispa stem extract on renal damage and hemolysis induced by p. berghei infection in mice . Fresh stems of tinospora crispa were collected from natural environment in suphanburi province, thailand, from march to april 2015 . Stems were minced into small pieces and dried in hot - air oven at 60c for 72 h, after which the dried plants were ground and then extracted in distilled water at the proportion of 1: 10 (w / v) using microwave at 360 w for 5 min . The extract was filtered through whatman number 1 filter paper, and evaporation was then performed to remove solvent . Powder extract was dissolved in distilled water to obtain appropriate doses for using in mice and volume should not be higher than 1 ml . Drug doses, expressed in mg / kg of body weight, were adjusted at the time of administration according to the weight of the mice . Pathogen - free, female icr mice, aged 46 weeks and weighting 2530 g, were purchased from the national laboratory animal center, mahidol university, bangkok, thailand . They were kept at 2225c with 12 h light / dark cycle and given standard mouse pellet and water ad libitum . All procedures of the animal experiments were approved by the ethical committee on animal experimentation, faculty of medical technology, western university . Pbanka was kept alive by intraperitoneal (ip) passage of 1 10 parasitized erythrocytes in nave mice . In addition, hematocrit (% hct) was also monitored by collecting tail vein into heparinized hematocrit tubes and centrifuged at 10,000 g for 10 min . Blood urea nitrogen (bun) and creatinine were used as indicators of renal function . Mouse blood was collected from tail vein into heparinized hematocrit tubes and centrifuged at 10,000 g for 10 min . Plasma was subsequently collected and used as subjects for measurement of bun and creatinine using an available commercial kit (biosystems) according to manufacturer's instruction . Protective effect of aqueous crude extract of t. crispa on renal damage and hemolysis during malaria infection was done based on standard 4-day suppressive test . Groups of icr mice (5 mice of each) were randomly divided, and infection was then performed with 1 10 parasitized erythrocytes of pbanka by ip injection . Two hours after infection, they were given orally by gavage with 500, 1000, and 2000 mg / kg of the extracts and every 24 h twice a day for 4 consecutive days . Three control groups were used; normal controls were given either distilled water or the extract while untreated control was given only distilled water . On day 4 of experiment, tail blood was collected to assess hemolysis by measurement of% hct . Parasitemia was first detectable on day 1 after infection with a parasitemia of 0.5% and reached 65% on day 10 (figure 1(a)). Next, we observed that bun and creatinine levels were markedly increased in infected mice, and first significant (p <0.05 and p <0.001) increases were found on day 4 after infection (figures 1(b) and 1(c)). Additionally, we also observed progressive decreases of% hct in infected mice in response to the presence of parasitemia (figure 1(d)). It was found that t. crispa stem extract showed protective effect on renal damage induced by pbanka infection in mice, and the highest activity was found at dose of 2000 mg / kg of extract treated group (figure 2). The bun and creatinine levels were increased significantly (p <0.01 and p <0.001, resp .) By malaria infection (untreated group) and infected mice treated with 500 mg / kg of the extract, compared to normal control . Interestingly, after oral administration of t. crispa stem extract, bun and creatinine levels declined significantly (p <0.01 and p <0.001, resp . ), especially at doses of 1000 and 2000 mg / kg, compared to untreated control . However, t. crispa stem extract could not be able to decrease parasitemia levels . T. crispa stem extract exerted antihemolytic effect against pbanka infected mice . As shown in figure 3, hemolysis was observed in untreated control and infected mice treated with 500 mg / kg of the extract as indicated by decreasing of% hct with significance (p <0.01), compared to normal control . Interestingly,% hct was significantly (p <0.01) increased in infected mice treated with 1000 and 2000 mg / kg of the extract, compared to untreated control . The highest effect was found at dose of 2000 mg / kg of the extract . Moreover, prolonged survival time of infected mice treated with this extract was also observed . In the current study, we provide evidence that describes the protective effect of t. crispa stem extract on renal damage and hemolysis induced by p. berghei infection in mice . The onset of renal damage and hemolysis in icr mice infected with pbanka come out from day 4 after infection and the incidence of renal damage and hemolysis were confirmed through manifestations such as increasing of bun and creatinine as well as decreasing of% hct in infected mice . Renal damage and hemolysis are proposed to be a consequence of parasite adhesion and exacerbated immune response against products of oxidative stress released during infection . In addition, parasitized erythrocyte sequestration at the microvascular site of renal has also been discussed to induce renal damage . The destruction of erythrocytes during blood stage infection accumulates high levels of toxic - free heme that has ability to induce oxidative stress from production of hydroxyl radicals via fenton / haber - weiss reaction . Moreover, heme - derived oxidative stress is considered to be a main factor in the iron - induced lipid peroxidation resulting in the formation of oxidized ldl . Therefore, these conditions derived by blood stage of infection result in renal damage and hemolysis . However, protective effects of renal damage and hemolysis induced by malaria infection were found in infected mice treated with t. crispa stem extract . Polyphenols and flavonoids show high antioxidant and anti - inflammation properties and then protect oxidative stress development . T. crispa stem extract has been shown to contain high amounts of polyphenols and flavonoids . Thus, the potent antioxidant activity of this extract might play a central role in protecting renal damage and hemolysis from oxidative stress induced by malaria infection . Moreover, t. crispa stem extract has been reported to have antimalarial activity against p. yoelii infection in mice . So, antimalarial activity to inhibit p. berghei growth and subsequently inhibit renal damage and hemolysis might also be mentioned . Taken together, our results suggest that t. crispa stem extract shows protective effects against p. berghei - induced renal damage and hemolysis . Appropriate pharmaceutical strategies might now be devised to increase the low bioavailability of this extract and to protect against rapid in vivo metabolic transformation, in such a way to make them more amenable as alternative antimalarial drug in combination therapies.
It is well known that patients struggle with the physical instillation of eyedrops . As many as 25% of patients who instill their own drops were unable to place a drop in their eye during a trial of drop instillation.1 when patients with visual impairment are taken into account, this percentage has been reported to be as high as 30%.2 in addition to struggling to place a drop in the eye, as many as 59% of patients have been observed to express multiple drops from the bottle in an attempt to get only one drop into the eye.1 furthermore, 80% of patients have been observed to make contact between the tip of the eyedrop bottle and the eye.3 defining successful eyedrop instillation as placing one drop in the conjunctival cul - de - sac without contaminating the tip of the bottle, one study found as few as 8.5% of patients to be successful.4 therefore, for many glaucoma patients who administer eyedrops, there is room for improvement in the arenas of efficiency, medication conservation, and contamination . If a patient is unable to actually place a drop in the eye, their disease will not be treated . If they place too many drops, the consequences can range from systemic side effects5 to increased expenses . The tip is often the most contaminated portion of eyedrop bottles.68 there have been multiple reports of eye infections that were associated with contaminated bottles.911 this leads to the possibility of eyedrops causing infection when instilled incorrectly . There have also been reports of inadvertent trauma from the tip of an eyedrop bottle, leading to conjunctival inflammation.12 one barrier that patients have reported in administering eyedrops is arthritis.13 cervical osteoarthritis can limit a person s ability to recline the head . Although there are several commercially available eyedrop aids that assist with aiming and squeezing the bottle, all require that the patient recline the head . We have developed an upright eyedrop bottle (ueb)14 to prevent the necessity of reclining the head . In an institutional review board (irb)-approved study with a small cohort of patients, the original device was tested for feasibility of use and to gather feedback . Based on the observations of the study team and feedback from participants, three subsequent prototypes were developed, each modified in an iterative process of design refinement and testing . The purpose of this study was to test the feasibility of use of the most recent iteration of the ueb (figure 1) among experienced eyedrop users . Secondary aims included studying bottle tip contamination rates and rates of excess drop usage using the ueb . Participants were recruited from a single academic glaucoma clinic from may 2014 to march 2015 in this duke medicine institutional review board - approved study . Included participants were at least 18 years of age, prescribed eyedrops for at least 6 months, reported regular self - administration of eye - drops, and answered yes to the question, do you ever have trouble getting your eyedrops in? Potential participants were excluded for best - corrected visual acuity <20/400 in both eyes and history of intraocular surgery within the preceding month . After giving written informed consent, participants were shown a multimedia presentation demonstrating the proper use of both the ueb as well as a standard eyedrop bottle . Following the presentation, participants filled out a questionnaire about their home eyedrop usage . Following the questionnaire, participants were taken to an exam room with an examination chair, mirror, sink, and tissues . Also in the room were two, closed sterile bottles containing saline solution, the ueb, and a trained observer to record the participant s results . The participants were randomized as to whether they would use the standard eyedrop bottle or ueb first . Each participant was instructed to place one drop in each eye from the first bottle, and afterward to place one drop in each eye from the second bottle . The observer recorded 1) time required for instillation, 2) if the participant placed at least one drop within the eye (accuracy), 3) if fluid rolled down the cheek indicating excess drops were used, and 4) if the participant contaminated the tip of the bottle by touching the eye or surrounding surfaces . Descriptive statistics were derived, including mean, median, range, and standard deviation . For the outcome of time to instillation of an eyedrop, the mcnemar s test for difference in paired proportions was used to test the difference between bottle types for the outcomes of accuracy, excess drop use, and contamination of the bottle . For all comparisons, in total, 40 participants were enrolled in this study . As illustrated in table 1, the average age of participants was 72.4 8.9 years, with the majority of participants being female and right - handed . More than half of the participants had visual acuity better than 20/40 in at least one eye, and 85% of the participants in the study had been using eyedrops for more than 1 year . As shown in table 2, the time required to instill drops with the ueb was shorter than with the conventional bottle in all three trials, with statistical significance in the last two trials . As noted in table 3, there was neither statistical significance nor pattern of differences in the number of participants able to instill a drop in their left or right eye across all three trials . Fluid rolled down the cheek significantly less with the ueb than the conventional bottle in all three trials of both eyes . Finally, the tip of the ueb was not contaminated in any trial by either eye; the tip was contaminated by 1620 patients per trial in all three trials with the conventional bottle . The purpose of this study was to address the feasibility of use of the ueb for eyedrop placement as well as to understand the rates of contamination and excess eyedrop use with the ueb . There was no statistically significant difference in accuracy of drop instillation between the conventional eyedrop bottle and ueb . Across all three trials and both eyes, approximately 70%90% of participants were able to accurately place a drop with both the conventional bottle and ueb . The ability to instill at least one drop in the eye in this study was within the same range reported by many other studies.13,1518 it is interesting that accuracy with the conventional bottle in this study was similar to other studies given that participants were specifically selected because they admitted to having trouble with self - administration of their eyedrops . They were not specifically asked what aspects of self - administration they struggled with and were not objectively screened for trouble instilling eyedrops either . These participants may have struggled more with other aspects of drop placement, rather than accuracy, which may explain why the ueb did not improve accuracy in this particular group of participants . The majority of participants in the study (34/40) had been taking drops for over a year, and thus had the opportunity to practice with the conventional bottle for over a year . The ueb was not known to patients prior to this study, and yet it still performed as well as the conventional bottle . In addition to performing as well as the conventional bottle, the ueb allowed participants to place their eyedrops faster than with the conventional bottle . Curiously, the time to instill drops decreased in each consecutive trial with not only the ueb but also the conventional bottle . It is possible that the conventional bottle used in the study differed from the one used at home (ie, different shape, size, or stiffness), and thus participants got faster as their comfort level with the bottle increased . Successful instillation of drops with the ueb with less time but not loss of accuracy was a surprising finding given that participants were using the ueb for the first time . Significantly fewer participants used excess drops with the ueb as compared to the conventional eyedrop bottle across all trials with both eyes . The percentage of participants using excess drops ranged from 63% to 74% with the conventional bottle and from 24% to 41% with the ueb . The percentage of participants expelling excess drops from the conventional bottle was slightly more than what stone et al1 found . Their research showed that up to 59% of patients used either more than one drop or instilled a stream of fluid.1 kass et al19 observed that 48% of patients used more than one drop . Brown et al3 recorded 21%, sleath et al15 found 30%, and hennessy et al16 observed 22% of patients used greater than one drop . Kass et al19 did find that the number of excess drops used correlated with increasing patient age . This may partially explain why more participants used excess drops from the conventional bottle than previous studies . The average age of participants was 72.4 8.9 years, older than the average age in any of the previously mentioned studies where fewer patients used excess drops . Given that glaucoma is more prevalent with advanced age, the age cohort of this study may represent the challenges faced with glaucoma eye - drop instillation more appropriately than studies with younger patients . In addition, participants were specifically selected for the study if they had difficulty with self - administration, which may include trouble applying only one drop . Finally, saline was used in both the conventional bottle and ueb in this study, which may have a lower viscosity than glaucoma medications, leading to a higher likelihood of it dribbling out of the eye . Less use of excess drops with the ueb compared to the conventional bottle may be related to the metered dose mechanism of delivery . The ueb has been designed with the intent that the volume that enters the fluid chamber of the device is the volume delivered . By pushing a button to deliver medication, patients deliver the same amount of medicine with every push . This is in contrast to squeezing a standard eye bottle, where a patient must estimate how much force to apply to expel a single drop . In addition, different bottles require different amounts of force,20 and as winfield et al21 found in their sample of patients, up to 13% cannot even generate enough force to expel one drop from a bottle.21 this difference in force may be due to bottle characteristics such as the dimensions, shape, wall thickness, or even bottle material . While the amount of force required to deliver medication was not tested with the ueb, it is possible that the metered dose mechanism may even allow patients who cannot squeeze a conventional bottle to deliver their own eyedrops . Examples of such side effects include bradycardia and bronchospasm due to ophthalmic solutions of -blockers.5 using excess drops can also lead to increased costs for patients . When patients use too many drops, this may cause them to run out of drops earlier than anticipated, leading to refills of medication that would otherwise be unnecessary, or running out of medication and being unable to obtain a refill prior to the authorized refill date . The most obvious area where the ueb differed from conventional eyedrop bottles was in contamination rates of the tip of the bottle . This is remarkable considering contamination rates for the conventional bottle in this study ranged from 42% to 53% . The contamination rates for the conventional bottle were similar to those found in other studies.19,22 the results suggest that the ueb will lower contamination rates . This is because the ueb design employs a physical barrier that keeps the tip of the bottle from touching the eye or its surrounding structures . Furthermore, the device is designed to be disposable after a single use, meaning the device does not need to be cleaned after use . One limitation of the prototype used in the study was that in order for the ueb to accept the conventional bottle, the tip of the conventional bottle needed to be popped off . This process has the potential to lead to contamination . In order to address this, a design has been developed that does not require the tip of the bottle to be removed . In addition to this limitation, the device cannot be used with single - use containers . One might speculate that patients will contaminate the tip of a conventional bottle less with experience, negating the need for a device to assist with this issue . Schwartz et al23 attempted to see if patients improved their eyedrop placement technique over a 12-week period . At the start of the 12-week period, 18.2% of patients contaminated the bottle compared to 18.5% at the end of the 12-week period.23 although the present study only observed participants over the course of three trials, the results agree with schwartz et al.23 contamination rates with the conventional bottle did not show a decreasing trend as trials progressed . Also important to note is that the majority (34/40) of the participants in this study had been using their drops for over a year and thus had experience already . This is in comparison to their lack of experience with the ueb, and yet they still did not contaminate the tip of the ueb bottle . Schein et al8 found that when patients conjunctiva were cultured for microorganisms, 34% of patients using topical ocular medications grew potentially pathogenic organisms compared to only 10% of patients who were not using medications . The same study found that 42% of patients had at least one of their medications contaminated, and of those patients with contaminated medications, 60% of them had the same organisms cultured from both their conjunctiva and their medication . Also, of those 42% of patients with a contaminated medication, 45% of them had a potentially pathogenic organism cultured from the medication . An interesting observation from this study was that when a patient had a potentially pathogenic organism grow from both their conjunctival and medication cultures, it was always a gram - negative organism.8 however, one must note that there are similar studies with different results . Geyer et al7 found no significant difference in positive conjunctival cultures between patients who were and were not using topical ocular medications . In that study, 28% of the medications had positive cultures, and of those, 22% had gram - negative organisms . None of the potentially pathogenic organisms were found in both the conjunctiva and medication of the same patient . It is interesting though, that in that study, 37 out of 37 patients who stated they touched their eyes while placing drops had at least one medication that was contaminated.7 while geyer et al7 and schein et al9 discuss the percentage of medications and conjunctiva with potentially pathogenic organisms, there have also been reports of eye infections associated with contaminated bottles.10,11 this is in addition to reports of inadvertent trauma leading to conjunctival inflammation.12 the ueb was able to decrease the number of tip contacts between the dropper and eye to zero, thus hopefully negating any degree of infection or inflammation that might occur as a result of tip contact . Other innovations and devices it is difficult to compare the performance of the ueb to these devices as information varies in the literature, but it is informative to understand examples of other eyedrop aids . Two such devices are the opticare eyedrop dispenser (cameron graham limited, huddersfield, uk) and xal - ease (pfizer ophthalmics, new york, ny, usa). The opticare eyedrop dispenser was designed to assist patients with conditions such as arthritis in squeezing their eyedrop bottles . When studied, it objectively improved patients ability to squeeze drops from a bottle from 48% to 90% and improved patients ability to get a drop into the eye with mild or no difficulty from 48% to 86%.24 it is however important to note that this device was studied in patients with rheumatoid arthritis . It would be interesting to see if improvements in accuracy were observed in patients without rheumatoid arthritis . One similarity between opticare and the ueb was that none of the patients contaminated the bottle with both opticare and the ueb . Subjectively, patients felt they were better able to squeeze and aim the bottle and dispense the proper amount of drops with opticare, but no objective measure of drops dispensed was presented.24 xal - ease delivery device was designed to help align the bottle over the eye and deliver only one drop using a button instead of squeezing the bottle . The device was only studied subjectively, but it was found that with the device patients felt they were less likely to need help with drop instillation . In addition, with the device, patients felt they contaminated the tip less (decreased from 35.6% to 3.2%), had increased accuracy (increased from 49.9% to 62.4%), and delivered less excess drops . There was no statistically significant difference in accuracy of drop instillation between the conventional eyedrop bottle and ueb . Across all three trials and both eyes, approximately 70%90% of participants were able to accurately place a drop with both the conventional bottle and ueb . The ability to instill at least one drop in the eye in this study was within the same range reported by many other studies.13,1518 it is interesting that accuracy with the conventional bottle in this study was similar to other studies given that participants were specifically selected because they admitted to having trouble with self - administration of their eyedrops . They were not specifically asked what aspects of self - administration they struggled with and were not objectively screened for trouble instilling eyedrops either . These participants may have struggled more with other aspects of drop placement, rather than accuracy, which may explain why the ueb did not improve accuracy in this particular group of participants . The majority of participants in the study (34/40) had been taking drops for over a year, and thus had the opportunity to practice with the conventional bottle for over a year . The ueb was not known to patients prior to this study, and yet it still performed as well as the conventional bottle . In addition to performing as well as the conventional bottle, the ueb allowed participants to place their eyedrops faster than with the conventional bottle . Curiously, the time to instill drops decreased in each consecutive trial with not only the ueb but also the conventional bottle . It is possible that the conventional bottle used in the study differed from the one used at home (ie, different shape, size, or stiffness), and thus participants got faster as their comfort level with the bottle increased . Successful instillation of drops with the ueb with less time but not loss of accuracy was a surprising finding given that participants were using the ueb for the first time . Significantly fewer participants used excess drops with the ueb as compared to the conventional eyedrop bottle across all trials with both eyes . The percentage of participants using excess drops ranged from 63% to 74% with the conventional bottle and from 24% to 41% with the ueb . The percentage of participants expelling excess drops from the conventional bottle was slightly more than what stone et al1 found . Their research showed that up to 59% of patients used either more than one drop or instilled a stream of fluid.1 kass et al19 observed that 48% of patients used more than one drop . Brown et al3 recorded 21%, sleath et al15 found 30%, and hennessy et al16 observed 22% of patients used greater than one drop . Kass et al19 did find that the number of excess drops used correlated with increasing patient age . This may partially explain why more participants used excess drops from the conventional bottle than previous studies . The average age of participants was 72.4 8.9 years, older than the average age in any of the previously mentioned studies where fewer patients used excess drops . Given that glaucoma is more prevalent with advanced age, the age cohort of this study may represent the challenges faced with glaucoma eye - drop instillation more appropriately than studies with younger patients . In addition, participants were specifically selected for the study if they had difficulty with self - administration, which may include trouble applying only one drop . Finally, saline was used in both the conventional bottle and ueb in this study, which may have a lower viscosity than glaucoma medications, leading to a higher likelihood of it dribbling out of the eye . Less use of excess drops with the ueb compared to the conventional bottle may be related to the metered dose mechanism of delivery . The ueb has been designed with the intent that the volume that enters the fluid chamber of the device is the volume delivered . By pushing a button to deliver medication, patients deliver the same amount of medicine with every push . This is in contrast to squeezing a standard eye bottle, where a patient must estimate how much force to apply to expel a single drop . In addition, different bottles require different amounts of force,20 and as winfield et al21 found in their sample of patients, up to 13% cannot even generate enough force to expel one drop from a bottle.21 this difference in force may be due to bottle characteristics such as the dimensions, shape, wall thickness, or even bottle material . While the amount of force required to deliver medication was not tested with the ueb, it is possible that the metered dose mechanism may even allow patients who cannot squeeze a conventional bottle to deliver their own eyedrops . Examples of such side effects include bradycardia and bronchospasm due to ophthalmic solutions of -blockers.5 using excess drops can also lead to increased costs for patients . When patients use too many drops, this may cause them to run out of drops earlier than anticipated, leading to refills of medication that would otherwise be unnecessary, or running out of medication and being unable to obtain a refill prior to the authorized refill date . The most obvious area where the ueb differed from conventional eyedrop bottles was in contamination rates of the tip of the bottle . This is remarkable considering contamination rates for the conventional bottle in this study ranged from 42% to 53% . The contamination rates for the conventional bottle were similar to those found in other studies.19,22 the results suggest that the ueb will lower contamination rates . This is because the ueb design employs a physical barrier that keeps the tip of the bottle from touching the eye or its surrounding structures . Furthermore, the device is designed to be disposable after a single use, meaning the device does not need to be cleaned after use . One limitation of the prototype used in the study was that in order for the ueb to accept the conventional bottle, the tip of the conventional bottle needed to be popped off . This process has the potential to lead to contamination . In order to address this, a design has been developed that does not require the tip of the bottle to be removed . In addition to this limitation, the device cannot be used with single - use containers . One might speculate that patients will contaminate the tip of a conventional bottle less with experience, negating the need for a device to assist with this issue . Schwartz et al23 attempted to see if patients improved their eyedrop placement technique over a 12-week period . At the start of the 12-week period, 18.2% of patients contaminated the bottle compared to 18.5% at the end of the 12-week period.23 although the present study only observed participants over the course of three trials, the results agree with schwartz et al.23 contamination rates with the conventional bottle did not show a decreasing trend as trials progressed . Also important to note is that the majority (34/40) of the participants in this study had been using their drops for over a year and thus had experience already . This is in comparison to their lack of experience with the ueb, and yet they still did not contaminate the tip of the ueb bottle . Schein et al8 found that when patients conjunctiva were cultured for microorganisms, 34% of patients using topical ocular medications grew potentially pathogenic organisms compared to only 10% of patients who were not using medications . The same study found that 42% of patients had at least one of their medications contaminated, and of those patients with contaminated medications, 60% of them had the same organisms cultured from both their conjunctiva and their medication . Also, of those 42% of patients with a contaminated medication, 45% of them had a potentially pathogenic organism cultured from the medication . An interesting observation from this study was that when a patient had a potentially pathogenic organism grow from both their conjunctival and medication cultures, it was always a gram - negative organism.8 however, one must note that there are similar studies with different results . Geyer et al7 found no significant difference in positive conjunctival cultures between patients who were and were not using topical ocular medications . In that study, 28% of the medications had positive cultures, and of those, 22% had gram - negative organisms . None of the potentially pathogenic organisms were found in both the conjunctiva and medication of the same patient . It is interesting though, that in that study, 37 out of 37 patients who stated they touched their eyes while placing drops had at least one medication that was contaminated.7 while geyer et al7 and schein et al9 discuss the percentage of medications and conjunctiva with potentially pathogenic organisms, there have also been reports of eye infections associated with contaminated bottles.10,11 this is in addition to reports of inadvertent trauma leading to conjunctival inflammation.12 the ueb was able to decrease the number of tip contacts between the dropper and eye to zero, thus hopefully negating any degree of infection or inflammation that might occur as a result of tip contact . Other innovations and devices have attempted to address the various aspects of eyedrop placement that patients struggle with . It is difficult to compare the performance of the ueb to these devices as information varies in the literature, but it is informative to understand examples of other eyedrop aids . Two such devices are the opticare eyedrop dispenser (cameron graham limited, huddersfield, uk) and xal - ease (pfizer ophthalmics, new york, ny, usa). The opticare eyedrop dispenser was designed to assist patients with conditions such as arthritis in squeezing their eyedrop bottles . It also serves to improve aim and decrease contamination . When studied, it objectively improved patients ability to squeeze drops from a bottle from 48% to 90% and improved patients ability to get a drop into the eye with mild or no difficulty from 48% to 86%.24 it is however important to note that this device was studied in patients with rheumatoid arthritis . It would be interesting to see if improvements in accuracy were observed in patients without rheumatoid arthritis . One similarity between opticare and the ueb was that none of the patients contaminated the bottle with both opticare and the ueb . Subjectively, patients felt they were better able to squeeze and aim the bottle and dispense the proper amount of drops with opticare, but no objective measure of drops dispensed was presented.24 xal - ease delivery device was designed to help align the bottle over the eye and deliver only one drop using a button instead of squeezing the bottle . The device was only studied subjectively, but it was found that with the device patients felt they were less likely to need help with drop instillation . In addition, with the device, patients felt they contaminated the tip less (decreased from 35.6% to 3.2%), had increased accuracy (increased from 49.9% to 62.4%), and delivered less excess drops . This study had several limitations in addition to those already mentioned in the discussion section . Participants were recruited from a single academic institution, and so it is possible that a different study population may have shown different results . Study participants were asked to instill their eyedrops in an artificial setting as they were in a clinic and possibly using bottles that were different from those they used on a daily basis . Eyedrop administration was scored by a trained observer but not videotaped, which would have allowed review to ensure fidelity . Up to 78% of patients welcome the idea of a compliance aid to assist in eyedrop administration.21 the ueb differs from other compliance aids in that it allows patients to place eyedrops without reclining their head . While the ueb does not improve accuracy of drop placement, it does lead to less drop wastage and less bottle contamination
Examples of these iatrogenic behaviors seen in pd include: binge eating, excessive spending, compulsive shopping, pathological gambling, and hypersexuality.1 hypersexuality in particular is a psychiatric issue that patients are often reluctant to discuss . Voon and colleagues found the lifetime prevalence of pathologic hypersexuality in pd to be 2.4%.1 hypersexual behavior typically entails an increase in premorbid sexual activities as well as an increase in the variety of sexual behaviors.2 while no direct cause has been identified, voon and colleagues found hypersexuality to be significantly associated with male sex and an earlier age of pd onset.1 treatment with dopamine agonist therapy and a history of impulse control disorders has also been proposed as possible risk factors.3 this study investigates prevalence and demographic characteristics of hypersexual behavior in pd patients residing in north central florida, usa . Four hundred packets were mailed to all patients diagnosed with idiopathic pd enrolled in the movement disorders center of the university of florida . The diagnosis was made by a fellowship trained movement disorders specialists and was based on fulfilling strict united kingdom brain bank criteria.4 as approved by the university of florida institutional review board, in the survey phase of this study, the subject s response to the questionnaire was considered an implied consent . In addition, all patients signed a separate informed consent to allow us to contact them, if needed, and to have their data analyzed . Information on demographics, health history, current therapies, and hypersexual behavior was obtained in addition to the neuropsychological inventory (npi) and beck s depression inventory (bdi). Patients were asked directly if they participated in hypersexual behavior on the hypersexual behavior questionnaire (see table 1). As part of the mail - in survey, the patients were asked if they would like to be further contacted in regard to this research survey . Only those patients who consented were subsequently interviewed for confirmation and classification to determine if they met the diagnostic criteria for subsybdromal or pathological hypersexuality as outlined by voon and colleagues (see table 2).1 the patient s significant other participated in the interview if the patient consented and was available at the time . Continuous demographic variables (eg, age of onset, pd duration, npi, bdi) were analyzed using unpaired t tests . Categorical demographic variables (eg, gender, handedness, marital status, times married, side of pd onset, smoking history, psychiatric history, family psychiatric history, dopamine agonist therapy, and levodopa therapy) were analyzed using fisher s exact tests and chi - squared tests . All statistical analyses were performed using graphpad prism (graphpad software, inc ., la jolla, ca, usa) software . One hundred forty - one from 400 (35.3%) patients returned completed research packets . This sample had a mean age of 68.0 years (10.2 yrs) and an average pd duration of 6.7 years (5.9 yrs). Only six of 15 patients met criteria for pathological hypersexuality (n = 5) or subsyndromal hypersexuality (n = 1). Whenever there was a question on the veracity of patient reports or reliability of their testimony, the subject s partner participated in the interviews, and in all cases these patients qualified as exhibiting pathological hypersexual behavior . In these six patients, the most common hypersexual behavior reported was increased pornography interest (magazine, video, or internet) (three of the six patients), and compulsive masturbation (two patients). Individually, patients also reported novel interests in a wide range of behaviors including sexual toys, sadomasochism, prostitution, and cross - dressing . The average age of pd onset was significantly lower in the hypersexual group (50.2 yrs 3.0) compared to the nonhypersexual group (61.5 yrs 1.0) (t(124) = 2.437; p <0.05). No significant association was found between hypersexual behavior and gender, handedness, marital status, times married, side of pd onset, psychiatric history, family psychiatric history, dopamine agonist therapy, levodopa therapy, pd duration, npi or bdi score . Interestingly, this sample showed a significant association between a history of smoking and not developing hypersexual behavior (fisher s exact test p <0.05). Given the innate sensitivity of the topic, it is very likely that hypersexual behavior is under - reported . Despite the limitations (and probable underestimation) of a mail - in survey, the prevalence of hypersexuality in our pd cohort (4.3%) was slightly higher than the 2.4% reported by voon and colleagues.1 patients with a younger age of pd onset were more likely to exhibit hypersexual behavior . Previously, hypersexuality was associated with males but not females.1 our current study failed to substantiate this finding . This highlights the possibility of hypersexual behavior in female patients that may be missed due to the inherently small sample size of hypersexual patients . Although the association between younger age of onset and hypersexual behavior might be thought to be due to a greater use of dopamine agonist therapy in these patients, no significant relationship was found between dopamine agonist therapy and hypersexual behavior in this sample . Klos and colleagues found that hypersexual behavior typically began after eight months of dopamine therapy and then resolved after discontinuing dopamine agonist therapy.5 it should be noted that a few patients in our study did mention a similar experience . Shaw and colleagues proposed dopamine therapy in general may cause hypersexuality both directly, by increasing libido and arousal, and indirectly by interacting with reward - center neural substrates.2 this is in line with the voon and colleagues finding that a higher total levodopa dose equivalent, but not dopamine agonist dose, was associated with hypersexuality.1 intuitively, it seems very likely that manipulation of the dopamine system would result in changes in reward - seeking behavior . Additional compulsive behaviors coincide with hypersexuality in up to 60% of hypersexual pd patients.5 given that only a relatively small percentage of pd patients exhibit hypersexuality or any type of impulse control disorder, it seems likely that susceptibility may be due to underlying heritable temperamental traits such as novelty - seeking.6 moreover, pontone and colleagues found an association between impulse control disorders and depressed mood, disinhibition, irritability, and appetite disturbance.7 table 3 shows demographic data from our current hypersexual population . While not statistically significant, five out of six patients had either a personal or family history of previous psychiatric disorders . Notably, none of the patients had a history of hypersexual behavior prior to pd onset . Also, there appears to be a trend of elevated depression scores, as measured by the bdi, in a majority of these patients . It should be noted that this study relied on a population of patients all from a single movement disorders center . Similar studies in subsequent populations would help to eliminate the possibility of a population bias . Also, due to survey limitations, as well as possible patient cognitive decline, since we relied on initial self - identification of hypersexual behavior before interviewing, it is quite possible that not all patients were able or willing to make this identification . In the future, it confirms previous reports that it occurs more commonly in the younger pd age group . Clinicians should be aware of possible hypersexual behavior in both sexes, regardless of dopamine agonist therapy, and be prepared to provide information and support to their patients and their patients caregivers.
Kleine - levin syndrome (kls) is a rare disorder which mainly affects adolescent boys and characterized by recurrent episodes of hypersomnia, and sometime along with hyperphagia, behavioral and cognitive disturbances, and hypersexuality . Several medications (stimulants, lithium, valproate, antipsychotics, and antidepressants) have been reported to provide variable benefit in different symptoms, with lithium being the most widely used drug . We are presenting a case of kls, who had complete remission with valproate and also reviewing the cases reported from india . A 17-year - old single male student of 12 standard, presented to our psychiatric outpatient clinic in september 2004 with hypersomnolence, low mood, decreased appetite and interest in studies, social and sexual disinhibition (such as singing obscene songs loudly at home, and touching unconsenting females including mother's body parts - limbs, face and genitalia). Onset was acute, without any elicitable precipitating factor and course was episodic with average 7 - 10 days episode in every month for last 4 months and he maintained completely well in inter episodic period . Provisional diagnosis of recurrent depressive disorder (brief episodes) was kept and he was started on sertraline (50 mg), on which he responded well . He remained asymptomatic for nearly 9 months, but started having similar episodes again from mid-2005, due to which sertraline were gradually hiked up to 150 mg / day, but of no use . Hence, he was admitted in our inpatient setting in march, 2006 for diagnostic evaluation and further management . After detailed evaluation, it was found that his sadness was not pervasive and depressive cognitions and associated disturbances were not present and hypersomnia remained predominant complaint as initially, he was sleeping 16 - 20 h / day . He was also not responding with these medications, hence differential diagnosis of kls versus depression was kept and later finalized to kls . His hemogram, renal functions, liver functions, blood sugar, routine urine, thyroid functions were within normal limits and chest x - ray, electrocardiography, electroencephalography (eeg), and magnetic resonance imaging brain were normal . In view of good literature support lithium was started from 600 mg / day and hiked to 900 mg / day (serum level 0.8 meq / l). On which he has shown significant improvement initially for 6 months, but later again started experiencing similar symptoms . He also had three episodes of fall, unresponsiveness and epileptiform discharge in eeg twice . Hence, in view of seizure disorder and lack of response, neurologist's consultation was sought, who opined to start antiepileptic medication . Hence, lithium was switched to valproate (750 mg / day) in december 2006, on which he maintained completely well for 4 years, except brief re - emergence of symptoms on discontinuing valproate, which improved completely on resuming the medication . Valproate was gradually tapered and stopped in january 2011 on insistence of patient and family with discussing its pros and cons . Now index case has been maintaining well - off valproate for last 3 years without any episode of hypersomnolence, sexual disinhibition, sadness, or epileptic seizure . Based on historical reports by kliene and levin, kls was essentially described and termed by critchley . Thereafter, many researchers have reported their cases and reviewed cases with kls . Here, we are reporting a case with kls, who responded well with valproate, after diagnostic dilemma and different psychotropic medications and also reviewing the other cases reported from india . In our electronic search for indian studies on kls, by using pubmed and google scholar, we could find 15 cases reported from india . Of them 13 were males and 2 females, similar to male preponderance reported in the literature . While presenting to psychiatric services their age was between 9 and 26 years and they had onset between 7 and 24 years of age . In two - third of patients (10 out of 15 patients), it was preceded with fever and their episodes of somnolence were lasted from 3 days to 10 weeks . Hypersomnia and hyperphagia were present in all, while two - third of patients also had social and sexual disinhibition (11 out of 15 patients). Other symptoms were cognitive disturbances (low intelligence quotient, impaired memory, confusion, and academic decline), irrelevant talk, and perceptual disturbances . Nearly one - third of patients improved spontaneously without any medication, while rest was given lithium, carbamazepine, methylamphetamine, dextroamphetamine, and modafinil . Longest asymptomatic follow - up period is reported for 2 years [table 1]. Reported cases with kls from india though literature supported lithium for higher response rate, but index patient had remarkable response with valproate, not with lithium, like earlier two reports . Like earlier report, index patient also improved on lower dose of valproate (divalproate 750 mg vs. 500 mg valproate). Compared to other cases reported from india, index patient had longest follow - up (7 years) and remained asymptomatic in this period, except small exacerbation on discontinuation of valproate treatment, which improved completely on resuming the drug . Similar to our patient, anticonvulsants (like valproate) are the preferred treatment for kls patient, and may also offer benefits in case of comorbid epilepsy . Valproate may be a good alternative to lithium in terms of efficacy as well as side - effect profile.
A 25-year - old female presented with right eye sudden and painless blurring of vision for 4 weeks, who visited a hilly area in the himalayas, north india . No history of headache, fever, stiff neck, malaise, nausea, redness, watering of eyes, floaters, and trauma was reported . Her visual acuity (va) in the right eye was 20/60 (unaided) and 20/20 in the left eye . Anterior chamber was normal, relative afferent pupil defect was present, and fundus examination showed signs of papillitis, superior temporal retinitis contiguous with disc, vasculitis involving large vessels near the disc [fig . 1]. Examination with + 78 d revealed vitreous cells in the vicinity of neuroretinitis . Fundus fluorescein angiography (ffa) and optical coherence tomography (oct) confirmed the diagnosis of neuroretinitis [fig . The patient was empirically started on oral steroids (prednisone 40 mg / day [1 mg / kg]). Fundus photograph showing papillitis, neuroretinitis, vasculitis (a) fundus fluorescein angiography showing active vasculitis . (b) optical coherence tomography showing vitritis rheumatoid factor, cytoplasmic antineutrophil cytoplasmic antibodies (c - anca), perinuclear anti - neutrophil cytoplasmic antibodies (p - anca) (anti - myeloperoxidase) antibodies, antinuclear antibody, anti - double - standard antibody, angiotensin converting enzyme, human leukocyte antigen b-27, rapid plasma reagin and fluorescent treponemal antibody test, treponema pallidum immobilization and t. pallidum hemagglutination assay for syphilis, toxoplasma titer, hiv elisa were within limits . Lyme disease antibodies, igg and igm serum were 0.3 (<0.90) and 9.1 (<0.90). Mantoux test (5 mm 5 mm), chest x - ray, magnetic resonance imaging head, and echocardiography revealed no abnormality . On follow - up examination after 1 week, her va dropped to hand movement close to face and signs of fluid retention were present . 3b]. Considering the worsening of signs, symptoms, and positive igm tire for borrelia burgdorferi, serum western blot analysis test confirmed lyme disease . Oral doxycycline 100 mg was administered twice daily for 6 weeks and prednisone was tapered over a period of 4 weeks . Fundus and oct showed settling of neurosensory detachment and reduction of intraretinal fluid [fig . (b) macular optical coherence tomography showing neurosensory detachment and vitritis (a) fundus photograph showing resolving lesions . (b) optical coherence tomography showing absence of neurosensory detachment and a normal foveal contour the infectious agent of lyme disease is b. burgdorferi, a spirochete, isolated successfully from blood, synovial fluid, spinal fluid, retina, and vitreous of the eye . It is transmitted by the vector ixodes tick bite and occurs in three chronological stages; primary, secondary, and tertiary table 1 . Over 50% of untreated, treated initially, and on treatment, patients may develop stage 3 lyme disease . Hence, long follow - up is recommended for early diagnosis of cardiac, neurological, and arthritic involvement . The centers for disease control and prevention made lyme disease a reportable disease in 1982 . Major manifestations individual case reports with positive lyme serology have reported papillitis, anterior ischemic optic neuropathy, optic neuritis, and neuroretinitis; false positives have been up to 10% among residents of endemic areas without any apparent signs of infection . The current definition of lyme disease includes a patient who develops erythema migrans (em) within 30 days of exposure in an endemic area or endemic area exposure, without em, but with signs involving one organ system and a positive laboratory test or no history of exposure, but with em as well as involvement of two organ systems or no exposure in an endemic area, but with em and a positive serology . Our patient was diagnosed as lyme disease based on history of exposure, involvement of one organ, i.e., eye and positive serology for b. burgdorferi . Neuroretinitis is a focal inflammation of the optic nerve and peripapillary retina or macula of either infectious or idiopathic etiology . Infectious causes are cat scratch disease, syphilis, lyme disease, toxoplasmosis, mumps, salmonellosis, tuberculosis, and histoplasmosis . Although rare in india, we need to consider lyme disease in the differential diagnosis of neuroretinitis, especially if the patient hails from a forested area . Babu and murthy and vanya et al . Have reported resolution of neuroretinitis with a combination of doxycycline and corticosteroids . Long - term use of antibiotics is successful in preventing disease progression to tertiary stage . We conclude that neuroretinitis can be the presenting and the only presentation of lyme disease, hence should be considered in the differential diagnosis of neuroretinitis . Documentation with fundus photography, ffa, and oct for response to treatment is recommended . Doxycycline and corticosteroids should be started at the earliest as it is a treatable disease.
In the present study, a simplified mathematical model of the dynamic activation of pka and erk was developed (fig . Inducer. Because activation of erk and pka are both required for ltf, inducer was proportional to the product of pka and erk activities . The peak levels of inducer, which corresponded to the peak synergistic interaction between pka and erk, were hypothesized to predict the efficacy of stimulus protocols . As a point of reference, the simulated peak level of inducer produced by five, 5-min pulses of 5-ht with uniform 20-min interstimulus intervals (isis) was selected (fig . This' standard' protocol has been widely used in experimental studies since its introduction in 1986 . To identify a protocol that produced the highest peak level of inducer, 9,999 alternative protocols were simulated . Each protocol included five, 5-min 5-ht stimuli, but the isis were chosen as multiples of 5 min, in the range from 550 min (supplementary movie 1). Thus, each of the four isis had 10 possible values (i.e., 10 total possible permutations, one of which represented the standard protocol). Simulation of these protocols revealed considerable variability in the peak level of inducer (supplementary fig . The protocol that maximized the peak level of inducer (the' enhanced' protocol) consisted of non - uniformly spaced 5-ht applications with isis of 10, 10, 5, and 30 min . 1c). To the extent that the model captured the salient features of the dynamics of pka and erk and the extent to which the synergism between pka and erk activity is essential for ltf, the enhanced protocol was predicted to induce more ltf than the standard protocol . Three groups of preparations were examined, the standard and enhanced protocols (see fig . 1b, c) and a vehicle (control) group, which received five 5-min applications of solution without 5-ht (20-min isis). In each group, excitatory postsynaptic potentials (epsps) were measured prior to 5-ht treatment (pretest) and 1, 2, and 5 d after 5-ht treatment (post - tests). Long - term facilitation was defined as a significant increase in the epsp amplitude in either the standard or enhanced groups as compared to epsps measured in the control group . Sample recordings are illustrated in figure 2a and summary data are illustrated in figure 2b . Statistical analyses (two - way anova) indicated significant overall differences in the amplitude of epsps among the three treatment groups (f2,73 = 22.06, p <0.001) and among the three time points (f2,73 = 7.28, p <0.002). Post hoc, pair - wise comparisons (student - newman - keuls tests, snk) indicated that 1 day after 5-ht treatment both the standard protocol (q2 = 4.49, n = 10, p <0.003) and enhanced protocol (q3 = 7.57, n = 11, p <0.001) induced ltf as compared to vehicle controls (n = 8). The ltf that was induced by the standard protocol, however, did not persist beyond the first day (day 2: q2 = 2.54, n = 10; day 5: q2 = 0.21, n = 9) as compared to controls on day 2 (n = 8) and on day 5 (n = 8). In contrast, the ltf that was induced by the enhanced protocol persisted for up to five days (day 2: q3 = 5.21, n = 9, p <0.002; day 5: q2 = 3.41, n = 9, p <0.02) as compared to vehicle controls . In addition, post hoc analyses indicated that the enhanced protocol induced significantly greater ltf at all time points as compared to the standard protocol (day 1: q2 = 3.18, p <0.03; day 2: q2 = 2.89, p <0.05; day 5: q3 = 3.73, p <0.03). The induction and consolidation of ltf in vitro depends on the activity of several transcription factors . For example, increased levels of creb1 phosphorylation are associated with the induction and consolidation of ltf at sensorimotor synapses . Because the enhanced protocol induced greater and longer - lasting ltf, the enhanced protocol was hypothesized to be associated with a greater level of creb1 phosphorylation . Three groups of sensory neurons cultures were examined, one group was treated with the standard protocol, the second group was treated with the enhanced protocol identical to that used in the electrophysiological experiments of figure 2, and the third group was a vehicle control . In each group, the level of creb1 phosphorylation was measured either immediately (0 h) or 18 h after treatment, and the values in the standard and enhanced groups were normalized to vehicle controls . Statistical analyses (two - way anova) indicated overall significant differences between the two 5-ht protocols (f1,18 = 30.12, p <0.001). Pair - wise comparisons (snk tests) indicated that compared to the standard protocol, the enhanced protocol induced significantly higher levels of creb1 phosphorylation immediately (q2 = 6.94, n = 4, p <0.001), and 18 h after treatment (q2 = 3.69, n = 7, p <0.02). Both long - term synaptic plasticity and modifications in creb1 are thought to be key processes underlying long - term memory . Having demonstrated that the enhanced protocol improved ltf and phosphorylation of creb1, the enhanced protocol was hypothesized to produce stronger learning than the standard protocol in behaving animals . 4). Statistical analyses (two - way anova) indicated significant differences among the three training groups (f2,82 = 11.77, p <0.001). Pair - wise comparisons (snk tests) indicated that both the standard protocol (q2 = 3.12, n = 12, p <0.04) and enhanced protocol (q3 = 4.13, n = 14, p <0.02) induced lts that lasted 1 d. the lts that was induced by the standard protocol, however, did not persist for five days (q2 = 0.38, n = 14). In contrast, the lts that was induced by the enhanced protocol persisted for at least five days (q3 = 5.48, n = 9, p <0.001). In the present study, a simplified mathematical model of the dynamic activation of pka and erk was developed (fig . Inducer. Because activation of erk and pka are both required for ltf, inducer was proportional to the product of pka and erk activities . The peak levels of inducer, which corresponded to the peak synergistic interaction between pka and erk, were hypothesized to predict the efficacy of stimulus protocols . As a point of reference, the simulated peak level of inducer produced by five, 5-min pulses of 5-ht with uniform 20-min interstimulus intervals (isis) was selected (fig . This' standard' protocol has been widely used in experimental studies since its introduction in 1986 . To identify a protocol that produced the highest peak level of inducer, 9,999 alternative protocols were simulated . Each protocol included five, 5-min 5-ht stimuli, but the isis were chosen as multiples of 5 min, in the range from 550 min (supplementary movie 1). Thus, each of the four isis had 10 possible values (i.e., 10 total possible permutations, one of which represented the standard protocol). Simulation of these protocols revealed considerable variability in the peak level of inducer (supplementary fig . The protocol that maximized the peak level of inducer (the' enhanced' protocol) consisted of non - uniformly spaced 5-ht applications with isis of 10, 10, 5, and 30 min . 1c). To the extent that the model captured the salient features of the dynamics of pka and erk and the extent to which the synergism between pka and erk activity is essential for ltf, the enhanced protocol was predicted to induce more ltf than the standard protocol . Three groups of preparations were examined, the standard and enhanced protocols (see fig . 1b, c) and a vehicle (control) group, which received five 5-min applications of solution without 5-ht (20-min isis). In each group, excitatory postsynaptic potentials (epsps) were measured prior to 5-ht treatment (pretest) and 1, 2, and 5 d after 5-ht treatment (post - tests). Long - term facilitation was defined as a significant increase in the epsp amplitude in either the standard or enhanced groups as compared to epsps measured in the control group . Sample recordings are illustrated in figure 2a and summary data are illustrated in figure 2b . Statistical analyses (two - way anova) indicated significant overall differences in the amplitude of epsps among the three treatment groups (f2,73 = 22.06, p <0.001) and among the three time points (f2,73 = 7.28, p <0.002). Post hoc, pair - wise comparisons (student - newman - keuls tests, snk) indicated that 1 day after 5-ht treatment both the standard protocol (q2 = 4.49, n = 10, p <0.003) and enhanced protocol (q3 = 7.57, n = 11, p <0.001) induced ltf as compared to vehicle controls (n = 8). The ltf that was induced by the standard protocol, however, did not persist beyond the first day (day 2: q2 = 2.54, n = 10; day 5: q2 = 0.21, n = 9) as compared to controls on day 2 (n = 8) and on day 5 (n = 8). In contrast, the ltf that was induced by the enhanced protocol persisted for up to five days (day 2: q3 = 5.21, n = 9, p <0.002; day 5: q2 = 3.41, n = 9, p <0.02) as compared to vehicle controls . In addition, post hoc analyses indicated that the enhanced protocol induced significantly greater ltf at all time points as compared to the standard protocol (day 1: q2 = 3.18, p <0.03; day 2: q2 = 2.89, p <0.05; day 5: q3 = 3.73, p <0.03). The induction and consolidation of ltf in vitro depends on the activity of several transcription factors . For example, increased levels of creb1 phosphorylation are associated with the induction and consolidation of ltf at sensorimotor synapses . Because the enhanced protocol induced greater and longer - lasting ltf, the enhanced protocol was hypothesized to be associated with a greater level of creb1 phosphorylation . Three groups of sensory neurons cultures were examined, one group was treated with the standard protocol, the second group was treated with the enhanced protocol identical to that used in the electrophysiological experiments of figure 2, and the third group was a vehicle control . In each group, the level of creb1 phosphorylation was measured either immediately (0 h) or 18 h after treatment, and the values in the standard and enhanced groups were normalized to vehicle controls . Statistical analyses (two - way anova) indicated overall significant differences between the two 5-ht protocols (f1,18 = 30.12, p <0.001). Pair - wise comparisons (snk tests) indicated that compared to the standard protocol, the enhanced protocol induced significantly higher levels of creb1 phosphorylation immediately (q2 = 6.94, n = 4, p <0.001), and 18 h after treatment (q2 = 3.69, n = 7, p <0.02). Both long - term synaptic plasticity and modifications in creb1 are thought to be key processes underlying long - term memory . Having demonstrated that the enhanced protocol improved ltf and phosphorylation of creb1, the enhanced protocol was hypothesized to produce stronger learning than the standard protocol in behaving animals . 4). Statistical analyses (two - way anova) indicated significant differences among the three training groups (f2,82 = 11.77, p <0.001). Pair - wise comparisons (snk tests) indicated that both the standard protocol (q2 = 3.12, n = 12, p <0.04) and enhanced protocol (q3 = 4.13, n = 14, p <0.02) induced lts that lasted 1 d. the lts that was induced by the standard protocol, however, did not persist for five days (q2 = 0.38, n = 14). In contrast, the lts that was induced by the enhanced protocol persisted for at least five days (q3 = 5.48, n = 9, p <0.001). The results indicated that computational approaches could be used to design a stimulus protocol that increases long - term synaptic plasticity, transcriptional activation, and long - term sensitization . Interestingly, the non - uniform distribution of isis in the enhanced protocol was in marked contrast to the fixed intervals used in previous protocols for behavioral training in aplysia . For example, a form of training with expanding isis appears to lead to enhanced performance in verbal recall tasks . The mechanistic underpinnings of this form of training are not well understood, and therefore provide little guidance for designing protocols that maximize retention . The present results suggested that modeling the dynamics of biochemical cascades could play an essential role in understanding why specific training protocols are more effective than others, and thereby, help guide the development of better learning paradigms . A goal of the present study was to develop training protocols that enhanced learning and memory . However, the model described in fig . 1a was also compatible with previous empirical results in which a massed training protocol (25 min continuous 5-ht) produced significantly less ltf than a spaced protocol (five, 5-min pulses of 5-ht). Simulations of this massed protocol produced a peak level of inducer of only 0.00016 m, which was at the low end of the distribution illustrated in supplementary fig ., the model was able to predict submaximal performance, which helped to increase confidence in its predictive power . These results indicate that the improvement found with the enhanced protocol did not represent a narrow range of model parameter values, but occurred for various combinations of values . Such behavior is expected for robust models of physiological processes, in which optima should not be sharply peaked at very specific parameter values . In humans, a number of cognitive models have been proposed to explain the superiority of spaced training or practice, but no consensus has emerged as to their usefulness or generality . It is likely that these models have had limited predictive ability, at least in part, because they are not based on known neurobiological processes . In contrast, the present model was based on well characterized biochemical cascades in individual neurons that participate in the induction and expression of ltm . Although the present model was limited to a description of the dynamics of biochemical cascades associated with the initial induction of ltm and to relatively brief training sessions (e.g., 1.5 h), the model successfully predicted a protocol that enhanced synaptic strength and memory for at least five days after stimulation . These results confirmed that important aspects of the induction of ltm occur during the initial training phase . For example, the slow dynamics of additional biochemical cascades contribute to ltm (e.g., changes in the synthesis of transcription factors such as creb1 and creb2)., the model did not include descriptions of postsynaptic protein kinase c, upregulation of local protein synthesis in the vicinity of the synapse or synthesis of the peptide sensorin in response to retrograde signals, which are involved in ltf . Finally, the model did not account for the differences in the time course and magnitude of ltf (fig . 4). Several factors may contribute to this discrepancy, including a nonlinear transformation of ltf into motor neuron activity and plasticity at other loci in the neural circuit that contribute to the behavioral enhancement . An expanded model that includes the dynamics of additional biochemical cascades and neural circuit elements would presumably have greater predictive capability . The present results indicated the feasibility of using computational methods to assist in the design of training procedures that enhance learning . It will be important to determine the extent to which the computational approaches can be applied to other memory systems, and ultimately, applied to training procedures used to improve human cognition . A detailed description of the methods is available in the online version of the paper at http://www.nature.com / natureneuroscience/. Note: supplementary information is available on the nature neuroscience website.
Traditionally, overnight fasting before elective surgery has been the routine in medical practice for risk reduction of pulmonary aspiration of gastric contents . But, some studies have shown that prolonged fasting does not reduce gastric contents, and instead causes an increase in patient discomfort . Consequently, several recent international guidelines have recommended to shorten the duration of preoperative fasting for enhancing recovery after surgery . Surgical trauma induces a' stress response' which involves a series of physiologic reactions and aims to promote recovery from impaired metabolic and physiological homeostasis . It also increases the release of stress hormones and inflammatory cytokines that lead the body into a catabolic state and insulin resistance . These hormones and cytokines induce the hepatic production of glucose by gluconeogenesis and glycogenolysis, and reduce glucose uptake in peripheral tissues, thus leading to postoperative hyperglycemia . In addition, they stimulate proteolysis and lipolysis . Increased insulin resistance can be sustained for 3 - 4 weeks after surgery and the degree of increased insulin resistance is affected by the extent of surgery, the amount of blood loss, and the duration of preoperative fasting . Prolonged preoperative fasting induces the utilization of nutrients stored in the body, and increases patient's discomfort and anxiety . It is well known that insulin resistance is related to delayed wound healing, increased morbidity / mortality, and prolonged hospital stay . Therefore, maintaining an anabolic state before surgery and reducing of postoperative insulin resistance are very important for enhancing recovery after the surgery . Several international anesthesiology societies recommend the reduction in preoperative fasting time in order to enhance recovery after the surgery . These new guidelines recommend the intake of solid foods for up to 6 hours before the induction of anesthesia and the intake of clear liquids up to 2 hours before the induction of anesthesia . Especially, enhanced recovery after surgery (eras) society and european society of anesthesiology (esa) recommend the intake of carbohydrate - containing fluids up to 2 hours prior to surgery (table 1). The purposes of preoperative carbohydrate loading are to maximize glycogen storage in the body as an energy source in order to minimize subsequent degradation of body tissues and reduce insulin resistance . A suitable carbohydrate - containing drink (chod) should contain 12.5% maltodextrin for gastric emptying . The recommendation is to intake 100 g of carbohydrate (800 ml of chod) in the evening before surgery, and 50 g of carbohydrate (400 ml of chod) until 2 hours before surgery . Insulin levels at the time are similar to those observed after normal diet and thus, the body can maintain an anabolic state . Based on these recommendations, a' preoperative cho loading diet (soft fluid diet and soft blended diet)' was developed to facilitate recovery from the illness after surgery and to increase patient's satisfaction in the national cancer center . The purpose of this case report is to share our experience of applying a preoperative carbohydrate loading prior to surgery . A' preoperative cho loading diet' was given to patients scheduled to undergo hepatobiliary - pancreatic (hbp) surgery, including pancreaticoduodenectomy, for which surgery cho loading is strongly recommended by the eras program . To evaluate the effect of preoperative carbohydrate loading on clinical outcome, it was necessary to estimate several factors including gastric residual volumes (grv) before surgery, nutritional factors, subjective well - being, and insulin resistance . This study was approved by the institutional review board of the national cancer center (ncc 2016 - 0104). A 52-year - old man was diagnosed with cancer of the common bile duct and admitted into the national cancer center for pancreaticoduodenectomy . The patient's height was 166.4 cm, body weight was 67.0 kg, body mass index was 24.2 kg / m . The patient did not have any underlying disease, including diabetes mellitus or gastro - esophageal reflux disease . Surgery was scheduled for 09:00 am on the following morning . A registered dietitian assessed the patient's nutritional status using the scored patient - generated subjective global assessment (pg - sga), the patient was classified as moderate malnourition (sga - b) due to weight loss and reduced food intake owing to abdominal discomfort . Also, a registered dietitian explained to the patient about the diet protocol, including chod before surgery (table 2). The patient consumed the entire of' preoperative cho loading diet (sbd)' without any problems . Cho, carbohydrate; sbd, soft blended diet; sfd, soft fluid diet . 100 ml of rice porridge with 800 ml of carbohydrate - containing drink; 400 ml of carbohydrate - containing drink . On the day of surgery, the patient was given the' preoperative cho loading diet (sfd)' including 400 ml of chod and allowed to drink this until 05:30 am . He was asked to score his subjective well - being with the 10 cm visual analog scale (vas) prior to surgery . The patient's subjective well - being scores were as follows: thirst-2; hunger-3; comfort-5; anxiety-1; fatigue-2 . Plasma glucose and insulin concentrations were measured and homeostatic model assessment - insulin resistance (homa - ir) was calculated in order to determine insulin resistance before the surgery . Plasma glucose and insulin concentrations were 112 mg / dl and, 8.2 u / ml, respectively, while homa - ir was 2.27 . The patient consumed 400 ml of chod up to 4 hours before surgery and grv was 5 ml . Following the surgery, the patient was sent to the surgical intensive care unit and received postoperative medical care for pancreaticoduodenectomy . The patient was provided with routine postoperative care . In the morning, the patient's plasma glucose and insulin concentrations were 187 mg / dl and 4.8 u / ml, respectively, and homa - ir was 2.22 (table 4). Compared to the day of surgery, the homa - ir on postoperative day #1 had not been increased . Homa - ir = [plasma insulin (u / ml) plasma glucose (mmol / l)] / 22.5 . The patient received' post - pancreaticoduodenectomy diet' and was recovered well in the general ward without any complications . The patient was discharged 14 days after the surgery and body weight was 61.2 kg . A registered dietitian assessed the patient's nutritional status using the scored patient - generated subjective global assessment (pg - sga), the patient was classified as moderate malnourition (sga - b) due to weight loss and reduced food intake owing to abdominal discomfort . Also, a registered dietitian explained to the patient about the diet protocol, including chod before surgery (table 2). The patient consumed the entire of' preoperative cho loading diet (sbd)' without any problems . Cho, carbohydrate; sbd, soft blended diet; sfd, soft fluid diet . 100 ml of rice porridge with 800 ml of carbohydrate - containing drink; 400 ml of carbohydrate - containing drink . On the day of surgery, the patient was given the' preoperative cho loading diet (sfd)' including 400 ml of chod and allowed to drink this until 05:30 am . He was asked to score his subjective well - being with the 10 cm visual analog scale (vas) prior to surgery . The patient's subjective well - being scores were as follows: thirst-2; hunger-3; comfort-5; anxiety-1; fatigue-2 . Plasma glucose and insulin concentrations were measured and homeostatic model assessment - insulin resistance (homa - ir) was calculated in order to determine insulin resistance before the surgery . Plasma glucose and insulin concentrations were 112 mg / dl and, 8.2 u / ml, respectively, while homa - ir was 2.27 . The patient consumed 400 ml of chod up to 4 hours before surgery and grv was 5 ml . Following the surgery, the patient was sent to the surgical intensive care unit and received postoperative medical care for pancreaticoduodenectomy . The patient was provided with routine postoperative care . In the morning, the patient's plasma glucose and insulin concentrations were 187 mg / dl and 4.8 u / ml, respectively, and homa - ir was 2.22 (table 4). Compared to the day of surgery, the homa - ir on postoperative day #1 had not been increased . Homa - ir = [plasma insulin (u / ml) plasma glucose (mmol / l)] / 22.5 . The patient received' post - pancreaticoduodenectomy diet' and was recovered well in the general ward without any complications . The patient was discharged 14 days after the surgery and body weight was 61.2 kg . Many recent studies have shown that preoperative carbohydrate loading does not influence gastric residuals and reduces patient discomfort . Moreover, jonas et al . Reported no difference in gastric residue two hours after drinking 400 ml of a 12.5% carbohydrate drink when compared to an equivalent volume of water . Yuill et al . Showed that the decrease in muscle mass after upper gastrointestinal surgery in preoperative carbohydrate loading group was less than that in fasting group . Also reported enhanced recovery of a hand grip strength and a reduced length of hospital stay in patients with colectomy after preoperative carbohydrate loading . According to a recent cochrane review, preoperative the case in our hospital showed almost no gastric residual volume and low scores of patient discomfort including thirst and hunger after preoperative oral carbohydrate loading . After surgery, blood glucose levels are increased due to the decrease in insulin secretion, and insulin resistance is also increased in response to stress . But, some studies showed that preoperative carbohydrate loading can reduce insulin resistance by glucose uptake in the muscle due to insulin secretion . Fujikuni et al . Showed that the postoperative insulin resistance in patients with gastrectomy was decreased by preoperative carbohydrate loading compared with fasting . In this patient, therefore, we think that preoperative oral carbohydrate loading may be helpful for the recovery of patients . Currently, preoperative oral carbohydrate loading is only recommended to patients undergoing hepato - biliary - pancratic surgery at our hospital, but we are on the research for the application of preoperative oral carbohydrate loading to the patients with other cancers . However, there are almost no studies on the amount of chod intake for korean subjects.
Since the identification of satellite cells on skeletal muscle fibers in 19611, the field of satellite cell biology has progressed immensely to its recent characterization as an adult stem cell and its implications in regenerative medicine . Although portrayal of postnatal myogenesis predominates the satellite cell biology field, what is less known is / are the factor(s) responsible for satellite cell activation and the myogenic response . In addition, there is no confirmation on the overall importance of satellite cells to muscle repair and growth following physiological stimuli . In recent years, notch and wnt signaling have been determined to be critical for postnatal myogenesis but limited muscle regeneration models have been utilized to test these pathways 2 - 6 . Employing a variety of muscle injury and growth techniques will result in a definitive understanding of satellite cell contribution to muscle repair and hypertrophy . With its systemic effects, physiological stimuli are an ideal muscle model as relates to investigating the myogenic properties of muscle regeneration and growth including satellite cell activation . Although the amount of muscle injury induced may not be as profound as artificial models, contraction - induced muscle injury and muscle overloading exercises are identified to up - regulate myogenic proteins . This review will summarize the literature to address the interest on the effects of physiological stimuli on notch and wnt signaling pathways during postnatal myogenesis . We will discuss the current knowledge on satellite cell regulation as well as the recent developments of the importance of notch and wnt signaling to muscle repair . We will also examine the usefulness of physiological stimuli to studying the regulation of satellite cell activation and muscle regeneration . Furthermore, we will enlighten the reader with what is currently known on the effects of physiological stimuli and muscle repair as relates to notch and wnt signaling . Skeletal muscle has the remarkable capability to completely repair within a few weeks of an insult 7 . Immediately following exposure to overt skeletal muscle injury (injurious contractions, chemical, freeze and crush), muscle degeneration occurs including ultrastructural damage to the contractile apparatus such as z - line streaming, a - band disruption, and altered sarcomere length 8, 9 . In addition, disruptions to the sarcolemma and increased cellular permeability renders the muscle fiber to be susceptible to the autogenetic response via activation of the non - lysosomal calpain system which further disrupts the contractile apparatus 10, 11 . In the hours to days following the initial mechanical injury, the acute inflammatory response is initiated within the injured muscle fiber and is characterized by vasodilation of the microcirculation, increased permeability of proteins accumulation and infiltration of phagocytes (neutrophils, monocytes and macrophages) 12 . Chemotactic signals derived from the muscle itself, resident muscle macrophages or the circulatory system direct the inflammatory cells to the injured site within the muscle fiber in which they function to destroy the necrotic tissue and prepare the site for muscle regeneration 13, 14 . The muscle regeneration process in postnatal skeletal muscle is mostly attributed to the small population of myogenic stem cells termed satellite cells . Located beneath the myofiber basal lamina, quiescent satellite cells comprise a small percentage of myonuclei (~5% at two months of age) 15 . Quiescent satellite cells are mitotically arrested at g0 state and are characterized with small cytoplasm, condensed chromatin and flatten shape 15 . Satellite cells have often been identified to be pax 7 +, cd34 +, cd45- and sca1- 16 . However, there are several markers identified to be associated with quiescent satellite cells such as cd34, 7-integrin, pax-7, abgc2, mcadherin, syndecan 3/4, c - met, cdh15, foxk1 (formerly myocyte nuclear factor), pax3, sox8, sox15, vcam1, cxcr4, 1 integrin, and sm / c-2.6 antibody, calcitonin receptor, caveolin-1and sphingomyelin 15, 17 - 26 . It is improbable to identify a single marker discriminating quiescent satellite cells from proliferating myogenic precursor cells since many of the aforementioned markers may only identify specific subpopulations of satellite cells or stage of satellite cell activation . Following muscle injury, quiescent satellite cells are exposed to signals derived either from the damaged muscle or elsewhere within the injured microenvironment and the cells become activated . Many factors have been suggested to be responsible for activation of satellite cells, such as muscle - derived growth factors including hepatocyte growth factor, fibroblast growth factor 2, 6, cytokines such as interleukin-6, heat stress, and developmental signaling pathways, but the molecular / cell mechanisms leading to awakening of satellite cells and from the unperturbed state remain to be fully identified 2, 7, 27, 28 . Upon activation, satellite cells undergo increased cytoplasmic volume, decreased heterochromatin and they become mitotically active as they transition from g0 to g1 cell cycle phase . Once in the first cycle satellite cells enter an intermediate progenitor stage and are known as myogenic precursor or progenitor cells 15 . Within hours following activation, satellite cells up regulate the expression of basic helix - loop - helix transcriptional activators of the myogenic regulatory factor (mrf) family (myod, myf5) and progress to the highly proliferative phase termed myoblasts . Myod (-/-) null mice and mice with either double - knockout for myod and myf5 genes or triple mutant of myf5, mrf4 and myod demonstrate a complete loss of skeletal muscle formation 29 - 32 . In the days post activation, proliferating myoblasts express the cell cycle arrest protein, p21 and late mrfs (myogenin and mrf4), resulting in withdrawal from the cell cycle and initiation of terminal differentiation to form multinucleated myotubes . Completion of the differentiation program results in fusion of myotubes with each other or with existing myofibers and is often identified with increased expression of muscle specific myosin heavy chain (mhc). Myogenin has been identified to be critical for myoblast differentiation as demonstrated in myogenin - deficient embryos that have myoblasts but not myofibers due to defective myotube formation 33, 34 . Micrornas (mirs), which function in post - transcriptional modification have recently been identified to participate in skeletal muscle repair and may play a role in the transition of proliferation to differentiation state of myoblasts 35, 36 . Exogenous application of mir1, mir-133 and mir-206 to lacerated rat ta result in enhanced muscle repair including increased expression of myod and pax7 35 . In embryos, mir-27b targets and down - regulates pax3 thereby promoting differentiation while mir-27b inhibitors result in continual pax3 expression and proliferation of satellite cells 36 . These data suggest that micrornas may provide a novel strategy for understanding the regulation of skeletal muscle repair particularly as relates to the transition of myoblast proliferation to the differentiation stage . The importance of pax 7 as relates to mrfs and postnatal myogenesis is a prevalent study topic in satellite cell biology . Upon activation and proliferation of satellite cells, pax 7 has been identified to up regulate myod through the recruitment of histone methyltransferase complex 37, 38 and as myoblasts progress to the differentiation stage, pax 7 expression is down regulated 39 . However, there is also a small population of activated satellite cells that retain pax 7 expression and do not express myod or myf5 . These cells function to maintain the progenitor pool by returning to a quiescent state instead of committing to myogenic differentiation 38, 39 . The importance of pax 7 to muscle regeneration may end by 21 days of age in rodents 32, 40 . 40 tracked the temporal fate of inactive pax7 gene expression and reports that in adult mice with mutant pax 7, myogenesis was able to ensue 40 . Juvenile age in which young skeletal muscle is developed and the remaining satellite cells are not needed and go into quiescence . There is much evidence supporting the notion that satellite cells possess stem cell properties such as their ability to generate non - muscle lineages . Satellite cells migrated from single muscle fibers cultured on matrigel formed myoblasts and myotubes but also expressed osteogenic and adipogenic protein markers suggesting that satellite cells have the potential to convert to other mesodermal derivatives including osteocytes and adipocytes 41 . Myoblasts treated with bone morphogenic protein (bmps) differentiate into osteocytes and suppress the expression of mrfs such as myod 42, 43 . In addition, myoblasts convert to adipocytes when cultured with thiazolidinedione or fatty acids 44 . C2c12s engineered for expression of ccaat / enhancer - binding proteins (c / ebps) (adipogenic protein) or bmps (osteogenic protein) result in differentiation of each respective fate 45 . Myod (-/-) null mice are not more susceptible to transition to osteogenic or adipogenic fates when treated with bmp or thiazolidinedione respectively relative to control mice suggesting that myod is not critical for possession of a myogenic fate 41 . In vivo models also show the conversion of muscle to non - myogenic lineages including the presence of adipose tissue and bone formation within muscle diseases such as duchenne muscular dystrophic patients and mdx mice 30, 46 - 54 . Another unique stem cell characteristic of satellite cells are their ability to self renew 20, 22, 55 - 59 . It has been suggested that a small population of activated satellite cells do not proliferate but assist in maintaining the quiescent satellite cell pool to ensure enough satellite cells are present for repairing of injured skeletal muscle 20, 22, 55 - 57 . Transplantation of genetically - labeled myofibers into mdx mice that were depleted of satellite cells result in replenishment of the satellite cell pool 58 . Activated satellite cells have been demonstrated to divide into 2 separate satellite cell populations, one faction that rapidly divides and another group that divides at a slower rate and may have the duty of replenishing the satellite cell pool 59 . Self - renewal may also occur through asymmetric division in which a daughter cell does not further divide and maintains stem - cell properties including expression of sca1, notch1and pax7 while the other daughter cell proliferates and progresses down the myogenic lineage by expressing the muscle differentiation marker desmin 2, 60 - 65 . Notch inhibitor, numb is asymmetrically expressed on the activated satellite cells and may regulate cell fate choices by promoting progression down the myogenic lineage 2 . Self renewal may also occur through symmetrical division in which both daughter cells maintain stem - cell properties 66, 67 . Cells that do not express myod but continue to express pax 7 are suggested to be refrained for self - renewal 32 . There is great advancement in postnatal myogenesis characterization as well as the recognition of the stem cell properties of satellite cells including their tactics to maintaining their numbers . The factors responsible for satellite cell regulation are under study and need to be determined . Ascertaining satellite cell regulation will have implications in muscle -associated ailments and disease as well as implications in stem - cell regenerative medicine . Notch and wnt signaling are well known to play a critical role in determining tissue specificity of stem cells by regulating cell fate decisions during embryonic development 68 - 72 . Since satellite cells are stem cells it is plausible to infer that notch and wnt are important for postnatal myogenesis . Activation of notch signaling occurs when the transmembrane notch receptor interacts with one of its cell - membrane - anchored dsl ligands (delta / jagged, serrate, or lag2) and undergoes a series of cleavages from metalloproteases and -secretases resulting in the intercellular domain of notch (nicd) translocating to the nucleus (figure 1). Within the nucleus, the now active nicd interacts with transcriptional repressors of the csl family (cbf1/rbp - j, suppressor of hairless (su[h]), and lag-1) and converts them to transcriptional activators (nicd - csl). With involvement of other proteins, the csl - nicd complex regulates target genes such as hey and he s genes, which encode proteins known to be involved in myogenesis 68 . Within hours to days following muscle injury there is increased expression of notch signaling components (delta-1, notch-1 and active notch) on activated satellite cells and neighboring muscle fibers2, 3 . Up - regulation of notch signaling promotes the transition of activated satellite cells to highly proliferative myogenic precursor cells and myoblasts, as well as prevents myoblast differentiation to myotubes 3 - 6, 73, 74 . Myoblast proliferation is decreased and differentiation is promoted when notch activity is inhibited in myoblasts with notch antagonist, numb,-secretase inhibitor, or with small - interfering rna (sirna) knockdown of presenilin-1 2, 4, 75 - 78 . In addition, mutations in delta - like 1 or csl result in excessive premature muscle differentiation and defective muscle growth 79, 80 . Multiple mechanisms for how notch signaling prevents myoblast differentiation have been proposed 81 - 87 . Hes1 or hey1 may form a complex with myod to repress its expression resulting in inhibiting critical proteins for myotube formation such as mef2d and myogenin 6, 81 - 83, 88, 89 . However, a recent study by buas et al . 84 does not show an interaction with hey1 and myod and instead proposes that hey1 prevents myod to bind to myogenin or mef2c promoters thereby inhibiting their transcription and ultimately repressing muscle differentiation84 . Another mechanism for notch preventing differentiation is a potential cooperative effort between notch and other signaling pathways to allow for sufficient myoblast proliferation prior to differentiation 85, 86 . Forkhead box o 1 transcription factor (foxo1) prevents muscle differentiation and is suggested to be critical for downstream notch signaling since sirna - induced inhibition of foxo1 represses notch - induced hes1 activity in c2c12 cells 85 . In addition, transforming growth factor signaling pathway (tgf) induces hes1 expression in c2c12 cells which may lead to inhibition of myod and progression of muscle differentiation 86, 87 . Similar to notch, canonical wnt signaling is critical for muscle repair 90 - 94 . The canonical wnt signaling cascade entails soluble wnt ligands to interact with frizzled receptors and low - density lipoprotein receptor - related protein co - receptors (lrp) (figure 2). This coordination stimulates phosphorylation of disheveled and inactivates gs3k's phosphorylation of catenin . With the assistance of axin, the de - phosphorylated and stable - catenin does not undergo ubiquitination and degradation and is instead translocated to the nucleus where it binds to tcf / lef1 transcription factors 95 . Tcf / lef1 transcription factors may directly activate myf5 and myod or may up regulate mrf co - activators such as c - jun n - terminal kinases 96 - 98 . There is a preponderance of evidence demonstrating that skeletal muscle injury up regulates wnt signaling 90 - 94 . Mrna expression of wnts5a, 5b, 7a, and 7b increases in muscle at four days post cardiotoxin - induced injury 90 . At four days post myofiber explantation, there is elevated expression of wnt3a, frizzled1 and 2, and axin2 93 . Using tcf - optimal promoter b - galactosidase mice (topgal mice) there is increased tcf reporter activity in myogenic cells at two and five days post freeze - induced muscle injury 93 . While wnt is critical for muscle development as evident in no dermomyotome formation or myf5 expression in embryonic wnt1 and wnt3a knockout mice, wnt also contributes postnatal muscle regeneration 90, 93, 98 . Activating wnt signaling by treating myoblasts with lithium chloride (licl, which inhibits gsk3 activity) results in increased mhc and myogenin expression; and treating myoblasts with wnt3a results in a ~ 30% increase in desmin expression 90, 93, 99, 100 . 94 reports a ~ 40% increase in muscle regeneration in myogenic cells with forced wnt expression, further demonstrating that wnt signaling promotes postnatal muscle repair 94 . Wnt's main role during muscle regeneration is myoblast differentiation and myotube fusion 99 - 103 . Furthermore, treatment of proliferation c2c12 myoblasts or single fiber cultures with wnt antagonists or secreted frizzled receptor proteins (sfrps) repress myotube formation, decreases myogenin and mrf4 rna expression by ~ 50% as well as inhibits desmin expression 93, 102 . Moreover, there is impaired myotube formation and decreased myogenin and mhc expressions in sirna - induced inhibition or knock - out mice of wnt co - activator bcl9 94 . Lastly, there is increased myogenic differentiation in cholesterol depleted- myoblasts identified to promote nuclear translocation of - catenin and tcf / lef activation 103 . Myogenesis consists of a series of orchestrated events that require communication of a variety of signaling pathways when transitioning from the phases of satellite cell activation to myoblast proliferation and differentiation and finally, myotube formation . Notch and wnt interact during muscle development as well as during postnatal myogenesis and it is plausible that this crosstalk contributes to the stage transitions during adult muscle repair 93, 104 . It is suggested that notch activity presides during myoblast proliferation after which there is a temporal switch to wnt signaling and subsequent myoblast differentiation and fusion into myotubes 93 . Inhibiting notch (with soluble jagged ligand or with a - secretase inhibitor) or activating wnt (by inhibiting gsk3 or adding wnt3a) decreases myf5 expression and promotes muscle differentiation providing evidence that notch signaling needs to be turned off and wnt turned on for differentiation to ensue 93, 100, 103 . When notch inhibitors are administered during the differentiation phase there is no acceleration of muscle regeneration, further supporting the notion that notch signaling does not participate in the later stages of muscle repair 93 . In addition, during the early stages of muscle repair (24 hours) following cardiotoxin - induced muscle injury, wnt inhibitor, sfrp2 is expressed in proliferating myoblasts and there is no wnt activity 105; furthermore, administration of wnt inhibitor, sfrp3 to single - fiber cultures at early stages of muscle repair (day 2 of culture) has no effect on wnt inhibition but when sfrp3 is added at later time points (day 3.5 of culture), wnt expression is inhibited and there is less myoblast differentiation . These data suggest that wnt activity does not occur during the early proliferative stages of muscle repair 93 . 93 recommends that low wnt signaling is needed for a sufficient quantity of proliferating myoblasts prior to myotube formation 93 . Notch and wnt orchestrate together for cell fate decisions in other systems but their roles have not been determined to be similar in the skeletal muscle model 106 - 108 . A co - activation of notch and wnt promote proliferation of progenitor intestinal epithelial cells 107 . When notch and wnt are both active in proliferating progenitor cells within the crypt region of the small intestine, the cells continue to proliferate and form the intestinal absorptive cells called in addition, administration of wnt3a to hematopoietic progenitors cultured in presence of notch ligand, delta results in increased hes1 and cd7 + cells (t cells) suggesting that hematopoietic progenitors cells transform into t cells in the presence of wnt and notch 108 . Determining if wnt and notch regulate each other will aid in delineating the mechanism of orchestration between notch and wnt during cell fate determination including postnatal myogenesis . Since notch appears to dominate during myoblast proliferation and wnt signaling leads during the differentiation / fusion stages of muscle repair, it is plausible to suggest that notch signaling is upstream of wnt . Transfection studies show that the intracellular domain of notch has binding sites on lef1 promoter that are different than - catenin sites and result in lef1 transcriptional activation 109 . Conversely, it should be noted that there is evidence suggesting wnt signaling is upstream of notch 110, 111 . The wnt target genes brachyury (t - box) and lef1/tcf1 have binding sites on notch1 and delta - like-1 (dll1) promoter and mice embryos with mutant tbx6 function (downstream t box target gene) or lacking brachyury do not express dll1 110, 111 . Furthermore, in the presence of exogenous wnt3a and delta1 there is increased hes1 and deltex1 expression in hematopoietic stem cells 108, 112 . Identifying the location for the point of transition between notch and wnt signaling would be beneficial in determining the mechanism of notch and wnt orchestration for cell fate decisions including postnatal myogenesis . Gsk3 directly binds to notch 1 intracellular domain (notch1ic) and facilitates notch signaling so it seems plausible that gsk3 may be a critical juncture between notch and wnt pathways during postnatal myogenesis 93, 113, 114 . While increased notch signaling corresponds with elevated gsk3 activity (gsk3 phosphorylated at tyrosine 216 gsk3) at one day post muscle injury, wnt contradicts gsk3 activity and is low at that time point 93 . However, as the time course of muscle repair progresses from one day to four days, wnt signaling increases and both gsk3 and notch activity declines 93 . Inhibiting gsk3 activity in mononucleated cells of topgal mice results in increased - catenin expression and wnt reporter activity 93 . In addition, treating myoblasts with notch inhibitor -secretase, results in decreased gsk3 activity 93 . These data further confirm the corresponding relationship between gsk3 and notch and inverse relationship between gsk3 and wnt as well as suggests that gsk3 activity may be pivotal for the transition between notch and wnt signaling . Presenilins, which assist in cleaving full - length notch to its activated state, have also been suggested to be major players in the coordination of the notch and wnt pathways 115, 116 . Presenilins have been identified to bind to - catenin and possibly participate in -catenin ubiquitination115 . In addition, there is increased tcf / lef1 transcription in presenilin1 knockout embryonic fibroblasts 116 . These date suggest that presenilins may function as mediator between notch and wnt by de - regulating wnt signaling . Besides gsk3 and presenilins, there is evidence that notch antagonist, numb may be a pivotal point of transition between notch and wnt since forced expression of numb on erythroid progenitor cells treated with exogenous wnt5a, results in increased tcf / lef transcriptional activity 106 . The orchestration of notch and wnt is crucial for cell fate decisions of a variety of tissues . However, there are conflicting reports on the mechanisms of their co - regulation during regenerative processes in skeletal muscle and other tissue models . Of particular interest, is understanding notch and wnt's role in satellite cell biology such as regulating myogenic proteins during postnatal myogenesis . What is even less known is the effect of different muscle injury models (including physiological stimuli) in regulating notch, wnt and myogenic protein expression during adult muscle repair . The current muscle injury models for examining the roles of notch and wnt in satellite cell regulation during postnatal myogenesis consist primarily of artificial techniques which do not consider the influences of other biological systems and are not easily applied to muscle function scenarios . Physiological stimuli such as contraction - induced muscle injury (injurious exercise) and growth models (overload hypertrophy, resistance exercises) are functionally relevant and involve the contribution of multiple active biological systems which are important to consider when investigating sources responsible for muscle regenerative processes . A common injurious exercise model, downhill running (dhr), uses eccentric muscle contractions in which the contracting muscle is forced to lengthen by an external force while producing tension 9, 117 - 119 . Dhr in rodents results in decreased isometric force production and elevations in muscle injury makers such as creatine kinase activity and muscle injury criteria identified with hematoxylin and eosin staining 118, 120, 121 . Dhr induce ~80% up regulation of mitogen - activated protein kinase (mapk) signaling such as phosphorylation of jnk, p38, and erk 129 . Myogenic markers myf5, myod, myosin heavy chain (mhc), mcadherin, desmin, and pax 7 increase expression within the first week following dhr 121 - 127 . There is increased myf5 transcriptional activity in myf5lacz reporter mice at 48 hours to 96 hours post dhr 128 . Overall, there is sufficient data to exhibit that dhr induces a myogenic response and may be worthy as a model for studying postnatal myogenesis regulation . One aspect to consider when using the dhr model to study myogenesis is that the time course of muscle repair with the dhr model may be different than with artificial muscle injury models . There is a delay in the onset of muscle regeneration in the dhr model relative to cardiotoxin injections (ctx) 122 . 122 reports that ctx - injured soleus fibers exhibit extensive myofiber damage at 24 hours to 72 hours post injury, while dhr - injured fibers are not apparent until 96 hours to 144 hours post exercise . At 120 hours post - ctx, muscle regeneration dominates while signs of muscle repair are not present until 144 hours to 168 hours post - dhr . Although dhr does not induce as profound of muscle injury as seen in the ctx model, this exercise injury model still induces a myogenic response . Besides the dhr model, other eccentric contraction models are available to study muscle regeneration 130 - 139 . Eccentric contractions on an isokinetic dynamometer results in muscle remodeling in the hours to days post exercise including increased expression of hepatocyte growth factor (hgf), myod, mechano growth factor, neural cell adhesion molecule, pax7 and insulin - like growth factor 1 (igf1) 134 - 138 . In situ muscle lengthening contraction injury model is a physiological test that uses a rigorous and precise control of muscle contraction by electrically stimulating motor neurons of associated muscles of interest 130 - 132, 139 . With its ability to quantify muscle function there is a 70%-80% decrease in isometric force production and a 15- 20% increase in injured fibers in mice edl at three days following exposure to electrically stimulated lengthening contractions 131 . Inflammation is also present at three days post injury with reports of a 7.9 fold and 51.2 fold increase in muscle associated neutrophils and macrophages respectively 140 . With the profound magnitude of muscle injury present in this physiological stimulus model as well as the presence of myogenic - associated proteins, it seems plausible that one could use this novel muscle injury model to study the regulation of myogenic repair as it relates to recovery of function . Muscle overload techniques (heavy resistance exercise, stretch hypertrophy and compensatory hypertrophy) are physiological stimuli capable of investigating the contribution of myogenic properties to growth . Overload - induced muscle hypertrophy is a type of a compensatory hypertrophy model in which muscles are ablated (such as the gastrocnemius, soleus or tibialis anterior) and the remaining muscles (such as the plantaris or extensor digitorum longus) work harder to maintain muscle function 141 . This compensation of the remaining muscles results in growth including increased dna synthesis, muscle mass and force production 141 - 148 . Overloaded rat soleus has a 40% increase in wet weight and ~ 6.76% to 12.74% increase in de novo myofibers 142 . The cross sectional area of a single myofiber as well as myonuclei number per millimeter of a myofiber increase at 46% and 44% respectively in an overloaded rat plantaris 149 . It is suggested that activation of satellite cells is a source of the new myonuclei rather than sole provision from differentiated myotubes 149, 150 . 151 reports that a 117% increase in satellite cells accompanies the 26% increase in myonuclei following heavy resistance exercises 151 . During the early hours and within the first week of compensatory overload there is evidence of satellite cell activation and myogenic response including up - regulated pax 7, m - cadherin, myod, desmin, myogenin, mhc as well as fibroblast growth factors 1 and 7, igf1 and hgf 142, 145, 147, 148, 152 - 155 . There is evidence supporting the notion that satellite cells are critical to muscle repair, recovery of muscle function as well as important to muscle growth 142, 149, 156 - 158 . Irradiation - induced inhibition of proliferating myogenic cells results in decreased force recovery following injurious eccentric contractions to rodent anterior crural muscles 156, 157 . Non - irradiated mice have complete force recovery at three weeks post exposure to an injurious lengthening protocol while irradiated mice exhibit a 35% force deficit 156 . In addition, myod and myogenin transcripts are increased at three, seven, 14, and 21 days post lengthening contractions in non - irradiated mice while irradiated mice do not show any mrf up - regulation 156 . There is evidence of the importance of satellite cells in the overload muscle growth models 142, 149 . Phelan et al . (1997) reports no hypertrophy following four weeks of overloading in irradiated rat soleus 142 . In addition, there is no increase in muscle mass, myofibrillar protein, dna content, or cross sectional area of a single myofiber at three months following overloading in irradiated rat hindlimb muscle 149 . However, it needs to be mentioned that there is data demonstrating the occurrence of muscle hypertrophy without up regulation of myogenic proteins following muscle overload and proponents that propose the critical component for muscle hypertrophy is protein synthesis and not satellite cells 159, 160 . Since igf1 induces muscle hypertrophy by stimulating protein production via signaling pathways such as pi3k / akt / mtor / s6k1 161 - 164 investigators from this camp manipulate igf to study the impact of protein synthesis on muscle hypertrophy . There is increased muscle hypertrophy in overloaded mice that over - express muscle igf1 or akt 165 - 167 . In addition, irradiated mice that are introduced with viral - mediated igf1 gene transfer have greater muscle growth than with just irradiation alone 162 . To add complication to the controversy, it is also proposed that both satellite cells and protein synthesis contribute to muscle repair and hypertrophy 162, 168, 169 . They suggest that igf1 could co - activate satellite cells and adult muscle fibers to induce hypertrophy so that in conditions in which irradiation depletes proliferating activated satellite cells the mature muscle fibers could be responsible for the increased protein synthesis 162 . Others suggest that the predominating mechanism of either satellite cell activation or increased protein synthesis may be due to the timeline of events following a hypertrophic stimulus 168, 169 . Perhaps early hypertrophy is due to increased transcription and translation while hypertrophy that occurs at later time points may be due to increased satellite cell activation, resulting in increased myonuclei number 168, 169 . More work is needed to delineate the importance of protein synthesis compared to satellite cells to muscle repair and growth . There are still many questions in regards to the contribution of satellite cells to muscle repair and growth . Using global forms of muscle injury and growth such as contraction - induced muscle injury models and overload - induced hypertrophy models will help identify whether the basis satellite cell activation is intrinsic, muscle - derived, or arise from other systems (nervous, circulatory, inflammatory, etc). Furthermore, studying the communication of cell signaling pathways important for muscle regeneration (such as notch, wnt) as relates to satellite cell activation will aid in delineating the source of satellite cell activation resulting in determining the importance satellite cells to muscle repair and growth . Research on the interaction of physiological stimuli, notch and wnt signaling and muscle repair and growth would aid in furthering the understanding of satellite cell regulation . Physiological stimuli up - regulates components of notch and wnt signaling 92, 127, 170 - 175 . Dhr increases notch1 and delta1 expression in myogenic cells at 72 hours through 120 hours post - injury 127 . A single injurious bout of resistance exercise increases notch1 and downstream regulator hes6 mrna 170 . Overload - induced hypertrophy also increases notch1 expression in pax7-positive myogenic cells 171 . In vivo exercise decreases gsk3 activity and - catenin phosphorylation which is required for wnt signaling activation 173, 174 . Overload - induced hypertrophy up - regulates total - catenin, receptor frizzled1, wnt regulator disheveled1, wnt downstream target genes cmyc and cyclin d1 as well as induce a 434% increase in nuclear - catenin and promotion of - catenin / lef1 complex formation 172, 175 . There is evidence that - catenin may be required for muscle hypertrophy since there is no hypertrophy in overloaded mice with conditional inactivation of - catenin 92 . There is an abundance of evidence stating that physiological stimuli up - regulates notch and wnt signaling but more research is needed on the effect of physiological stimuli on the orchestration and regulation of these signaling pathways during muscle repair . With the development of tools available to identify satellite cells, it is feasible to study the characterization and regulation of postnatal myogenesis . More work is needed to discern the factor(s) responsible for re - awakening these normally quiescent skeletal muscle stem cells as well as to determine the collaboration of the various signaling mechanisms as relates to regulating the myogenic proteins . The revelation of the importance of notch and wnt signaling to during muscle repair is recognized in artificial muscle injury models but is largely unknown in physiological stimuli models . Contracting skeletal muscle releases a plethora of signals that induces autocrine and paracrine effects that may influence the regulation of postnatal myogenesis differently than localized muscle injury models . Therefore, understanding the intrinsic and systemic response of contracting muscle will aid in deciphering the regulation of satellite cells and muscle repair as well as understanding the molecular mechanisms responsible for the beneficial protective effects of exercise to cardiovascular disease and cancer.
Malignant glaucoma, or aqueous misdirection syndrome, is a condition characterized by sudden intraocular pressure (iop) elevation, and it is usually unilateral and induced by ocular surgical intervention or by medical therapy . Here, we report a case of simultaneous bilateral malignant glaucoma in a young patient with no history of any ocular diseases . A case of a 24-year - old female with no apparent previous history of ocular medical or surgical conditions was referred to our hospital because of recent bilateral iop elevation associated with a severe drop in vision and shallow anterior chamber with no posterior segment anomalies detected by ocular ultrasound in both eyes . Yttrium aluminum garnet (yag) laser iridotomy dropped the iop only temporarily and the patient received topical atropine treatment with combined trabeculectomy and anterior vitrectomy . In this case, the patient had a typical presentation of bilateral malignant glaucoma and her iop dropped only temporarily following laser iridotomy to rise again shortly thereafter . Also, deepening of the anterior chamber and iop decrease after topical atropine was very supportive of the diagnosis of malignant glaucoma . This is a very rare case of bilateral malignant glaucoma in a young adult without any prior eye conditions; only one similar case has been reported in the literature . We propose our own theory regarding this simultaneous occurrence of the pathology based on previously published studies about the presence of communication between the two eyes along the cerebrospinal fluid pathways . Malignant glaucoma, or aqueous misdirection syndrome, was first described by von graefe in 1869 as an aggressive form of postoperative glaucoma that is resistant to treatment and can result in blindness.1 this entity is characterized by intraocular pressure (iop) elevation, with shallowing to complete flattening of the anterior chamber (ac) in the presence of a patent peripheral iridectomy and in the absence of posterior segment anomalies, particularly suprachoroidal hemorrhage.2 malignant glaucoma is thought to occur in anatomically predisposed eyes,2 with one of the proposing mechanisms being posterior misdirection of the aqueous humor within or behind the vitreous substance . This produces a continuous expansion of the vitreous cavity and increased posterior segment pressure, leading to anterior displacement of the lens iris diaphragm in phakic and pseudophakic eyes, or forward displacement of the anterior hyaloid face in aphakic eyes.3 this generates a vicious cycle, in that the higher the pressure in the posterior segment, the more firmly the lens is held forward . The iop is classically markedly increased, but it may also be normal.4 malignant glaucoma has been reported in different situations, such as following cataract surgery, laser iridotomy, neodymium: yttrium aluminum garnet (nd: yag) cyclophotocoagulation or sclerotomy; following glaucoma drainage device implantation, viscoelastic use, intravitreal injection of triamcinolone acetonide; with aspergillus flavus intraocular infection; and with the ingestion of topiramate or sulfa drugs.5,6 only a few cases of isolated malignant glaucoma have been reported in eyes without any antecedent eye diseases or surgery, and only one case of bilateral simultaneous idiopathic malignant glaucoma has already been reported in the literature.7 here, we report a case of spontaneous, simultaneous, bilateral malignant glaucoma in a young female with no systemic or eye diseases and no prior medical or surgical eye treatments . We also propose a postulated mechanism that might explain the simultaneous bilaterality of this case . The patient is a 24-year - old female who was referred to our eye center in mediclinic dubai mall, dubai, united arab emirates, because of recent bilateral iop elevation with a severe drop in vision . She had been hospitalized elsewhere 1 week earlier because of severe bilateral frontal headache, nausea, and vomiting . Blood tests and brain magnetic resonance imaging (mri) were performed and revealed no abnormalities . During her stay after 5 days in hospital, the patient complained of loss of vision in the right eye, and after approximately 6 hours, also in the left . The ophthalmology consultant suspected bilateral acute angle - closure glaucoma as a possible cause for the severe bilateral loss of vision . Goldmann applanation pressure was> 60 mmhg in both eyes; the patient presented with severely miotic pupils and flat chambers, with almost iridocorneal touch centrally, cloudy corneas, and severe bulbar conjunctival injection . The patient s past history was negative for any medical or surgical diseases and her eye history was also negative . Of note, a routine eye examination had been performed a few months earlier and was reported to be normal . Initial management at our center consisted of multiple topical antiglaucoma medications and intravenous acetazolamide (500 mg). Ultimately, we managed to perform bilateral nd: yag laser peripheral iridotomy despite the corneal clouding . After the peripheral iridotomy, the iop dropped in both eyes to around 45 mmhg, but it rose back to its original level after less than half an hour . The diagnosis of bilateral malignant glaucoma was raised and the patient was admitted to hospital for surgical management . Topical atropine 1% drops and intravenous mannitol (1 g / kg of body weight) were started, and the patient was put under continuous monitoring . In less than 2 hours, the acs started to deepen in both eyes, with partial clearing of the corneas . The iop dropped again to 45 mmhg in both eyes . At that stage, the possibility of bilateral malignant glaucoma as a diagnosis was more likely . The next day, the patient was scheduled for trabeculectomy with mitomycin - c (0.02% for 2 minutes) combined with pars plana vitrectomy . Informed consent was obtained from the patient prior to surgery . In the meantime, overnight treatment with topical atropine and antiglaucoma medications was continued, along with intravenous acetazolamide . A second dose of intravenous mannitol was repeated 1 hour before the planned surgery . At the time of surgery, surgery was performed in the right eye as planned, and the same procedure was carried out in the left eye 1 day later . In brief, the surgical procedure was performed in both eyes as follows: a limbus - based conjunctival flap was performed, followed by topical 0.2% mitomycin - c application for 2 minutes; prior to the dissection of a 33 mm half - thickness trabeculectomy flap, one - port limited core vitrectomy was performed through a pars plana approach 4 mm from the limbus . After completion of the vitrectomy, the trabeculectomy was completed by entering the ac through a small sclerotomy opening using a super blade . The trabeculectomy flap was sutured back using two 10 - 0 nylon titrated sutures, and the conjunctiva was closed using running 8 - 0 vicryl (johnson & johnson, new brunswick, nj, usa). Postoperatively, the patient was put on a topical combination of tobramycin (3 mg / ml) and dexamethasone (1 mg / ml), and atropine was continued once a day for 2 weeks . Both eyes had well - formed superior blebs and, surprisingly, visual acuity returned to 20/20 without correction, and there was no optic nerve (on) damage in either eye . The iop remained <18 mmhg all throughout the postoperative follow - up period, which extended for more than 1 year, with both eyes maintaining deep chambers and reactive pupils . The study was approved by the review board / ethics committee of the beirut eye specialist hospital, beirut, lebanon . The main features of malignant glaucoma (or aqueous misdirection syndrome) include: increased iop; the presence of shallowing to complete flattening of the ac; the absence of posterior segment anomalies particularly suprachoroidal hemorrhage; no response to peripheral iridectomy; and adequate response to topical atropine.2,7 malignant glaucoma is difficult to treat and characteristically progresses to blindness.2 in our case, the patient had a typical presentation of malignant glaucoma and the iop dropped only temporarily after laser iridotomy, to rise again shortly thereafter . Also, deepening of the ac and iop decrease after topical atropine was very supportive of the diagnosis of malignant glaucoma . Although malignant glaucoma typically occurs in predisposed eyes, especially among those subjected to surgical or medical therapy, only a few cases have been reported where isolated malignant glaucoma occurs without a previous history of any kind of treatment and, to the best of our knowledge, only one case of bilateral simultaneous idiopathic malignant glaucoma has been reported in the absence of any history of previous systemic or eye diseases.7 as such, our case is considered as the second report of bilateral idiopathic malignant glaucoma . The rare phenomenon of malignant glaucoma occurring simultaneously in both eyes of young patients who do not have any predisposing factors is highly unusual and warrants exploration . Malignant glaucoma is thought to occur in anatomically predisposed eyes,2 with one of the proposed mechanisms being posterior misdirection of the aqueous humor within or behind the vitreous body, which leads to anterior displacement of the iris lens diaphragm and anterior hyaloid face.3 in our case, the occurrence of malignant glaucoma in one eye (most probably the right eye first, as deduced from the patient s own story) was in an anatomically predisposed eye, and the aqueous entrapped behind the vitreous established a pressure gradient between the on head and the cerebrospinal fluid (csf) in the subarachnoid space (sas) of the on . This pressure gradient encouraged fluid movement toward the sas of the on and subsequently to the sas of the brain . Migration of fluid to the sas of the controlateral on could occur directly through the optic chiasm, or it could be mediated by the elevated csf pressure within the sas of the central nervous system through the phenomenon of bidirectionality of csf flow.8 this proposed mechanism of aqueous humor migration from the vitreous cavity to the on and csf of the brain is also supported by the observation of silicone oil (so) migration from the vitreous cavity to the on and to the brain in vitrectomized eyes.9,10 also, espinosa et al11 reported the migration of so into the on of the contralateral eye causing optic neuritis . This migration is facilitated if there is a gradient of pressure between the vitreous cavity and the csf, or in a predisposed on head, such as in the presence of an on pit.12 in conclusion, one possible explanation for the occurrence of simultaneous bilateral malignant glaucoma is that the entrapment of aqueous humor behind the posterior vitreous face can establish a pressure gradient between the vitreous cavity and the fluid around the orbital on . This facilitates the migration of fluid to the sas of the on, and then to the sas of the contralateral orbital on, either directly through the optic chiasm or due to increased intracranial csf pressure . The fluid subsequently migrates to the vitreous cavity of the contralateral eye and pushes the total vitreous core anteriorly; this precipitates forward movement of the anterior hyaloid face and lens iris diaphragm, resulting in malignant glaucoma in the contralateral eye . This postulated mechanism could also explain the observed phenomenon of immediate constriction of the contralateral pupil when a strong miotic agent is injected into the ac of the first eye upon completion of the phakic posterior chamber intraocular lens (piol) implantation in a planned bilateral piol surgery . Given this observed phenomenon, we avoid the injection of miotic agents into the ac upon completion of surgery on the first eye in the setting of bilateral implantation of phakic piol . Although it is very rarely indicated in our surgical practice of cataract surgery, the same phenomenon is observed during bilateral cataract surgery . One more lesson to learn from this case is that severe iop elevation causes symptoms that can mimic those of certain central nervous system disorders . Finally, the diagnosis of idiopathic malignant glaucoma is challenging because, on the one hand, it is an uncommon entity, and on the other hand, early intervention with vitrectomy could possibly maintain good visual acuity and prevent blindness.
Their identification is an important method of risk stratification in asymptomatic patients with low to intermediate risk for coronary artery disease (cad). The absence of coronary calcium is associated with a very high event - free probability . Conversely, the detection of coronary calcium indicates an increased risk of incident coronary heart disease above that predicted by standard risk factors, from 2-fold for scores of up to 10011-fold for scores above 1000 . In reality, only 20% of coronary plaques are calcified, and not all calcified plaques contribute to flow - limiting lesions . Hence, contrast computed tomography coronary angiogram (ctca) is more accurate than coronary calcium score (ccs) in diagnosing or excluding a significant coronary lesion, and it is considered appropriate to perform a ctca in low and intermediate pretest probability (ptp) patients based on their age, gender, and symptoms . However, a high calcium score precludes ctca as calcium in the coronaries overestimates lesions due to blooming effect and interferes with a valid interpretation . The purpose of this study was to evaluate the conventional angiogram findings in those patients in whom a ctca is considered appropriate but could not be performed in view of coronary calcium high enough in its extent and location to preclude a valid interpretation . Fifty - two patients who were considered ineligible for undergoing a ctca (64 row multidetector ct) or had compromised interpretation of ctca because of high coronary calcium were included in the study . Noncontrast - enhanced prospectively electrocardiogram - gated ct calcium score was performed using a 250 mm field of view and 2.5 mm slice thickness . The calcium scores for individual vessels were computed using the inbuilt software supplemented by manual identification of the calcified locations defined by areas with attenuation> 130 hu along the course of the coronary arteries . Noncoronary calcification (sinuses of valsalva, mitral annulus) was cautiously excluded while measuring calcification . These values were the sum of the area and density of the coronary calcium giving the unitless agatston score . Radiation dose range was 56.065.5 mgycm and average was 59.84 mgycm . For analysis purpose, a ccs cut off 1000 is used to define those with very calcium score> 1000 (very high ccs group) from those with high ccs between 350 and 1000 (high ccs group). Based on individual vessel calcium score, they are divided into the following four groups, namely, none or noncalcified (ccs - 0), low or mildly calcified (ccs - 1100), moderately calcified (ccs - 101300), and heavily calcified (ccs ->300). Fifty of the 52 patients underwent conventional ca . The syntax score (ss) known to represent the global atherosclerotic burden was calculated for all patients using the online calculator version 2.11 (http://www.syntaxscore.com) by two separate cardiologists blinded for the calcium score . Concordant scores were accepted, whereas a discordant score was evaluated by a third cardiologist for validation . The lesions in individual coronary arteries are coded on an ordinal scale from normal to total occlusion with insignificant lesions defined as those ranging from any luminal irregularity to <70%, significant lesions with> 70% luminal narrowing, and total as those with 100% occlusion . The 2008 framingham risk score (frs) was calculated for individual patients from their history, physical examination findings, and blood investigations, and they are divided into a low, intermediate, and high risk based on the cut off 10%, 10%20%, and> 20% 10-year predicted risk for a cardiovascular event, respectively . The coronary stenosis, frs, and fluoroscopic calcium were studied as ordinal data, whereas the calcium score and the ss are studied as a continuous data . The association between continuous variables is studied using the pearson's correlation coefficient, whereas the association between ordinal data is studied using nonparametric tests (spearman's co - efficient). Standard error of mean was calculated for continuous variables between the groups with and without significant stenosis using the independent sample t - test if they are normally distributed; else a nonparametric test like kruskal wallis test was used . A receiver operator curve (roc) was plotted for the total ccs and individual arterial calcium scores against the presence of a flow limiting stenosis for the corresponding patient or vessel to derive the sensitivity and specificity cut off for ccs in predicting a significant stenosis . The coronary stenosis, frs, and fluoroscopic calcium were studied as ordinal data, whereas the calcium score and the ss are studied as a continuous data . The association between continuous variables is studied using the pearson's correlation coefficient, whereas the association between ordinal data is studied using nonparametric tests (spearman's co - efficient). Standard error of mean was calculated for continuous variables between the groups with and without significant stenosis using the independent sample t - test if they are normally distributed; else a nonparametric test like kruskal wallis test was used . A receiver operator curve (roc) was plotted for the total ccs and individual arterial calcium scores against the presence of a flow limiting stenosis for the corresponding patient or vessel to derive the sensitivity and specificity cut off for ccs in predicting a significant stenosis . The mean age of the study group was 62 years with 40% represented by females . Seventy percent of the study population had a low to intermediate ptp for cad based on their age, gender, and symptoms, whereas the rest of the 30% had high ptp but were either self - referred or had denied an invasive angiogram as the initial modality of evaluation . Based on the frs, 70% had a high risk, 20% had an intermediate risk, and only 10% had a low risk . Baseline characteristics all patients except for three had a calcium score> 400 . In these three patients, the score was between 300 and 400, but were still feared to interfere with contrast ctca . Right coronary artery (rca) had the highest amount of calcium (467 524), followed by left anterior descending (lad; 458 397), left circumflex (lcx; 181 274), left main (lm; 70 170), and posterior descending artery (pda) had the lowest calcium . Nine patients (18%) of this cohort with high ccs had an angiogram without significant disease in any of the vessels [table 2 and figure 1]. Of these, four patients had a ca that was considered to be normal and all four had a ccs <1000 . Four of the other five patients with insignificant disease had scores <1000, whereas one had a score> 1000 . The average calcium for a normal or an abnormal ca with insignificant disease was 611 256, and for an abnormal angiogram with significant disease, it was 1318 885, and this distribution of ccs among patients with and without significant stenosis is statistically significant (p = 0.014) [figure 2]. The correlation between ccs and significant stenosis is moderate (r = 0.3, p = 0.005) for the whole cohort, but become insignificant when analyzed separately for the high and very high ccs groups . In the roc analysis, an individual's calcium score of 777 has a sensitivity and specificity of 77% to predict a flow - limiting lesion [figure 3]. Coronary calcium score versus significant lesion at patient level coronary calcium score versus significant lesion at patient level . Both high and very high coronary calcium score group a subset of patients in the high coronary calcium score group has normal to insignificant disease . Mean and standard error of calcium scores at patient and vessel levels with and without significant flow - limiting lesions . Patients and vessels with flow - limiting lesions have significantly higher coronary calcium score than those with normal or insignificant disease . A total coronary calcium score of 777 and above has a sensitivity and specificity to predict significantly . Of the 250 vessels analyzed, there were 168 vessels without significant stenosis and 82 vessels with significant stenosis . Of the 168 normal or vessels with insignificant lesions, only 52 had moderate to high calcium, whereas 61 out of the 82 vessels with significant lesions had moderate to high calcium [table 3 and figure 4]. When the individual vessel calcium scores were studied for association with any coronary lesion, there was a strong correlation with significant p value (r = 0.526, p <0.01); however, the correlation for a significant lesion is weak (r = 0.29, p <0.05). Conversely, the mean calcium scores for vessels without and with significant lesion were 129 240 and 454 497, respectively, which were significantly higher for vessels with flow - limiting lesions (p = 0.05). In the roc analysis, an individual vessel score of 121 has a sensitivity and specificity of 70% to predict a flow - limiting lesion in the same vessel [figure 5]. Coronary calcium score versus lesion severity for individual vessels coronary calcium score versus lesion severity for individual vessels . At lower calcium scores per vessel, more number of vessels was normal, as the score increases more number of vessels have significant disease . A score above 120/vessel has a sensitivity and specificity of 70% to predict a significant stenosis in that vessel . The mean ss for the whole study group was 15.9 9.4; when stratified into high <1000 and very high> 1000 groups, the very high group had a ss of 22.7 6.9 and the high group had 9.5 6.7 . On comparing the ccs and the ss for association, there was strong significant correlation between the two among the whole cohort (r = 0.68, p <0.01) [figure 6] and the very high calcium group (r = 0.53, p <0.01, n = 30) but not for the high calcium group (r = 0.1, p = 0.62, n = 20). The frs correlated moderately with ss (r = 0.38, p = 0.006) and ccs (r = 0.46, p = 0.001). There is no significant gender - associated difference in the correlation between ccs and ss score (0.66 for females and 0.64 for males). The mean age of the study group was 62 years with 40% represented by females . Seventy percent of the study population had a low to intermediate ptp for cad based on their age, gender, and symptoms, whereas the rest of the 30% had high ptp but were either self - referred or had denied an invasive angiogram as the initial modality of evaluation . Based on the frs, 70% had a high risk, 20% had an intermediate risk, and only 10% had a low risk . All patients except for three had a calcium score> 400 . In these three patients, the score was between 300 and 400, but were still feared to interfere with contrast ctca . Right coronary artery (rca) had the highest amount of calcium (467 524), followed by left anterior descending (lad; 458 397), left circumflex (lcx; 181 274), left main (lm; 70 170), and posterior descending artery (pda) had the lowest calcium . Nine patients (18%) of this cohort with high ccs had an angiogram without significant disease in any of the vessels [table 2 and figure 1]. Of these, four patients had a ca that was considered to be normal and all four had a ccs <1000 . Four of the other five patients with insignificant disease had scores <1000, whereas one had a score> 1000 . The average calcium for a normal or an abnormal ca with insignificant disease was 611 256, and for an abnormal angiogram with significant disease, it was 1318 885, and this distribution of ccs among patients with and without significant stenosis is statistically significant (p = 0.014) [figure 2]. The correlation between ccs and significant stenosis is moderate (r = 0.3, p = 0.005) for the whole cohort, but become insignificant when analyzed separately for the high and very high ccs groups . In the roc analysis, an individual's calcium score of 777 has a sensitivity and specificity of 77% to predict a flow - limiting lesion [figure 3]. Coronary calcium score versus significant lesion at patient level coronary calcium score versus significant lesion at patient level . Both high and very high coronary calcium score group a subset of patients in the high coronary calcium score group has normal to insignificant disease . Mean and standard error of calcium scores at patient and vessel levels with and without significant flow - limiting lesions . Patients and vessels with flow - limiting lesions have significantly higher coronary calcium score than those with normal or insignificant disease . A total coronary calcium score of 777 and above has a sensitivity and specificity to predict significantly . Of the 250 vessels analyzed, there were 168 vessels without significant stenosis and 82 vessels with significant stenosis . Of the 168 normal or vessels with insignificant lesions, only 52 had moderate to high calcium, whereas 61 out of the 82 vessels with significant lesions had moderate to high calcium [table 3 and figure 4]. When the individual vessel calcium scores were studied for association with any coronary lesion, there was a strong correlation with significant p value (r = 0.526, p <0.01); however, the correlation for a significant lesion is weak (r = 0.29, p <0.05). Conversely, the mean calcium scores for vessels without and with significant lesion were 129 240 and 454 497, respectively, which were significantly higher for vessels with flow - limiting lesions (p = 0.05). In the roc analysis, an individual vessel score of 121 has a sensitivity and specificity of 70% to predict a flow - limiting lesion in the same vessel [figure 5]. Coronary calcium score versus lesion severity for individual vessels coronary calcium score versus lesion severity for individual vessels . At lower calcium scores per vessel, more number of vessels was normal, as the score increases more number of vessels have significant disease . A score above 120/vessel has a sensitivity and specificity of 70% to predict a significant stenosis in that vessel . The mean ss for the whole study group was 15.9 9.4; when stratified into high <1000 and very high> 1000 groups, the very high group had a ss of 22.7 6.9 and the high group had 9.5 6.7 . On comparing the ccs and the ss for association, there was strong significant correlation between the two among the whole cohort (r = 0.68, p <0.01) [figure 6] and the very high calcium group (r = 0.53, p <0.01, n = 30) but not for the high calcium group (r = 0.1, p = 0.62, n = 20). The frs correlated moderately with ss (r = 0.38, p = 0.006) and ccs (r = 0.46, p = 0.001). There is no significant gender - associated difference in the correlation between ccs and ss score (0.66 for females and 0.64 for males). Calcium in the coronary arteries is believed to represent a dystrophic process, where the worn - out tissues of the aging vessels with plaques are replaced by calcium . This process is mediated by the calcium - binding glycophosphoprotein osteopontin . According to the american heart association, staging of the coronary atherosclerotic plaques calcification is a feature of stage v, but with refined microscopic techniques micro calcification can be identified from stage iii onward . The location of calcification is also variable, which may be intimal, medial, or adventitial . As macroscopic coronary calcification is a late stage in the progress of atherosclerosis, in any cross - sectional autopsy series that included patients from all age groups, only 20% of plaques are found to be calcified . Furthermore, the process of calcification indicates stabilization of plaques along with positive remodeling leading to compensation for the lumen loss caused by the plaque . It is for these reasons that the identification of coronary calcium though predicts the vascular age and the global atherosclerotic burden of the patient, and it is not specific for a vulnerable plaque or a significantly stenotic lesion . Supporting this fact, there are many reports of documented absent coronary calcium that was followed by acute coronary syndromes within months this has led to the discouragement of recommending calcium scoring as diagnostic rather than a screening test for risk stratification . Any ctca protocol includes a plain scan at low radiation, and when that shows sufficiently high calcium known to interfere with final interpretation, the procedure might be abandoned . In such patients, published literature on calcium scoring are mainly for risk stratification in asymptomatic subjects . Based on large population - based studies, it has been shown to be of benefit in predicting cardiovascular events . In our study, we have shown that a high coronary calcium correlates strongly and significantly with the global coronary plaque burden as derived by the ss . It was also found that the individual vessel calcium scores were correlating strongly with the presence of any lesion that may be anything above 30% narrowing, but only weakly with a significant stenosis defined by a luminal narrowing of 70% [figure 7]. The sensitivity and specificity of calcium score to predict a significant lesion is higher at patient level than at vessel level which again reinforces the fact that ccs is a marker of global atherosclerotic burden rather than a marker of a focal significant lesion [figure 8]. It is also plausible with the pathogenesis of coronary calcification which occurs in older plaques rather than a significant plaque, and the majority of the coronary plaques are not significant but may be older and usually outnumber significant plaques by 35-fold . The ss mandates scoring for any lesion 50% stenosis in vessels that are 1.5 mm . As a result, borderline lesions also contribute to the overall score and hence correlating with ccs irrespective of the severity of lesion, not necessarily significant one . We have also found that ss correlated strongly with the frs which is also pathogenetically plausible as frs represents the vascular age based on the patients risk profile . The limitations of our study include the small number of patients included, but this could be compensated by the number of vessels studied, as both calcium scoring and lesions on ca were computed and analyzed for individual coronary arteries (lm, lad, lcx, rca, and pda). The second limitation of our study could be that 70% of the studied population were high risk according to the 2008 frs and so they are expected to have a higher vascular age . Hence, extrapolating this to the low and intermediate risk group may not hold true . It is also plausible that the high frs would have confounded the strong correlation of the ccs with the ss, but the r value for ccs and ss is stronger than the r value for frs and ss . This again reinforces the fact that ccs may have incremental value over and above the frs for any given patient in predicting the atherosclerotic burden . We have a subtle evidence for the same in the form of loss of significant correlation of the ccs to ss in those with a total score <1000 . The strength of our study includes the following: (1) the ccs is done by an experienced radiologist . (2) the ca was interpreted by two cardiologists with a third one validating any dispute . Axial plain computed tomography images in a 45-year - old male show extensive calcification in the left anterior descending (a) and left circumflex (b) indicated by arrow; catheter angiogram (c) showing normal left anterior descending (long arrow) and left circumflex (short arrow). Axial plain computed tomography images in a 53-year - male show extensively calcified left anterior descending (a) and left circumflex (b) indicated by arrow; catheter angiography image (c) showing multiple foci (arrows) of insignificant and significant stenosis in left anterior descending . High ct calcium scores in this cohort of intermediate to high (framingham score) risk patients correlated strongly with the subject's global burden of the cad as derived by the ss, more so for subjects with very high scores . Similarly, high ccs predicted significant stenosis at both individual and arterial levels; also, the arterial vessel scores correlated strongly for any lesion but only weakly for the presence a significant stenosis . In spite of this association between ccs and the flow - limiting disease on conventional angiogram, a proportion of patients may still have insignificant disease when ccs is between 350 and 1000.
With ever - improving imaging technologies and high - performance computational power, the complexity and scale of brain imaging data have continued to grow at an explosive pace . Recent advances in imaging technologies, such as molecular imaging using positron emission tomography (pet), structural imaging using high - resolution magnetic resonance (mr), as well as quantitative electrophysiological cortical mapping using electroencephalography (eeg), have allowed an increased understanding of normal and abnormal brain structures and functions [14] it is well understood that normal brain function is dependent on the interactions between specialized regions of the brain which process information within local and global networks . Consequently, there is a need to integrate the acquired multimodality data in order to obtain a more detailed understanding about process interaction in a complex biological system . In addition to integration, advanced computational tools need to be developed which allow quantitative analysis of a variety of functional patterns as well as the design of a software environment that allows quantitative assessment of relationships between diverse functional and anatomical features . Although this is an area of active research, current state - of - the - art technologies are still suboptimal with respect to multimodality integration and quantitative assessment of qualitatively distinct neuroimaging datasets in patients with structurally abnormal brains . This paper presents a multimodality database for neuroimaging, electrophysiological, as well as clinical data specifically designed for the assessment of epileptic foci in patients with epilepsy, and can serve as a standard template for other diagnostic procedures which employ multiple imaging technologies . We discuss previous work by other investigators and how it relates to the proposed framework . In section 3, we introduce a novel image analysis method able to assess quantitatively the relationship between imaging and electrophysiological data . In section 4, we describe the integration of diverse data sets into an extendable database structure and describe available queries for quantitative analysis of multimodality data . Section 5 details the implementation issues and shows the applications of the presented environment . Finally, in section 6 we discuss the strength and limitations of the proposed system and conclude with potential future directions . The accurate segmentation of anatomical brain structures from medical images and their compact geometrical representation in a rigorous computational framework is difficult due to the complexity and physiological variability of the structures under study . In the past, a large number of techniques have been applied to achieve spatial standardization of the cortical surface within a group of subjects, but only a few methods are currently available for the definition of homotopic volume elements within the whole brain . For example, thompson et al ., lohmann, and vaillant et al . Used a manually labeled atlas brain, which was then warped to fit an individual subject's brain surface with subsequent transfer of labels onto the subject's cortical surface . Another related method is the 3d stereotactic surface projection (3d - ssp) technique, which is based on the extraction of subcortical functional data onto a predefined set of surface pixels following stereotactic anatomical standardization of an individual brain . More recently, conformal mapping techniques have gained a wider application in brain mapping . For example, hurdal and stephenson proposed a discrete mapping approach that uses spherical packing in order to produce a flattened image of the cortical surface onto a sphere yielding maps that are quasiconformal approximations to classical conformal maps . Moreover, gu et al . Proposed optimization of the conformal parameterization method by composing an optimal mbius transformation so that it minimizes the landmark mismatch energy . Based on the work of the above investigators as well as on our previous work with landmark - constrained conformal surface mapping, we have developed a landmark - constrained conformal mapping of the brain where the mapping accuracy is achieved through matching of various cortical landmarks (e.g., central sulcus, sylvian fissure, interhemispheric fissure). These landmarks are manually defined in the subject's native space and subsequently aligned in the spherical domain using landmark - constrained conformal mapping . Finite cortical elements are defined geometrically in the spherical domain and subsequently reversely mapped into the subject's native space where all data analyses take place . As the position of cortical landmarks is affected by structural abnormalities, this method incorporates both physiological variations as well as structural abnormalities into the mapping process . The creation of brain image databases which allow storage, organization, and the sharing of processed brain data between investigators with different scientific backgrounds in easily accessible archives is one of the main goals of the newly emerging field of neuroinformatics . This objective is becoming increasingly important for researchers in the brain imaging field as an unprecedented amount of brain data is now acquired using complementing modalities which need to be analyzed based on an overarching strategy [1315]. Following the model of other scientific fields which were revolutionized through the implementation of powerful database structures such as the genbank (http://www.ncbi.nlm.nih.gov) for the field of genomics or the protein data bank (http://www.rcsb.org/pdb) for the field of proteomics, several neuroimaging databases were generated in the past decade in order to fully mine the information present in the acquired data . The visible human project website http://www.nlm.nih.gov/research/visible/visible_human.html researchers to view high - resolution image sections of two human cadavers and is widely used in education and training . More comprehensive human brain databases which attempt to combine the expertise of computer programmers, statisticians, and basic researchers in an attempt to develop neuroinformatics tools for interdisciplinary collaboration are the icbm database http://www.loni.ucla.edu which includes pet, mri, fmri, eeg, and meg modalities as well as the brainmapdbj http://www.brainmapdbj.org and the echbd databases which both integrate pet and fmri image data . In comparison to these multiinstitutional large - scope archives, our database is specific to the task of understanding the mechanisms of epileptogenesis in patients with epilepsy who are evaluated for resective surgery . Whereas the above - mentioned archives integrate the data within the concept of a probabilistic atlas of the human brain, a major consideration of our database design was the analysis and integration of data in native space . As a consequence, our approach not only allows the integration of complementing modalities (pet, mri, eeg) in native space of each individual patient, but also provides software tools able to quantify the relationship between these diverse modalities . Consequently, the developed framework has the potential to generate quantitative measures which characterize the state of the brain in more detail than would be possible with each individual modality . The main objective of presurgical evaluation of patients with medically refractory epilepsy is to define the boundaries of epileptogenic brain regions to be resected . Towards this goal, definition of the epileptogenic cortex by intracranial subdural eeg recording however, the accuracy of foci localization using subdural electrodes depends greatly on location of electrodes placed on the brain surface, and selection bias (i.e., area of cortex sampled) is a major limitation . In order to guide the placement of subdural electrodes, a combination of noninvasive anatomical and functional imaging, such as mr and pet imaging, is frequently used . These modalities provide the epilepsy surgery team with important information to guide placement of subdural electrodes over epileptogenic brain regions based on the position and extent of anatomical and functional abnormalities [1820] as well as the seizure semiology (i.e., symptoms of seizures). Furthermore, after the intracranial eeg electrodes are implanted, each electrode records a number of electrophysiological parameters that need to be related to the imaging information in order to decide on the most effective course of surgical intervention . To this end, we designed an extendable multimodality database which allows assessment of relationships among various modalities across a large patient population . A focal point of our design was the extensibility of the database scheme in order to allow easy inclusion of data originating from newly developed pet tracers or the inclusion of emerging modalities such as diffusion tensor imaging (to incorporate a measure of brain connectivity) or susceptibility weighted imaging (to assess venous vasculature in brain tissue). Pet imaging using the tracers [f-18]deoxy - l - glucose (fdg) and [c-11]flumazenil (fmz) was performed as part of the clinical management of patients undergoing evaluations for epilepsy surgery . Pet studies were performed using the cti / siemens exact / hr scanner (knoxville, tenn, usa) with a reconstructed image resolution of about 5 mm fwhm . All subjects was fasted for 4 hours prior to the pet procedure and eeg was monitored using scalp electrodes during the whole study duration . Static pet images were obtained based on coincidence data acquired between 4060 minutes post injection for the fdg and between 2040 minutes post injection for the fmz tracer . Mri studies were performed on a ge 1.5 tesla signa unit (milwaukee, wis, usa). High - resolution t1-weighted images were performed using a fast spoiled gradient echo (spgr) sequence . The spgr technique generates 124 contiguous 1.5 mm sections of the entire head using a 4.6/1.3/450 (tr / te / ti) pulse sequence, flip angle of 12 degrees, matrix size of 256 256, and fov of 240 mm . Following coregistration of the pet image volumes to the high - resolution mr image volume, all extracerebral structures were removed from the mr image volume using an in - house developed software package . Following initial thresholding, the software performs multiple erosion and dilation operations in order to delete connections between the brain and the skull in subdural space . Finally, a connected component analysis is performed and all but the largest component (brain) is deleted, resulting in an mr image volume where all extracerebral structures are removed and cortical sulci and gyri become visible . The standard set of cortical landmarks consisted of the central sulcus, sylvian fissure, and parieto - occipital sulcus, which were subsequently used to define the spatial extent of the four brain lobes (frontal, parietal, temporal, and occipital). In order to find the border of the occipital cortex on the lateral cortical surface, both the parieto - occipital sulcus as well as the medial border between the cerebellum and the occipital lobe were defined on the midplane and then projected normal to the midplane onto the lateral surface (figure 1). We have recently developed a landmark - constrained conformal surface mapping technique [12, 21] which allows accurate and reproducible transformation of each patient's cortical surface to a canonical spherical domain (the conformal brain model (cbm), see the appendix). Once the cortical surface of a subject is mapped to the cbm, finite surface elements can be defined geometrically on the spherical surface at various resolution levels (8, 32, 128, 512, and 2048) (figure 2). The set of surface elements can be subsequently reversely mapped into native space where they represent homotopic surface elements in brains of individual patients . These surface elements scale proportionally to the size and shape of individual brains . Moreover, in order to integrate pet and mri data, a normal fusion approach is applied in native space of each subject . The extracted mr brain surface is smoothed using a roller - ball algorithm based on alpha - shapes where the parameter alpha is the radius of a ball rolled over the surface . Smoothing of the cortical surface is achieved as all points deep inside cortical sulci are recovered which cannot be reached by the ball . Following triangulation of the brain surface, the normal vector to the surface is calculated in each surface voxel . By averaging the pet tracer concentration along the inverse normal vector in the coregistered pet image volume, the average pet tracer concentration within a 10 mm cortical mantel is calculated . By comparing the pet tracer concentration in these cortical elements between a group of normal control subjects and an individual patient, functionally abnormal increases or decreases of pet tracer concentration can be determined in finite elements of the cortical mantel . Decision about the functional abnormality of a cortical element of a patient at a given location can be then made based on a severity index calculated as (1)severityi=(mii)i, where mi is the mean tracer concentration at cortical location i of a patient and i and i are the mean tracer concentration and standard deviation (sd) at cortical location i derived from a control group . Values of the severity index within 2 sd are assumed to represent normal cortex . The severity of detected abnormalities (outside 2 sd) is then color - coded and mapped onto the cortical surface allowing assessment of functional abnormalities relative to anatomical cortical landmarks . Our approach attempts to merge the advantages of voxel - based (such as spm) and region - of - interest - based strategies, but at the same time tries to avoid the pitfalls associated with either of these methods . The semiautomated geometric parcellation procedure creates cortical elements that are much larger than individual voxels of the image volume, but circumvents the time consuming and subjective definition of large surface - based regions of interest . Moreover, as this method does not rely on asymmetry measures between homotopic cortical volume elements, it is well suited to detect bilateral cortical abnormalities . Finally, the most prominent advantage of our method is the fact that all data samplings and analyses are performed in the subject's native space . By avoiding spatial warping, we allow application of this method to brains that are very different from the normal adult brain, such as brains of patients with tuberous sclerosis (with a large number of tubers deep inside the brain) or children during various developmental stages . All patients with intractable focal epilepsy included in the present study underwent chronic eeg monitoring with subdural electrode grids as part of their presurgical evaluation . Subdural electrode placement was guided by seizure semiology, scalp eeg recordings, and cortical glucose metabolism abnormalities on pet . During the chronic subdural eeg monitoring, more than two habitual seizures were captured and analyzed using stellate's sensa 5.0 software, yielding for each grid electrode the mean interictal spike frequency as well as the normalized interictal spike frequency (normalized to the electrode with highest spike frequency). Identification of electrodes involved in seizure onset and seizure spread of habitual clinical seizures was determined by chronic subdural eeg monitoring (see, e.g.,). In the past, we have implemented a method that allows the localization of subdural grid electrodes on the cortical surface [25, 26]. In short, this method relies on the accurate alignment of a lateral planar x - ray image (figure 3(b)) of the patient's head with the electrode grid in place with a three - dimensional surface rendering of the head (figure 3(a)). Upon completion, a surface view of the cortex is created which corresponds to the planar x - ray image and where the spatial coordinates of the four corner grid electrodes of each rectangular eeg grid can be determined on the brain surface (figure 3(c)). The accuracy of this method was reported to be 1.24 0.66 mm with a maximal misregistration of 2.7 mm . Our previous results indicated that intracranial subdural electrodes showing electrophysiological ictal (seizure onset and spread) or interictal (spike frequency) abnormalities are either overlapping or in close proximity to functional abnormalities measured with pet imaging . Because classical receiver operator characteristic (roc) analysis is suboptimal in describing spatially related measures, we have previously developed a spatial proximity index (spi). This index yields an overall quantitative measure of the spatial relationship between electrophysiological and functional pet image data and is calculated based on the position of intracranial grid electrodes relative to abnormal pet tracer concentration in cortical areas (figure 4). The spi is a continuous variable that equals zero for perfect overlap between seizure onset electrodes and pet - defined abnormal cortical elements and increases in value proportional to the distance between eeg - defined onset electrodes and pet - defined abnormal cortical elements . The subdural eeg electrode array defines electrodes that are either eeg positive (e+) or negative (e) for seizure onset . Moreover, electrodes located within the pet abnormality are designated as pet positive (p+) and those outside the pet abnormality are designated as pet negative (p). Using these definitions, the (unitless) spatial proximity index (spi) is computed as the ratio of the penalized total weighted distance between eeg positive and pet positive electrodes and the total number of seizure onset electrodes, (2)spi=i=1mwidi(ei+\|pi+)+i=1kpi+(ei+) i=1m(ei+), where m is the number of eeg positive electrodes, k is the number of pet positive electrodes, and wi is a weighting factor which accounts for the varying interictal spike frequency at different electrode locations . The weighting factor is either 1 or 0 for both seizure onset and spread, whereas for spike frequency the weighting factor is derived as the quotient between the spike frequency at a particular electrode location and the maximal spike frequency in the whole brain . In (2), the first term in the numerator represents the total weighted distance between all seizure onset electrodes and the nearest pet positive electrode, whereas the second term represents the number of all false positive pet electrodes (i.e., pet positive electrodes which are not eeg positive). Finally the denominator reflects the total number of eeg positive electrodes . Because subdural electrodes are always arranged in a rectangular lattice, the city - block it is important to note that all characteristics of the euclidian metric are preserved in the city - block metric . In this metric, the distance between two adjacent electrodes is 1 and the distance between two diagonal electrodes is 2 . Spi values were calculated separately for seizure onset (spionset), seizure spread (spispread), and interictal spike frequency (spispike). In order to demonstrate the computation of spi values, figure 4 shows a (5 4) electrode grid with seizure onset electrodes (red) and electrodes which are not seizure onset electrodes (yellow). In figure 4(a), the pet abnormality is adjacent to the seizure onset electrodes resulting in an spionset value of 2.66, while in figure 4(b) the pet abnormality is remote to the seizure onset electrodes resulting in an spionset value of 5.66 . In this example, the lower spi value indicates a closer spatial proximity between (complementing) pet and eeg modalities and indicates that low spi values are associated with situations in which pet imaging is successfully guiding the placement of an intracranial eeg grid (despite being nonoverlapping). Conceptually, the database is structured in three hierarchical levels: a patient level, a cluster level, and a cortical element level . At the highest level (patient level), clinical variables are stored such as the age of the patient, age at first seizure, seizure type, seizure severity, and seizure duration . An index of seizure severity was derived as the product of average monthly seizure number and average seizure duration . As the cortical distribution of each pet tracer (e.g., fdg, fmz, etc .) Is distinct, the database includes separate row for different pet tracers (exam_type in pet_exam table). The lowest level is the cortical element level, which provides information with regard to the location of functional data in cortical elements . Cortical elements at different resolution levels are represented by the coordinates of the three vertices building the corners of a surface triangle (corner_a, corner_b, corner_c in the cortical_element table). In addition, the pet table stores for each cortical element the severity (severity_index), and the spi table stores spi values for seizure onset, seizure spread, and interictal spiking for the whole brain at each resolution level . Moreover, the surface coordinates of each eeg grid electrode are stored at this lowest level in the eeg_grid table . This table stores for each of the potentially multiple electrode grids placed onto the cortex the location of the corner electrodes (corner_i - corner_iv in the eeg_grid table). The location of all other electrodes within the eeg grid can be then computed from the location of the corner electrodes given a 10 mm spacing between individual electrodes . Electrophysiological parameter such as the seizure category (onset, spread, spiking), interictal spike frequency, or normalized interictal spike frequency can be then mapped to a particular grid electrode . Given the location of each electrode, the software then automatically determines the corresponding cortical element at various resolution levels . Finally the intermediate cluster level (cluster table) allows the grouping of cortical volume elements either into anatomical territories such as the prefrontal, motor - frontal, parietal, temporal, and occipital cortices, or into functional clusters according to pet or eeg data . To accommodate the flexibility of an evolving database schema, the database allows graceful inclusion of additional modalities at each of the three levels . The xml - based approach does not only accommodate the incorporation of additional data structures into the database, but ensures also the scalibility of the system . This was achieved through following design criteria: (1) an efficient mapping algorithm that generates corresponding relational schemas from xml schemas in linear time, (2) the implementation of two efficient linear data mapping algorithms in order to store xml data in the database, and (3) the implementation of a linear xml subtree reconstruction algorithm able to reconstruct the xml subtree from the database . The database was initially populated with control data determined in control subjects in order to establish a normative pattern . The control group consisted of 15 young adult controls (mean age 27.6 4.5 years) who were not taking any medication, and had no history of neurological or psychiatric disorder . The purpose of the database is to provide an integrated framework able to present relationships between functional and electrophysiological parameters in an organized and, for the user, easy - to - navigate fashion . A focus of our design was to merge an efficient database structure with a display module, so that users can grasp the results of their queries in an intuitive way . Figure 6 shows the main control panel allowing access to all data structures within the database and the link to an integrated 3d image display module . For example, the user can request to highlight all eeg grid electrodes with spike frequency greater than 20% of the maximal spike frequency and to calculate the corresponding spi values . This is highly relevant in light of our previous reports indicating that in patients with nonlesional neocortical epilepsy, seizures arose from areas adjacent to pet - defined abnormalities, rather than from within the pet - defined abnormalities . In essence, this query retrieves the electrophysiological data from table electrode and the pet data from table cluster, respectively, based on the input patient id($pid). After the location of an electrode with (normalized) spike frequencies> 0.20 is determined, data representing the current electrode is integrated with data characterizing the distribution of abnormal pet clusters using the join true only in the case when the current electrode is adjacent to an abnormal cortical region . In addition, our design supports advanced graphical structure mining techniques in order to discover nontrivial associations between multiple electrophysiological parameters and their spatial relationship to the pet - defined abnormalities . One of these data mining techniques, the association rule mining, is used to determine whether certain clinical patterns result in larger spi values or whether different pet tracers produce larger spi values given the same clinical pattern . This information is important in order to determine the clinical performance of newly developed pet tracers . Two measurements that are used in the association mining process are support and confidence defined as follows: support(condition c) = #of patients satisfying c / total #of patients, confidence(condition c1, condition c2) = support(c1 & c2)/support(c1) the association rule mining procedure returns all rules in the form of condition 1 condition 2, with a support and a confidence greater than some user - specified thresholds . The two measurements in conjunction quantify the association strength between condition 1 and condition 2 . One example of an association rule is (3) (#of patients (seizureduration> 3 .and . This indicates that if a patient has had seizures for more than 3 years and the severity score of these seizures was on average higher than 2, then there is greater than 80% likelihood that the patient's spionset will be greater than 1.5 . Support(condition c) = #of patients satisfying c / total #of patients, confidence(condition c1, condition c2) = support(c1 & c2)/support(c1) we have utilized vtk / opengl for rendering, oracle 9i for database applications, and c / c++ for implementation of computationally intensive algorithms . Furthermore, we separated the computational components from other functionality and created a standalone programming library consisting of the following modules: (1) conformal mapping; (2) objective definition of pet abnormalities; (3) eeg grid overlay; (4) calculation of spi values; (5) extendable database component; and (6) graphical output module . With the expected growth of knowledge about the evolution of epileptic foci as well as the emergence of new computational methods that require diverse information inputs, it is impractical to design a fixed database schema . As new requirements arise, it is critical that the database schema adjusts gracefully to these new requirements . In contrast to traditional database systems, where evolution of the database schema to new requirements is problematic for dynamic applications, we applied a flexible data model based on xml (extensible markup language) [29, 31, 32] due to its extensibility and flexibility nature . While in the past we have successfully developed an xml storage and query system, in which the database schema was automatically created from an xml schema, this technique needed to be extended to include support for the processing and storage of spatial information . Twelve children (mean age 5.2 4.3 years, age range 1 to 14.8 years) with medically intractable partial epilepsy were analyzed . All children were diagnosed with unilateral seizure foci based on seizure semiology, scalp ictal, and intracranial eeg as well as fdg pet, which were performed as part of their presurgical evaluation . The region of epileptic focus as verified by eeg showed hypometabolism in all cases . No cortical or subcortical lesions on mri scans were observed; however, patients with pure hippocampal atrophy were included . All studies were performed in accordance with guidelines stipulated by the ethics committee of wayne state university . Our application provides integration of anatomical, functional, and electrophysiological data as shown in figure 7 . Upon retrieval from the database, the software displays a surface rendering of the selected patient's brain with functional data derived from pet imaging mapped directly onto the cortical surface . In addition, each eeg electrode is color coded according to its electrophysiological characteristics (red = seizure onset, yellow = seizure spread, green = normal) and is displayed directly on the cortical surface . As the display is fully three - dimensional, the user can interactively rotate and zoom the brain in order to inspect the spatial relationship between eeg electrodes and pet abnormalities from various viewing angles . In addition to the multimodality display, our software also allows calculation of spi values in order to allow quantitative comparison between localizing information based on clinical readings (onset / spread) and localizing information obtained based on a semiquantitative analysis of interictal spike frequency . Figure 8 shows images determined in a patient studied with both fdg as well as fmz pet, and reports spi values which were calculated with regard to seizure onset electrodes and electrodes with high interictal spike frequency (> 40% of maximal spike frequency). The user is able to select various thresholds of spike frequency and interactively assess the spatial relationship between frequently spiking cortical tissue, the location of seizure onset, and areas of functionally abnormal cortex . The spatial relationship between these areas is then encoded in spi values and stored for further statistical assessment . Usually, eeg abnormalities are located adjacent to extensive pet abnormalities and might be reversible after surgical intervention . In addition, remote areas of pet abnormality might exist, which possibly indicate secondary epileptic foci initially triggered by the primary focus but which might mature with time and become independent . This phenomenon is at present time poorly understood and possible mechanisms are discussed elsewhere . There are strong indications, however, that the relationship between electrophysiology and molecular function is complimentary and that the eeg and pet modalities characterize different aspects of brain tissue epileptogenicity . With ever - improving imaging technologies and boost in computational power, the medical imaging field has experienced a tremendous increase in the amount of information collected . Although medical imaging modalities provide diverse quantitative and qualitative information, each modality has its distinct strengths and limitations . While t1-weighted mr images yield high - resolution anatomical images, the obtained functional information is limited . In contrast, molecular imaging using a variety of pet tracers provides accurate functional information, unfortunately with significantly less anatomical detail as compared to mr imaging . Nevertheless, both modalities provide invaluable clinical information to the physicians even when utilized qualitatively . In the past, our group has successfully used data from pet, mr and eeg in the presurgical evaluation of epileptic children and showed a significant improvement in surgical outcome when using such multimodality approach [33, 34]. It appears that further improvement in the localization of epileptogenic brain tissue might be derived from a tighter integration of all presurgical data (anatomical, functional, electrophysiological, and clinical) within a comprehensive database . Once data from multiple imaging and nonimaging modalities are combined within a rigorous computational framework, the information entailed in one modality may be used to enhance or reinterpret information derived from a complementing modality . Thus, the information content in such a database is not simply additive, but is likely to have an amplifying effect . Large - scale database structures containing various brain disorders can be then constructed with a common frame of reference, allowing meta - analysis of data patterns distributed over several modalities . Obviously, such analyses require a well - developed visual interface which allows the researchers to grasp the relationship between data patterns in an intuitive way . We believe that such a visual database has the potential to provide physicians with additional important clues regarding the pathological state of cortical areas and is likely to be of relevance for the clinical management of epilepsy patients . As with every method, a few limitations need to be considered when applying this method to images of children with intractable epilepsy . As indicated above, we used young adults to create a normal database, and this database was implicitly assumed to characterize accurately normal tracer concentration patterns in children . This approach is inevitable due to ethical guidelines which sanction only the study of children who may derive direct benefit from studies using administration of radioactive substances . Although the application of adult tracer concentration patterns to children represents a potential source of error, it appears that at least for fdg the tracer concentration patterns might be very similar between children and adults . Secondly, integration of brain pet and mr image volumes requires their spatial alignment, a potential source of error . Because the brain is encased by the skull and can be therefore regarded as a rigid body, alignment between the mr and pet brain image volumes is likely to be accurate within a few millimeters, especially if anatomical landmarks inside the brain are used . Moreover, the localization of the electrode grid on the cortical surface might result in a maximal misregistration of about 3 mm . Thus, despite the fact that misalignment effects associated with various alignment procedures might be additive, their total effect is likely to be less than 5 mm . Finally, the initial processing step of our method requires the manual definition of cortical landmarks, guided by a detailed surface rendering of cortical sulci and gyri . Although high - resolution mr images provide detailed features of the cortex, reproducible definition of cortical landmarks is not trivial and might contribute to the misalignment of presumably homotopic cortical elements in different patients . In order to keep this source of error to a minimum, we chose only anatomically well - defined cortical features for our standard landmark set . Nevertheless, the definition of cortical landmarks contributes the largest error to possible misalignment between homotopic cortical elements and future studies are warranted to assess their clinical relevance . The creation of a multimodality extendable database structure accessible through an advanced 3d visualization interface holds promise of providing new insights into the formation and identification of epileptic foci . A better understanding of the relationship between functional and electrophysiological data might lead to new approaches in epilepsy surgery and will likely improve clinical management of a large number of patients suffering from intractable epilepsy.
Rice (oryza sativa) is not only the most important staple food feeding a large part of the world population, but also an important model organism for biological studies of crops as well as other related plants (1,2). For this reason, rice was chosen as the first crop for whole genome sequencing . The availability of genome sequences of the two most common cultivated rice subspecies, indica 93 - 11 and japonica nipponbare (3,4), along with the resequencing of cultivated and wild rice accessions (5), enables the in - depth characterization of rice genes (6), exploration of agronomically important traits (7) and investigation of rice diversity and domestication (8). However, building standardized rice reference genomes with comprehensive and accurate annotations remains a formidable challenge ., conducted manually by dedicated experts . To perform curation, expert curators often administer raw biological data, conduct a thorough literature search, extract essential information from multiple publications, curate the information using structured and controlled vocabularies and then submit the information to a knowledge database [e.g., the reference sequence database (11) at the national center for biotechnology information] to make it public . However, with the exponentially exploding volume of rice knowledge and other relevant data, expert curation is becoming more laborious and time - consuming . Keeping biological knowledge in expert - curated databases comprehensive, up - to - date and accurate is increasingly lagging behind knowledge creation, or worse, not being done at all in fields where insufficient funds can be allocated to curation (14). Simply put, although expert - curated databases have traditionally proven important for scientific research, they are struggling with the flood of knowledge . Explosive growth in the volume of rice data requires a large number of people getting involved in knowledge curation, viz . Community curation exploits the whole power of the scientific community for knowledge curation (1519). A case in point that harnesses community intelligence in knowledge integration is wikipedia (http://www.wikipedia.org). Wikipedia is an online encyclopedia, allowing any user to create / edit any content . It features collaborative knowledge curation, up - to - date content, huge coverage and low cost for maintenance (20). Despite fears that the openness of editorial capacity could lead to incorporation of significant flawed content (21), broad participation in wikipedia not only increases knowledge coverage and keeps knowledge up - to - date, but also improves knowledge accuracy (with enough eyeballs, all bugs are shallow; http://oreilly.com/web2/archive/what-is-web-20.html). Owing to the extraordinary success of wikipedia, it has been advocated that biological databases go wiki (23). As a consequence, more than a dozen biological wikis (2442) have been constructed to exploit the full potential of the scientific community for knowledge curation, such as ecoliwiki for the model bacteria escherichia coli (27), gene wiki for human gene annotation (28) and pdbwiki for protein structures (31). To date, however, there is no community - curated resource for rice . To establish a platform for community integration of rice data and to facilitate efficient management of community knowledge on rice, here we develop ricewiki (http://ricewiki.big.ac.cn), a wiki - based, publicly editable and open - content platform for community curation of rice genes unlike extant relevant databases, ricewiki features harnessing community intelligence in curation of rice genes, quantifying users contributions in each curated gene and providing explicit authorship for each contributor in any given gene, with the aim to attract more participation from the scientific community in collaborative and collective curation of rice genes . Ricewiki has been implemented using mediawiki (http://www.mediawiki.org; a free and open source wiki engine; version 1.18.4), mysql (http://www.mysql.org; a free and popular relational database management system; version 5.1.58) and php (http://www.php.net; a widely used general - purpose scripting language; version 5.2.17) on a red hat enterprise linux server . The wide adoption of community intelligence in knowledge curation is primarily attributable to free wiki software such as mediawiki that provides a collaborative framework for knowledge collection, management and dissemination (that powers wikipedia). Mediawiki allows any user to add, modify or delete any content (with customized permission control for editing) via a web browser without any extra add - ons and thus enables web content to be edited easily, swiftly and collaboratively by multiple different users . History; every change made to a page can be stored; the user responsible for every change can be identified and every history revision can be reviewed or recovered . Built on mediawiki, ricewiki enables users to be involved in an ongoing process of creation and collaboration that constantly changes the contents . Thus, ricewiki can significantly ease the process of knowledge collection, curation and sharing, befitting the exploding volume of biological data . In addition, mediawiki allows any user to develop customized functionalities by packaging a bunch of codes as mediawiki extensions . Authorreward (http://www.mediawiki.org/wiki/extension:authorreward) in ricewiki, with the aim to attract more participation from the scientific community for collaborative curation of rice genes . A wiki page can be collaboratively curated by multiple users and thus may have different edit versions . For each version that is contributed by a specific person, authorreward quantifies his / her contribution by factoring edit quality as well as edit quantity; the edit quantity amounts to the edit distance in comparison with its previous version (i.e. The minimum number of edit operations required to transform one string into the other), and the edit quality corresponds to whether the edit persists in comparison with the last version, ranging from 1, when the edit is entirely reverted (short - lived), to 1, indicating that the edit is totally preserved in the last version (long - lived). Because one person may perform many discontinuous edits for a wiki page, his / her contribution score in this page is the sum of quantified contributions over all contributed edits . Authorreward provides ricewiki with an authorship metric; it quantifies users contributions and yields explicit authorship for each wiki page according to their quantitative contributions (described later in the text). Thus, authorreward bears the potential to increase community participation in ricewiki and to achieve community curation of massive biological knowledge . All extensions as well as software installed in ricewiki can be found at http://ricewiki.big.ac.cn/index.php/special:version . In ricewiki, users can access any content, but only registered users can perform the edits . This restriction is due to a trade - off between simplicity for users to make contributions and reliability of the edits provided . Open identity provided by registration not only improves content reliability and increases users collaborations and communications, but it is also supportive to reward community - curated efforts by giving explicit authorship . Thus, although the requirement of registration poses an unpleasant obstacle to community curation, it is of crucial significance for bio - wikis that would like to give credit to all contributors in reward for community - provided content . Additionally, it is beneficial to greatly avoid vandalism or spammed content in bio - wikis, albeit, of course, there are multiple solutions for vandalism detection (43). Ricewiki incorporates the two common cultivated rice subspecies (o. sativa indica 9311 and o. sativa japonica nipponbare) and covers> 66 000 rice genes . As implemented on mediawiki, ricewiki inherits its features: each content page is associated with a discussion page (where users can discuss content or leave a comment), a history page (where revision as well as its contributor can be recognized) and category terms (that increase the usability for information management). The central objects of ricewiki are rice genes and thus each gene corresponds to a specific wiki page . To provide easy access to gene - specific pages, they are built based on gene identifiers . Although they can also be based on gene names, it is noted that only a small part of rice genes has been well studied . With the ongoing studies on the biological functions of rice genes, a gene name often comes and goes as our knowledge about that entity increases, which would bring uncertainty of designations and synonyms of rice genes . Thus, gene identifiers are relatively stable to refer to specific genes, as they do not change with the accumulation of novel information . The gene information in ricewiki was initially seeded from ncbi refseq (11), ensembl (44), rap - db (12) and msu rice genome annotation project (http://rice.plantbiology.msu.edu). The content of every gene in ricewiki is structured into multiple sections, namely, annotated information, structured information, labs working on this gene and references, as well as a one - sentence summary for gene description at the top of each page (figure 1). Annotated information is organized as free text and is helpful to users who share their knowledge and contribute edits without training in the curation or wiki techniques, significantly simplifying edits provision and lowering technological entrance barriers for wider community participation in curation . It can also fall into several sub - sections, such as function, evolution and expression, making it convenient to direct users to the sub - section(s) of interest . Although these sub - sections are preset, new sub - sections can be easily added and irrelevant sub - section(s) can be deleted . Such arrangement with multiple sub - sections enables not only automatic entry of information via application programming interface but also facilitates users to intuitively edit the information by clicking an edit link available to each sub - section . On the contrary, structured information is organized structurally in the form of a table, including gene symbol, gene description, sequence information, expression profile, external links to other related databases and a set of images from gbrowse (45) showing the genomic context of the gene and the gene structure . Albeit the structure of this table is preset, it also allows users to provide updates and additions . Labs working on this gene is a list of laboratories throughout the world working on this gene, facilitating communication and collaboration in curation of this gene . Figure 1.screenshots of the ricewiki page for the rice semidwarf-1 (sd1) gene, available at http://ricewiki.big.ac.cn/index.php/os01g0883800 . This gene was collaboratively curated by nine researchers, yielding 89 versions as of 1 august 2013 . (b) brief authorship information in reward for community curation and a one - sentence summary for description of this gene . It is a list of laboratories working on this gene derived from references and provided by the community . (e) references, which are automatically generated and formatted with the help of the it is organized structurally in the form of a table, including gene symbol, gene description, sequence information, gene structure, etc . Authorreward extension that quantifies researchers contributions and provides explicit authorship according to their quantitative contributions . The cutoff score for awarding authorship is configurable and set to 1 (by default) in ricewiki . Screenshots of the ricewiki page for the rice semidwarf-1 (sd1) gene, available at http://ricewiki.big.ac.cn/index.php/os01g0883800 . This gene was collaboratively curated by nine researchers, yielding 89 versions as of 1 august 2013 . (b) brief authorship information in reward for community curation and a one - sentence summary for description of this gene . It is a list of laboratories working on this gene derived from references and provided by the community . (e) references, which are automatically generated and formatted with the help of the it is organized structurally in the form of a table, including gene symbol, gene description, sequence information, gene structure, etc . Authorreward extension that quantifies researchers contributions and provides explicit authorship according to their quantitative contributions . The cutoff score for awarding authorship is configurable and set to 1 (by default) in ricewiki . The major focus of ricewiki is to exploit the full potential of the scientific community in collaborative curation of rice genes . It should be noted, however, that one crucial essential to making knowledge aggregation successful in ricewiki and also in other bio - wikis is sufficient participation (19), which requires a large number of people from the scientific community to curate biological knowledge collectively and collaboratively . Unfortunately, the current status is that, despite the presence of well - constructed bio - wikis, most researchers seldom make direct contributions . The reason is well known: most people in areas covered by bio - wikis are in academic research fields, and the currency of academic research careers is authorship, and bio - wikis do not offer attributable authorship . It is an especially severe issue for young researchers, who are most open to new technologies such as wikis, but for whom one s publication record is the significant determinant to his / her academic professional success (41). It is inevitably time - consuming and poorly motivating for researchers to spend time performing curation without career - advancing credit (46) as opposed to writing their next paper although, arguably, contributing to a common and open resource such as a bio - wiki may have much broader scientific impacts . There is no mechanism to reward people who perform knowledge curation in bio - wikis . It has been recognized that the major limitation deterring researchers from active participation in bio - wikis is the lack of explicit authorship and thus no credit for their contributions (17,19,41). It should be also noted, however, that not all people care about authorship, e.g., long - term wikipedia editors . Despite this, authorship might be essential to academic - based researchers . To attract more participation from the scientific community for ricewiki and to make it a vivid platform for community curation of rice genes authorreward (47), an extension to mediawiki, which provides a standard practice to reward community - provided contents in bio - wikis by quantifying researchers contributions and providing explicit authorship according to their quantitative contributions . For each gene - centric page in ricewiki, community curation is quantified as a contribution score for each contributor in which both edit quantity and quality are taken into account (figure 1). Accordingly, authorship in ricewiki is awarded only to a contributor whose contribution score is> 1 (by default; this cut - off score is configurable). At the top of each page, we provide the authorship information, displaying contributor name(s), gene i d, hyperlink to this gene and last update time, which is aimed to encourage community participation and to show clearly how to cite community - curated efforts . Additionally, at the bottom of each page, we present the detailed information, including a pie chart to visualize quantified contributions of multiple contributors who were involved in curating this gene, a histogram to depict the edit quantity and quality for each contributor and a table summarizing contributor name, contribution score, edit count, edit quantity, edit quality, last edit time and edit details . As one person may perform curations for multiple different pages, we define the total contribution of this person to ricewiki as the sum of multiple contribution scores in all participated pages . With explicit authorship as a reward for community curation, ricewiki has the potential to attract more people to share their expertise and to provide edits on genes of their interest . Wikiomics in action (48). Considering the ever - growing volume of rice data and related literature and contrastingly the relatively small number of expert curators working on rice, it should be noted that community curation is an important complement to expert curation, and community - based bio - wikis, like ricewiki, are not aimed for replacement of traditionally expert - driven databases . Ricewiki harnesses community intelligence in curating a wide range of rice - related topics and thus can save considerable time and efforts of expert curators . To encourage collaborative curations of rice genes in ricewiki, expert curators can conduct quality control for community - contributed information and provide a variety of training (e.g., webinars, online tutorials and open discussion) for the community on how to perform curation in an accurate and standard manner . Meanwhile, journals can be also involved in curation by building a mechanism to regard community curation as a compulsory post - publication process and by providing obligations or incentives to authors submitting relevant information to ricewiki . Such a mechanism has already been put into practice in the journal plant physiology, partnering with the arabidopsis information resource to increase the curation of arabidopsis . Wider adoption of this practice would be enormously beneficial to curatorial efficiency, accuracy and reliability (as testified by the admirable annotations for arabidopsis). Therefore, the community as well as expert curators, authors, and journals should collaborate together to make ricewiki more influential and to achieve community curation of massive rice knowledge . Future directions for ricewiki include establishment of close collaborations with laboratories in the world working on rice . Rice knowledge and related data developed by many laboratories and researchers should be added to ricewiki and shared with the whole scientific community . We will also promote, as best as we can partnership with journals to require community curation as a compulsory post - publication . In addition, we encourage investigators / teachers to incorporate community curation of rice genes in ricewiki as student assignments . For example, n students collaborate to curate n rice genes, where n 3, and contribution score for each student should be> 1 . Ricewiki will continue to integrate more types of data (e.g., mutants, repetitive elements, expression and phenotyping) from different resources and improve the connections with existing relevant databases . We have collected 40 000 rice - related publications from pubmed (http://www.ncbi.nlm.nih.gov/pubmed/), including title, author(s), affiliation(s), abstract and hyperlinks to the full text (if available), and currently are attempting to dig out the . Therefore, ricewiki will also integrate tools particularly for literature mining (49,50) and incorporate literature - based curated annotation, to realize automatic information retrieval and improve the credibility of community - provided contents . In response to an update of literature collection, ricewiki will build a mechanism to send an invitation to authors with recent publications for curation of specific genes (51). In sum it exploits the whole power of the scientific community in collaborative curation of rice genes by rewarding community - provided content through contribution quantification and explicit authorship . Such a collaborative community - contributed and contribution - rewarded resource would make it possible to build a rice encyclopedia by and for the scientific community (52) that harnesses collective intelligence for collaborative knowledge curation, covers all aspects of biological knowledge and keeps evolving with novel knowledge . 100-talent program of chinese academy of sciences [y1slxb1365 to z.z . ]; the national basic research and development program [973 program; 2010cb126604 to j.x . ]; national programs for high technology research and development [863 program; 2012aa020409 to z.z . And j.x . ], the ministry of science and technology of the people s republic of china . Funding for open access charge: national programs for high technology research and development [863 program; 2012aa020409], the ministry of science and technology of the people s republic of china.

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