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autophagy is primarily a process that delivers cytoplasmic component to lysosomes for degradation. in its original definition, autophagocytosis and the latter heterophagocytosis, although these terms are less frequently used in modern parlance. the two processes can be distinguished according to what the cell eats, namely, itself in the case of autophagy or foreign bodies such as bacteria in the case of phagocytosis. we now know, however, that these processes are not entirely distinct from one another. the molecular apparatus identified during early studies in autophagy turns out also to be involved in the processes associated with the infecting bacteria from the cells of mammals and other organisms such as insects. for example, once salmonella enterica serovar typhimurium invades nonphagocytic cells such as epithelial cells, a subpopulation of the bacteria becomes decorated with autophagic marker proteins. many studies have been performed on these phenomena, which some have termed xenophagy [13 ]. in this paper, we will discuss the mechanism by which the autophagic machinery is recruited, focusing especially on the case of salmonella. salmonella enterica serovar typhimurium, a gram - negative bacterium, infects small intestinal epithelial cells and develops as an intracellular bacterium within this niche, where it causes gastroenteritis [4, 5 ]. therefore, in order to better control salmonella infection, it is important to understand the mechanisms by which salmonella develops into an intracellular bacterium in host cells. salmonella possesses a type iii secretion system (ttss), which employs a needle - like structure to inject effector proteins into the host cell 's cytosol [4, 6 ]. by injecting a number of effector molecules into the host cytosol, salmonella can invade epithelial cells via a type of endocytic pathway. following invasion, the bacteria form a specialized single membrane organelle, the salmonella - containing vacuole (scv), by modifying endosomal structures. in the early phase, the scv temporarily displays early endosome markers, such as rab5, and eea1 ; subsequently, these makers are replaced by late endosomal proteins, such as lamp1 [79 ]. scv succsessively develops into a variety of long tubular structure, termed spacious vacuole - associated tubules (svats), snx3 tubules, and the salmonella - induced filaments (sifs), after a few hours following infection ; thereafter, the bacteria can slowly propagate in scv and the related structures [710 ]. meanwhile, a subpopulation of salmonella is targeted for xenophagy, beginning with the recruitment of autophagic machinery to the vicinity of the infecting bacteria. in an experimental system using mouse embryonic fibroblasts, 40% of infected salmonella is decorated by autophagic marker proteins one hour after infection [11, 12 ]. in the absence of autophagic capacity in host cells, the salmonella replicates more extensively [11, 12 ]. xenophagy may serve as a backup system to limit the growth of infections in situations in which the scv is somehow malformed. it was suggested that scv is damaged by the action of ttss, resulting in the induction of autophagy toward it. the first identified specific marker of the mammalian autophagosome, microtubule - binding protein light chain 3 (lc3) is localized on the autophagosome, or its immediate precursor structure, the isolation membrane. therefore, cells expressing gfp - tagged lc3 exhibit a punctate fluorescence pattern when autophagy is induced. although the exact function of lc3 in autophagy remains to be precisely understood, it has been proposed to play a role in selective autophagy. in contrast to nonselective autophagy, which targets general cytosolic materials, the targets of selective autophagy range from organelles such as mitochondria and peroxisomes to large protein complexes. it is generally understood that each target possesses a specific tag, such as atg32 for yeast mitochondria [14, 15 ]. adaptor proteins such as p62/sqstm, alfy and nbr1 recognize the target - specific tags [1619 ]. because it can bind both p62/sqstm and nbr1, lc3 has been proposed to be involved in the recruitment of autophagic machinery at the target, [1620 ]. however, recent studies of xenophagy against salmonella have led to another interpretation regarding this recruitment mechanism. lc3 and its paralogues are orthologs of yeast atg8 ; they are ubiquitin - like proteins, but are modified at the carboxyl terminus with phosphatidylethanolamine (pe) instead of proteins [21, 22 ]. in yeast, atg7 and atg3 serve in these process as e1 and e2 enzymes ; in their absence, the pe modification does not occur and starvation - induced autophagy is defective. this is also the case in salmonella xenophagy. in atg7- and atg3-knockout mef cells, gfp - lc3 is not recruited to the vicinity of salmonella enterica serovar typhimurium ; as a result, the bacteria replicate overwhelmingly, leading to host cell death. when mouse embryonic fibroblast cells expressing gfp - atg5 are challenged with salmonella, gfp signal can be observed around some of the intracellular bacteria, in a pattern reminiscent of lc3. in the atg3 knockout mef, the efficiency of gfp - atg5 recruitment is not significantly different. therefore, even in the absence of lc3 recruitment, salmonella can be recognized by the autophagic apparatus. a number of adaptor proteins, including p62/sqstm, ndp52, and optineurin, bind to lc3 and are involved in xenophagy against a variety of bacteria [2325 ]. these interactions with lc3 may be more important for functions rather than recruitment of other atg proteins. two reports, including ours, have revealed detailed phenotypes by depleting the function of all lc3 paralogues involved in starvation - induced autophagy. one group knocked out atg3 genes in mouse embryonic fibroblasts ; another exogenously expressed mutant atg4b. atg4 is protease involved in the cleavage of the carboxyl termini of nascent atg8/lc3 family members and their pe conjugates. overexpression of atg4b containing a point mutation at its catalytic center aminoacid, cysteine, titrates out lc3 and its homologues by binding them strongly and preventing the pe conjugation reaction. both approaches yielded essentially the same results : accumulation of incomplete and unsealed autophagosomes in the cytosol [26, 28 ]. in these experiments, significant proportions of the autophagosome membranes were mostly, but not completely, closed [26, 28 ]. based on these results, we proposed a reverse - fusion model in which lc3 functions in the closing process by directly catalyzing membrane hemifusionmembran- like process. beyond this role in closing, it remains controversial whether lc3 is involved in the elongation of the autophagosomal membrane [26, 28, 31, 32 ]. there is also a report that atypical autophagy, which does not require this lc3 system, exists. in mouse embryonic fibroblast cells expressing gfp - lc3, a punctate gfp signal appears in the proximity of infecting salmonella cells and elongates along the surface of the bacterium, just as the isolation membrane elongates to become the autophagosome. using fluorescence microscopy and electron microscopy correlation, the membrane structure corresponding to gfp - positive salmonella was observed ; a double - membrane structure resembling the canonical autophagosome surrounded the salmonella cells. inside the double - membrane structure, another single membrane thought to be the scv could also be observed. therefore, at least in the case of xenophagy in this system, salmonella is surrounded by autophagosome in addition to the scv [12, 34 ]. in mefs lacking atg7, the e1 enzyme for the lc3 lipidation reaction, a double membrane surrounds the scv, though it may not be completely closed. atg5 is a subunit of the e3 involved in lc3 lipidation, and the mutant is defective in this process [35, 36 ]. these observations clearly indicated that lc3 function is required neither for membrane elongation nor for recruitment of the autophagic membrane to the target. the next question arising is the identity of the alterative factors that actually do recruit the autophagic machinery. good candidates for these factors are found among other atg proteins that function in starvation - induced autophagic process. atg9 is a six - transmembrane protein, essential for autophagy [38, 39 ], whose precise role remains to be determined. mammalian cells have two atg9 homologues, atg9l1 and atg9l2, but the latter is expressed only in placenta and pituitary. in mammalian cells, atg9 travels around the golgi and endosome and potentially the autophagosome. knockout of only atg9l1 brings about severe defects not only in canonical autophagy but also in salmonella xenophagy, evidenced by observations that salmonella replication dramatically increases within infected atg9l1- knock out cells, just as in atg7-knock out cells [12, 41 ]. even in atg9l1-knockout mef cells, gfp - lc3 is efficiently recruited to salmonella enterica serovar typhimurium at levels comparable to those observed in wild - type cells ; however, in these mutant cells, gfp - lc3-positive salmonella is not surrounded by an autophagosome - like double membrane. thus, atg9l1 is required for membrane formation in autophagy, but indispensable for lc3 recruitment. the atg16l complex consists of two sets of atg16l1 and atg12atg5 conjugate, bound by a ubiquitination - like reaction. atg12 binds to atg3, the e2 enzyme of the lc3 lipidation reaction, and the lipidation reaction occurs where atg16l is localized. based on these observations, atg16l complex serves an e3-like role by linking e2 to the target (pe in membrane) in the lc3 lipidation reaction [36, 43 ]. the atg16l complex is exclusively localized on forming autophagosome, the isolation membrane in starvation - induced autophagy [35, 44 ]. in the case of salmonella xenophagy, however, even in the absence of an autophagosome - like double membrane, atg16l complex can localize to the vicinity of infecting salmonella. this implies that the atg16l complex can be recruited to scv independent of the existence of a double - membrane structure. it remains to be determined whether the same mechanism is also applicable to the wild - type cell, but it is highly likely that some targeting mechanism exists that is independent of both the double membrane and lc3. ulk1 is a mammalian orthologue of yeast atg1 protein kinase, which is essential for autophagy [45, 46 ]. ulk1 forms a protein complex with fip200, atg13, and atg101. in mef cells lacking fip200, salmonella xenophagy is defective, as is starvation - induced autophagy. in the fip200 knockout, phenotypes pertaining to gfp - lc3 localization and autophagosome - like double - membrane generation were quite similar to those of atg9l1-knockout cells : gfp - lc3 is efficiently recruited around salmonella enterica serovar typhimurium, and the double membrane is not observed. one plausible explanation for this result is that one of the proteins is responsible for the recruitment of the other to the vicinity of salmonella, but this is not the case. in fip200 knock out cells, atg9 is recruited to salmonella, whereas in atg9l - knock out cells, ulk1 is recruited. thus, localizations of the two proteins are independent of each other. on the contrary, atg9l1 accumulates to a greater extent near salmonella in fip200-knock out cells ; likewise, ulk1 accumulates in atg9l1-knock out cells. ulk1 complex and atg9l1 are potentially recycled between the vicinity of salmonella and other cytosolic pools ; detachment of either protein from salmonella appears to be dependent on the other. these two players seem to play quite important roles in membrane biogenesis, and it is likely that their functions are tightly coupled with their recycling. there are both similarities and differences between these models and what has been observed in yeast autophagy. in yeast, atg1 (ulk1 homologue) is also required for recycling of atg9 from the pas, the site of autophagosome formation, to other pools. however, targeting of atg9 to the pas is dependent on atg17, a potential counterpart of fip200, through direct binding. this fip200-independent atg9l1 localization may be explained by the fact that a part of atg9l is transiting early endosome, which is closely associated with scv at steady state, even in the absence salmonella infection. pi3p plays critical roles in canonical starvation - induced autophagy.when cells are treated with wortmannin, a potent inhibitor of pi3-kinase, lc3 localization to autophagosome is completely defective. in the case of salmonella xenophagy, however, wortmannin treatment does not affect lc3 targeting to the vicinity of salmonella enterica serovar typhimurium, although another study showed some reduction in the efficiency. this does not necessarily mean, however, that pi3p is indispensable for salmonella xenophagy. for starvation - induced autophagy, there exists a specific pi3-kinase protein complex, consisting of vps34, vps15, beclin-1, and atg14l [5254 ]. the knockdown of atg14l, the sole complex - specific subunit, leads to salmonella overgrowth in infected cells. wipi-1, a pi3p - binding protein involved in autophagy, is also observed there, and this localization is sensitive to wortmannin treatment. thus, similar to the case of atg9l1 and ulk1 complexes, autophagy - specific pi3-kinase activity is involved in salmonella xenophagy, but is dispensable for lc3 targeting. this is easily understandable in light of the fact that localization of atg14l to salmonella becomes defective in cells lacking either atg9l1 or fip200. this implies that both atg9l1 and ulk1 complexes are upstream determinants of autophagy - specific pi3-kinase localization. in the case of starvation - induced autophagy, autophagy - specific pi3-kinase is targeted to the endoplasmic reticulum, where it forms foci (the omegasome) in order to form the autophagosome. this omegasome is marked by dfcp-1 through its pi3p - binding capacity, whose function in autophagy is still unclear. dfcp-1 is closely associated with salmonella xenophagy, so this may take place in close proximity to the er (see figure 1). it is now clear that there exist at least three independent axes for the recruitment of autophagic machinery to the vicinity of salmonella enterica serovar typhimuriuma : atg16l complex, atg9l1, and ulk1 complex. in the case of yeast autophagy, atg17, a subunit of atg1 complex, is proposed to be a fundamental determinant of the recruitment of other atg proteins to the pas. in the case of mammalian starvation - induced autophagy, however, both cases of starvation inducue autophagy lack the existence corresponding to scv, which can become an alternative membrane target of atg16l complex (i.e., instead of the autophagosome). therefore, the possibility that an ulk1 complex - independent atg16l recruitment mechanism is also involved in starvation - induced autophagy can not be eliminated. in that case, what factors exist upstream of atg16l and ulk1 complexes ? involvement of adaptor proteins is highly likely, although their direct binding to lc3 is indispensable. ubiquitin and several adaptor proteins are recruited to the vicinity of salmonella, so they must play critical roles [2325 ]. it is possible that these adaptor proteins also bind other atg proteins, such as ulk1 and atg16l complexes. in this regard, it is noteworthy that tecpr1, a novel adaptor protein involved in xenophagy, binds to atg5. combining with other important players such as diacylglycerol, understanding the direct trigger for salmonella xenophagy represents the next important step for this field. | salmonella enterica serovar typhimurium enter epithelial cells and take up residence there. within epithelial cells, a portion of the bacteria are surrounded by an autophagosome - like double - membrane structure, and they are still residing within the salmonella - containing vacuole (scv). in this paper, we will discuss how the autophagy machinery is recruited in proximity to salmonella. the formation of this double membrane requires atg9l1 and fip200 ; these proteins are important for autophagy - specific recruitment of the pi3-kinase complex. in the absence of atg9l1, fip200, and pi3-kinase activity, lc3 is still recruited to the vicinity of salmonella. we propose a novel model in which the mechanism of lc3 recruitment is separate from the generation of the isolation membrane. there exist at least three axes in atg recruitment : ulk1 complex, atg9l1, and atg16l complex. |
spinal cord injury harms the physical and mental condition of patients and causes substantial economic burden to society. therefore, the prevention and treatment of spinal cord injury has become an important topic in medicine. scientists are investigating various avenues for treatment of spinal cord injury ; however, aside from methylprednisolone, none of these developments have reached even limited use in the clinical care of human spinal cord injury patients in the usa. microarray data analysis features high throughput and high sensitivity, which has made it possible to test the expression changes of the whole genome. therefore, the development of microarray analysis has provided new insights into diagnosis and treatment of sci [79 ]. toshiya, using complementary dna microarray analysis, identified 3 up - regulated and 7 down - regulated genes that may play a role in response to tissue damage or repair following sci. however, most of these reports only concentrated on several individual genes, lacking overall data. in addition, the degs of interest were rarely the same in different reports, and results of analysis of chips varied due to many factors, such as samples, platforms, and analysis methods. thus, many experts have made great efforts to obtain reliable results from chips by use of meta - analysis ; however, many essential issues, such as annotation and comparison of cross - platforms, could not be solved appropriately. in the present study we identified 113 differentially expressed mirnas between normal and spinal cord injury patients. then we constructed a ppi network by use of the string database to explore 3 function modules to determine the biological processes and signal pathways they were involved in. we found no significant differences in signal pathway between genes participating in function module 1 and all 113 degs. eventually, we found that a critical protein, actc1, could function as a prognostic and predictive marker in clinical treatment of sci. the microarray expression profiles of spinal cord injury were extracted from the geo (gene expression omnibus, http://www.ncbi.nlm.nih.gov/geo/) database under the accession number of gse33886. a total of 7 specimens divided into 2 groups were available for the analysis, including 4 samples from normal vastus lateralis leg tissues and 3 specimens from leg tissues of patients with spinal cord injury. the original data are preprocessed by rma function in the affymetrix package of r language. the original cel files were converted into probe expression measures, and the probe - level data were converted into gene names by an annotation package based on the bioconductor platform. probes matching more than 1 gene were eliminated and the average value was used for probes matching the same single gene. degs that were significantly differentially expressed among the mesenchymal stem cell samples from sci patients and the matched normal tissues were screened out by use of the limma package in r language. fold change > 2 was used as the threshold to determine the significance of gene expression difference. we used cluster analysis and the corresponding cluster figure to ensure that the screened genes perfectly expressed the differences between samples of sci patients and the matched normal tissues. the david (database for annotation visualization and integrated discovery) database was used to perform the go functional and kegg pathway analysis. significantly enriched go terms and kegg pathways with fdr (fold discovery rate) 2 was used as the threshold to determine the significance of gene expression difference. we used cluster analysis and the corresponding cluster figure to ensure that the screened genes perfectly expressed the differences between samples of sci patients and the matched normal tissues. the david (database for annotation visualization and integrated discovery) database was used to perform the go functional and kegg pathway analysis. significantly enriched go terms and kegg pathways with fdr (fold discovery rate) 5 and p - value 5 and p - value < 0.05 by clusterone plug in cytoscape (table 3, figure 5). eight genes were enriched in 29 biological pathways in the first module, while 10 genes were enriched in 18 pathways related with molecule functions. several pathways associated with biological process were enriched, but no obvious signaling pathway or molecular function was enriched in the second or the third module (table 5). twelve biological processes, 9 cell constitution, 2 molecular function, and 3 kegg pathways were enriched in 3 function modules and all degs (figure 6), except for 5 biological processes that were enriched in the degs but not in the function module. this result revealed that there was no obvious difference in signaling pathways between the 11 genes that participated in module 1 and the 113 differentially expressed genes. in this study we identified 113 differentially expressed genes from the most significantly altered genes using data from 2 datasets on the same platform of the geo dataset, which can effectively avoid differences resulting from many factors, such as different samples, platforms, and analysis methods. much of the previous research concentrated on the several genes they selected instead of the overall data. although much effort was made, such as using meta - analysis to obtain more reliable results, critical problems could not be solved, such as annotation and comparison of trans - platforms. our study effectually overcomes the above defects by using 2 datasets on the same platform, thus enhancing the reliability of our results. as 2-gene interaction is more sensitive and accurate in clinical prognosis and diagnosis of sci compared to use of a single gene, we selected 40 interacting genes by string method to construct a ppi network for better analysis of data, which turned out to be a network with biological functionality. the ppi network of degs in our study appeared to be highly clustered, which is the essential character of a biological network, reflecting a high degree of modularization. we divided the network into 3 separate modules and performed functional enrichment analysis on these 3 modules. the ppi network we constructed in the research was larger and more reliable than the previous ones. three function modules cardiac muscle contraction (hsa05410), hypertrophic cardiomyopathy (hcm) (hsa05414), and dilated cardiomyopathy (hsa04260) were explored using clusterone. actc1 was found to be up - regulated, while myosin heavy - chain 6 (myh6), myosin heavy - chain 7 (myh7), and myosin light - chain 3 (myl3) were down - regulated. in addition, the availability and rationality of these functions were supported by the fact that there was no obvious difference between the signal pathway participation of genes in module 1 and those of all the degs, which make our results more reliable. our results suggest a potential application of target genes and the proteins they express as prognostic and predictive markers in development and improvement of sci therapies. stat3 (signal transducer and activator of transcription 3) was reported to be involved in scar formation after spinal cord injury by regulating astrocyte activity, which can in turn restrict the spread of inflammatory cells after sci. in another study, the rho signaling pathway was reported to be a potential target for therapeutic interventions after spinal cord injury. however, these studies concentrated primarily on pathways after spinal cord injury, and there has been little research into the mechanisms that regulate the progression and development of sci. in this study, through comparing functions between module 1 and all the degs, we obtained 3 pathways the myocardium contraction pathway, hypertrophic cardiomyopathy pathway, and the dilated cardiomyopathy pathway that may function as predictors of clinical outcome in sci patients. evidence from previous research showed that sci has many common complications, and cardiac dysfunctions were among the most important complications following sci. cardiovascular disturbances were proven to be the leading causes of morbidity and mortality in both acute and chronic stages of sci. therefore, we suggest that the 3 significantly enriched pathways found this study may be associated with the occurrence of spinal cord injury because cardiovascular deconditioning may lead to loss of skeletal muscle pumping activity. actc1 protein, the critical protein in these 3 pathways, was significantly up - regulated, while myosin heavy - chain 6 (myh6), myosin heavy - chain 7 (myh7), and myosin light - chain 3 (myl3) were down - regulated. therefore, it is important to study the regulation mechanism of actc1 to help understand its role in predicting targets in the injury process in sci patients. the up - regulation of actc1 can affect the myofilament contraction in the myocardium contraction pathway, and the state of striated muscle in hypertrophic cardiomyopathy pathway and the dilated cardiomyopathy pathway. in fact, in the last decade, actc1 has increasingly been recognized as an important property of muscle state. it mediates a variety of essential biological functions in all eukaryotic cells, providing a structural framework around which cells shape themselves. our results in the present study suggest its momentous role in the cytoskeleton and a potential role as a prognostic factor and predictor in clinical management of sci patients. in addition, we discovered that degs were significantly enriched in cytoskeleton - dependant intracellular transport (go : 0030705) and actin filament - based movement (go : 0030048), confirming the role of actin in sci development and progression. we also that found myosin heavy - chain 6 (myh6), myosin heavy - chain 7 (myh7), and myosin light - chain 3 (myl3) were down - regulated. recent studies have discovered that the regulation of mhc isoform expression involves a complex interaction of multiple control mechanisms, including myogenin and calcineurin, and indicated that other intracellular signaling pathways were also likely to contribute. the enriched pathway in the present study muscle tissue morphogenesis (go : 0060415) is likely involved in the progression of sci by participating in this process. additionally, it has been reported that mutations in myh6 can cause a spectrum of phenotypes, ranging from hcm (hypertrophic cardiomyopathy to dcm (dilated cardiomyopathy), which is correlated with the pathways that were found to be enriched in our study the hypertrophic cardiomyopathy pathway and the dilated cardiomyopathy pathway. thus, we hypothesize that down - regulation of myhc is related with sci and has a potential to function as a new marker in clinical diagnosis and development of therapies for sci. three function modules were explored using clusterone and 3 enriched signaling pathways were found : the myocardium contraction pathway, the hypertrophic cardiomyopathy pathway, and the dilated cardiomyopathy pathway. we found no differences among the signal pathways between genes participating in module 1 and all the degs, confirming its potential role as a prognostic and predictive indicator for use in sci patients. moreover, critical proteins, such as actc, myh6, myh7, and myl3, in the enriched pathways may also provide useful information for better understanding of genetic changes in sci. | backgroundspinal cord injury (sci) is the most critical complication of spinal injury. we aimed to identify differentially expressed genes (degs) and to find associated pathways that may function as targets for sci prognosis and therapy.material/methodsseven gene microarray expression profiles, downloaded from the geo database (i d : gse33886), were used to screen the degs of leg tissue and to compare these between sci patients and corresponding normal specimens. then, go enrichment analysis was performed on these selected degs. afterwards, interactions among these degs were analyzed by string database and then a ppi network was constructed to obtain topology character and modules in the ppi network. finally, roles of the critical proteins in the pathway were explained by comparing the enrichment results of the genes in sub - modules and all the degs.resultsa total of 113 degs were determined. we found that 21 up - regulated genes were enriched in 7 biological processes, while 9 down - regulated genes were significantly enriched in 4 kegg pathways. the ppi network was constructed, including 40 interacting genes and 73 interactions. three obvious function modules were identified by exploring the ppi network, and actc1 was identified as the critical protein in the 3 enriched signal pathways. however, no obvious difference was found in the signal pathway in which both the 11 genes in module 1 and all 113 degs participated.conclusionscore proteins in the signal pathway associated with spinal cord injury may serve as potential prognostic and predictive markers for the diagnosis and treatment of spinal cord injury in clinical applications. |
we combined single molecule fluorescence resonance energy transfer (smfret) with single particle tracking in live cells to detect the in vivo conformation of individual proteins. we site - specifically labeled recombinant snare proteins with a fret donor and acceptor before microinjecting them into cultured cells. we observed that individual proteins rapidly incorporated into folded complexes at the cell membrane, demonstrating the potential of this method to reveal dynamic interactions within cells. |
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nonalcoholic fatty liver disease (nafld) affects 10% to 39% of the world population, 50% of diabetic patients, 57% to 74% of obese people, and up to 90% of the people with morbid obesity. its prevalence in obese adolescents is between 22.5% and 52.8% [1, 2 ]. when not appropriately treated, nafld can progress to cirrhosis [1, 2 ]. thus, treatment of nafld is a relevant issue in clinical hepatology, and several therapeutic approaches have been tested in uncontrolled and controlled pilot studies. obesity and type 2 diabetes are considered the most powerful predisposing risk factors for the development of severe manifestations of nafld. it is important to note that adipose tissue is recently considered endocrine organ strictly related to several comorbidities. the expansion of visceral fat is a determinant risk factor for the development of nafld in obese individuals. moreover, a new study reported that the extent of hepatic inflammation and fibrosis is augmented incrementally with increases in visceral fat. for each 1% increase in visceral fat, the odds ratios for increasing liver inflammation and fibrosis were 2.4 and 3.5, respectively. visceral fat remained an independent predictor of advanced steatohepatitis and fibrosis even when the model controlled for insulin resistance and hepatic steatosis. considering the relation between obesity and nafld, an adequate treatment must include strategies to reduce all metabolic syndrome predictor factors, including gradual body mass reduction and efforts to control blood glucose and lipid levels. a cornerstone of the strategy to treat obese adolescents with nafld is a long - term multidisciplinary intervention that utilizes exercise, clinical, psychological, and nutritional therapies. although an optimal management program for nafld in obesity and insulin resistance has not been established, some strategies are based on hypoglycemic and triglyceride - lowering agents and on lifestyle intervention through a low fat diet, vitamin e supplementation, and exercise. metformin is a well - established oral hypoglycemic agent in the treatment of adults with type 2 diabetes mellitus and other conditions of insulin resistance. it acts by decreasing hyperinsulinemia and improving hepatic insulin resistance. in pediatric obese patients with insulin resistance, it was observed that short - term use of metformin is well tolerated by obese children and that it has a beneficial effect on body mass index (bmi) and cardiovascular autonomic control as well as a trend toward the improvement of insulin sensitivity. thus, long - term treatment with metformin may provide a means to ameliorate the metabolic profile of obese adolescents. the role of metformin in the reduction of insulin resistance and visceral in the pediatric nafld obese population has not been adequately studied. therefore, the main aim of this study was to assess the effect of metformin in obese adolescents with nafld who participated in a multidisciplinary lifestyle modification program. furthermore, it aimed to identify the association between insulin resistance and the degree of steatosis at us in obese patients. obese adolescent boys were recruited for this study from the multidisciplinary obesity intervention program outpatient clinic of the federal university of so paulo paulista medicine school between january and february of 2008. the boys included in the study were between 15 and 19 years old, presented with primary obesity (body mass index > 95th percentile of the cdc reference growth charts), and had reached the initial postpubertal tanner stage five. the initial cohort of the present study that met the above criteria included 80 individuals. subsequently, 35 obese boys who had a positive ultrasound diagnosis of nafld were selected and randomized into two groups : lifestyle modification plus metformin (n = 21) and lifestyle modification plus placebo (n = 14). the adolescents and their families signed an agreement to participate, on a voluntary basis, in a long - term multidisciplinary therapy. all subjects performed an ergometric test before starting the program and had the approval of a physician. the exclusion criteria were an identified genetic disease, metabolic or endocrine disease, chronic alcohol consumption (> 20 g / day), and previous drug utilization or other causes of chronic liver disease. all patients had normal age - specific growth and sexual development, and none had marked hirsutism or severe acne. this study was carried out in accordance with the principles of the helsinki declaration and was formally approved by the ethical committee of the federal university of so paulo paulista medicine school (no. informed consent was obtained from all subjects and/or their parents. after being diagnosed with nafld diagnosis, obese male adolescents were submitted to a long - term multidisciplinary therapy (12 months) that aimed at promoting changes in their sedentary lifestyles and nutritional habits. the basic requirement for participation was the motivation to follow the therapy, presenting high attendance (> 75%) at the sessions. the multidisciplinary therapy consisted of aerobic exercise, nutritional, psychological assistance and clinical management of obesity, and nafld. the short - term intervention corresponded to 6 months and the long - term intervention to 12 months. this model of multidisciplinary therapy was suggested by the world health organization (who). this kind of treatment attempts a more integrated overview of the key role of these emerging strategies for therapeutic intervention. the program consisted of four physicians, two dietitians, four physiologists, and one psychologist. subjects were medically screened, had their pubertal stage assessed, and had their anthropometrics profile measured (height, weight, and body mass index). a blood sample was collected and analyzed for glucose and insulin concentrations, as well as alanine aminotransferase (alt), aspartate aminotransferase (ast), and gamaglutamil aminotransferase (ggt). the patients were randomized to the metformin - treated group (500 mg twice daily) according to freemark and bursey and to the placebo group. the results were compared at baseline and after short- and long - term interventions. to remove any influence of diurnal variations, the procedures were scheduled for the same time of day and at least 15 hours after the last training session. thereafter, all 35 obese male adolescents with a diagnosis of nafld started the multidisciplinary long - term weight loss therapy. two placebo - treated subjects and four metformin - treated subjects withdrew from the study within the first 12 weeks for reasons unrelated to drug toxicity or complications of the trial. wearing light clothes and no shoes, subjects were weighed to the nearest 0.1 kg on a filizola scale. height was measured to the nearest 0.5 cm using a wall - mounted stadiometer (sanny, model es 2030). bmi was calculated as body weight (kg) divided by height (m) squared (kg / m). blood samples were collected in the outpatient clinic around 08:00 h after an overnight fast. insulin resistance was assessed by the homeostasis model assessment insulin resistance index (homa - ir). homa - ir was calculated using the fasting blood glucose (fbg) and the immunoreactive insulin (i) : [fbg (mg / dl) i (mu / l)]/405. the homa - ir data were analyzed according to reference values described by schwimmer.. all abdominal ultrasonographic procedures and measurements of visceral and subcutaneous fat tissue and fatty liver were performed before and after the intervention in a double - blinded manner by the same specialized physician. a 3.5 mhz multifrequency transducer (broad band) ultrasound - determined subcutaneous fat was defined as the distance between the skin and external face of the rectoabdominal muscle. visceral fat was defined as the distance between the internal face of the same muscle and the anterior wall of the aorta. the definition of ultrasonic fatty liver was based on previously reported diagnostic criteria. detected liver steatosis was classified as degree 1 (liver attenuation slightly less than spleen), degree 2 (more pronounced difference between liver and spleen and intrahepatic vessels not seen or slightly higher attenuation than liver), or degree 3 (markedly reduced liver attenuation with sharp contrast between liver and intrahepatic vessels). medical followup included initial medical history, physical examination, and appropriate tests following regular clinical surveillance. energy intake was set at the levels recommended by the dietary reference intake for subjects with low levels of physical activity of the same age and gender. they were encouraged to reduce their food intake and follow a specifically balanced diet, reducing saturated fats and refined sugars. once a week, adolescents had a dietetics lesson (food pyramid, recordatory inquiry, weight loss diets, dietetic versus low calorie foods, fat and cholesterol, nutrition facts). additionally, individual nutritional counseling was recommended when patients had problems making changes in their dietary consumption. at the beginning and after the long - term multidisciplinary therapy, each adolescent filled in a three - day dietary record with the help of his / her parents. these dietary data were transferred to a computer by the same nutritionist, and the nutrient composition was analyzed by a pc program developed at the federal university of so paulo paulista medicine school (nutwin software, for windows, 1.5 version, 2002) based on western and local food tables. in addition, the parents were encouraged to call if they needed further information. during the multidisciplinary intervention, adolescents followed a personalized aerobic training program comprised of 60-minute sessions, three times a week (180 minutes / week) under the supervision of a physiologist. the program was developed according to the results of an initial oxygen uptake test for aerobic exercises (cycle ergometer and treadmill). the intensity was set at a workload corresponding to ventilatory threshold 1 (50% to 70% of oxygen uptake test). after 6 weeks, aerobic tests were performed to assess physical capacities and to individually adjust physical training intensity. during the aerobic sessions the exercise program was based on the american college of sports medicine statement (2001). to assess improvement in cardiovascular function, the subjects performed the same exercise test protocol used at the beginning of the study after 6 weeks and again at the end of the short- and long - term therapies. obesity has been associated with several psychological problems, such as depression, disturbances in body image, anxiety, and a lowered self - esteem. validated questionnaires designed to assess these problems were used to diagnose psychological problems in the study participants. during the multidisciplinary intervention, the adolescents had weekly psychological orientation group sessions in which they discussed body image and alimentary disorders such as bulimia, anorexia nervosa, and binge eating and their signs, symptoms, and consequences for health. the relation between feelings and food and more familiar problems, such as alcoholism, and other topics were also discussed. all data were analyzed using statistica version 6 for windows with the significance level set at p 2.0) according to schwimmer. indeed, in the metformin group, three individuals had normalized homa - ir, and five had normalized alt values. the pathogenesis of nafld is still not clear ; however, there is evidence that insulin resistance and visceral fat play an important role in this disease. according to park., patients with central adiposity and insulin resistance have a higher risk of nafld, reinforcing the strict association between these two parameters. in fact, it was observed that both groups analyzed had altered homa - ir and insulin levels at baseline. indeed, a positive correlation between the degree of steatosis at us with insulin concentrations and homa - ir was shown, corroborating the findings of previous studies [21, 22 ] (figures 1 and 2). given the close relations between the metabolic syndrome and nafld, an adequate treatment must be included for all risk factors associated with obesity. in this way, metformin use can be an important strategy to control insulin sensitivity in patients with nafld. the mechanism by which metformin improves the clinical parameters of nafld might involve the effect of the drug on the inhibition of intestinal glucose absorption, on the reduction of hepatic glucose production, and on the improvement of insulin sensitivity in peripheral target tissues [24, 25 ]. in the present investigation, only the metformin - treated group presented a significant reduction in body mass, bmi, insulin concentration, homa - ir, and visceral fat after short- and long - term interventions (table 1). although we did not observe a statistical decrease in all parameters in the placebo group, this group did have a significant reduction in visceral fat tissue. these findings suggest that multidisciplinary therapy in combination with metformin is important to improve these variables, as well as alt enzyme levels, which are clearly correlated with the development of nafld [26, 27 ]. our data corroborated with those of fu., suggesting that metformin therapy and lifestyle modification improve parameters indicative of metabolic syndrome and nafld. a study with obese adolescents demonstrated that insulin resistance and the associated compensatory hyperinsulinemia are usually associated with obesity, and it can be considered an initial factor of metabolic syndrome development. the effects of metformin on nafld were recently compared with a lipid and calorie - restricted diet in a 6-month, open label, randomized study. the mean changes in insulin, insulin resistance (homa - ir), and aminotransferase concentrations were greater in the experimental arm than in the group given only dietary advice. a previous randomized, controlled trial of metformin in an obese pediatric population with insulin resistance demonstrated that metformin therapy is safe and well tolerated. indeed, the patients had improved insulin sensitivity with metformin, similar to data verified in the present investigation. other relevant findings in this study were that short - term lifestyle intervention plus metformin was efficient to promote a significant improvement in most of the parameters analyzed, and the long - term intervention improves nafld control. similar results were observed by nadeau., suggesting that taking metformin in combination with making lifestyle changes is important to nafld management and that lifestyle improvement by itself may not be as effective. however, both treated groups showed an important decrease in the prevalence of nafld. a recent study indicates that metformin may have antiinflammatory and lipolytic effects mediated through adipocytokines, suggesting that researchers should consider these effects in new studies. further clinical trial investigations involving a larger population, a placebo - control group, a double - blind format, and a long - term followup of metformin treatment are needed to improve our findings. the lack of a histological endpoint is a limitation of this study ; however, we used the degree of steatosis observed at us diagnosis and the hepatic transaminases as a noninvasive score of liver damage in nafld. in conclusion, taking metformin in combination with lifestyle intervention was more effective than intervention alone to promote an improvement in insulin resistance, visceral adiposity, and other commonly observed clinical parameters. | objective. to assess the long - term effects of metformin in combination with lifestyle intervention and its association between insulin levels and the degree of steatosis at ultrasonography (us) in obese adolescents. methods. thirty - five postpubertal obese boys were randomized into two groups : one receiving metformin in combination with a multidisciplinary lifestyle intervention versus a placebo group, which also received the same intervention. the visceral, subcutaneous fat and degree of steatosis were measured by ultrasonography. fasting blood samples were collected to analyze glucose, insulin, insulin resistance, and aminotransferases. repeated anova measures were used to compare changes over time and between groups, and spearman 's correlations were used to identify an association between insulin and the degree of steatosis at us. results. there was a positive correlation between the degree of steatosis at us with insulin concentrations and homa - ir. long - term therapy plus metformin significantly reduced body weight, body mass index, insulin, homa - ir, and visceral fat. conclusions. metformin was more effective than the placebo in improving clinical parameters associated with obesity and steatosis. |
the opercular syndrome, also known as foix - chavany - marie syndrome was first reported in 1926, but was described first by magnus in 1837. this term is usually used in relation to adults as it refers to damage that is acquired later in life. controversy exists in the literature regarding the nomenclature of these various developmental and acquired disorders with characteristic clinical presentation, caused by lesions involving the peri - sylvian opercular cortex, which is the area on the motor homunculus responsible for motor control of the face and mouth. a 9-year - old male presented with unclear and slurred speech, drooling of saliva and inability to protrude tongue and difficulty swallowing since early childhood. he was born with emergency caesarean section (due to fetal distress) and birth weight was 2.5 kg. he developed seizures at 6 hours of life due to perinatal asphyxia, for which he was admitted in neonatal intensive care unit for a week. he had motor and cognitive delay with involvement of speech domain greater than social and motor domain. at the age of 6 years ; he developed seizures which were brief, tonic - clonic, involving right side of body with loss of consciousness. seizures were poorly controlled on multiple anti - epileptic drugs regimen consisting of sodium valproate, lamotrigine, and clobazam. he had dysarthria with nasal twang in voice, mouth half open and not able to close volitionally. bilateral trigemino - facio - linguo - pharyngeal palsy was noticed on volitional control but automatic and emotional motor movements were well preserved. he was able to blink, close his eyes completely while sleeping, and move the jaw and facial muscles with spontaneous emotional responses like laughing or crying. there was hypertonia in all four limbs and deep tendon reflexes were exaggerated with normal jaw jerk along with extensor bilateral plantars. electroencephalography (eeg) findings consisted of focal left fronto - temporal epileptiform discharges and magnetic resonance imaging (mri) brain revealed bilateral fronto - temporal and parietal cortical and subcortical region encephalomalacia, with involvement of opercular region [figure 1 ]. induced sleep eeg did not reveal any continuous spike and wave discharges as seen in continuous spike and wave discharges in sleep (csws) or landau - kleffner syndrome (lks). (a) axial ct scan (b) axial t1 mri (c) axial t2 mri (d) flair coronal mri images showing bilateral fronto - temporal and parietal cortical (left > right) and subcortical (right > left) region encephalomalacia, with predominant involvement of anterior frontal opercular region based on above clinical findings, a diagnosis of acquired bilateral opercular syndrome due to perinatal asphyxia was considered. the opercular syndrome consists of the clinical syndrome of bilateral corticobulbar involvement with facio - labio - pharyngo - glosso - laryngo - brachial paralysis on voluntary movement of these muscles, but well - preserved automatic and reflex movements of the same muscles due to lesions at cortical or subcortical region involving the anterior opercular area surrounding the insula formed from gyri of the frontal, temporal, and parietal lobes. mouth and the term often refers to the fold that is clearly visible on each side of the perisylvian region of cerebral hemispheres. it is often grouped as perisylvian - opercular syndrome which could be congenital or acquired ; transient, intermittent or persistent type with heterogeneous etiologies. it has been classified into anterior and posterior subtypes and unilateral and bilateral types [table 1 ]. clinical types of opercular syndrome voluntary control of face, tongue, and pharynx movement requires intact primary motor cortices and pyramidal pathways between the areas and the brain - stem, containing the relevant cranial nerve nuclei. any lesions involving the motor cortices, subcortical areas, or pyramidal pathways bilaterally produce a selective palsy of voluntary use of the facial, pharyngeal, and masticatory muscles, on the other hand spontaneous, emotional control of the same muscles requires intact extrapyramidal pathways and probably portions of the thalamus and hypothalamus. bilateral opercular lesions typically impair the voluntary movements of the facial, lingual, pharyngeal, and masticatory with preservation of the automatic - emotional movements which is the classical presentation of this syndrome. this phenomenon often referred to as facio - labio - pharyngeo - linguo - masticatory paralysis with automato - voluntary dissociation helps in clinically differentiating the opercular syndrome from the pseudo bulbar palsy associated with bilateral supra - bulbar lesions [table 2 ]. differentiating opercular syndrome from bulbar and pseudobulbar syndrome in patients with dysphagia and dysarthria the actual incidence is unknown. case reports with varied etiologies including perinatal insults, cerebrovascular disease, trauma, surgery, tumor, herpes encephalitis, toxoplasmosis, other meningoencephalitis, inflammatory demyelinating lesions, and developmental malformations like poly - microgyria or heterotopias are found to be associated. acquired epileptiform opercular syndrome is a distinct epileptic syndrome, equivalent to the landau - kleffner syndrome (where language is impaired), has also been described where acquired dysarthria is associated with electrographic status epilepticus in the form of continuous spike and wave discharges during sleep (csws). congenital bilateral perisylvian opercular syndrome described by kuzniecki, is usually caused by fetal developmental abnormalities has been found to be associated with xq27.2-q27.3, xq2822q11.2 mutation, but may also occur from early fetal insults like fetal vascular insult or intra - uterine infections in early pregnancy. its severest form presents in neonates, with difficulty sucking breast milk and recurrent aspiration. usual symptoms are dysarthria, dysphagia, and difficulty in chewing, blowing, moving the tongue, and swallowing difficulty which are affected more than motor and social milestones. developmental delay is seen in 60% cases and mild - moderate mental retardation seen in 50 - 80% cases. epilepsy is seen in 87 - 90% cases, focal seizures with secondary generalization are common, but all types of seizures are reported, and are refractory to treatment in two - thirds. the prognosis is variable, dependent on the severity of clinical disease, the type of the etiology, and the time of diagnosis. in disorders like meningo - encephalitis, vascular or traumatic insult, the epileptic opercular syndrome may be transient and reversible ; while developmental and perinatal insults are likely to be permanent. | we present a 9-year - old boy with history of perinatal asphyxia and neonatal seizures ; who presented with delayed development of speech, with predominant dysarthria, dysphagia, and drooling of saliva and unable to protrude tongue along with delayed motor and mental milestones. he had complex partial seizures since last 3 years requiring multiple anti - epileptic drugs. he had dysarthria, nasal twang, and drooling of saliva with difficulty in chewing and swallowing. hearing and understanding were normal. bilateral trigemino - facio - linguo - pharyngeal palsy was noticed on voluntary movements with normal jaw jerk with preserved automatic and emotional motor movements. electroencephalography revealed focal left fronto - temporal epileptiform discharges and brain imaging was suggestive of bilateral cortical and subcortical region encephalomalacia, predominantly involving bilateral opercular region. the clinical and neuroimaging features correspond to bilateral opercular syndrome which could have resulted from the perinatal insult in this case. |
laser - assisted in situ keratomileusis (lasik) is a widely used technique in refractive corneal surgery. many complications can occur during the preparation of the corneal flap, resulting in costly retreatments. a meta - analysis of 30 trials reported an incidence of 31% for intraoperative complications, of which 19% were microkeratome associated. these were cutting failures such as buttonholes, free caps, or irregular or incomplete cuts. the complications required retreatments, comprising the majority of postoperative costs.1,2 application of femtosecond lasers (fs - lasers) is safe and predictable. nevertheless, specific complications, such as transient light sensitivity syndrome, rainbow glare, and complications concerning gas bubbles, may arise. the laser devices have been found to be as good as or better than mechanical microkeratomes, but long - term results are still needed.3 therefore, refinement of cutting quality with regard to morphology of cutting surfaces and edges is ongoing. a smooth cutting surface is considered to be one of the main quality markers in lasik. surface irregularities can cause haze and result in lower visual outcome ; furthermore, predictability is reduced because the later excimer laser ablation profile is altered.4,5 sharp cut edges are relevant for the reduction of postoperative epithelial ingrowth, less flap dislocations, and less postoperative inflammation.69 today, different instruments can be used for corneal preparation such as occurs in lasik. one recent option is use of an fs - laser system,10 which is, however, a very expensive surgical tool for lamellar keratotomies. despite the increasing use of fs - lasers, microkeratomes are still common for cutting flaps.3 as long as fs - lasers do not offer a main advantage in terms of visual outcome or complications, microkeratomes can continue to be regarded as a cost - saving alternative. hence it is also necessary to refine the technical parameters of mechanical microkeratomes. in this study, we examined the influence of different oscillation rates, head advances, and cutting depths on the quality of corneal flap cuts. in this experimental study, freshly isolated porcine eyes (n=32) obtained from a local slaughterhouse were used. prior to surgery, eyes were held at room temperature for 2 hours to allow deswelling of the corneal stroma. only macroscopically perfect eyes not showing any epithelial defects were used. after removing adnexa (lids, extraocular muscles, and conjunctiva), the eyes were placed in a suction device in which the intraocular pressure was set to 20 mmhg by vacuum. flaps were created using the carriazo - pendular (cp) microkeratome (schwind eye - tech - solutions gmbh & co, kg, kleinostheim, germany). this instrument relies on a pendulum - like cutting action being controlled by a console giving acoustic and visual signs. blade speed can be set within a range of 2 to 5 mm / second, and oscillation frequencies can be adjusted from 9,000 to 15,000 rpm. cutting heads of different sizes (90 m, 110 m, 130 m, 150 m, and 170 m) are also available.11 four flap - cut series including eight cuts each were performed. for each cut, a new blade was used. microkeratome settings were changed so that four different setups (cp 14) could be tested (table 1). to compare the different head advances, a quotient was created containing the oscillation frequency and head - advance - speed. this quotient was named the oscillation / advance ratio (oar) in cuts / mm. with an oscillation frequency of 15,000 rpm, 30,000 cuts per minute or 500 cuts per second were performed, equating to 100 cuts per mm when a head advance of 5 mm per second is applied. after cutting, flaps were lifted using a spatula (g-16193 ; geuder ag, heidelberg, germany), cut at the hinge, and discarded. the interface was washed with 5 ml bss - solution (alcon pharma gmbh, freiburg i m breisgau, germany). with an 11 mm franceschetti trephinator, the corneal beds, including the cutting edges, were removed from the rest of each globe and prepared for scanning electron microscopy (sem). the corneas were immediately immersed in 3% glutaraldehyde, 100 mm hydroxyethyl piperazineethanesulfonic acid (hepes) (ph 7.4), 1 mm cacl2, 1 mm mgcl2, and 25 mm nan3 and left at room temperature for 2 hours, and then at 4c overnight. the tissues were treated with 2% tannic acid for 1 hour, 2% osmium tetroxide for 2 hours, and 2% uranyl acetate for 1 hour, with washing steps in between. afterwards, the samples were dehydrated in a graded series of aqueous ethanol solutions (20%100%). finally, samples were mounted on aluminum stubs, sputtered with gold, and examined by sem in a dsm 940 a (carl zeiss ag, oberkochen, germany). the description of the morphology of the corneal stromal bed as well as the evaluation of their surface quality was carried out on the basis of the electron micrographs. for evaluation, a semiquantitative score system was applied to the results observed in the sem micrographs.12 different criteria were evaluated, with points given corresponding to the cutting quality. therefore, cutting edges (table 2) and cutting surfaces (table 3) were evaluated separately. for evaluating the cutting edges, three different graduations were available (criteria i iii). a clearly visible cutting edge with no irregularities and a sharp epithelial a visible but partly inhomogeneous rim was evaluated as 2 points (criterion ii). if the edge was barely locatable, it was evaluated as 1 point (criterion iii). an electron micrograph with ten - fold magnification was used to evaluate the cutting surface (criterion a). regularity of surface structure (criterion b), portion of surface irregular (criterion c), and position of the irregular area (criterion d) were evaluated on the basis of 50-fold magnification of the sample. each of the eight specimens per series was scored up to a maximum of 3 points for the evaluation of the cut edges (possible maximum of 24 points) and 11 points for the evaluation of the cut surfaces (possible maximum of 88 points). as we examined eight cuts per setting, a maximum of 112 points could be achieved per series. statistical analysis was performed using spss software (v 16 for windows ; ibm corporation, armonk, ny, usa). a p - value less than 0.05 was considered to be statistically significant. for each microkeratome setting, the results were averaged and compared using excel software (microsoft corporation, redmond, wa, usa). as new blades were used for each keratotomy and a complex preparation for sem was needed, sample size was limited. regarding cutting edges, the carriazo - pendular microkeratome creates a serrated rim, showing the typical shape (figure 1a - a). another criterion was the visibility of the crossing from the epithelium to the stroma of the cornea (figure 1a - b). in this area, irregularities such as tissue ruptures appeared (figure 1a - c). according to the sharpness of the cutting edges, different scores were available (criteria i iii), as shown in table 2. the cutting surface was observed with regard to any surface roughness on the basis of ten - fold magnification electron micrograph (table 3, criterion a). in case of a homogenous surface, the interface could show waves (figure 1b - a), tissue roughness (figure 1b - b), or tissue ruptures (figure 1b - c). depending on the location of these irregular morphologies, centrally or peripherally, scores from 13 points regarding cutting edges, the carriazo - pendular microkeratome creates a serrated rim, showing the typical shape (figure 1a - a). another criterion was the visibility of the crossing from the epithelium to the stroma of the cornea (figure 1a - b). in this area, irregularities such as tissue ruptures appeared (figure 1a - c). according to the sharpness of the cutting edges, different scores were available (criteria i iii), as shown in table 2. the cutting surface was observed with regard to any surface roughness on the basis of ten - fold magnification electron micrograph (table 3, criterion a). in case of a homogenous surface, the interface could show waves (figure 1b - a), tissue roughness (figure 1b - b), or tissue ruptures (figure 1b - c). depending on the location of these irregular morphologies, centrally or peripherally, scores from 13 points no cutting complications, such as free flaps, buttonholes, or incomplete cuts, occurred. figure 2 shows representative samples of cutting edges and stromal surfaces for each of the series. the amount of the inhomogeneous areas led to different evaluations, as shown in table 4. evaluating criterion a, which characterized the overall appearance of the interface, there was a tendency toward more irregularities in series cp 1 and 2, with the 110 m and the 130 m cutting head, respectively. the other cutting surface criteria (b d) were taken together as the regularity index. more specifically, it was the percentage amount of irregular areas (criterion c) and the integrity of the surface regularity (criterion b) that exhibited the main differences. comparing the surface scores of the different series series cp 4 achieved a total of 77 points, equating to 88% of the theoretically possible maximum points. in comparison, cp 3 scored 72 points (p=0.407) and series cp 1 and 2 scored 65 points each (p=0.055 and p=0.108, respectively). the sharpness of the transition between epithelium and stroma, in particular, was inspected. comparing the four series with their different cutting heads, deeper keratotomies led to a sharper cutting edge. the 150 m cutting head (series cp 3 and 4) scored significantly higher than the 110 m cutting head used in series cp 1 (p<0.05). the group with a higher blade cutting rate (cp 4) also scored slightly higher. our investigations revealed that different head - advance speeds and different cutting heads did not influence the surface of the keratotomies significantly, although there was a trend toward better surface quality using cutting heads performing a deeper keratotomy (figure 4b). further, higher head - advance speeds (lower oar) seemed to be advantageous for a smoother interface. as such, we observed a higher surface regularity in the series with 5 mm / second advance speed (figure 4a). this trial was scored 77 points, whereas the same settings, except with a 3 mm / second head advance, scored 72 points. overall, the scores of the different series ranged from 65 to 77 points (theoretical maximum 88 points). on the basis of scanning electron micrographs, certain surface morphologies, such as tissue ruptures or parallel waves, could be visualized. changing the cutting heads from 110 m over 130 m to 150 m, the standard deviation decreased (0.77 over 0.73 to 0.66). the surface scored a mean of 8.125 points for the 110 m and 130 m cutting head. the 150 m head scored 9 points (theoretical maximum of 11 points). after reducing the oar, the amount of the inhomogeneous areas led to different evaluations, as shown in table 4. evaluating criterion a, which characterized the overall appearance of the interface, there was a tendency toward more irregularities in series cp 1 and 2, with the 110 m and the 130 m cutting head, respectively. the other cutting surface criteria (b d) were taken together as the regularity index. more specifically, it was the percentage amount of irregular areas (criterion c) and the integrity of the surface regularity (criterion b) that exhibited the main differences. comparing the surface scores of the different series series cp 4 achieved a total of 77 points, equating to 88% of the theoretically possible maximum points. in comparison, cp 3 scored 72 points (p=0.407) and series cp 1 and 2 scored 65 points each (p=0.055 and p=0.108, respectively). the sharpness of the transition between epithelium and stroma, in particular, was inspected. comparing the four series with their different cutting heads, deeper keratotomies led to a sharper cutting edge. the 150 m cutting head (series cp 3 and 4) scored significantly higher than the 110 m cutting head used in series cp 1 (p<0.05). the group with a higher blade cutting rate (cp 4) also scored slightly higher. our investigations revealed that different head - advance speeds and different cutting heads did not influence the surface of the keratotomies significantly, although there was a trend toward better surface quality using cutting heads performing a deeper keratotomy (figure 4b). further, higher head - advance speeds (lower oar) seemed to be advantageous for a smoother interface. as such, we observed a higher surface regularity in the series with 5 mm / second advance speed (figure 4a). this trial was scored 77 points, whereas the same settings, except with a 3 mm / second head advance, scored 72 points. overall, the scores of the different series ranged from 65 to 77 points (theoretical maximum 88 points). on the basis of scanning electron micrographs, certain surface morphologies, such as tissue ruptures or parallel waves, the reproducibility increased with the depths of keratotomy. changing the cutting heads from 110 m over 130 m to 150 m, the standard deviation decreased (0.77 over 0.73 to 0.66). the surface scored a mean of 8.125 points for the 110 m and 130 m cutting head. the 150 m head scored 9 points (theoretical maximum of 11 points). after reducing the oar, involved instruments have to provide a maximum of safety, accuracy, and simplicity with regards to handling and performance. this study showed morphologic characteristics of keratotomies performed by the carriazo - pendular, a modern mechanical microkeratome. this study assessed the influence of variable depths of keratotomies and different head - advance speeds while using a constant oscillation frequency. the carriazo - pendular microkeratome offers a unique and modern design for preparation of lamellar keratotomies such as lasik or descemet s stripping automated endothelial keratoplasty (dsaek). it is one of the most used devices for mechanical flap preparation in lasik procedure in germany.13 an advantage is its special type of cutting (molding technique). realized by the cutting head design fulfilling a convex applanation of the cornea, the created flap shows a planar configuration.11,14 this offers advantages for the later ablation by the excimer laser. because of the constant pressure on the cornea, fewer buttonholes appear.15 a homogenous stromal surface after flap preparation leads to better visual outcome, and high oscillation frequencies combined with a fast blade rate lead to higher reproducibility,4,5 while an elevation of head advance using the same oscillation frequencies created worse surface qualities. using lower oars, better cutting results were observed.16 higher oscillation frequencies combined with lower advance speed possibly increases the horizontal tissue dislocation because of more blade movement without real cutting the collagen fibers. on the other hand, if the head advancement per oscillation is lower, the tissue is cut more cleanly and with less dragging and chattering.17 contrary to our results, no influence of different head advance speeds on the interface smoothness has been previously revealed in porcine eyes.18 characteristic serrations have been observed on the cutting edges, as described in earlier studies.12,19 reducing the cutting depth, the edge configuration becomes increasingly blurred. the trial series with the 110 m head (3 mm / second head advance) was scored 10 out of the theoretical maximum of 24 points (42%). in comparison to the 150 m head (3 mm / second head advance), a cutting head of 130 m scored 12 points, which is 50% of the theoretical maximum. the head advance did not affect the cutting edge quality significantly, but there was a trend toward better results in the trial using 5 mm / second head advance ; here, 19 of 24 points were achieved (79%). the series with 3 mm / second scored 17 of 24 points (71%). to our knowledge, clinical studies proving the impact of keratotomy depth on postoperative complications such as flap dislocation are not available to date. nevertheless, it is essential to leave a residual stromal thickness of at least 250 m after cutting and laser ablation in order to avoid iatrogenous corneal ectasia. it would be desirable to investigate the lowest cutting depth with which cutting edges present the best morphology.20 sarayba investigated nine human corneas using sem micrographs and showed a smoother stromal bed after application of a 30 khz fs - laser when flaps with 110 m thickness were created.21 this may have been due to the fact that they used human corneas and different parameters of photodisruptive preparation technique. it seems very probable that fs - lasers can generate smoother cutting surfaces, although thinner flaps are created. the small sample size in sarayba s study reduces its validity.21 in this context, fs - lasers might be advantageous over microkeratomes as they can produce very thin flaps while the cutting edges remain extremely sharp.22 porcine corneas are established models simulating human tissue;12,19,22,23 however, it would have been desirable to investigate human eyes because there are ultrastructural differences that limit the comparability of both tissues. the human cornea, having a strictly organized bowman s layer, is more rigid. as the porcine corneal tissue is smoother it might visualize cutting characteristics better.24 considering this aspect, porcine eyes could be regarded as a sensitive model showing the characteristics of lasik instruments on surface quality of the cuts. in contrast, there are investigations demonstrating more surface irregularities in human corneas than in porcine corneas.18 the animal model can serve its purpose for investigation of different technical parameters, especially since large numbers of samples are available. the next step could be in situ investigations, such as in the study of ramrez, who studied reflective particles in the interface after either bilateral lasik by fs - laser or microkeratome on one eye using confocal microscopy, found that there was no statistically significant difference in the numbers of particles between each eye.25 the scoring system used in this study provided a very detailed description of cutting quality, but depended on the subjective evalu ation of the surgeons. to avoid this bias iatrogenic keratectasia is an ongoing problem, even if the residual stroma thickness is more than 300 m.26,27 the preparation of ultrathin flaps may lead to problems like microstriae, tissue particle dislocation into the interface, or flap dislocation.11,26 a sharp cutting edge is regarded as advantageous, avoiding corneal erosion, epithelial ingrowth into the interface, and scarring, as well as flap dislocation.19 serrations of the cutting edge could further support the flap adaptation. the steeper the cut, the less epithelial cells are altered, which could decrease epithelial ingrowth and unreasonably wound healing reaction. stronger tissue alteration caused by blurred cutting can cause more inflammation leading to stronger scaring as well as myopic regression.7,9 additionally, the cutting head design has an important effect concerning the development of epithelial defects.8 another possible application for microkeratomes is the preparation of corneal grafts for dsaek, in which it is desirable to create a thin and homogenous lenticule for transplantation. the carriazo - pendular microkeratome device in combination with an artificial anterior chamber is available for this type of surgery. in comparison to fs - laser - assisted preparation, the microkeratome seemed to be superior in regards to postoperative visual outcome.28,29 further developments in dsaek are made in creating an ultrathin flap by a microkeratome. after a first cut of 300 m, a second cut formed a 100 m lenticule, which is used for dsaek. with this technique, comparable visual outcomes to descemet s membrane endothelial keratoplasty could be reached, requiring less time and with fewer difficulties with donor tissue preparation.3032 good cutting results while preparing deeper keratotomies, in particular, could be advantageous. a reason for the better outcome after micro - keratome preparation in comparison to fs - laser preparation might be less tissue bridges.23,33 material and equipment costs play an important role in daily medical routine. microkeratomes such as the carriazo - pendular device could be a cost - saving alternative to the very expensive and vulnerable fs - laser devices. in a prospective randomized clinical study of visual outcome, higher - order aberrations and flap thicknesses of fellow eyes treated with fs - laser- or mechanical keratome - created flaps during laser in situ keratomileusis in myopes was investigated.34 besides more predictable flap thicknesses in the fs - laser group, there were no significant differences concerning visual outcome, whereas higher - order aberrations were lower in the micro - keratome group. in a meta - analysis, there were more epithelial defects after microkeratome use, but more cases of deep lamellar keratitis after fs - laser application.10 a deeper keratotomy enables a safer sliding of the blade in the corneal stroma. microkeratomes may offer a cost - saving alternative to fs - lasers. it still remains a wide field for indications for micro - keratome use such as lasik or dsaek. further studies should compare both techniques to provide more information about their advantages and disadvantages. | purposefemtosecond lasers have become the standard for laser - assisted in situ keratomileusis (lasik) flap creation, but advanced mechanical microkeratomes are still an alternative, more cost - effective way to create the flap. the schwind carriazo - pendular microkeratome is one of the most commonly used microkeratomes. the influence of different cutting parameters (head - advance speeds, cutting heads) on morphology of lasik cuts was investigated.settingexperimental study performed at the university eye hospital of the martin luther university halle / wittenberg, halle (saale), germany.methodsthe carriazo - pendular microkeratome was used on freshly enucleated porcine eyes for lamellar keratotomy. after flap removal, the cutting edge and stromal bed were evaluated from scanning electron micrographs using an individualized scoring system. four different settings of microkeratome parameters were compared. for each setting, eight cuts were evaluated (n=32).resultsdifferent oscillation frequencies and head - advance speeds did not influence the cutting qualities. a higher oscillation / feed rate ratio seemed to be advantageous for a smoother interface. concerning different cuttings heads, a deeper keratotomy led to sharper cutting edges. the thinner the flap, the more irregularities in the stromal bed appeared. complications did not occur.conclusionthe carriazo - pendular microkeratome is a safe tool with which to create a lasik flap and is a good alternative to a costly femtosecond laser. deeper keratotomies, as well as the use of a higher oscillation / feed rate quotient, improve the cutting quality. |
our attempts to match memory performance across ieeg (patient) and fmri (control) groups (by varying the number of sources, retention interval and encoding list length ; see methods) were successful (table 1). a pr measure (probability of correct minus probability of incorrect old decisions to studied items) showed that overall recognition memory was significantly above chance (0), (fmri : pr = 70% (+ / 4), t19 = 16.18, p.11). for further analyses, trials in which an item was recognized (hit), but either a do nt know response or an incorrect source response was given, were collapsed, and are referred to as item recognition (ir). mean reaction times (rts) for correct rejection (cr), item recognition (ir) and source recognition (sr) are shown in table 1. all pairwise comparisons were significant (fmri : all t19 > 3.47, p 3.93, p 2.41, p 2.18, p 2.91, p 3.27, p 4.61, p.11). for further analyses, trials in which an item was recognized (hit), but either a do nt know response or an incorrect source response was given, were collapsed, and are referred to as item recognition (ir). mean reaction times (rts) for correct rejection (cr), item recognition (ir) and source recognition (sr) are shown in table 1. all pairwise comparisons were significant (fmri : all t19 > 3.47, p 3.93, p 2.41, p 2.18, p 2.91, p 3.27, p 4.61, p ir). the first role (item recognition through decreased activity, or conversely, increased activity for experimentally - novel stimuli) has received considerable support from a variety of methods. for instance, neurons in the primate perirhinal cortex have been shown to decrease their firing rates as a function of stimulus repetition, and lesions to the primate perirhinal cortex result in marked object recognition deficits. moreover, human fmri studies have reported reduced bold signals for old relative to new items, while previous ieeg studies in human epilepsy patients have reported reduced n400 components for old relative to new items. thus, our finding of a reduced perirhinal response for ir relative to cr is compatible with this region s established role in (familiarity - based) item recognition. importantly, however, a more recent series of findings also suggest a role of perirhinal cortex in memory processes beyond simple item recognition. for example, single neurons in primate perirhinal cortex have been shown to code for object - object associations (pair - coding neurons). also, recent fmri studies in humans have shown that perirhinal cortex can support, in conjunction with the hippocampus, successful associative encoding as well as associative retrieval. thus, converging evidence suggests that the role of perirhinal cortex in episodic memory may encompass both item- and source memory. however, if item- and source memory are indeed separate functions of perirhinal cortex, one might expect these functions to be mediated by separate cell populations and/or to show different temporal / dynamic profiles. indeed, we found that the item recognition effect (ir vs. cr) lasted from 200ms until ~800ms post - stimulus onset, whereas the associative recognition effect (sr vs. ir) started at 400ms (figs. 3b and 4) and was sustained throughout the remainder of the trial period, terminating with the memory response. thus, not only do the current data show both item- and source effects in perirhinal cortex, but they suggest that item recognition and source retrieval may be partially - separable processes supported by this region (possibly in conjunction with the hippocampus ; see below). our data reveal that the item effect in perirhinal cortex precedes the onset of the source effect in the hippocampus, which in turn precedes the onset of the source effect in perirhinal cortex (fig. this temporal pattern is suggestive of a signaling loop between perirhinal cortex and hippocampus, in which a rapid item recognition signal in perirhinal cortex triggers source retrieval processes in the hippocampus, which in turn entrain the perirhinal cortex in the service of retrieving and/or maintaining an associated source detail. a recent study obtaining intralaminar recordings from primate perirhinal cortex during a paired associate task showed a reversal of the signal flow from (i) a forward signal during the cue period (item processing) to (ii) a backward signal during the delay period (when retrieving the paired associate). although that study did not record from the hippocampus, our current data suggest that this reversal might be triggered by the hippocampus. indeed, a critical role of the hippocampus in initiating source retrieval is consistent with single unit data showing that successful free recall is preceded by a gradual increase of hippocampal firing rates. this idea that hippocampus and perirhinal cortex need to interact during successful source retrieval is further substantiated by our finding of enhanced functional coupling among these regions during sr relative to ir (fig., the temporal sequence of an early perirhinal item effect / familiarity signal followed by a hippocampal source effect / recollection signal lends strong support to current models that emphasize a functional separation between hippocampus and perirhinal cortex during episodic memory processes. importantly, however, the subsequent source effect in perirhinal cortex, along with increases in functional coupling between hippocampus and perirhinal cortex, also highlight the intricate interaction of these regions that underlies the retrieval of episodically rich memory traces. | in the endeavor to understand how our brains enable our multifaceted memories, much controversy surrounds the contributions of the hippocampus and perirhinal cortex (prc). here we recorded functional magnetic resonance imaging (fmri) in healthy controls and intracranial electroencephalography (eeg) in patients during the same recognition memory paradigm. although conventional fmri analysis showed indistinguishable roles of the hippocampus and prc in familiarity - based item recognition and recollection - based source retrieval, event - related fmri and eeg time - courses revealed a clear temporal dissociation of memory signals within and across these regions. whereas an early source retrieval effect was followed by a late, post - decision item novelty effect in hippocampus, an early item novelty effect was followed by a sustained source retrieval effect in prc. although factors like memory strength were not experimentally controlled, the temporal pattern across regions suggests that a rapid item recognition signal in prc triggers a source retrieval process in the hippocampus, which in turn recruits prc representations / mechanisms evidenced here by increased hippocampal - prc coupling during source recognition. |
in march 2014, a 47-year - old woman was hospitalized at the bernhard nocht clinic (hamburg, germany) with chills, nausea, and fever (temperature 5,000 ng / ml) and cocaine (> 1,000 ng / ml) (reference values < 20 ng / ml for both substances). three blood cultures were collected before empiric antimicrobial therapy with intravenous ceftriaxone (2 g 1/d) was given on day 1. her leukocyte count increased to 27.6 10 cells/l, and c - reactive protein level increased to 112 mg / l. she was highly febrile and became hypotensive and tachycardic, which are compatible with severe sepsis. the patient responded rapidly to fluid substitution, became hemodynamically stable, and did not need vasopressors. antimicrobial therapy was changed empirically to meropenem (1,000 mg 3/d) on day 2. after 18 h of culture incubation, microbial growth was detected in 3 aerobic blood culture bottles inoculated at admission. gram staining showed gram - negative rods, and matrix - assisted laser desorption ionization / time - of - flight mass spectrometry fingerprinting with direct sample deposition without extraction (bruker daltonim gmbh, bremen, germany) identified a. ursingii (score 2.19). a blast search (http://www.ncbi.nlm.nih.gov/blast) of partial 16s rrna gene sequence was performed by using a taxonomy browser (http://www.ncbi.nlm.nih.gov) and showed 99% identity with the a. ursingii type strain nbrc110605 (genbank accession no. antimicrobial susceptibility testing was performed by using vitek 2 (biomrieux, nrtingen, germany). results showed susceptibility to imipenem (mic < 0.25 mg / ml), meropenem (< 0.25 mg / ml), gentamicin (< 1 mg / ml), and ciprofloxacin (< 0.25 mg / ml) and resistance to ceftriaxone (32 mg / ml). the patient became afebrile the next day, her clinical symptoms improved, and laboratory parameters of inflammation returned to reference values. 2 months later, the patient had no symptoms and was attending group - counseling sessions to maintain drug abstinence. after the first description of a. ursingii as a new species (2), for which 13 of 29 a. ursingii isolates were obtained from patients with nosocomial bloodstream infections, few cases of a. ursingii bloodstream infections have been reported (table). we were not able to identify cases of community - acquired a. ursingii bloodstream infections in the literature. reported a case of a. ursingii bloodstream infection in a severely immunocompromised patient with pulmonary adenocarcinoma who received chemotherapy and corticosteroids (3). in that study, a. ursingii was associated with an implanted port device and considered of low pathogenic potential. however, in the past few years, there have been reports of serious a. ursingii bloodstream infections in immunocompromised and immunocompetent patients. reported nosocomial a. ursingii bloodstream infections in 2 pregnant women who had no concurrent illnesses (4), but the route of transmission was not elucidated. the authors hypothesized that infections might have been acquired from intravenous catheters from the ward shower bath, where a. ursingii and 2 different strains of a. junii were found. a. ursingii was also found associated with nosocomial bloodstream infections among newborns, as demonstrated by 2 outbreaks in neonatal intensive care units that had high mortality rates. in both outbreaks, the source of infection (5) and mder. (6), catheter - related infections were assumed. dortet. (8) reported 10 a. ursingii infections, which included 3 cases of nosocomial bloodstream infections, although severe infections with a. ursingii are rare, identification of a. ursingii by molecular methods, such as 16s rrna gene sequencing, rpob gene sequence cluster analysis, and amplified fragment length polymorphism fingerprinting, indicates a relatively high prevalence of a. ursingii (range 2.6%4.5%) among clinical acinetobacter spp. isolates (1013). in a study from the united kingdom, isolates collected over a 20-month period during 20082009 ; a total of 17 (71%) of 24 a. ursingii isolates were recovered from blood cultures, but clinical details were not reported (12). bloodstream infections in norway ; 3 (2.6%) were with a. ursingii, but clinical details of these patients were not provided. this relatively high incidence is consistent with results from the netherlands (10) and northern ireland (11). in most reports, a. ursingii isolates were more susceptible to antimicrobial drugs than a. baumannii isolates (4,6,8,12,13). however, endo. (9) reported an imp-1producing, carbapenem - resistant a. ursingii isolate from a patient in japan with a nosocomial bloodstream infection. a. ursingii isolates are usually resistant to cephalosporins (3,5,8), as was the isolate from the patient we report, which explains the progression from severe sepsis when she was empirically given ceftriaxone to rapid clinical improvement after administration of meropenem. (6) reported a premature infant with an a. ursingii bloodstream infection also given a cephalosporin (ceftazidime). these findings suggest that a. ursingii is an emerging and serious clinical pathogen that is often involved in nosocomial bloodstream infections and associated with use of intravascular catheters. its natural habitat remains unclear, but so far, it has only been isolated from human clinical sources. the frequency and clinical role of a. ursingii might have been underestimated because species identification by phenotypic and semiautomated identification methods is unreliable. for example, vitek 2 has misidentified a. ursingii as bordetella bronchiseptica (6,8). the most recent software version for vitek 2 enabled unambiguous identification of a. ursingii (h. seifert, pers. we report a case of community - acquired bloodstream infection and severe sepsis caused by a. ursingii that was probably associated with intravenous drug use in an immunocompetent patient. this report also highlights the usefulness of matrix - assisted laser desorption / ionization time - of - flight mass spectrometry for identification of acinetobacter spp., including a. ursingii, in routine clinical practice and help elucidate the prevalence and clinical role of this emerging pathogen. | we report a community - acquired bloodstream infection with acinteobacter ursingii in an hiv - negative woman who injected drugs. the infection was successfully treated with meropenem. species identification was performed by using matrix - assisted laser desorption / ionization time - of - flight mass spectrometry. improved identification of acinetobacter spp. by using this method will help identify clinical effects of this underdiagnosed pathogen. |
poland is a central european country with a population of more than 38 million inhabitants. from the beginning of the hiv epidemic in 1985 to 2004, 8491 cases of hiv infection, 1421 aids cases, and 676 hiv / aids - associated deaths have been reported and confirmed. at the beginning of 2004, more than 2000 hiv - positive individuals were receiving antiretroviral treatment. in silesia, which has 4.7 million citizens and is the second largest population among polish provinces, the number of hiv infections from the beginning of the epidemic to 2004 was 1123, which constitutes 13.2% of the total number of hiv infections detected in poland. in that time, 185 aids cases and 87 hiv / aids associated deaths have been recognized in silesia. the mean number of newly diagnosed hiv cases during this time was less than 60 per year in our region. the epidemiologic and clinical situation regarding hiv infections in silesia seems to be similar to that observed in other parts of poland. inability of the viral reverse transcriptase (rt) to proofread nucleotide sequences during replication results in a high degree of hiv-1 genome variability, which together with rapid viral turnover, contributes to drug - resistant mutant development. in the absence of antiretroviral treatment, antiretroviral drugs incompletely suppressing viral replication exert selective pressure that results in resistant - strain dominance. drug selection is not the only possible way of the resistant variants development, because the transmission of drug - resistant mutants to treatment - nave subjects has been reported in many cases [612 ]. to date, hiv isolates resistant to each class of antiretroviral drugs were identified, and drug resistance is considered a major contributor to treatment failure. currently approved antiretrovirals are targeted against viral rt, protease, integrase, and envelope glycoprotein. the nucleoside inhibitors of hiv-1 rt were introduced as the first antiretroviral drugs in 1987, and they are still the most widely used drug class. for this reason, screening for the occurrence of rt inhibitors resistance mutations in the hiv-1 pol gene seems to be a suitable tool for presenting retrospective drug resistance studies. such retrospective investigations were undertaken to enable comparisons with the present situation and to follow the dynamics of possible future changes in the drug resistance patterns. although knowledge of the global situation concerning drug resistance mutation frequencies and types is permanently growing, in many local populations, such information is still rather limited and unsatisfactory., we have undertaken retrospective studies on drug resistance mutations among the 101 hiv-1positive silesian individuals who acquired infection before 2004. our studies have focused on estimations of the drug resistance mutations types, frequencies, and the level of their influence on drug effectiveness, in the group with almost 35% treatment - nave subjects. enrollment of patients not administered with antiretroviral drugs in the studied population sheds some light on a potential transmission of drug - resistant mutants in the history of hiv-1 epidemic in silesia. presented results may serve as an indispensable starting point for the further analysis of hiv-1 drug resistance and possible changes in this field in our region. we included a group of 101 hiv-1 seropositive individuals infected before 2004 (table 1). all patients were silesian residents and were attending the department of diagnostics and therapy for aids in chorzw, poland. antiretroviral therapy was introduced before samples collection in 66 patients (65.3%), 7 of them (10.6%) were treated with the nucleoside reverse transcriptase inhibitors (nrtis) exclusively, 12 (18.2%) received nrtis with nonnucleoside reverse transcriptase inhibitors (nnrtis), 30 (45.5%) were using nrtis and protease inhibitors (pis), and 17 patients (25.7%) were treated with the drugs from nrtis, nnrtis, and pis classes. thirty - five subjects (34.7%) had received no antiretroviral treatment by the time of specimen collection. eighty individuals (79.2%) were intravenous drug users (idus) and 21 (20.8%) reported no drug addiction, with 11 being heterosexuals (10.9%), and 10 being homosexual men (9.9%). blood samples were obtained after patients signed informed consent ; the study fell under the agreement of the medical university of silesia bioethics committee (nn-6501 - 191/i/05/06). peripheral blood mononuclear cells (pbmcs) were separated from edta - treated blood samples by ficoll - histopaque density gradient centrifugation (1.077 g / cm) and immediately stored at 80c. genomic dna extraction was performed from uncultured pbmc by the routine proteinase k and phenol - chloroform method. proviral dna was amplified in the part of the pol gene covering the first 256 codons of the reverse transcriptase by nested polymerase chain reaction (pcr) using previously described primer pairs. briefly, the pol gene fragment of 2017 bp spanning nucleotides 23774393 according to the hiv hxb2 reference sequence (genbank accession number k03455) was amplified with the use of rt1 and rt2 as outer primers. in the second step, rt3 and rt4 inner primers were used to amplify 775-bp pol region located between 2545 and 3319 nucleotides in the hiv hxb2. the cycling parameters of the first amplification round with the rt1 and rt2 primers were an initial denaturation step at 94c/5 minutes, followed by 35 cycles of 94c/1 minute, 55c/1 minute, 72c/2.5 minutes, with the final extension at 72c/10 minutes, in a final volume of 50 l. rt3-rt4 amplification was conducted with the following conditions : an initial denaturation step at 94c/5 minutes, followed by 35 cycles of 94c/30 seconds, 54c/30 seconds, 72c/1 minute, with the final elongation at 72c/5 minutes, in a final volume of 50 l. purified nested - pcr products were further subjected to direct sequencing using the abi prism big dye terminator version 3.1 cycle sequencing kit (applied biosystems, foster city, ca, usa) with the rt3-rt4 primers for the pol gene fragment recognition. sequences of both strands were determined separately using an abi prism 377 automated dna sequencer (applied biosystems). the nucleotide sequences reported in the presented study were submitted to the genbank under accession numbers eu910734eu910834. reverse transcriptase inhibitors resistance mutations in the pol gene fragments were determined and interpreted with the hivdb : genotypic resistance interpretation algorithm available at the stanford university hiv drug resistance database. according to this algorithm, determining the hiv-1 genetic variant was performed with the ncbi genotyping tool, a program designed to identify the subtype sequence homologies with the use of the basic local alignment search tool (blast) algorithm. reference sequences representing all hiv-1 genetic groups, subtypes, subsubtypes, and circulating recombinant forms were applied during ncbi genotyping tool analysis. hypermutated sequences were recognized with the hypermut version 2.0 program. to compare data between patients who received antiretroviral therapy with treatment - nave subjects, the mann - whitney u test was used for analyses of quantitative variables as age, and the 2-tailed fisher exact test was used to compare qualitative variables such as sex and transmission risk group. to estimate the statistical values, we included a group of 101 hiv-1 seropositive individuals infected before 2004 (table 1). all patients were silesian residents and were attending the department of diagnostics and therapy for aids in chorzw, poland. antiretroviral therapy was introduced before samples collection in 66 patients (65.3%), 7 of them (10.6%) were treated with the nucleoside reverse transcriptase inhibitors (nrtis) exclusively, 12 (18.2%) received nrtis with nonnucleoside reverse transcriptase inhibitors (nnrtis), 30 (45.5%) were using nrtis and protease inhibitors (pis), and 17 patients (25.7%) were treated with the drugs from nrtis, nnrtis, and pis classes. thirty - five subjects (34.7%) had received no antiretroviral treatment by the time of specimen collection. eighty individuals (79.2%) were intravenous drug users (idus) and 21 (20.8%) reported no drug addiction, with 11 being heterosexuals (10.9%), and 10 being homosexual men (9.9%). blood samples were obtained after patients signed informed consent ; the study fell under the agreement of the medical university of silesia bioethics committee (nn-6501 - 191/i/05/06). peripheral blood mononuclear cells (pbmcs) were separated from edta - treated blood samples by ficoll - histopaque density gradient centrifugation (1.077 g / cm) and immediately stored at 80c. genomic dna extraction was performed from uncultured pbmc by the routine proteinase k and phenol - chloroform method. proviral dna was amplified in the part of the pol gene covering the first 256 codons of the reverse transcriptase by nested polymerase chain reaction (pcr) using previously described primer pairs. this gene region is known for harboring most of the rt inhibitors resistance mutations. briefly, the pol gene fragment of 2017 bp spanning nucleotides 23774393 according to the hiv hxb2 reference sequence (genbank accession number k03455) was amplified with the use of rt1 and rt2 as outer primers. in the second step, rt3 and rt4 inner primers were used to amplify 775-bp pol region located between 2545 and 3319 nucleotides in the hiv hxb2. the cycling parameters of the first amplification round with the rt1 and rt2 primers were an initial denaturation step at 94c/5 minutes, followed by 35 cycles of 94c/1 minute, 55c/1 minute, 72c/2.5 minutes, with the final extension at 72c/10 minutes, in a final volume of 50 l. rt3-rt4 amplification was conducted with the following conditions : an initial denaturation step at 94c/5 minutes, followed by 35 cycles of 94c/30 seconds, 54c/30 seconds, 72c/1 minute, with the final elongation at 72c/5 minutes, in a final volume of 50 l. purified nested - pcr products were further subjected to direct sequencing using the abi prism big dye terminator version 3.1 cycle sequencing kit (applied biosystems, foster city, ca, usa) with the rt3-rt4 primers for the pol gene fragment recognition. sequences of both strands were determined separately using an abi prism 377 automated dna sequencer (applied biosystems). the nucleotide sequences reported in the presented study were submitted to the genbank under accession numbers eu910734eu910834. reverse transcriptase inhibitors resistance mutations in the pol gene fragments were determined and interpreted with the hivdb : genotypic resistance interpretation algorithm available at the stanford university hiv drug resistance database. according to this algorithm, the resistance levels were specified as potential low, low, intermediate, and high. determining the hiv-1 genetic variant was performed with the ncbi genotyping tool, a program designed to identify the subtype sequence homologies with the use of the basic local alignment search tool (blast) algorithm. reference sequences representing all hiv-1 genetic groups, subtypes, subsubtypes, and circulating recombinant forms were applied during ncbi genotyping tool analysis. to compare data between patients who received antiretroviral therapy with treatment - nave subjects, statistical analyses were performed. the mann - whitney u test was used for analyses of quantitative variables as age, and the 2-tailed fisher exact test was used to compare qualitative variables such as sex and transmission risk group. to estimate the statistical values, all investigated samples collected from 101 hiv-1positive patients were successfully amplified and sequenced in the part of the pol gene spanning the first 256 codons for the rt. according to the ncbi genotyping tool, all viral nucleotide sequences were classified as group m, subtype b. one viral sequence was recognized as hyper - mutated according to the hypermut program and was excluded from further analyses. hypermutated sequence was derived from an idu who had experienced no anti - retroviral therapy. in the analyzed sequences all of them were identified exclusively in the hiv-1 proviral dna obtained from patients receiving antiretroviral therapy. among all detected mutations corresponding to the drug resistance, 7 (m41l, a62v, d67n, k70r, m184v, t215y, and t215s) were related to nrtis / ntrtis resistance. the percentage of hiv-1 strains carrying any of the following substitutions : m41l, k70r, m184v, and t215y was 2%. mutations : a62v, d67n, and t215s were present in 1% of hiv-1 strains. the frequency of all nrtis / ntrtis resistance - associated mutations in the analyzed sequences was 10.9%. further, 3 substitutions (k103n, v179d, and y181c) were related to the nnrtis resistance. in the studied population, the v179d change occurred in 1% of the investigated hiv-1 pol sequences. in the analyzed group of sequences, all rt inhibitors resistance - related mutations mentioned above were detected in 10 (9.9%) of 101 pol gene sequences examined. in 7 analyzed pol gene fragments, only single resistance - associated mutations were identified, in 2 sequences, 2 drug resistance mutations were present, and in 1 fragment, 5 different substitutions were found. to evaluate the potential influence of recognized resistance - related substitutions on the level of drug resistance sequence examinations revealed different extents of resistance to nrtis / ntrtis for 5 hiv-1 strains (table 3). these viral strains were found among 5 patients who were receiving antiretroviral drugs for 116 months on average. next, 5 strains exhibited different levels of nnrtis resistance, and they were present among 5 individuals with mean of 49 months of therapy (table 3). we observed no hiv-1 strains with dual resistance to nrtis / ntrtis and nnrtis. in the nrtis / ntrtis drug classes, a high level of resistance hiv-1 strains with mutations decreasing nnrtis susceptibility were highly resistant to delavirdine, nevirapine, and efavirenz. the hiv-1 drug resistance development is currently one of the main factors limiting the efficacy of antiretroviral therapy. selection and transmission of drug - resistant mutants restricts the number of possible antiretroviral drug combinations and reduces the chance for life - long therapy responsiveness of individual patients. identification of the mutations related to the drug resistance in the viral genome is widely used method for the drug resistance estimation. in europe, drug resistance testing is recommended especially among patients with therapy failure and among pregnant women, as well as their hiv-1 infected children. it is also strongly suggested to perform a test on hiv-1 drug susceptibility among subjects with the acute hiv-1 infection or recent seroconverters before introducing the first anti - retroviral therapy regimen to facilitate the optimal choice of the drug combination. despite the fact that the most drug resistance investigation is focused on hiv-1 subtype b genotype, it has been shown that particular hiv-1 genetic variants may exhibit differences in natural susceptibility to the antiretroviral drugs. for instance, group o is naturally resistant to nnrtis because of harboring the y181c and a98 g mutations in the rt coding region of the pol gene. innate resistance to nnrtis is also carried by some group m isolates, for example subtype c or d. there also are indications that subtype - specific pathways for the development of resistance against certain drugs may exist. although drug resistance mutations that were described for subtype b also have been observed among other viral genetic forms, it has been shown that some resistance - related substitutions may appear among various genetic variants with different frequency. this is true for the nnrti resistance mutation, v106 m, which is much more frequent in the pol gene of subtype c than b, as development of this substitution may be dependent on the pre - existing genomic background. for these reasons, it was important to determine genetic variants present among the investigated population. by 2004, the only hiv-1 genetic form that was recognized among the silesian patients was subtype b. this hiv-1 genetic variant also remains the most - prevalent form in the majority of the european countries. we analyzed 101 pol gene fragments covering the first 256 codons of a total of 440 for rt protein. seven of 10 recognized different drug resistance mutations were involved in the reduced nrtis / ntrtis susceptibility. they were m41l, a62v, d67n, k70r, m184v, t215y, and t215s. presence of the m41l, d67n, k70r, and t215y mutations in hiv-1 pol gene results in the emergence of the phosphorolytic activity of the rt, allowing the enzyme to excise nucleotide analogues already incorporated to the dna chain. the t215s substitution has been reported as a partial reversion change at codon 215, conferring increased risk of virologic failure of azt and d4 t in antiretroviral - nave patients. in the presence of these drugs, strains harboring the t215s mutation may exhibit rapid development of the t215y mutation. the a62v substitution, together with unobserved in the analyzed pol gene fragments the q151 m mutation, is responsible for the cross - resistance between nrtis drugs. in the presence of a62v and q151 m rt can discern between the cellular dntps and activated nrtis molecules, which prevent nrtis incorporation to the growing proviral dna polymer. strains with m184v substitution possess rt with decreased affinity to the nrtis molecules, which leads to resistance to lamivudine, emtricitabine, and to lesser extent, to abacavir. on the other hand, this mutation may increase viral susceptibility to zidovudine, stavudine, and tenofovir. another 3 identified pol gene mutations (k103n, v179d, and y181c) are described to be involved in nnrtis resistance. all these substitutions generate changes in the hydrophobic site of rt where nnrtis are bound. this results in the decreased affinity of rt to nnrtis and consequently, in the lack of the nnrtis antiretroviral activity. based on the identified and described resistance mutations, we could establish that 10 viral strains in the investigated silesian population were, to a different extent, resistant to the rt inhibitors (table 3). because in the presented study hiv-1 strains with the drug resistance mutations were found only among persons receiving antiretroviral drugs on the other hand, as the time between hiv-1 infection diagnosis and collecting samples was long (on average 47 months), we can not exclude that the treatment - nave patients could have been infected with drug - resistant hiv-1, but reversion to the sensitive wild - type virus could have occurred over time. however, there is some evidence that drug - resistant viruses can persist for years in the plasma of treatment - nave hiv-1-positive individuals. the presence of such viruses among individuals without therapy experience may act as an indicator of the drug resistance transmission in a given population. in this view, the lack of resistant viruses among the treatment - nave silesian patients hiv-1-infected before 2004 can indicate that there was no transmission of the antiretroviral drug - resistant viruses in the studied population to that time. this finding may be meaningful for hiv-1 drug resistance testing strategies in our region, outlining the usefulness of storing the earliest sample available, to test the drug resistance before planned treatment introduction. the possibility of superinfection, however, should be taken into account during interpretation of test results in such cases. the frequency of the resistant viral strains in the studied population of hiv-1-positive individuals who acquired infection before the year 2004 was 9.9%, and as we noted earlier, drug - resistant viruses were not observed among the treatment - nave subjects. compared with our results, in 19 european countries, a 10.4% frequency of drug resistance was observed among persons with the hiv-1 infection recognized between 1996 and 2002 who had never received antiretroviral drugs. this rate remained stable in the next study, which included a european population of treatment - nave persons, with the hiv-1 infection diagnosed in 20022003. surprisingly, in poland, the frequency of drug - resistant strains among untreated individuals with the infection detected between 2004 and 2006 has reached 14.7%. mutations of this kind, such as k70r / e, t69s / n, and t215d / e were present in 2.6% samples. all recognized nnrtis resistance mutations (a98 g, k101e, k103n, v108i, and m230l) were identified in 1.7% hiv-1 strains. in our studies, we have observed only 2 mutations from those described above. they were the k70r and k103n substitutions, each present in 2% of viral strains ; however, we found them exclusively in the samples from subjects with therapy experience. the authors of the earlier quoted investigation described the group of patients that could have been hiv-1-infected later than subjects included in our retrospective studies. this fact may be responsible for the different frequencies of the drug resistance among treatment - nave patients. both studies taken together may represent a shift in the drug resistance frequencies that has occurred in a relatively short time. we think that the wider access to the antiretroviral drugs results in the increased selective pressure on the virus. this selective pressure seems to be the main factor responsible for the wider spread of the drug resistant variants causing the observed shift. the second possibility is that relevant differences in drug - resistant mutants frequencies among drug - nave patients may exist in different parts of poland. because we found no drug - resistant viruses among treatment - nave silesian patients hiv-1 infected before 2004, it is highly possible that there was no drug - resistant viruses transmission in the studied population to that time. forthcoming studies including recent hiv-1 infections will address the question if this unusual situation among the european regions is stable over time or if it changes as more antiretroviral drugs are applied in the population. | summarybackgroundthis study was a retrospective analysis of drug resistance mutations among hiv-1 strains prevalent in silesia, poland, from the origin of the epidemic to 2004. the investigations included both type and frequency of the reverse transcriptase inhibitors resistance mutations and estimation of the drugs resistance levels.material/methodsproviral dna, obtained from peripheral blood mononuclear cells of the 101 hiv-1infected patients, was amplified and sequenced in the pol gene fragment covering the first 256 codons of the reverse transcriptase (rt). reverse transcriptase inhibitors resistance mutations were determined and interpreted with the hivdb : genotypic resistance interpretation algorithm available from the stanford university hiv drug resistance database. in the examined population, 35 subjects (34.7%) received no antiretroviral treatment by the time of specimen collection.resultsthe overall frequency of the rt inhibitors resistance mutations in the studied population was 15.8%. substitutions related to the reverse transcriptase inhibitors resistance were identified in 10 pol gene sequences (9.9%), all of them were present in the hiv-1 sequences obtained from persons receiving antiretroviral therapy.conclusionslack of drug - resistant viruses among treatment - nave silesian patients hiv-1-infected before the year 2004 may indicate that there was no transmission of the drug - resistant viruses in the studied population to that time. |
bipolar ii disorder is a chronic affective disorder characterised by episodes of major depression and hypomania. a hypomanic episode is characterised by symptoms such as flight of ideas, a decreased need for sleep, elation and talkativeness. these symptoms occur within an elevated or irritable mood which lasts for at least 4 days. there are a number of important diagnostic issues regarding the effective diagnosis of bipolar disorder, such as the minimum duration of a hypomanic episode required for diagnosis. it has been suggested that manic and hypomanic symptoms are not recognised as readily as depressive symptoms for a number of reasons, including a lack of subjective suffering, a lack of elevated mood and seasonal rhythmicity. perhaps due to these diagnostic controversies, a number of studies have documented high levels of previously unrecognised hypomanic symptoms in those with a unipolar depression, suggesting possible incorrect diagnosis [4 - 9 ]. for example ghaemi. found that 37% of those who received a diagnosis of unipolar depression met the diagnostic criteria for bipolar disorder. incorrect diagnosis as unipolar depression may have a number of negative consequences, such as treatment with antidepressants rather than mood stabilisers, which can cause rapid cycling. on the other hand, early identification and treatment of bipolar disorder it is important to increase the detection of hypomania in order to better distinguish between unipolar depression and bipolar ii disorder. systematic evaluation of hypomania has been found to double the estimated prevalence of bipolar ii disorder. consequently it has been suggested that bipolar ii disorder is in fact as prevalent as, or more prevalent than, unipolar major depression. in order to optimise detection of hypomania and bipolar ii disorder, developed a structured clinical interview (sci - moods), and hirschfeld. developed a self - report measure, the mood disorder questionnaire, to screen for affective disorders including bipolar disorder. angst. have developed a self - report questionnaire, the 32-item hypomania checklist (hcl-32), to detect hypomania in those with depression in order to optimise diagnosis of bipolar ii disorder. a number of studies have documented high levels of depression in undergraduate students. however, no studies have examined levels of hypomanic symptoms in undergraduate students with depression. this study therefore aimed to examine scores on the hcl-32 in a sample of female undergraduate students reporting a formal diagnosis of unipolar depression. participants were also recruited via posters in trinity college dublin, and some took part as part of a research credit scheme. the participants described here represent a small subset of this initial data set, and consist of female undergraduate students of british, irish, u.s., new zealand or canadian nationality, who self - reported that they had, at some point in their lives, been formally diagnosed with unipolar depression. using these criteria, ages ranged from 18 - 42, with a mean age of 24.3 (sd=6.2). nationalities were 35.7% (n=10) british, 21.4% (n=6) irish, 14.3% (n=4) u.s., 7.1% (n=2) canadian, 14.3% (n=4) australian, and 7.1% (n=2) new zealander. participants completed a brief demographics questionnaire created by the researchers for the purposes of this study, which included questions about previous formal diagnoses of mental illness. this is a self - report questionnaire of 32 questions, which measures the severity of previous hypomanic episodes by asking participants to try to remember a period when you were in a the hcl-32 has two subscales ; active or elevated and risk - taking or irritable. total scores range from 0 to 32, with a score of 14 or more distinguishing major depressive disorder from bipolar disorder with a sensitivity of.80 and a specificity of.51. additional studies have validated the hcl-32 in a number of different countries, demonstrating good psychometric properties [16 - 20 ]. ethical approval was granted by the trinity college dublin school of psychology research ethics board. scores on the hcl-32 ranged from 10 to 30 (out of 32), with a mean of 19.9 (sd=5.4). fig. (1) demonstrates the cumulative percentage of total scores on the hcl-32. overall, 85.7% (n=24) scored equal to or above the cut - off point of 14 for bipolar ii disorder as suggested by angst. in their original validation of the hcl-32. a total of 92.9% (n=26) scored equal to or above the cut - off point of 12 for bipolar disorder suggested by carta.. table 1 displays the percentage of the sample that scored equal to or above the various hcl-32 total score cut - off points as suggested by previous validation studies. scores on the active or elevated subscale ranged from 7 to 17, with a mean of 14.2 (sd=2.9). scores on the risk - taking / irritable subscale ranged from 0 to 7 with a mean score of 2.8 (sd=1.9). report that a score of two or more on this subscale distinguished between bipolar i disorder and major depressive disorder with a specificity of 62% and a sensitivity of 62%. in the current sample, a total of 67.9% (n=19) scored 2 or more on the risk - taking / irritable subscale. this study examined scores on the hcl-32 in a sample of female undergraduate students who reported having been formally diagnosed with unipolar depression at some point in their lives. the results suggest high scores on the hcl-32 in this group, with the vast majority of the sample scoring equal to or above the cut - off point suggested by angst.. many also scored equal to or above the various other cut - off points suggested by other validation papers. these cut offs were variously suggested to discriminate major depression from bipolar i and/or bipolar ii disorder, bipolar i from bipolar ii disorder, and bipolar (i or ii) disorder from other axis i disorder or controls. scores were also high on the two subscales, with the majority scoring equal to or above a cut off on the risk / taking - irritable subscale designed to discriminate bipolar i disorder from unipolar depression. the means noted here are similar to the work of meyer. in a non - clinical population, with if the cut off points suggested by previous studies are used, then the current results suggest that the majority of the undergraduate students in the current sample were diagnosed with unipolar depression when they may, in fact, have high levels of hypomanic symptoms, and thus potentially meet the criteria for bipolar disorder. if the cut off point of 14 given in the original validation paper by angst. is taken, then 85.7% of the current sample have sufficiently high scores to suggest the possible presence of bipolar ii disorder. however it should be noted that self - report measures such as the hcl-32 are often intended for screening followed up by a clinical interview, and thus scores on this measure alone are not necessarily indicative of bipolar disorder, but rather suggest that further examination is required. although previous studies have found high levels of hypomanic symptoms in those with depression, suggesting potential misdiagnosis, the levels here appear to be particularly high. previous estimates of the proportion of those diagnosed with unipolar depression who meet the diagnostic criteria for bipolar disorder are 37.5%, 56.8% and 61.3%. this suggests that high levels of undergraduate students have been diagnosed with unipolar depression though they may experience hypomanic episodes and thus it is possible that they meet the criteria for bipolar ii disorder. therefore clinicians at university health clinics should be careful to systematically screen for hypomania in those presenting with depression. the hcl-32 may serve as a useful clinical tool for initial testing, to be followed up by a structured clinical interview. the sensitivity and specificity of the hcl-32 for discriminating unipolar depression from bipolar disorder has been variously reported at.80 and.51,.8 and.54,.68 and.83, and.82 and.67, and.85 and.79. these studies have demonstrated moderate specificity ; nonetheless some false positives are expected. on the other hand, thus it is important to consider the possibility that the suggested cut off point may be too low for use with an undergraduate population. this may be due to the lower age of the sample ; meyer. the mean age of the undergraduate sample in this study was 24.3, whereas the other validation studies had a much higher mean age. for example, the italian sample used in the original validation by angst. had a mean age of 43.2 years. thus undergraduate students in general, as well as those diagnosed with depression, may score higher on the hcl-32 on average than other populations. therefore for undergraduate students a higher cut off may be necessary in order to optimise sensitivity and specificity. angst. estimated that around 50% of those with major unipolar depression in fact belong to the bipolar spectrum. if it is assumed that 50% of the current sample are bipolar, then a cut off point of 21 or above on the hcl-32 would appear to be appropriate for undergraduate students, as exactly 50% of the sample scored equal to or above this. however, a study comparing scores on the hcl-32 in undergraduate students with bipolar disorder and unipolar depression comparatively is required to confirm what cut - off is appropriate for this population. first, the sample size is small, in particular when compared with previous validation studies on the hcl-32 [13, 16 - 20 ]. second, the sample is entirely female, whilst the various cut off points were designed for both male and female patients. similarly the sample is international, and thus the cut off points may not be accurate for all of the nationalities represented, as previous work has demonstrated higher mean scores in different nationalities. third, a depression diagnosis was determined only via self - report, and thus there is a problem with validity of this diagnosis. an alternative sampling method using university a related point is that the sampling method used here is prone to a number of problems and biases, in particular due to the self - selection that may have occurred. despite the limitations of this study, these results may be important as they suggest one of two possibilities, both of which have important implications for those working with students with depression. firstly, there appear to be high levels of hypomanic symptoms in female undergraduate students with a diagnosis of unipolar depression. this in turn suggests that many of these individuals may meet the diagnostic criteria for bipolar disorder. alternatively, the cut off points on the hcl-32 suggested by previous research with older adults may not be appropriate for use in this population, as high scores are common. as earlier research has documented high levels of depression in undergraduate students, either of these possibilities future research using larger samples and validated depression diagnoses is needed to further study the area of recognition of bipolar disorder in undergraduates presenting with depression. no funding was obtained for this research, except for approximately 30 for printing from the school of psychology, trinity college dublin. | background : a number of studies have documented high levels of hypomanic symptoms in those diagnosed with depression, suggesting a potential misdiagnosis of bipolar disorder as unipolar depression. research suggests that undergraduate students have high levels of depression, but whether such misdiagnosis occurs in this population has not been examined. the aim of this study was therefore to examine levels of hypomania in undergraduate students reporting diagnosed depression.methods:an international sample of undergraduate students completed the 32-item hypomania checklist (hcl-32). a cohort was analysed for this study, consisting of female undergraduate students reporting a formal diagnosis of depression (n=28).results : participants scored high on the hcl-32, with a mean total score of 19.9 (sd=5.4) out of 32. overall, 85.7% (n=24) scored equal to or above the original cut off point of 14 suggested for bipolar ii disorder.conclusions:two possible conclusions are suggested by this study. firstly, there are high levels of hypomanic symptoms in undergraduate students diagnosed with depression, suggesting that a formal diagnosis of bipolar disorder should be pursued in those with high scores. alternatively, the cut - off points previously suggested for the hcl-32 may not be accurate for use with undergraduate students. |
it is susceptible to neovascularization - inducing intervention, that is, microbial infection or ocular surface damage. the process of the new vessel formation in an injured cornea is orchestrated by a complex system of various growth factor signaling [15 ]. resident corneal cells and inflammatory cells invaded to an injured tissue express growth factors and cytokines involved in injury - induced neovascularization. such factors include vascular endothelial growth factor (vegf), transforming growth factor (tgf), and fibroblast growth factor (fgf) [610 ]. members of the transient receptor potential (trp) channel superfamily are polymodal receptors that are activated by a host of stimuli to mediate sensory transduction. the family is divided into 7 different subfamilies and composed of 28 different genes [1114 ]. trp vanilloid subtype 1 (trpv1), the capsaicin receptor, is a nociceptor and one of the prototypes of trpv subfamily. it elicits responses to a variety of noxious stimuli including chemical irritants besides capsaicin, inflammatory mediators, tissue injury, an alteration in ph, and moderate heat (43c). trpv1 activation leads to nociception and evokes pain or pain - related behaviors and reportedly induces release of tachykinin neuropeptides from sensory nerves, inducing neurogenic inflammation in the surrounding area [1517 ]. various nonneuronal cell linages, that is, epidermal keratinocyte or corneal epithelium and keratocytes, also express trpv1, which presumably exerts a variety of biological responses to external stimuli [1824 ]. we previously reported that lacking trpv1 counteracted inflammatory and fibrogenic reactions in corneal stroma following an alkali burn. the phenotype of less - inflammation / fibrosis was found to depend on the loss of keratocytes in the affected stroma, but not on inflammatory cells as revealed by reciprocal bone marrow transplantation experiments. stromal neovascularization is also a component of the biological reaction observed in an injured cornea. in our previous study on an alkali - burned cornea, however, we failed to extract the effects of the loss of trpv1 on injury - induced neovascularization in an alkali - burned cornea due to complex tissue reaction in the stroma. it was reported that capsiate and piperine, both trpv1 agonists, suppress angiogenic behaviors of vascular endothelial cells cultured in the absence of inflammatory components in vitro [25, 26 ]. in vivo role of trpv1 signal in modulation of neovascularization is to be assessed in in vivo condition. in the present study we examined the roles of trpv1 signal in the activity of neovascularization development by using trpv1-null (ko) mice and in vitro human umbilical vein endothelial cell (huvec) culture model of neovascularization. in vivo experiments were approved by the dna recombination experiment committee and the animal care and use committee of wakayama medical university and performed in accordance with the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research. the degree of tube - like structure formation by huvecs on a fibroblast feeder layer was employed to assess the effects of each agent on neovascularization activity of the cells. huvecs were seeded on the fibroblast feeder layer as the manufacture suggested (nv kit, kurabo, tokyo, japan). then the culture was maintained in the routine culture condition in the presence of vascular endothelial cell growth factor- (vegf-) a (10.0 ng / ml, kurabo, tokyo, japan) as an angiogenesis inducer in the presence or absence of a trpv1 antagonist, sb366791 (10 m, sigma - aldrich). huvecs did not develop tube - like structure in the absence of vegf (data not shown). after 11 days of culture the cells were processed for immunohistochemistry for cd31 (a marker for vascular endothelium) according to the manufacture 's protocol. color development was performed by 3,3-diaminobentidine (dab) reaction [8, 27 ]. average length and the average number of bifurcations (the number of branching points) were counted in three central fields in each well in a blinded fashion by an investigator. the mean value of the data from these three fields represented the data of each well. statistical analysis of the data from eight wells was conducted by employing tukey - kramer test and p < 0.05 was taken as significant. we then performed an in vivo neovascularization assessment experiment by using a wild - type (wt) of c57bl/6 (n = 52) or ko mouse of c57bl/6 background (n = 65) as previously reported. there was no difference in the histological findings in an uninjured cornea between a wt and ko mice as previously reported. corneal stromal neovascularization from the limbal vessels was induced by cauterization of the central cornea of an eye of both wt and ko mice by disposable cauterization tool as previously reported. we first observed the morphology of neovascularization at day 3 after cauterization in whole - mounted specimens by using cd34 immunostaining. mice were sacrificed at day 3 after cauterization in the central cornea by co2 asphyxia. the eyes were fixed in 4% paraformaldehyde for 48 hrs. after washing in phosphate - buffered saline (pbs), the specimens were treated in 0.5% triton x for 1 hr to facilitate the antibody penetration into the tissue. after rinsing in pbs, the samples were allowed to react with a monoclonal anti - cd31 antibody (1 : 100 in pbs, santa cruz biotechnology inc., santa cruz, ca, usa) for 24 hrs at 4c. after washing the antibody the tissues were then treated with a fitc - labeled secondary antibody (southern biotechnology, birmingham, alabama, usa) for 12 hrs at 4c, mounted in fluoromount - g after another wash in pbs, and observed under carl zeiss apotome we then examined the length of neovascularization from limbus toward the center of the cornea following cauterization in histological section. for this purpose, 15, 16, or 8 wt mice and 21, 21, or 10 ko mice were used for assessment at day 3, 7, or 14, respectively. mice were sacrificed at days 3, 7, and 14 after cauterization in the central cornea by co2 asphyxia. the eye was enucleated, processed for cryosections, and used for immunohistochemistry for cd31 (monoclonal, 1 : 100 in pbs, santa cruz biotechnology inc.) as previously reported. the length of corneal stromal neovascularization was measured as follows : length between limbus and the tip of neovascularization was measured in both sides of the limbus in three cryosections produced from one eye. statistical analysis was conducted with the use of mann - whitney u test, and p < 0.05 was taken as significant. cornea of three eyes of each genotype of mice was also cauterized and then processed for paraffin section immunohistochemistry for active form of tgf1, vegf, substance p, and f4/80 macrophage antigen as previously reported. as described below we semiquantified mrna expression of tgf1 in treated corneas and tgf1 exerts its action after processing to the active form. we therefore used an antibody that reacts the active, but not inactive, form of tgf1 in the current study [24, 29 ]. we examined the expression of mrnas of neovascularization - related growth factors and the degree of inflammation in in vivo mouse cornea. we considered mrna level quantification was essential because our preliminary immunohistochemistry for vegf and tgf1 showed very faint staining with minimal difference of the staining in central corneal stroma between a wt and a ko mouse. centrally cauterized cornea (n = 6 in each of wt or ko group) was excised at day 3. total rna was extracted from these tissues and processed for taqman real - time reverse transcription - polymerase chain reaction (rt - pcr) for vegf, tgf1, myeloperoxidase (mpo) and f4/80 macrophage antigen and as previously reported. delta / delta ct method by applied biosystem inc. was employed with the internal control of glyceraldehyde 3-phosphate dehydrogenase (gapdh) expression. primers used (applied biosystem inc.). consider vascular endothelial growth factor : mm01281447_ml ; transforming growth factor 1 : mm03024053_ml ; substance p : mm01166996_ml ; interleukin-6 : mm01210732_gl ; myeloperoxidase : mm01298422_gl ; f4/80 : mm00802524_ml. vascular endothelial growth factor : mm01281447_ml ; transforming growth factor 1 : mm03024053_ml ; substance p : mm01166996_ml ; interleukin-6 : mm01210732_gl ; myeloperoxidase : mm01298422_gl ; f4/80 : mm00802524_ml. dense cd31 immunoreactivity was detected in tissue where huvecs formed a vessel - like tube structure. without exogenous vegf, huvecs grown on the fibroblast feeder layer did not form a vessel - like tube tissue. the huvec culture was processed for cd31 immunocytochemistry at day 11 (figure 1(a)). the angiogenic behaviors of huvecs were evaluated by measurement of the mean total length of the structure (figure 1(b)) and of the men number of bifurcations (figure 1(c)) in randomly selected fields of the culture as described above. in the culture with vegf - a (10 g / ml) cd31-labeled tube - like structure was observed. supplementation of a trpv1 antagonist, sb366791 (10 m), did not affect vegf - a action on tube - like structure formation by huvecs (figure 1). we first observed the morphology of neovascularization in whole - mounted specimens by employing cd31 immunostaining. figure 2 shows the morphology of limbal vasculature of wt and ko corneas at day 3. in both wt (figures 2(a) and 2(b)) and ko (figure 2(c)) corneas loop - like distributions of blood vessels were observed. in wt corneas neovascularization was observed in the stroma apart from the limbus, although the staining procedure did not figure the continuous elongation of the vessels (figures 2(a) and 2(b)). cd31 immunostaining was performed in cryosections of the mouse cornea (figure 3(a)). in wt mouse corneas, development of the cd31-labeled neovascularization from the limbus in the corneal stroma was detected in the peripheral cornea as early as at day 3 (figure 3(a)). the length of the neovascularization between the limbus (arrowheads) and the tip (arrows) of new vessels in the corneal stroma was measured at each timepoint. the length of neovascularization was shorter in ko mice as compared with wt mice at day 3 and day 7, but not at day 14 (figure 3(b)). immunohistochemistry showed that active forms of tgf1, vegf, and substance p were not detected in untreated cornea of both genotypes of mice (not shown). active form of tgf1 was detected in stroma just beneath the epithelium in the area of cauterization at days 3 and 7 in wt mice (figures 4(a) and 4(c)). its immunoreactivity was quite less marked in cornea of ko mice (figures 4(b) and 4(d)). vegf was also not detected in uninjured corneas of both genotypes of mice. at day 1 after cauterization very faint vegf immunoreactivity was observed in basal cells of corneal epithelia in cauterization area of both wt and ko mice (figures 4(e) and 4(f)). at day 3 the basal epithelial cells with vegf immunoreactivity were more frequently observed in a wt cornea as compared with a ko mouse (figures 4(g) and 4(h)). immunoreactivity for substance p was detected in basal layer of corneal epithelium with no obvious difference in intensity between wt and ko mice at days 1 and 3 (figures 4(i), 4(j), 4(k), and 4(l)). we then examined mrna expression of vegf and tgf1, the major two growth factors reportedly involved in corneal neovascularization in day 3 specimens by using real - time rt - pcr. expression of mrnas of both vegf (figure 5(a)) and tgf1 was significantly less in a ko cornea as compared with a wt cornea at day 3 (figure 5(b)). trpv1 signal is reportedly involved in expression of substance p and interleukin-6 (il-6), both involved in local tissue inflammation. however, in the present study the loss of trpv1 did not affect mrna expression level of substance p (figure 5(c)) and il-6 (figure 5(d)) in the centrally cauterized cornea at this timepoint. we previously reported that cauterization in the central cornea induced inflammation in the affected area of tissues. we therefore first examined distribution of f4/80-labeled macrophages by using immunohistochemistry and saw difference of distribution of f4/80-labeled macrophages (figure 6(a)). we then semiquantified the invasion of neutrophils and macrophages in tissues by conducting real - time rt - pcrs for mrnas of mpo, a neutrophil marker, and f4/80. the loss of trpv1 exhibited no remarkable effect on mrna expression of these cell markers (figures 6(b) and 6(c)). the present experiments first showed that lacking trpv1 cation channel receptor suppressed stromal neovascularization in an in vivo mouse cornea following receiving a cauterization injury at the central cornea. neovascularization was found to sprout from the loop - shaped vessels of the limbus toward the center of the corneal stroma in whole - mounted samples of wt tissues, while such neovascularization was much less in a ko cornea at day 3. the distance between limbus and the tip of the neovascularization in the stroma was significantly shorter in a ko cornea and compared with a wt mouse. cell culture experiment showed that blockage of trpv1 receptor did not affect vegf angiogenic action on huvecs. huvecs do not reportedly express trpv1, and thus the present in vivo finding of less angiogenesis observed in a ko cornea was not attributed to the direct effects of the loss of trpv1 on vascular endothelial cells. although various growth factors that potentially affect neovascularization activity are expressed in an injured cornea during tissue repair [2932 ], expression of such components is reportedly modulated by ion channel receptor signaling, that is, signaling derived from trp family members. to analyze the mechanism of antiangiogenic effect of lacking trpv1 in in vivo corneal stroma we conducted further experiments. because trpv1 signal is involved in inflammation in response to external stimuli, which potentially affect formation of neovascularization, we ran real - time rt - pcr for inflammatory / angiogenic growth factors and inflammation / wound healing - related components. the results showed that mrna expression of vegf and tgf1 in cauterized cornea was suppressed by lacking trpv1, but expression of mrna of il-6 was not affected by the loss of trpv1. immunohistochemistry also showed that deposition of active form of tgf1 in the stroma beneath the regenerated epithelium and vegf expression in the basal epithelial cells in the central cornea both seemed less in amount in a ko tissue as compared with a wt mouse. we reported that tgf1 is expressed in corneal epithelium and is deposited in stroma beneath the regenerated epithelium as an active form. thus, reduced accumulation of active form of tgf1 in the ko cornea might be attributable to the suppression of tgf1 expression in epithelium after cauterization by the loss of trpv1. il-6 was not detected by immunohistochemistry presumably because the protein might be secreted out from the cells. the present assessment of inflammatory cell markers, that is, mpo and f4/40, indicated that following cauterization in the central cornea the loss of trpv1 did not affect infiltration of neutrophil leukocytes and macrophages. data from these real - time rt - pcr results suggest that less expression of angiogenic growth factors, that is, vegf and tgf1, is unattributed to the alteration of the level of inflammatory cell infiltration following cauterization in a ko tissue and presumably is dependent on the effects of lacking trpv1 on the gene expression in resident corneal cells, that is, corneal epithelial cells. involvement of keratocytes in the suppression of neovascularization in the ko mouse is to be further investigated. trpv1 in sensory nerve fibers reportedly mediates expression of neuroinflammatory mediators, for example, substance p [34, 35 ]. in the present study protein and mrna expressions of substance p were similar in wt and ko corneas after cauterization. in conclusion, | to investigate the effects of loss of transient receptor potential vanilloid receptor 1 (trpv1) on the development of neovascularization in corneal stroma in mice. blocking trpv1 receptor did not affect vegf - dependent neovascularization in cell culture. lacking trpv1 inhibited neovascularization in corneal stroma following cauterization. immunohistochemistry showed that immunoreactivity for active form of tgf1 and vegf was detected in subepithelial stroma at the site of cauterization in both genotypes of mice, but the immunoreactivity seemed less marked in mice lacking trpv1. mrna expression of vegf and tgf1 in a mouse cornea was suppressed by the loss of trpv1. trpv1 gene ablation did not affect invasion of neutrophils and macrophage in a cauterized mouse cornea. blocking trpv1 signal does not affect angiogenic effects by huvecs in vitro. trpv1 signal is, however, involved in expression of angiogenic growth factors in a cauterized mouse cornea and is required for neovascularization in the corneal stroma in vivo. |
male and female lmna, lmna, and lmna mice were bred as described (12, 15). genotyping of mice was performed by pcr with genomic dna from tail biopsies (12, 15). mice were fed a chow diet and housed in a virus - free barrier facility with a 12 h light - dark cycle. an fti, abt-100 (18), was administered to groups of male and female lmna, lmna, and lmna mice (n = 12 mice / group). abt-100 was mixed in drinking water containing 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol at a concentration of 0.4 mg / ml, so as to deliver an approximate dose of 52 mg / kg / day. vehicle - treated mice were given drinking water with 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol. the fti was initiated at 4 weeks of age and was continued for up to 38 weeks of age (at that time point, any mouse that had not yet succumbed to the disease was euthanized). body weights were assessed weekly, and body weight curves were compared with repeated - measures anova and the log rank test. the number of surviving mice was recorded weekly and expressed as a percentage of the total number of mice. body fat depots (reproductive, inguinal, and mesenteric) were measured when each mouse died or was euthanized. the number of spontaneous rib fractures in lmna and lmna mice was documented when each mouse died. after opening the thoracic cavity and removing the heart and lungs, the interior of the thorax was photographed and rib fractures were counted (12, 15). numbers of rib fractures in fti- and vehicle - treated lmna and lmna mice were compared with a two - tailed student 's t - test. mean bone density and bone cortical thickness in the ribs were measured with computer - assisted tomographic scans. procedures for preparing liver extracts and western blotting techniques have been described previously (12, 14, 15). to detect lamins a and c and progerin, we used a 1:400 dilution of a goat anti - lamin a / c igg (santa cruz biotechnology, santa cruz, ca) and a 1:5000 dilution for an irdye800-conjugated donkey anti - goat igg (li - cor ; lincoln, ne). to detect prelamin a, we used a 1:2000 dilution of a rabbit anti - prelamin a antiserum (6, 8, 13) and a 1:5000 dilution of an hrp labeled anti - rabbit igg (ge healthcare, piscataway, nj). to detect hdj-2, we used a 1:500 dilution of a mouse monoclonal antibody against hdj-2 (neomarkers ; fremont, ca) and a 1:5000 dilution of an hrp - labeled anti - mouse igg (ge healthcare). for both prelamin a and hdj-2 western blots, antibody binding was detected with the ecl plus chemiluminescence system (ge healthcare) and exposure to x - ray film. to assess protein farnesylation in fibroblasts, the cells were incubated for 48 h with an analog of farnesol, 8-anilinogeraniol (ag) (100 m in dmso) (19) in the presence and absence of abt-100 (1 m). ag is incorporated by cells into ag pyrophosphate, a farnesyldiphosphate analog, which is used by protein farnesyltransferase as a substrate to farnesylate caax proteins. ag incorporation into cellular proteins was detected by western blotting with a mouse monoclonal antibody specific for ag, diluted 1:5000 (19). male and female lmna, lmna, and lmna mice were bred as described (12, 15). genotyping of mice was performed by pcr with genomic dna from tail biopsies (12, 15). mice were fed a chow diet and housed in a virus - free barrier facility with a 12 h light - dark cycle. an fti, abt-100 (18), was administered to groups of male and female lmna, lmna, and lmna mice (n = 12 mice / group). abt-100 was mixed in drinking water containing 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol at a concentration of 0.4 mg / ml, so as to deliver an approximate dose of 52 mg / kg / day. vehicle - treated mice were given drinking water with 0.4% hydroxy methyl propyl cellulose and 1.0% ethanol. the fti was initiated at 4 weeks of age and was continued for up to 38 weeks of age (at that time point, any mouse that had not yet succumbed to the disease was euthanized). body weights were assessed weekly, and body weight curves were compared with repeated - measures anova and the log rank test. the number of surviving mice was recorded weekly and expressed as a percentage of the total number of mice. body fat depots (reproductive, inguinal, and mesenteric) were measured when each mouse died or was euthanized. the number of spontaneous rib fractures in lmna and lmna mice was documented when each mouse died. after opening the thoracic cavity and removing the heart and lungs, the interior of the thorax was photographed and rib fractures were counted (12, 15). numbers of rib fractures in fti- and vehicle - treated lmna and lmna mice were compared with a two - tailed student 's t - test. mean bone density and bone cortical thickness in the ribs were measured with computer - assisted tomographic scans. procedures for preparing liver extracts and western blotting techniques have been described previously (12, 14, 15). to detect lamins a and c and progerin, we used a 1:400 dilution of a goat anti - lamin a / c igg (santa cruz biotechnology, santa cruz, ca) and a 1:5000 dilution for an irdye800-conjugated donkey anti - goat igg (li - cor ; lincoln, ne). to detect prelamin a, we used a 1:2000 dilution of a rabbit anti - prelamin a antiserum (6, 8, 13) and a 1:5000 dilution of an hrp labeled anti - rabbit igg (ge healthcare, piscataway, nj). to detect hdj-2, we used a 1:500 dilution of a mouse monoclonal antibody against hdj-2 (neomarkers ; fremont, ca) and a 1:5000 dilution of an hrp - labeled anti - mouse igg (ge healthcare). for both prelamin a and hdj-2 western blots, antibody binding was detected with the ecl plus chemiluminescence system (ge healthcare) and exposure to x - ray film. to assess protein farnesylation in fibroblasts, the cells were incubated for 48 h with an analog of farnesol, 8-anilinogeraniol (ag) (100 m in dmso) (19) in the presence and absence of abt-100 (1 m). ag is incorporated by cells into ag pyrophosphate, a farnesyldiphosphate analog, which is used by protein farnesyltransferase as a substrate to farnesylate caax proteins. ag incorporation into cellular proteins was detected by western blotting with a mouse monoclonal antibody specific for ag, diluted 1:5000 (19). we administered an fti, abt-100 (52 mg / kg / day), or vehicle alone to groups of 12 male and female lmna, lmna, and lmna mice, starting at 4 weeks of age. our goal was to determine if the fti would ameliorate progeria - like disease phenotypes in lmna mice in addition to lmna mice. the plasma levels of abt-100 in treated mice were similar to those in earlier studies (1214), ranging from 0.3 to 0.7 g / ml. when we incubated lmna and lmna fibroblasts with the same concentration of abt-100 that we achieved in mice (0.5 g / ml), the farnesylation of b - type lamins and progerin was inhibited, as judged by metabolic labeling experiments with a farnesol analog (8-anilinogeraniol) (fig. 1). also, we observed, as expected, an accumulation of prelamin a and nonfarnesylated hdj-2 in liver extracts from fti - treated mice (fig. interestingly, there were lower levels of mature lamin a, relative to actin, in liver extracts from fti - treated mice (fig. long - term treatment of lmna and lmna fibroblasts with abt-100 also lowered levels of mature lamin a, consistent with the findings in fti - treated mice (fig. western blot testing the effect of abt-100 on the farnesylation of the b - type lamins and progerin in lmna and lmna fibroblasts. for these studies, we used the same concentration of abt-100 that we achieved in the plasma of mice (0.5 g / ml). the top panel shows the merged images of western blots with an antibody specific for lamin a / c (red) and an antibody specific for the farnesol analog ag (green) ; the middle panel shows the signal for the antibody against ag ; the bottom panel shows a loading control (actin). in this experiment, lmna and lmna fibroblasts were incubated with ag (100 m) in the presence (+) or absence () of abt-100. the electrophoretic mobility of progerin, lamin b1, and lamin b2 are virtually identical, and all are farnesylated ; hence, the antibody against ag detected a farnesylated protein in both cell lines (in the absence of an fti). when progerin and lamin a / c were immunoprecipitated with an antibody against lamin a / c, the progerin in lmna fibroblasts, but not lmna fibroblasts, stained with the antibody against ag. treatment with an fti lowers steady - state levels of mature lamin a. a : western blots of liver extracts from lmna, lmna, and lmna mice that had been treated with a protein farnesyltransferase inhibitor (fti) or vehicle (veh) alone. western blots were performed with antibodies against lamin a / c and prelamin a. lmna fibroblasts, treated with either the fti or the vehicle, were used as controls. b : quantification of lamin a levels in liver extracts of lmna, lmna, and lmna mice (n = 3 mice / group ; each sample was analyzed on two independent western blots). lamin a / actin ratios in liver extracts of fti - treated mice were expressed relative to those in vehicle - treated mice. in the livers of lmna, lmna, and lmna mice, the lamin a / actin ratio in fti - treated mice was lower than in vehicle - treated mice (p < 0.0001). long - term treatment of fibroblasts with abt-100 lowers steady - state levels of mature lamin a, relative to lamin c or actin. lmna and lmna fibroblasts were treated with either fti (abt-100, 10 m) or dmso (as control) for 2 days or 14 days. (12, 14), the fti improved body weight curves in lmna mice (p < 0.0001 for both males and females when compared with lmna mice treated with vehicle alone) (figs. in contrast, the body weight curves of fti- and vehicle - treated lmna mice were not different. this was the case for both male and female lmna mice (p = 0.27 and 0.54, respectively). also, there were no differences in the body weight curves of fti- or vehicle - treated lmna mice (p = 0.36 for males and 0.52 for females). an fti improves body weight curves and survival in lmna mice but not in lmna mice. a and b : effect of the fti treatment on body weight curves in male (a) and female (b) mice. lmna (squares), lmna (circles), and lmna (triangles) mice were given the fti (closed symbols) or vehicle alone (open symbols), beginning at 4 weeks of age. body weight curves for the fti - treated lmna mice were improved (p < 0.0001 for both males and females). the fti treatment did not improve the body weight curves in lmna or lmna mice. c : kaplan - meier survival plots for lmna, lmna, and lmna mice treated with an fti or vehicle alone (n = 24 mice / group). fti treatment significantly improved survival in lmna mice (p < 0.0001). the fti had no significant effect on survival in lmna mice (p = 0.45). kaplan - meier survival curves revealed that the fti improved survival in lmna mice (p < 0.0001), extending survival by 68 weeks (fig. in contrast, the fti had no effect on survival of lmna mice (p = 0.45) (fig. consistent with the improvement in body weight curves in fti - treated lmna mice, the fti significantly increased body fat stores in lmna mice, compared with vehicle - treated mice (p = 0.002) (fig. 5). in contrast, the fti had no effect on fat stores in lmna mice (fig. 5). body fat weights in lmna and lmna mice treated with the fti or the vehicle alone. the fti significantly increased the weight of body fat in lmna mice (p = 0.002), but not in lmna mice (p = 0.21, n = 24 mice / group). we assessed the impact of the fti treatment on spontaneous rib fractures in both male and female mice. the fti clearly reduced the number of rib fractures in lmna mice (p < 0.0001) (fig. in contrast, the drug had no significant effect on the number of rib fractures in lmna mice (fig. the fti improved mean bone density and bone cortical thickness in lmna mice (p < 0.0001 for both males and females) (fig. in contrast, the fti had no effect on these bone phenotypes in lmna mice (fig. a : numbers of rib fractures in fti - treated (closed circles) and vehicle - treated (open circles) lmna and lmna mice at the time of death. all mice that survived to 38 weeks of age were euthanized at that time point. the fti - treated male and female lmna mice had fewer rib fractures (p < 0.0001) than vehicle - treated lmna mice. the numbers of rib fractures in fti- and vehicle - treated mice were similar (n = 24 mice / group). fti treatment improved the bone density (b) and cortical thickness (c) in the ribs of male and female lmna mice (n = 24 mice / group, p < 0.0001) but not in lmna (wt) and lmna mice (n = 24 mice / group). (12, 13) showed that an fti ameliorates disease phenotypes in a mouse model of hgps. although the results were significant and reproducible, putting the findings into perspective is challenging, particularly with the discovery that lmna mice develop disease (15). lmna have all of the same progeria - like disease phenotypes as lmna mice, albeit somewhat milder, and they invariably succumb to the disease. the discovery that nonfarnesylated progerin elicits disease prompted us to consider the possibility that the beneficial effects of the fti in lmna mice might have little to do with a direct effect on the farnesylation of progerin. instead, we wondered whether the effect of the fti might be more indirect, perhaps secondary to inhibiting the farnesylation of other cellular proteins. if the effects of the fti were indirect, one would predict the fti might be equally efficacious in lmna and lmna mice. fti treatment had no effect on body weight, survival, the number of rib fractures, fat stores, bone cortical thickness, or bone density of lmna mice. in contrast, the fti improved all of these phenotypes in lmna mice, and did so in a highly significant fashion. the fact that the fti improved disease in lmna but not lmna mice suggests that the beneficial effect of the fti in lmna mice is likely a consequence of inhibiting the prenylation of progerin, rather than an indirect effect of the drug on other cellular proteins. the beneficial effects of the fti on disease phenotypes in lmna mice were very apparent, and most of the differences were significant at a p < 0.0001 level. in contrast, the fti had no significant effects in lmna mice. one could argue that there may have been a slight trend toward improved body weights and lower numbers of rib fractures in fti - treated lmna mice, but these differences did not achieve statistical significance. however, if one were forced to suggest a potential explanation for the trend, one could point to the lower levels of mature lamin a in the setting of fti treatment. genetic studies have suggested that lower levels of lamin a synthesis reduce the disease phenotypes elicited by a lmna allele (20). the finding of lower lamin a levels in association with fti treatment was also observed in earlier studies (8, 14, 15). the mechanism for the fall in lamin a levels is unclear but we suspect that sustained blockade of prelamin a farnesylation leads to the eventual turnover of nonfarnesylated prelamin a, reducing the production of mature lamin a. the current study reinforces the idea that an fti can ameliorate disease in lmna mice, but important questions remain about the utility of ftis in the treatment of humans with hgps. indeed, questions were raised about whether an fti is the most appropriate drug for inhibiting the prenylation of progerin (21). 21) reported data suggesting that progerin can be geranylgeranylated in the setting of an fti, and they proposed that a combination of a statin and a bisphosphonate (which would theoretically inhibit both farnesylation and geranylgeranylation) might be more useful for treating hgps. they went on to show that a statin / bisphosphonate combination improved progeria - like disease phenotypes in zmpste24-deficient mice ; however, they provided no evidence that the combination actually inhibited the prenylation of prelamin a (or any other protein) in mice. until such evidence is in hand, doubts will remain about the rationale for the statin / bisphosphonate combination, and in particular, whether the combination ameliorates disease indirectly (perhaps secondary to the bone - strengthening properties of bisphosphonates) or more directly by inhibiting the prenylation of prelamin a. for those interested in investigating the utility of a bisphosphonate / statin combination, the approach outlined in the current study should be of interest. if a statin / bisphosphonate combination were to improve disease phenotypes in lmna mice, that would suggest that the mechanism for the combination was likely indirect, perhaps due to the bone - strengthening properties of the bisphosphonates, and not to a specific effect on the prenylation of prelamin a. in summary, an fti ameliorates disease phenotypes in lmna mice, but not lmna mice. the failure of the fti to improve disease in lmna mice suggests that the beneficial effects of the drug in lmna mice are likely due to blocking the farnesylation of progerin. | hutchinson - gilford progeria syndrome (hgps) is caused by the accumulation of a farnesylated form of prelamin a (progerin). previously, we showed that blocking protein farnesylation with a farnesyltransferase inhibitor (fti) ameliorates the disease phenotypes in mouse model of hgps (lmnahg/+). however, the interpretation of the fti treatment studies is open to question in light of recent studies showing that mice expressing a nonfarnesylated version of progerin (lmnanhg/+) develop progeria - like disease phenotypes. the fact that lmnanhg/+ mice manifest disease raised the possibility that the beneficial effects of an fti in lmnahg/+ mice were not due to the effects of the drug on the farnesylation of progerin, but may have been due to unanticipated secondary effects of the drug on other farnesylated proteins. to address this issue, we compared the ability of an fti to improve progeria - like disease phenotypes in both lmnahg/+ and lmnanhg/+ mice. in lmnahg/+ mice, the fti reduced disease phenotypes in a highly significant manner, but the drug had no effect in lmnanhg/+ mice. the failure of the fti to ameliorate disease in lmnanhg/+ mice supports the idea that the beneficial effects of an fti in lmnahg/+ mice are due to the effect of drug on the farnesylation of progerin. |
the septic cavernous sinus thrombosis (cst) due to odontogenic infection is uncommon and leads to substantial morbidity and mortality. the peculiar anatomy of cervicofacial planes, dental structures, and its direct communication with cavernous sinus predisposes individual to development of septic cst in background of dental infections. the negligence of dental hygiene and dental infections can lead to disastrous consequences. in this illustration, we intend to present an immunocompetent female having widespread pulmonary and dental infection who developed bilateral cst with facial palsy. the timely referral from dentistry department to neurologist and prompt management led to complete recovery at discharge after a month. in nutshell a 50-year - old, previously healthy, female patient presented with complaints of fever, pain, and pus discharge from right maxillary molars and swelling of right cheek since 15 days. she was mildly febrile. on examination, there was diffuse inflamed swelling over right maxillary region along with bilateral exophthalmos with chemosis and congestion of conjunctiva. bilateral ptosis with external ophthalmoplegia was present along with palsy of lower half of face [figure 1 ]. anterior view photograph of the patient at the time of presentation showing bilateral exophthalmos with chemosis along with palsy of lower half of face on right side her baseline investigations revealed the following results : total leukocyte count : 17000/mm, differential leukocyte count : p80l18e1m1, erythrocyte sedimentation rate : 46 mm / h, serum na136 meq / l, serum k3.4 meq / l, blood urea 20 mg / dl, serum creatinine 0.6 mg / dl, and random blood glucose 106 mg / dl. her chest x - ray revealed multiple thin - walled abscesses (pneumatoceles) [figure 2 ]. chest radiogram showing multiple pneumatoceles the patient was first seen by oral and maxillofacial surgeon. her orthopantomogram was done which showed opacity at the tips of right second and third maxillary molars [figure 3 ]. drainage of the dental abscess and extraction of the right maxillary molars was done immediately. her magnetic resonance imaging (mri) brain was done which showed bilateral mastoiditis along with bilateral cst with abscess in right cerebellar and right cerebellopontine angle region with infarct in right parietooccipital and right high parietal region [figures 4 and 5 ]. magnetic resonance (mr) the patient was started on intravenous fluids and antibiotics (vancomycin 2 g / day, ceftriaxone 2 g / day, and clindamycin 1200 mg / day). orthopantomogram showing opacity at the tips of right second and third maxillary molars (arrow) coronal contrast - enhanced t1wi showing multiple defects in the enhancing bilateral cavernous sinuses (arrows) mri - brain showing right cerebellar abscess (arrow) the patient improved during the hospital stay of 4 weeks and was discharged satisfactorily. at follow - up after 3 months, she had complete recovery. however, less commonly it may be caused by pharyngeal, otogenic, and odontogenic infections. blood from brain is drained by a complex system of veins and dural sinuses into internal jugular vein. the most important dural sinuses are cavernous sinuses, sagittal sinuses, straight sinus, transverse sinuses, and sigmoid sinus, which are all interconnected. the right and left cavernous sinuses are situated on the lateral aspect of the sella turcica. they extend from the superior orbital fissure to the petrous apex of the temporal bone. cavernous sinuses receive blood from the ophthalmic veins, the superficial middle and inferior cerebral veins, and the sphenoparietal and sphenoid sinuses. the cavernous sinuses in turn drain into the pterygoid venous plexus via emissary veins, and into the internal jugular vein and the sigmoid sinus via the inferior and the superior petrosal sinuses respectively. absence of valves in the cavernous sinuses and their connections favor bidirectional spread of infection. most commonly, cst results from spread of infection from the sinuses, especially the sphenoid, ethmoid, and frontal sinuses, or from the middle one third of the face. less commonly infection from teeth, nose, tonsils, soft palate, and ears may constitute primary source of the infection. the dental infection most commonly spreads via the pterygoid venous plexus, where an infected thrombus may extend or disseminate septic emboli. in 1989, ogundiya., reported case of a patient with dental infection which got complicated leading to orbital abscess, unilateral blindness, and cst. in 1991, yun., reported cst in a 60-year - old diabetic male about 38 days after extraction of an infected upper third molar tooth. in 2000, feldman., reported a case of 69-year - old male who was being treated for dental abscess. computed tomography of brain did not show any intracranial abnormalities at the time of admission, but there was presence of a right parapharyngeal abscess. repeat computed tomography (ct) scan after 48 h revealed a right parapharyngeal abscess and a bilateral cst. the patient underwent a tracheotomy, incision, and drainage of the parapharyngeal abscess, and extraction of the right third molar. the patient was found to be completely recovered during his follow - up. in 2007, embong., described a case of 43-year - old man who presented with a history of right - sided toothache, progressively increasing painful left orbital swelling, and diminution of left eye vision. ct head was suggestive of left orbital cellulitis, right parapharyngeal abscess, tooth abscess over right upper alveolar region with bilateral maxillary, and posterior ethmoidal sinusitis. jones and arnold reported a case of dental infection complicating with left facial abscess, cst, bilateral internal jugular thrombosis, and multiple lung abscesses. the signs of cst result from venous congestion due to impaired venous drainage from the orbit and eye. the onset is acute, usually with unilateral periorbital edema and proptosis associated with headache and photophobia. the infection can spread to the contralateral cavernous sinus via intercavernous sinuses, usually within 1 to 2 days of the initial presentation. the diagnosis of cst is usually done on clinical grounds and can be confirmed by appropriate radiographic studies. it may show a heterogeneous signal from the abnormal cavernous sinus along with deformity of the cavernous portion of the internal carotid artery, and an obvious hyperintense signal of thrombosed vascular sinuses. s. aureus is the most frequently cultured organism in these infections (70%), followed by streptococcus species (20%). empirically, patients should be started on antibiotics directed at the most common pathogens (gram - positive, gram - negative, and anerobes). early institution (within 5 - 7 days) may help in reducing morbidity, but delayed use provides no benefits. serious complications like septic pulmonary embolism, meningitis, carotid thrombosis, subdural empyema, and brain abscess may occur. with the availability of good broad - spectrum antibiotics, the prognosis of septic cst has improved reducing mortality from near 100% to 20 - 30%. residual neurological deficits in the form of squint and numbness and paresthesia in the region of 5 nerve supply have been noted. the interesting aspect of this case is the association of septic cst with the facial palsy. to the best of our knowledge, only lower half of the face on right side is involved. however, there is no corresponding intracerebral lesion that could explain it. mechanical compression of lower branches of the facial nerve seems to be more likely possibility causing facial palsy. | septic cavernous sinus thrombosis (cst) related to dental infection is a rare clinical event. the septic cst is a disease of high morbidity and mortality. the prompt diagnosis and timely treatment of septic cst is cornerstone of successful outcome. the dental infection should be given due attention, as to prevent cst. in this case report of immunocompetent female, we highlighted the role of dental abscess in producing bilateral cst and facial palsy. the close collaboration between dentist and neurologist and early institution of antibiotics led to complete recovery at follow - up after 3 months. the dental infection should never be neglected as it is the interface of serious intracranial complication like cst. |
a 48-year - old man was incidentally detected as having a well - defined, heterogeneous enhancing soft tissue density lesion in the bilateral retroperitoneum. the lesions were found during ct while the patient was undergoing evaluation for a gallbladder polyp that was incidentally detected on ultrasonography. the ct showed bilateral masses in the anatomical position of the adrenal glands with densities indicating adipose tissue. the maximum diameter was 8.0 cm for the left mass and 1.8 cm for the right mass. 1). he had a 4-year history of hypertension, for which he was receiving antihypertensive medication. he had no specific symptoms and the results of a physical examination were also nonspecific. the biochemistry profile showed an elevated level of plasma epinephrine of 67.8 pg / ml (range, 1 - 20 pg / ml), norepinephrine of 340.8 pg / ml (range, 15 - 80 pg / ml), and acth of 158 pg / ml (range, 10 - 90 pg / ml), and urinary 17-ks of 48.5 mg / day (range, 8 - 22 mg / day) and 17-ohcs of 55 mg / day (range, 5 - 23 mg / day). plasma cortisol, renin, aldosterone, and 24-hour urinary cortisol, free cortisol, metanephrine, vanillylmandelic acid, and homovanillic acid were all within normal limits. in the abdominal mri, the left tumor measured 8.2 cm and the right tumor measured 2.0 cm in maximum diameter, had high signal intensity on both t1- and t2-weighted images, and low signal intensity on t1 fat - suppressed images and chemical - shift images (fig. a differential diagnosis for malignant tumors was necessary, so the patient was subjected to transperitoneal laparoscopic left adrenalectomy. on macroscopic examination of the surgically removed specimen, the tumor was measured as 9.14.9 cm, weighed 128 g, and was yellowish - brown in color. microscopic examination revealed that the tumor was composed of mature fat mixed with hematopoietic elements including megakaryocytes (fig. the adrenal gland is the most common site, but myelolipomas are also rarely present in extra - adrenal sites, including the pelvis, mediastinum, retroperitoneum, and paravertebral region, as an isolated soft tissue mass. adrenal myelolipoma, affecting both sexes in similar rates, usually presents late in adult life, and most cases are diagnosed during the fourth to seventh decade. the association of myelolipoma with obesity, hypertension, chronic disease, and malignancies has been described. adrenal myelolipomas are in the majority of cases unilateral and often less than 4 cm in diameter. the term giant myelolipoma is preferred when the size exceeds 8 cm. in the literature, the largest adrenal myelolipoma (diameter 31 cm, weight 6.0 kg) was described by akamatsu and colleagues. to our knowledge, 10 cases of adrenal myelolipoma were reported in korea. however, these cases were all unilateral. adrenal myelolipoma, which is usually asymptomatic, is generally diagnosed during surgical interventions and imaging procedures performed for other purposes. our patient also performed ct for evaluation of a gallbladder polyp that was incidentally detected on ultrasonography. in symptomatic cases, the most common symptom is nonspecific abdominal pain, probably due to hemorrhage, tumor necrosis, or mechanical compression of adjacent organs from large tumor bulk. adrenal myelolipomas are generally hormonally inactive, although there are case reports of their association with overproduction of adrenal hormones. they have been associated with overproduction of dehydroepiandrosterone - sulphate (dheas), congenital adrenal hyperplasia caused by 21-hydroxylase deficiency, congenital adrenal 17 -hydroxylase deficiency, cushing disease, conn 's syndrome, adrenal insufficiency, and pheochromocytoma. some of these tumors coexist with adrenal adenomas, which may in part account for the hormonal activity. among them, congenital adrenal hyperplasia and cushing syndrome appear to be the two most common endocrine disorders described. in our case, the levels of plasma epinephrine, norepinephrine, and acth were elevated, but there was no evidence of a functioning pheochromocytoma in the mibg scan or other hormone - producing tumors in the histopathology. the pathogenesis of myelolipoma is uncertain, with the prevalent hypothesis suggesting that they arise from the zona fasciculate of the adrenal cortex from metaplasia of undifferentiated stromal cells. more specifically, they are assumed to arise from metaplasia of either previously uncommitted adrenal cortical mesenchymal cells or of groups of choristomatous hematopoietic stem cells that transmigrate during intrauterine life to the developing adrenal gland. other theories, less favored, propose embolism of bone marrow cells and development from intra - adrenal embryonic rests of bone marrow. diagnosis of myelolipoma is based on imaging, with ultrasonography, ct, and mri being effective in more than 90% of cases. the classic myelolipoma is radiolucent on plain films and avascular on angiography. on ultrasonography, a myelolipoma is hyperechoic if it consists mostly of fat and hypoechoic if it is composed primarily of myeloid cells. ct is the most sensitive imaging modality, and the appearance of myelolipoma depends on its histological composition. myelolipoma often has a discrete capsule and appears as well - delineated heterogeneous masses with regions of less than -30 hounsfield units that correspond to low - density mature fat. in mri, fat tissue has high signal intensity in both t1 and t2 images, whereas myeloid tissue has low signal intensity in t1 and moderate signal intensity in t2 images. differential diagnosis includes other fat - containing adrenal masses such as teratoma, lipoma, and liposarcoma, which are less common, and even rarely angiomyolipoma, mass - forming extramedullary hematopoiesis, and adenoma. in case a diagnosis has not been established, fine needle aspiration can be helpful to definitively rule out malignancy. previously, most patients with myelolipoma underwent surgical resection for a suspected malignant neoplasm. nowadays, with the frequent detection of these myelolipomas incidentally, the treatment has been a matter of much debate. when the tumor is 4 cm or smaller and asymptomatic, follow - up with ct if symptoms occur, surgery should be carried out promptly, especially for a large myelolipoma, because spontaneous rupture of the lesion with hemorrhage is possible. previously, a tumor size exceeding 5 to 6 cm was thought to be a contraindication for the laparoscopic approach. but several recent studies have shown that the laparoscopic approach is technically feasible, safe, and comparable with the open approach in these patients. in the case of bilateral adrenal myelolipomas, a staged tumor removal is preferable, removing the larger one and continuing to observe the contralateral myelolipoma as long as possible in an effort to avoid adrenal insufficiency and a lifetime of steroid replacement. our patient had bilateral adrenal myelolipomas in which the left tumor was larger than the right, measuring 8.2 cm vs. 2.0 cm in maximum diameter on the mri. however, if there were bilateral, giant, symptomatic adrenal myelolipomas in a patient with congenital adrenal hyperplasia, bilateral surgical resection should be done. | adrenal myelolipoma is a rare, nonfunctional benign tumor that is composed of mature adipose tissue and hematopoietic elements. in the past, these tumors were accidentally discovered at autopsy. today, they are found much more frequently and incidentally, mainly because of the widespread use of noninvasive imaging with ultrasonography, computed tomography, and magnetic resonance imaging. most lesions are asymptomatic, small, and unilateral, but a number of bilateral tumors have been reported. we report here on a case of a 48-year - old man with incidentally found myelolipomas of both adrenal glands. in particular, a giant myelolipoma of the left adrenal gland was treated by transperitoneal laparoscopic adrenalectomy. |
frontal fibrosing alopecia (ffa) is a scarring alopecia characterized by progressive recession of the frontotemporal hairline. current treatment is aimed at stopping progression, and the combination of dutasteride and pimecrolimus is the most effective therapy. side effects associated with dutasteride are erectile dysfunction as well as breast tenderness and enlargement, while pimecrolimus produces a burning sensation. we present a 57-year - old postmenopausal female with a 3-year history of a scarring alopecic plaque in her frontotemporal region. biopsy confirmed the diagnosis of ffa, and she was started on dutasteride 0.5 mg p.o. q.d. eight months after initiating treatment, she showed hyperpigmentation on her metacarpophalangeal and interphalangeal joints, as well as on the cheeks and on the chin ; dutasteride and pimecrolimus were discontinued. after 5 months of follow - up, her hyperpigmentation improved by 80% only by using photoprotection. because of the variable clinical course of ffa, treatment is focused on halting its progression. several therapeutic agents have been evaluated and the combination of dutasteride and pimecrolimus has shown a high response rate. there is no reported evidence of hyperpigmentation associated with this combination. frontal fibrosing alopecia (ffa) is a scarring alopecia that affects postmenopausal women and is considered a clinical variant of lichen planopilaris. currently, there is no treatment of choice and because of the scarring nature, the aim is to stop progression. studies have shown that the combination of dutasteride and pimecrolimus is by far the most effective therapy for this condition, and even hair regrowth has been reported [2, 3 ]. we present a 57-year - old postmenopausal female with a 3-year history of scarring alopecic plaques on both her frontotemporal regions, which she developed after an upper third face lift with aptos threads. skin biopsy revealed the presence of a perifollicular lymphocytic infiltrate and lamellar fibrosis, a decrease of hair follicles and apoptotic cells in the external root sheath, therefore confirming the diagnosis of ffa (fig. q.d. at the fifth month of follow - up, we added topical pimecrolimus 1% b.i.d. at this point, eight months after the treatment was started, the patient presented hyperchromic macules in the metacarpophalangeal and interphalangeal joints of both hands as well as on the cheeks and chin (fig. five months after the treatment was discontinued, the patient showed an 80% improvement of her hyperpigmented lesions (fig. ffa was described in 1994 by kossard in postmenopausal women, but it can also affect premenopausal women and males. it is considered a clinical variant of lichen planopilaris ; histologically, both entities show a lymphocytic infiltrate and fibrosis between the follicular epithelium and the dermis, involving the upper mid - portion of the follicle, but ffa shows less inflammatory infiltrate than lichen planopilaris. the pathogenesis is unknown, but a t - cell - mediated inflammatory reaction with the destruction of stem cells and hormonal changes as well as increased levels of androgens are thought to play an important role. several therapeutic agents have been evaluated and topical and systemic corticosteroids have been the most commonly used agents ; with a high risk of relapse upon discontinuation, other treatments have been studied, e.g. antimalarials, mycophenolate mofetil, doxycycline, and topical minoxidil with variable results. treatments with 5-reductase inhibitors have shown good results supporting the role of androgens in the pathogenesis of ffa. dutasteride is a 5-reductase inhibitor, it is 3 times more potent than finasteride at inhibiting type ii enzyme and 100 times more potent at inhibiting type i enzyme, making it a leading option in treating ffa. pimecrolimus, a topical calcineurin inhibitor that blocks t - cell activation and cytokine production, has been used effectively in combination with dutasteride. reported side effects associated with dutasteride and finasteride use are erectile dysfunction as well as breast tenderness and enlargement [7, 8 ]. for finasteride, there are reports of cutaneous vasculitis, drug - related folliculitis, erythema annulare centrifugum, solitary fixed drug eruption, urticarial rash and acute generalized exanthematous pustulosis ; the last one occurred 3 months after it was initiated. for pimecrolimus, the reported adverse effects are a burning sensation and pruritus, and there is one report of induced lentiginosis on the application site of this drug ; however, in this case, the hyperpigmentation did not appear at the sites of application of pimecrolimus. there is no reported evidence of hyperpigmentation associated with this combination or with the use of dutasteride. another described cause of hyperpigmentation in the presence of alopecia is the cronkhite - canada syndrome. it is a rare syndrome involving the skin and the gastrointestinal tract, showing hyperpigmentation, alopecia, onychodystrophy, diarrhea, hamartomatous polyposis and weight loss [11, 12 ]. the alopecia in these patients is a non - scarring alopecia, with telogen hair follicles without miniaturization or atrophy of the hair follicle and absence of inflammation, which can start in patches and then become total ; the hyperpigmentation is observed diffusely in the upper extremities. it shows onychodystrophy which includes a thinning of the nail plate, onycholysis and onychomadesis. in our patient 's case, this diagnosis is discarded because she does not have gastrointestinal symptoms, she has a scarring alopecia, and the hyperpigmentation occurred mainly on her face. because of the variable clinical course of ffa, treatment is focused on halting its progression. several therapeutic agents have been evaluated ; the combination of dutasteride and pimecrolimus has shown a high response rate. this case is of particular interest because the patient developed hyperpigmentation following the use of dutasteride and a remission after its discontinuation. there is still no clear evidence that points to dutasteride as the sole precipitating agent ; therefore, more studies are required to further evaluate this association. | introductionfrontal fibrosing alopecia (ffa) is a scarring alopecia characterized by progressive recession of the frontotemporal hairline. current treatment is aimed at stopping progression, and the combination of dutasteride and pimecrolimus is the most effective therapy. side effects associated with dutasteride are erectile dysfunction as well as breast tenderness and enlargement, while pimecrolimus produces a burning sensation.case reportwe present a 57-year - old postmenopausal female with a 3-year history of a scarring alopecic plaque in her frontotemporal region. biopsy confirmed the diagnosis of ffa, and she was started on dutasteride 0.5 mg p.o. q.d., and later, topical pimecrolimus 1% b.i.d. was added. eight months after initiating treatment, she showed hyperpigmentation on her metacarpophalangeal and interphalangeal joints, as well as on the cheeks and on the chin ; dutasteride and pimecrolimus were discontinued. after 5 months of follow - up, her hyperpigmentation improved by 80% only by using photoprotection.conclusionbecause of the variable clinical course of ffa, treatment is focused on halting its progression. several therapeutic agents have been evaluated and the combination of dutasteride and pimecrolimus has shown a high response rate. there is no reported evidence of hyperpigmentation associated with this combination. |
limited rest time, high intensity, and competition rate of strenuous sports emphasize on the necessity of following the most suitable approach to manage the functional overload of professional athletes. a proper nutritional strategy is helpful in order to achieve proper recovery, with multiple competing periods and several times per day. the content and time of nutrient consumption impact the resynthesis of fuel supply, reduction of muscle injury, and optimizing the competition performance. liquids are more tolerable in suppressed appetite of instant post - exercise period and they help in cell rehydration and substituting lost electrolytes due to sweating. beverage micro and macronutrient content and their utilization, during or post - exercise period, are effective in fuel restoration. studies reflect that the consumption of carbohydrate - protein beverages during post - exercise recovery periods can facilitate glycogen restore and muscle turnover speed. chocolate milk composition is similar to common sports drinks, and it can enhance blood sugar level, speed of muscle glycogen repletion, and protein turnover. its branched chain amino acids, carbohydrates, electrolytes, and easily absorbed casein and whey protein help athletes muscle stores. dough or persian salty yogurt drink with a consistency similar to milk which contains high amount of whey protein and critical electrolytes such as sodium and calcium can affect athletes performance. moreover, non - alcoholic beer as a source of carbohydrate, minerals, and vitamins is a popular and available supplement fluid. choosing the most proper recovery beverage can be useful for nursing and medical members of sports medicine to guide athletes. in the present study, we compared the effects of dough, non - alcoholic beer, and carbohydrate replacement drink on lactate dehydrogenase enzyme level, f2-isoprostane, lipid, and glucose blood level. the professionals were asked to stop their exercises for 24 hours before initiation of the intervention program. they were also asked to note their food intake in one - day food recall questionnaires. five milliliter venous blood was collected, and after 10-min warming up exercises, athletes followed standard protocol of running - based anaerobic sprint test (rast). blood lactate was tested after running the protocol and 1-h post - rast by a calibrated lactometer (scout company, germany). athletes at a 4 day interval, received 500 cc isocaleric beverages as dough, non - alcoholic beer, and chocolate milk. after taking the first beverage then, the participants venous blood was taken ; the other two beverages were consumed following the same guidelines. in other words, 21 participants who enrolled in one experimental study group consumed all three beverages. indirect vo2 max was determined by harvard step test and their 24-h recalls were assessed using a nutritionist iv software (version 7.0 ; n - squared computing, salam, or, usa). serum triglyceride, total cholesterol level, and blood sugar assessed with enzymatic kits (pars azmoon ins, tehran, iran). data were compared between different times using a simple repeated - measures analysis, and post - hoc comparisons were also performed too. all the analyses were done using the statistical package for the social sciences software (spss inc, chicago, il, usa) (version 20). paired t - test and analysis of variance (anova) test were also performed. ethics committee of iums approved the study process, and informed consent was gained from all of the subjects. ethics committee of iums approved the study process, and informed consent was gained from all of the subjects. the taekwondo players mean age was 23 2.7 years. levels of lipid profile and blood sugar before and after drinking beverages during pre and post - recovery periods are shown in table 1. total cholesterol levels decreased after the three intervention periods, however, this reduction was not significant. comparison of total cholesterol change after intervention did not reflect a significant difference (p > 0.05). plasma concentrations of profile lipids and blood sugar before and after ingestion of beverage plasma triglyceride was lower after dough and carbohydrate replacement drink intake. the mentioned decrease was marginally significant in taekwondo players after dough consumption (p = 0.076), whereas there was a non - significant difference after carbohydrate drinking periods. in addition, non - alcoholic beer intake non - significantly increased triglyceride level. between groups was marginally significant after consuming non - alcoholic beer (p = 0.083), however, the mean change of plasma glucose did not show a significant increase for the three beverages. moreover, lactate dehydrogenase level reduced after all the intervention cycles. mean change of this plasma enzyme level was statistically significant after non - alcoholic beer consumption (p = 0.048). in addition, no significant increase was observed between mean pre and post - recovery f2-isoprostane values and between groups comparison did not show any statistically significant difference (p > 0.05). participants did not complaint about any side effects. mean and standard error of oxidative stress and muscle damage biomarkers before and after drinking beverages are shown in table 2. in this study, we compared the effects of various types of beverages including dough, non - alcoholic beer, and carbohydrate - rich beverages on blood sugar, lipid profile, lactate dehydrogenase, and f2-isoprostane levels of elite taekwondo players. findings show that all three beverages at pre- and post - recovery periods enhanced blood sugar and f2-isoprostane levels, whereas these fluids supplements intake decreased plasma total cholesterol and lactate dehydrogenase levels. non - alcoholic beer increases the triglyceride level, and the other liquids lowered plasma triglyceride level. in a study by bishop., plasma glucose levels reduced in the placebo group in comparison to the group that received carbohydrate - rich beverages, both at fatigue and at 1 hour after exercise. it seems that stress hormone release was accompanied with post - exercise blood glucose reduction. moreover, it can balance the fatigue perception, lowering quality of athletes performance. nutrient profile of milk and its products as carbohydrate, whey, electrolytes, and water can be effective on glucose level in athletes recovery periods. the observed effect of fluid drink on triglyceride and total cholesterol levels are comparable with previous findings. in a 6-week intervention trial with fermented milk product, ageraek. observed a significant reduction in cholesterol level of 58 healthy participants, whereas plasma triglyceride showed no significant change. cholesterol and triglyceride levels reduction shows an approximately similar trend, however, its strength is affected by sample size of the studied participants. the effect of yogurt, as a milk product, on lowering serum total cholesterol can be explained by its lactobacillus acidophilus content. bioactive compounds, calcium, conjugated linoleic acid fermentation bacteria, and probiotic components can play critical roles in reducing plasma cholesterol and triglyceride levels. exercise leads to a higher lactate dehydrogenase concentration as a converting enzyme with fuel supplying roles and also its level reflected the increased free radical concentration which is caused by stress of sport. in addition, its level affects lactate concentration of athletes muscle and their performance ability. karp. on comparing the effects of chocolate milk, fluid replacement drink, and carbohydrate replacement drink consumption in highly - trained cyclists observed increased post - exercise lactate level ; exhaustion time and glycogen - depleting exercise of participants can be managed by milk chocolate beverages. however, the mentioned effects were non - significant in a comparison of within - subject difference in the thomas trial on male trained cyclists. the non - significant within and between comparison of our supplement beverages on f2-isoprostane as a muscle injury and free radical arachidonic acids peroxidation indices can be explained using steensberg findings. they observed that plasma f2-isoprostane level decreases significantly in response to sport stress, however, this reduction is compensated in 1 hour after the recovery period. the trevor trial involving 127 men and women in the age group 30 - 65 years showed that low fat diet containing a daily 3-rich fish meal can reduce cell lipid peroxidation rate and lower urinary f2-isoprostane excretion. this reduction was higher in participants following aerobic exercises in addition to the mentioned diet. lack of reported studies on the effects of fluid supplements on f2-isoprostane level makes more comparison impossible. these findings can help nursing and medical team members of sports medicine to guide elites and professional athletes in rapid and most proper recovery periods. the limitations of our study were small sample size of participants and a before - after study design. moreover, measuring detailed nutrient and electrolyte content of beverages can be effective in our assessment. in regards to the strong points, the present study assessed the effects of isocaloric volume of dough intake in comparison to other available fluid supplements on lipid profile, blood sugar, muscle damage, and oxidative stress markers in professional athletes for the first time. in conclusion, we observed that dough, non - alcoholic beer, and carbohydrate replacement drink consumption at pre- and post - recovery periods can decrease plasma total cholesterol and lactate dehydrogenase level. non - alcoholic beer increases triglyceride level, and the consumption of other liquids was accompanied with lower plasma triglyceride in elite taekwondo players. | background : athletes recovery is important in improving their performance. nutritional strategies can be effective in enhancing recovery rate. choosing the best food items in appropriate intervals can play effective roles in resynthesis of fuels and recovery of muscle injury. beverage micro and macronutrient content are helpful in fuel restoration. in this study, we assess the effects of various kinds of beverages on oxidative stress, muscle injury, and metabolic risk factors in taekwondo players.materials and methods : this quasi - experimental study was performed on 21 taekwondo players of isfahan. after collecting fasting blood, they performed runningbased anaerobic sprint test (rast). blood lactate was tested again and participants were divided into 3 intervention groups, that is, receiving 500 cc dough, non - alcoholic beer, and chocolate milk at 4 day intervals. after a 2-h recovery period, blood sampling was repeated. elites consumed other beverages in later phases. dietary intake and fasting triglyceride, cholesterol, blood sugar, lactate dehydrogenase, and f2-isoprostane concentrations were determined. data were analyzed with a simple repeated - measures test and post - hoc tests using the statistical package for the social sciences software.results:data showed that cholesterol levels non - significantly decreased after intervention. triglyceride level was lower after taking dough and carbohydrate replacement drink. blood glucose concentration increased after intervention periods, however, this increase was significant only after non - alcoholic beverage consumption. lactate dehydrogenase levels reduced after all cycles, however, f2-isoprostane level showed no significant change. there was not significant change in lactate dehydrogenase and f2-isoprostane levels.conclusions:non-alcoholic beer consumption can reduce lactate dehydrogenase concentration ; however, it leads to blood sugar increase. moreover, dough consumption significantly reduced triglyceride level in taekwondo players. |
von hippel - lindau disease (vhl ; mim # 193300) is a hereditary multi - systemic tumour syndrome that pre - disposes affected individuals to haemangioblastomas of the central nervous system and retina, pheochromocytomas, clear - cell renal carcinomas, adenomas and carcinomas of the pancreas, paragangliomas, renal and pancreatic cysts, papillary cystadenomas of the epididymis and, rarely, cystadenomas of the endolymphatic sac and broad ligament. vhl affects approximately 1 in 36,000 newborns and is transmitted in an autosomal dominant manner with a penetrance of more than 90% by the age of 65 years [1, 2 ]. vhl is a tumour suppressor gene located on chromosome 3p2526 [3, 4 ]. the gene consists of three exons, is highly conserved across species, and is ubiquitously expressed in both foetal and adult tissues [5, 6 ]. expression of the vhl gene is not restricted to the organs affected in vhl [3, 79 ]. the vhl protein pvhl) has been implicated in a variety of functions, including transcriptional regulation, posttranscriptional gene expression, extracellular matrix assembly, protein folding and ubiquitination as reviewed by kaelin. an increasing number of germline mutations have been reported in vhl - affected individuals [1114 ], and genotype - to - phenotype correlations are now emerging. somatic mutations in vhl have also been detected in several types of sporadic and hereditary tumours [1618 ]. phenotypes vary among families, reflecting genotypic differences [1, 12, 14, 19 ]. clinically, vhl is classified in type 1 or type 2 based on the absence or presence of pheochromocytomas. the occurrence of renal cell carcinoma (rcc) allows a further distinction between type 2 a (low risk of rcc) and type 2 b (high risk of rcc). some type 2 families develop pheochromocytomas only, with no other neoplastic findings of vhl (vhl - type 2 c). vhl tumours, including pheochromocytomas and paragangliomas, may appear clinically to be sporadic but represent milder cases of vhl, with the attenuated phenotype resulting from either a mild impairment in function of the mutated pvhl or somatic mosaicism. the latter is a condition in which genetically different cells coexist in tissues of the same individual, and the intratumoural mixture of vhl - mutated and vhl - non - mutated cells clearly can modulate the resulting phenotype. we have analysed the vhl gene in the available members of a vhl family in which the pro - band presented with bilateral pheochromocytomas and multiple paragangliomas. her father showed what we believe is a very mild and relatively late - onset vhl phenotype. in this study, we describe the somatic mosaicism of the pro - band 's father and we reviewed the literature for all the described cases of vhl - mosaicism. the clinical features of the female pro - band have been reported in our previously published study, briefly, the young girl now 26 years old, underwent surgery at 11 years of age to resect a pheochromocytoma associated with hypertension. at age 18 years, she underwent further surgery to remove a pheochromocytoma in the contralateral adrenal gland and two concurrent paragangliomas of the abdominal aorta and urinary bladder. one year ago, she was also found to have a right - sided, extra - axial, 1.6-cm supratentorial frontal meningioma (fig. post - operatively, neuroendocrine serum markers (plasma free metanephrines, chromogranin a, neuron- specific enolase, and gastrointestinal hormones carcinoembryonic antigen [cea ] and calcitonin) have remained negative. a family history obtained from her parents, one brother and one sister was uninformative, except for the father 's history. mr image of the pro - band 's brain.the arrow indicates a 1.6-cm - diameter right - sided supratentorial frontal meningioma. the patient 's father, now 51 years old, was found to have an angioma of the glans penis and had had surgery for a mandibular cyst and epididymal cystadenomas at age 43 years. indeed, abdominal ultrasonography and total - body magnetic resonance imaging (mri) revealed a 2.3-cm cyst in his right kidney. his blood pressure and levels of plasma free metanephrines, fractionated urinary metanephrines, chromogranin a were normal. genomic dna was extracted from peripheral blood lymphocytes (pbls) of the pro - band and her four first - degree relatives. to assess the possibility that the pro - band 's father had an attenuated vhl phenotype caused by mosaicism, somatic dna was extracted from his oral epithelial cells, hair roots and skin fibroblasts. the entire coding sequence of the vhl gene was pcr - amplified with 36-cycle reactions using conditions and primers, with minimum variations, described previously. pcr products were analysed by denaturing high - performance liquid chromatography (dhplc) using a wave 2100 dna fragment analysis system (transgenomic wave system, omaha, ne) at column temperatures recommended by the wavemaker version 4.1.31 software (transgenomic) and at melt temperatures determined by the dhplc melt software (http://insertion.stanford.edu/melt.html). nucleic acids were separated in the column according to size and degree of denaturation in a gradient of two buffers (a : 0.1 m triethylammonium acetate, [teaa, ph 7 ] ; b : 0.1 m teaa, 25% acetonitrile). dhplc analysis was performed at a melt temperature of 60c at a constant flow rate of 0.9 ml / min using a linear gradient of acetonitrile. to 13.75%, increased over 5 min. to 16.25%, was kept constant for 1 min., increased over 1 min. to 25%, was kept constant for 1 min. (wash), decreased over 1 min. to 8.75%, and was kept constant for 1 min. heteroduplex molecules can be detected as an additional peak, or shoulder, in the chromatogram. amplimers with abnormal denaturing profiles were purified (microcon pcr, millipore, bedford, ma) and sequenced bidirectionally using an abi bigdye terminator cycle sequencing kit v.3.1 and an abi prism 310 genetic analyser (both from applied biosystems, foster city, ca, usa). sequencing results were analysed using the sequencing analysis v.3.6.1 and autoassembler v.2.1 software packages (both from applied biosystems). for semiquantitative analysis, the pcr products were cloned into a vector (topo ta cloning, invitrogen carlsbad, ca, usa) according to the manufacturer 's instructions, directly amplified from bacterial colonies, and sequenced. the clinical features of the female pro - band have been reported in our previously published study, briefly, the young girl now 26 years old, underwent surgery at 11 years of age to resect a pheochromocytoma associated with hypertension. at age 18 years, she underwent further surgery to remove a pheochromocytoma in the contralateral adrenal gland and two concurrent paragangliomas of the abdominal aorta and urinary bladder. one year ago, she was also found to have a right - sided, extra - axial, 1.6-cm supratentorial frontal meningioma (fig. post - operatively, neuroendocrine serum markers (plasma free metanephrines, chromogranin a, neuron- specific enolase, and gastrointestinal hormones carcinoembryonic antigen [cea ] and calcitonin) have remained negative. a family history obtained from her parents, one brother and one sister was uninformative, except for the father 's history. mr image of the pro - band 's brain.the arrow indicates a 1.6-cm - diameter right - sided supratentorial frontal meningioma. the patient 's father, now 51 years old, was found to have an angioma of the glans penis and had had surgery for a mandibular cyst and epididymal cystadenomas at age 43 years. indeed, abdominal ultrasonography and total - body magnetic resonance imaging (mri) revealed a 2.3-cm cyst in his right kidney. his blood pressure and levels of plasma free metanephrines, fractionated urinary metanephrines, chromogranin a were normal. genomic dna was extracted from peripheral blood lymphocytes (pbls) of the pro - band and her four first - degree relatives. to assess the possibility that the pro - band 's father had an attenuated vhl phenotype caused by mosaicism, somatic dna was extracted from his oral epithelial cells, hair roots and skin fibroblasts. the entire coding sequence of the vhl gene was pcr - amplified with 36-cycle reactions using conditions and primers, with minimum variations, described previously. pcr products were analysed by denaturing high - performance liquid chromatography (dhplc) using a wave 2100 dna fragment analysis system (transgenomic wave system, omaha, ne) at column temperatures recommended by the wavemaker version 4.1.31 software (transgenomic) and at melt temperatures determined by the dhplc melt software (http://insertion.stanford.edu/melt.html). nucleic acids were separated in the column according to size and degree of denaturation in a gradient of two buffers (a : 0.1 m triethylammonium acetate, [teaa, ph 7 ] ; b : 0.1 m teaa, 25% acetonitrile). dhplc analysis was performed at a melt temperature of 60c at a constant flow rate of 0.9 ml / min using a linear gradient of acetonitrile. to 13.75%, increased over 5 min. to 16.25%, was kept constant for 1 min., increased over 1 min. to 25%, was kept constant for 1 min. (wash), decreased over 1 min. to 8.75%, and was kept constant for 1 min. heteroduplex molecules can be detected as an additional peak, or shoulder, in the chromatogram. amplimers with abnormal denaturing profiles were purified (microcon pcr, millipore, bedford, ma) and sequenced bidirectionally using an abi bigdye terminator cycle sequencing kit v.3.1 and an abi prism 310 genetic analyser (both from applied biosystems, foster city, ca, usa). sequencing results were analysed using the sequencing analysis v.3.6.1 and autoassembler v.2.1 software packages (both from applied biosystems). for semiquantitative analysis, the pcr products were cloned into a vector (topo ta cloning, invitrogen carlsbad, ca, usa) according to the manufacturer 's instructions, directly amplified from bacterial colonies, and sequenced. the vhl gene sequence was altered in both the proband and her father, though to different extents in each. dhplc analysis of vhl exon 3 pbls dna showed quantitative differences in the dna elution profiles between daughter and father (fig. this finding suggested a heterozygous mutation in both relatives and mosaicism in the father. to test for mosaicism dna exon 3 amplicons extracted from the father 's buccal mucosa, hair roots, and skin fibroblasts showed different levels of intensity of the same altered dhplc peaks, although the intensity of the peaks in these tissues was comparable to that seen in the pbl dna (fig. 2b). denaturing high - performance liquid chromatography (dhplc) analysis of exon 3 of the von hippel - lindau disease (vhl) gene in all the family members. (a) dhplc analysis of the pcr products of exon 3 of the vhl gene in the pro - band (dhplc 3), her father (dhplc 1), her mother (dhplc 2) and her brother and sister (dhplc 4, 5). in the first - degree pedigree of this family, the pro - band is indicated by number 03, while her father, mother, sister and brother are indicated by the numbers 01, 02, 04 and 05, respectively. dhplc analysis of the pro - band 's dna shows an extra peak that is barely visible (but reproducibly so) in her father 's dna. (b) dhplc analysis of dna extracted from a normal control (nc) sample, the father 's pbls, and the father 's oral mucosa (tissue 1), hair roots (tissue 2) and fibroblasts (tissue 3). sequence analysis demonstrated that the abnormal elution profile (abnormal peak) seen in the proband and at various levels in the different cells from the pro - band 's father was the result of a missense mutation in exon 3. this mutation was a g - to - a substitution at cdna nucleotide 695, predicting the replacement of wild - type arginine with glutamine codon 161 of pvhl (r161q). the mutation - related peak in the pbls dna of the father was smaller than the same peak in the daughter 's pbls dna (compare fig. 3a with fig. amplicon cloning revealed that only a few clones contained the g - to - a mutant allele. in other words, in the dna extracted from the father 's circulating pbls, the ratio of wild - type to mutated gene was approximately 85:15, instead of the 50:50 ratio expected in the absence of mosaicism. sequencing analysis of exon 3 of the vhl gene in dna from pbls of the pro - band (a) and her father (b). to summarize, direct sequencing analysis suggested that only a small fraction (about 15%) of pbls contained the vhl mutation, lower than the typical 50% observed in a heterozygous individual. furthermore, only in the dna extracted from buccal cells, hair roots and cultured skin fibroblast cells was this mutation evident. the mosaic individual we described here presented at age 51 years with an angioma of the glans penis and a renal cyst. at age his daughter, in contrast, had a full - blown germline vhl gene mutation at a much younger age (11 years). in the mosaic subject, the late disease onset and mild vhl phenotype might have been mediated by the presence of two different cell populations, a prevailing population with a normal vhl gene and a smaller one with a mutated vhl gene. very few abnormal cells are likely to be present in the father 's chromaffin cells, thus explaining the absence of pheochromocytomas / paragangliomas, because decreased / lower likelihood to have a so - called second hit event with subsequent movement of mutated cells more towards homozygosity, as it has been shown in multiple endocrine neoplasia type 2 associated tumours [23, 24 ]. the inaccessibility of the chromaffin cells in the father 's adrenal medulla and paraganglia precluded the experimental quantification the ratio of normal to mutated vhl. as a consequence of the atypical phenotype of vhl in the father, the presence of familial vhl was not recognized initially. in recent years, the role of pvhl in the regulation of hypoxia - inducible genes through the targeted ubiquitination and degradation of hif1 has been elucidated, leading to a model of how disruption of the vhl gene can result in highly vascularized tumours. when pvhl is absent or mutated, hif1 subunits accumulate, resulting in cell proliferation and neovascularization in vhl tumours. vhl is inherited in an autosomal dominant fashion, with about 80% of cases being familial and about 20% sporadic as a result of a de novo mutation. the family history can sometimes be falsely negative because of failure to recognize the disorder in some family members, reduced penetrance, intrafamilial variability of clinical expression, death of the affected parent before the onset of symptoms or late onset of the disease in the affected parent. another reason why vhl may go unrecognized in either parent, so that the disease in the child is erroneously considered to be sporadic, is somatic mosaicism. first described vhl mosaicism in 2 (5%) of 42 unrelated families, both of which lacked a history of vhl. the two patients (one man and one woman) had clinical evidence of vhl, but no vhl mutations were detected in the initial genetic test performed on dna from their pbls ; in contrast, their clinically affected offspring tested positive for vhl mutations in their pbls. another case of parental mosaicism was described by murgia. in kindred in whom this pro - band presented at age 26 years with cerebellar haemangioblastoma, retinal haemangioma, multiple bilateral renal cysts and bilateral pheochromocytomas. in contrast, his asymptomatic (mosaic) mother, whose pbls dna showed a barely visible single - strand conformation polymorphism bandshift identical to that seen in her son, presented with only a small pheochromocytoma and renal microcysts by age 48 years. in both of the above studies, dna was also extracted from tissues, such as buccal cells and skin fibroblasts to confirm the somatic mosaicism. in similar families, the mosaic individual may be mildly or minimally affected, generally tends to have less severe disease than his or her offspring, and may be asymptomatic or present with less severe features of the disease. disease severity varies among mosaic individuals depending on whether mutations occur early or late in embryogenesis. asymptomatic carriers have been described in a number of heritable tumour syndromes reviewed by zlotogora and in many other heritable diseases, such as hutchinson - gilford progeria. mosaicism has also been demonstrated to be the cause of many cases of clinical recurrence. in a mosaic individual, the co - existence of mutated cells with even a small population of normal cells is an important parameter in predicting phenotype and overall prognosis and can increase the difficulty of obtaining a correct diagnosis of vhl. vhl in a mosaic individual may be difficult to recognize merely on clinical grounds, but it should always be considered when evaluating patients with isolated vhl - related tumours or parents of affected individuals. under these conditions, such individuals should be analysed for low - level mutations by emerging dna analysis techniques. it is presumed that the genotype - phenotype correlation in vhl reflects the degree to which the functions of pvhl are quantitatively and qualitatively altered by different mutations. a number of mutation carriers have been described, but not in sufficient numbers to define mutation - based phenotypes. furthermore, the percentages of symptomatic and asymptomatic vhl mutation carriers and the most important variable affecting disease penetrance and severity (age at diagnosis, sex distribution, genetic co - factors or environmental modifiers) have yet to be evaluated. this would allow the development of unified surveillance guidelines for vhl patients or those at risk for this disease. in summary, we have provided molecular evidence of somatic mosaicism in the father of a patient with full vhl. because of the incomplete penetrance of the disease the diagnosis of vhl was first not recognized. because of penetrance age related in vhl, a long - term follow up is warranted. counselling of patients and closely related family members must take a central place in the management of hereditary multi - organ cancer syndromes, such as vhl. a careful and complete clinical examination in the parents of each patient affected by an apparently de novo vhl germline mutation is warranted. the evaluation of the parents of a pro - band with an apparent de novo vhl gene mutation should include molecular genetic testing if the vhl disease - causing mutation in the pro - band is known. if the disease - causing vhl mutation in the pro - band is unknown, both parents should be offered a complete and extensive clinical and images examination, such as neurological test including mri of the craniospinal axis ; ophthalmologic evaluation ; measurement of plasma free metanephrines, chromogranin a, and fractionated urinary metanephrines ; and abdominal ultrasonography, mri or computed tomography. the real incidence of mosaicism in vhl remains uncertain, but such a phenomenon has important consequences for molecular testing, clinical diagnosis and genetic counselling, in terms of prediction of phenotype and risk of recurrence after the initial diagnosis. the real incidence of mosaicism will hopefully yield better data on the real incidence of de novo vhl mutations. | abstractvon hippel - lindau disease (vhl) is an autosomal dominant, familial neoplastic disorder with variable interfamilial and intrafamilial expression. vhl is characterized by pre - disposition to development of a combination of benign and malignant tumours affecting multiple organs. we provide molecular evidence of somatic mosaicism in nearly asymptomatic man whose daughter had vhl. the mosaic subject was found to have a cyst of the kidney and an angioma of the glans penis and had had surgery for a mandibular cyst and epididymal cystadenomas. mosaicism could provide a genetic explanation for the clinical heterogeneity and variable severity of vhl. the real incidence of mosaicism is still unclear and the identification of mosaicism has important consequences in genetic counseling of vhl patients who appear to have de novo vhl mutations and should be considered when evaluating patients with isolated vhl - related tumours. our results strongly suggest a complete and extensive clinical examination in the parents of each patient affected by an apparently de novo vhl germline mutation.we recommend performing a mutation screening of both parents of a proband with techniques that permit detection of low percentages of mosaicism before concluding that the proband has a de novo vhl mutation. |
self - monitoring of blood glucose (smbg) provides real - time glucose readings for insulin and diet / exercise adjustments, and thus plays an important role in diabetes management for insulin - treated patients. its role in type 1 diabetes is well recognized and is recommended to practice at a certain frequency in different guidelines. however, as a voluntary behavior, the practice of smbg among patients with type 1 diabetes is not satisfactorily implemented. many studies have shown a significant gap between recommended smbg utilization and the real - world practice. previous studies shown that many factors have been associated with the frequency of smbg practice, including gender, age of onset, length of time since diagnosis, insulin regimen, economic status, and insurance coverage. however, these observational studies were not conceptually integrated and did not reveal the ultimate elements generating smbg behavior, thus hardly suggested any actionable intervention target. a more integrated and systemic method is required to better understand smbg behavior and to provide directions for practical interventions. the information - motivation - behavioral skills (imb) model of health behavior is a well - researched theoretical model derived from behavioral science. the model has been widely applied in both observation and intervention studies of hiv risk behaviors and antiretroviral therapy adherence and is known to be an effective tool in this field. recently, other fields of health care related to health behaviors, especially those have a lot to do with self - management like diabetes, have been exploring the use of this successful model. guided by imb model of health behavior, this questionnaire - based survey was designed to study smbg behavior systemically among a sample of chinese adult patients with type 1 diabetes. participants were selected from the guangdong type 1 diabetes mellitus translational medicine study, of which our diabetes clinic is one of the 16 registry centers. adult (older than 18 years) patients with type 1 diabetes visiting our diabetes clinic from january to march 2012 (n = 63) were consecutively interviewed, except for those newly diagnosed patients (duration 0.50 for 70% of the items, p 0.50 for 70% of the items, p < 0.001) than the other two. questionnaire administration all respondents completed the questionnaire at the same clinical setting, which was a separate quiet examination room with desks and chairs. two interviewers were trained before the study to standardize the interview process and interviewed the 55 patients (70 person - times) throughout the study. brief introduction and necessary explanations about the study purpose and design were given first, written consent was obtained afterward, and then the questionnaire was delivered to the patient for self - completion. negative (incorrect) and neutral responses to statements in information and motivation sections were coded as for each item, proportions of patients that were deficient in that particular information / motivation / skill were described. spearman 's correlation was used to analyze relationships between each module and smbg frequency as well as interrelationships between the three modules. multiple correlation analysis was used to evaluate the impact of smbg information, motivation, and behavioral skills as a whole on smbg frequency. the compliance rate of the american diabetes association recommendation (to test at least three times daily) was 36.4%, 27.3% of participants tested less often than once a day, 5.4% (n = 3) reported they barely practice smbg. characteristics of the survey participants bmi, insulin dosage (u / kg), and hba1c were normally distributed variables : mean sd ; age, years since diagnosis were nonnormally distributed variables : median (25 percentile, 75 percentile) ; gender, education level, and insulin program were categorical variables : n (%). bmi : body mass index ; csii : continuous subcutaneous insulin infusion ; mdi : multiple daily injection ; sd : standard deviation. smbg information deficits, motivation obstacles, and behavioral skill limitations were identified in a substantial proportion of participants [table 4 ], of which the most prevalent deficits / obstacles / limitations included : the meaning of high blood sugar before exercises (not understood in 50.9% of participants), kind of food that should be taken when blood sugars was low (47.3%) ; views of the cost of testing being too expensive (85.5%) or painful (72.7%) if adhered to the doctor 's recommendation ; feeling difficult to talk with colleges about diabetes (63.6%) and to buy test strips conveniently (58.2%). smbg information, motivation, and skills deficits among adult patients with type 1 diabetes smbg : self - monitoring of blood glucose. scores of smbg motivation (r = 0.299, p = 0.026) and behavioral skills (r = 0.425, p = 0.001) were significantly correlated with smbg frequency while score of smbg information was not (r = 0.255, p = 0.060). figure 1 shows the relationship between each module and the frequency of smbg and interrelationship between the modules. the multiple correlation of smbg information, smbg motivation, and smbg behavioral skills with smbg frequency was r = 0.411 (r = 0.169, p = 0.023). smbg information, motivation, and skills together accounted for 16.9% of the variation in smbg frequency among our participants. correlations between modules of the imb - smbg model and smbg frequency (p < 0.05, p < 0.01). imb : information - motivation - behavioral skills ; smbg : self - monitoring of blood glucose. the compliance rate of the american diabetes association recommendation (to test at least three times daily) was 36.4%, 27.3% of participants tested less often than once a day, 5.4% (n = 3) reported they barely practice smbg. characteristics of the survey participants bmi, insulin dosage (u / kg), and hba1c were normally distributed variables : mean sd ; age, years since diagnosis were nonnormally distributed variables : median (25 percentile, 75 percentile) ; gender, education level, and insulin program were categorical variables : n (%). bmi : body mass index ; csii : continuous subcutaneous insulin infusion ; mdi : multiple daily injection ; sd : standard deviation. smbg information deficits, motivation obstacles, and behavioral skill limitations were identified in a substantial proportion of participants [table 4 ], of which the most prevalent deficits / obstacles / limitations included : the meaning of high blood sugar before exercises (not understood in 50.9% of participants), kind of food that should be taken when blood sugars was low (47.3%) ; views of the cost of testing being too expensive (85.5%) or painful (72.7%) if adhered to the doctor 's recommendation ; feeling difficult to talk with colleges about diabetes (63.6%) and to buy test strips conveniently (58.2%). smbg information, motivation, and skills deficits among adult patients with type 1 diabetes smbg : self - monitoring of blood glucose. scores of smbg motivation (r = 0.299, p = 0.026) and behavioral skills (r = 0.425, p = 0.001) were significantly correlated with smbg frequency while score of smbg information was not (r = 0.255, p = 0.060). figure 1 shows the relationship between each module and the frequency of smbg and interrelationship between the modules. the multiple correlation of smbg information, smbg motivation, and smbg behavioral skills with smbg frequency was r = 0.411 (r = 0.169, p = 0.023). smbg information, motivation, and skills together accounted for 16.9% of the variation in smbg frequency among our participants. correlations between modules of the imb - smbg model and smbg frequency (p < 0.05, p < 0.01). imb : information - motivation - behavioral skills ; smbg : self - monitoring of blood glucose. guided by the imb model, the current study developed an smbg questionnaire, carried out a survey among adult patients with type 1 diabetes in our hospital, and disclosed considerable deficiencies in smbg information, personal attitudes, and social support in regard to smbg, and smbg performing skills among surveyed population. over half of the patients deemed it was ok to increase exercises when blood glucose is very high. nearly half of the patients had wrong ideas about the kind of food that should be taken when blood glucose is low and were not against the idea of feel the blood sugar without testing. one - fifth did not understand the different meanings of hba1c and smbg results and the necessity to practice both. more than half of the patients found that practicing smbg as recommended would be too expensive, painful, unpleasant, causing anxiety, or interfering with their work. a considerable proportion of patients did not feel support of regular smbg from surrounding and/or important people. behavioral skill obstacles mainly included difficulties in talking about having diabetes with workmates and friends, buying test strips conveniently, practicing smbg painlessly, and keeping glucose meter available. correlation analysis showed significant correlation between smbg motivation and frequency as well as between smbg skill and frequency, and the latter correlation was stronger, suggesting that behavior skills were ultimately the most closely associated factors with smbg behavior. the deficiencies revealed were similar with the findings in an earlier study carried out among the us citizens with type 1 diabetes, but with a substantial higher proportion of patients having them, especially in the sections of motivation and behavioral skills. in that particular study, mean adherence to recommended smbg frequency was 90% (n = 208), much higher than the 36.4% recommendation adherence in this study. as could be expected, the mean hba1c level was lower in the us study than that in this study, 7.3% (56 mmol / mol) versus 7.7% (61 mmol / mol). the imb model emphasizes the subjective perspectives from the patients without studying the objective requisite conditions in performing the behavior, making it less fitting in study settings where requisite requirements can not be met at first place. for smbg behavior, such requirements might include possessing a glucose meter, having been recommended a proper blood glucose monitoring pattern and frequency by a professional, having the basic economic condition or insurance coverage to pay for the monitoring supplies, and reach ability of a diabetes doctor to discuss over the glucose monitoring results. thus, future studies in attempt to observe or improve smbg practice among diabetes patients, these and maybe other prerequisites should be investigated in the beginning, especially in developing areas. most of the participants in this survey (54/55) were from guangzhou city, a relatively developed district in china. the study may be relatively more helpful for diabetes educators in our clinic and other clinics in guangzhou. the results of this study can not be extrapolated to patients from other districts with different economic and health - care environments. interventional research in this area remains to be carried out. in conclusion, the study shown that adult patients with type 1 diabetes in our clinic had substantial smbg information deficits, motivation obstacles, and skill limitations. these deficiencies accounted for a respectable proportion of the variation in smbg frequency, which may provide potential - focused education targets for diabetes health - care providers. this work was supported by grants from the sun yat - sen university clinical research 5010 program (no. 2015a030401034), and the chinese national natural science foundation (no. 81100556). this work was supported by grants from the sun yat - sen university clinical research 5010 program (no. 2015a030401034), and the chinese national natural science foundation (no. 81100556). | background : the information - motivation - behavioral skills (imb) model of health behavior is an effective tool to evaluate the behavior of diabetes self - management. the purpose of this study was to explore behavioral factors affecting the practice of self - monitoring of blood glucose (smbg) within the frame of imb model of health behavioral among adult patients with type 1 diabetes in a single diabetes clinic in china.methods:a questionnaire with three subscales on smbg information, motivation, and behavioral skills based on imb model was developed. validity and reliability of the measures were examined and guaranteed. adult patients with type 1 diabetes visiting our diabetes clinic from january to march 2012 (n = 55) were consecutively interviewed. the self - completion questionnaires were administered and finished at face - to - face interviews among these patients. both descriptive and correlational analyses were made.results:fifty-five patients finished the questionnaires, with the median duration of diabetes 4.5 years and the median of smbg frequency 2.00. specific smbg information deficits, motivation obstacles, and behavioral skill limitations were identified in a substantial proportion of participants. scores of smbg motivation (r = 0.299, p = 0.026) and behavioral skills (r = 0.425, p = 0.001) were significantly correlated with smbg frequency. the multiple correlation of smbg information, smbg motivation, and smbg behavioral skills with smbg frequency was r = 0.411 (r2 = 0.169, p = 0.023).conclusions : adult patients with type 1 diabetes in our clinic had substantial smbg information deficits, motivation obstacles, and skill limitations. this information provided potential - focused education targets for diabetes health - care providers. |
dogs are the source of several zoonotic diseases and their close contact with humans may increase the risk of zoonotic disease transmission particularly when dogs are free to roam. dogs in urban and rural areas can be used as sentinels in humans and wildlife to evaluate the diversity and environmental contamination with parasites. backyard pigs in tropical mexico are important for domestic consumption [2, 3 ]. however, their sanitary management is very poor or nonexisting [3, 4 ] representing a risk for the transmission of zoonotic parasitic agents. are capable of producing systemic and ocular disease in dogs and humans and share soil ingestion as common mode of exposure. however, the protozoa t. gondii are more commonly transmitted by the ingestion of contaminated food or water. domestic dogs are the principal animal reservoir hosts for toxocara canis, a common nematode throughout the world producing severe anemia and systemic disease in puppies which in humans elicits various syndromes currently characterized as generalized (visceral larva migrans and covert toxocariasis) and compartmentalized as well as ocular and neurologic, with more clinical manifestations on kids. diverse epidemiological studies have demonstrated that exposition to t. canis varies from countries and regions reaching up to 83% of seropositivity in people from the caribbean region. another important foodborne nematode trichinella spiralis is transmitted to humans generally by the consumption of undercook contaminated pork meat. information related to both animal and human zoonotic parasite infections in rural communities of mexico is scarce. to establish an effective parasite prevention and control strategies, it is necessary to understand the biology of parasites circulating in endemic regions, monitoring their presence and the risk factors associated in humans, reservoir animals, and vectors for a better planning of strategies for prevention and control of zoonotic parasitic diseases. the objective of this study was to determine the seroprevalence of humans to t. canis, t. gondii, and t. spiralis in a rural community of yucatan, mexico, together with the seroprevalence of t. gondii and detection of t. canis in their dogs. the study was conducted in the rural community of molas yucatan, mexico (2049 and 2048n and 8937 and 8938w). a sectional, descriptive, and prospective study was conducted considering for convenience 90 dwellings reporting tenure of at least one dog. the community was divided into four sectors (north - south and east - west) ; 20 houses in each sector were selected. samples of blood and feces of at least one dog per household were taken. in dogs, blood samples were obtained from the cephalic vein and were processed to obtain serum which was frozen at 20c until tested. feces were taken directly from the rectum ; then, rabies vaccine was applied and dogs were dewormed. additionally, blood samples from an adult person per household were taken. in humans, blood samples were collected by venipuncture of the brachial vein prior to written consent of the participants and according to the rules of the bioethics committee of the campus of biological and agricultural sciences at the autonomous university of yucatan (cb - ccba - i-20014 - 001). a questionnaire was applied to all sampled persons including questions about their age, feeding habits, water sources, presence of eye lesions, and previous deworming procedures. fecal samples were processed by centrifugation - flotation technique ; positive samples were examined quantitatively by mcmaster technique to estimate the number of eggs per gram of feces (epg) with particular attention to t. canis. human sera were evaluated for the measurement of igg t. canis and t. spiralis antigens using elisa kit (dgr laboratories international inc. the presence of specific igg antibodies was determined by indirect elisa (international gmbh re58241, hamburg, germany) containing tachyzoites of t. gondii. serum samples were diluted to a ratio of 1 : 100 in pbs and secondary antibody anti - human igg hrp - labeled anti - human igm hrp were also marked. the intensity of infection (elimination of epg) was classified as low (50100 epg), median (150500 epg), or high (> 550 epg). the overall prevalence of igg - positive people to t. canis, t. canis, and t. spiralis was also estimated. a primary screening of humans seropositive to t. canis was assessed using 2 2 contingency tables of exposure variables ; odds ratio and confidence interval were determined. since very few negative cases of t. gondii in human and dogs and few positive cases of t. spiralis were found, no statistical analysis was performed. odds ratios (or) and 95% confidence intervals were calculated using epi - info software (version 6.0 ; cdc atlanta, ga, usa). a total of 52 viable fecal samples were collected from adult dogs of which 24 (46.2%) had at least some order / species kind of intestinal nematode. the nematode genera found in dog feces were ancylostoma caninum (70.8%), toxocara canis (12.5%), trichuris vulpis (12.5%), and the order eucoccidiorida (4.2%). the intensity of infection of nematode order / species is presented in table 1. in most cases, intensity of the infection was low followed by median intensity ; no cases of high infection load were seen ; from the infected dogs, all of them had only a single order / species of nematode present. regarding the serology from human samples, 26 (29.2%) from the 89 persons were positive to igg anti - t. none of the risk factors studied were significantly associated with seropositivity in humans (table 3). all participants owned at least one dog and none of them collected the feces from their animals. from the questionnaire, 36 persons claim vision disorders such as cloudy vision and increased tearing. gondii serological results, from 89 human blood samples collected, 90.6% were positive to igg t. gondii (table 2). for dogs, since very few negative cases were found, no statistical inferences were feasible. from the interviews, 93% drink water from commercial carafes, 5% drink tap water, and 2% drink from both sources. participants reported that the ingestion of meat is usually well cooked in 82% of cases and 7% claim to eat semicooked meat. 88.8% of the interviewed persons eat sausages and from the 10 persons (11.2%) who do not eat sausages 2 were seronegative. only 6 (6.9%) persons were found seropositive to t. spiralis (table 2). the prevalence of dogs infected with gastrointestinal parasites in this study is considered high and similar to the reported one in other regions of the world, that is, 52.4% in argentina, 53% in hungary, and 68% in nigeria. the high variation observed has been associated with various weather conditions, idiosyncrasies, and sanitary control of dogs. a previous study in the same community was performed four years ago and prevalence of gastrointestinal parasites in dogs was 80% with a high egg output of a. caninum, t. canis, and t. vulpis found ; in this study, deworming of dogs was performed and talks to children, adolescents, and adults about health management of dogs were given. these actions could explain the lower prevalence and intensity of infection reported at this time categorized between low and medium intensity of infection. in the specific case of t. canis, a higher prevalence (12.5%) the annual variation in the number of infected puppies in the region may influence the disposal and spreading of t. canis eggs. the presence of infected puppies can increase the number of eggs and larvae deposited in the environment in which they can remain infective for several months infecting other dogs and with great potential to infect humans, especially children. human toxocariasis is worldwide distributed and occurs when embryonated eggs from the environment are ingested. this is more critical when dog overpopulation is present without the practice of preventive medicine (deworming). children with pica are found at increased risk particularly in regions of low socioeconomic status and with proliferation of dogs and/or those who have puppies at home. adults are also at risk of infection as shown by several studies where prevalence of 26.8% in brazil was reported and 19% in lebanon was reported. in argentina, seroprevalence of 2038% in children and 1039% in general population [18, 19 ] is reported. in mexico, 38% of people suffering from acute nongranulomatous uveitis were seropositive for t. canis, with a prevalence of 4.6% to 17.59% reported in children less than 16 years of age. results of this study indicate a high spread of infective eggs in the rural community probably because of the high density of dogs having free access to the street and lack of health programs including a deworming program. although deworming has been introduced in health management of dogs in the studied community, still spreading of infected eggs and contamination of the area are present ; thus, efforts to intensify the deworming of dog and cat are necessary for better control results. the ocular larva migrans syndrome is one of the major health problems associated with t. canis. however, eye lesions were not statistically associated with seropositive persons ; this syndrome is more critical in children where severe problems have been reported, so studies to evaluate the impact of this parasite on children should be designed and performed. ocular larva migrans in the case of toxocariasis may cause visual impairment as a response of granulomatous inflammatory reaction with consequences such as blindness and secondary glaucoma [7, 24 ]. in the present study, however, other symptoms not evaluated in the present study may involve headache, muscle pain, influenza - like syndrome, and diarrhea in seropositive patients. in a previous study conducted in mexico, young children, < 5 years, are most affected with toxocariasis with clinical signs of fever, abdominal pain (probably due to hepatomegaly), and lower respiratory symptoms. further studies should be focused on children in the studied region examining the different body systems to evaluate the impact of this zoonotic agent. the high prevalence of t. gondii found in dogs and their owners demonstrates a high spread of oocysts in the studied area with a probably common source of infection. the prevalence found in dogs from brazil was 26.9% with a higher risk for dogs over five years of age. however, in the present study, due to the very high prevalence found, all animals are expected to seroconvert since young ages with frequent episodes of reinfection. in mexico, free roaming dogs are indicators of the high contamination of the environment with t. gondii oocysts as they may be sentinels of exposition. in humans, toxoplasmosis has been reported previously in the yucatn peninsula with seroprevalence of 25% in non - cat owners and also associated in patients with recent cases of spontaneous abortion [30, 31 ]. in humans, the main route of infection of t. gondii is by the ingestion of tissue cysts. in the present study, the oral route by consumption of undercooked meat (tissue cysts) is more likely but also the ingestion of infective oocysts from contaminated water as previously demonstrated could occur. considering the 100% t. gondii seroprevalence found in 50 studied cats from the same region, the density of cats and constant new born kittens in the village with excretion of high numbers of oocysts probably occurred, contaminating the environment and probable water sources. even when 93% of the interviewed persons drank commercial bottled water, tap water and water from artisanal wells are also used for several other activities and may be considered as a potential source of infection with t. gondii oocysts. however, since dogs in the studied area are fed with human leftovers, contaminated meat and sausages may also be involved in the transmission cycle. t. spiralis is a nematode capable of infecting any mammal ingesting infected raw or undercooked meat. infection in humans with this nematode is described worldwide and outbreaks have been reported in developed countries infecting over 30% of people ingesting wild boar meat. exposition of consumers to infected meat can reach up to 65.1% of incidence, especially when meat is not tested prior to consumption when slaughter is made in households. in mexico, consumption of pork meat coming from backyard is very common in rural villages without any kind of quality test. in mexico, the ministry of health (nom-194-ssa1 - 2014) makes the inspection of pork meat in search of t. spiralis compulsory. t. spiralis in mexico has been reported since 1975 in zacatecas state where several outbreaks have been reported in cases with mortality rates of 33% in some cases ; in those outbreaks, transmission was most commonly caused by the ingestion of raw pork products or undercooked meat. more recently, a prevalence of 1% using elisa was reported in semirural county areas of mexico. in yucatan, however, the toxocara larval excretory antigen is normally used in serological tests that may exhibit low specificity due to cross - reaction between related helminthes, including t. canis. further research is needed to investigate the epidemiology of these zoonotic helminthes using more accurate techniques for diagnosis including the search of the agent in farm and backyard pigs from the region. the main limitation of the present study was the small sample size used and consequently results may be biased or may not be representative of the rural communities from the studied area. however, this study may be a good indicator of the zoonotic situation and establish the bases for further studies including a bigger and targeted sample size from both animals and humans. it is concluded that the dogs in a rural community of yucatan are reservoirs of some helminthes parasites specially t. canis. this implies a high risk of the presence of parasites in the environment of the village and a potential risk of infection for the human community. it is important to improve the dissemination and promotion of educational programs on this important zoonosis and establish effective control measures to reduce the emission of eggs and avoid contact of human with areas contaminated with dog feces. | a cross - sectional study was made on 89 inhabitants and their dogs from a rural community of yucatan, mexico, to determine the serological prevalence of some zoonotic parasitic agents. samples were taken to monitor the presence and intensity of infection with gastrointestinal parasites in dogs. in humans, the serological prevalence of t. canis, t. gondii, and t. spiralis was 29.2%, 91.0%, and 6.7%, respectively. no associations were found between positive cases and studied variables. from the total of blood samples taken from dogs, 87 (97.6%) were seropositive to t. gondii ; only 52 viable fecal samples were collected from dogs of which 46.2% had the presence of gastrointestinal parasites with low to moderate intensity ; from those, 12% had the presence of t. canis. this study demonstrates the presence of the studied zoonotic agents in the area particularly t. gondii which suggest a common source of infection in dogs and humans and a high number of oocyts present in the environment. preventive measures must be designed towards good prophylactic practices in domestic and backyard animals (t. canis and t. spiralis). contaminated sources with t. gondii (food and water) should be further investigated in order to design effective control measures. |
renal arteriovenous malformations (avms) are rare anomalous arterial and venous connections commonly presenting with spontaneous hematuria and recurrent flank pain. a liquid embolizer composed of ethylene - vinyl alcohol (evoh) dissolved in dimethyl sulfoxide (dmso), mixing with tantalum powder for radio - opacity, is emerging as a new treatment modality for visceral avms due to its inherent benefit of obtaining good nidal penetration and ease of embolizer control. we present a case of renal avm after onyx embolization, which is the first reported case of hyperdense striations along the renal medulla, secondary to tantalum powder on computed tomography (ct). (chang gung memorial hospital) and waived the need for the patient 's inform consent. a 65-year - old woman, without a significant history, presented to our institution with intermittent gross hematuria and left flank pain for 10 days. physical examination, noncontrast ct, cystourethroscopy, and laboratory examinations were all unremarkable, except for hematuria (color of urine : red and red blood cell count > 500 /l) and blood clot in the left renal pelvis. blood test showed no obvious anemia (red blood cell count 4.43 million/l and hemoglobin level 13.6 g / dl). subsequent renal artery ct angiography demonstrated a tangled cluster of vessels in the left peripelvic region with arterial feeders and early draining vein, consistent with a cirsoid - type renal avm (figure 1a, ct image) and confirmed by conventional transcatheter angiography (figure 1a, angiography image). two arterial feeders arising from posterior segmental artery of the left kidney supply the renal avm. one is originated from the proximal portion of the posterior segmental artery, and the other one is from the distal portion (figure 1a, ct image). (a) pretreatment image (ct image) ct angiography (cta) with volume - rendering reformatted image of the left kidney (a) demonstrated a cirsoid - type renal arteriovenous malformation (avm) at the upper pole of the left kidney with arterial feeder (bold arrow) and draining vein (triangular arrowhead). oblique coronal image with maximal - intensity projection showed two arterial feeders (thin arrows, proximal and distal) arising from posterior segmental artery of the left kidney supply the renal avm (angiography image). transcatheter angiography of the left kidney confirms the cta findings of renal avm (bold arrow). (b) post - treatment image (left) noncontrast - enhanced ct performed on the 5th postembolization day shows fine linear hyperdense striations in the left renal parenchyma (bold arrow). note the confinement of the hyperdense striations within the renal contour (red triangular arrowhead) and also the height difference between the peripelvic embolizer (small arrowhead) and the striations (bold arrow) exclude beam - hardening artifact. (right) control angiography after the embolization showed complete obliteration of the renal avm with resultant infarction at the lateral aspect of the mid portion of the kidney (bold arrows). ct, computed tomography. as an effort to achieve a satisfactory distal penetration of the nidus, we used onyx instead of coil and n - butyl cyanoacrylate (nbca). to ensure adequate mixing and homogenous radiopacity of the onyx mixture, the vials were kept on a shaker (vortex - genie ; scientific industries, inc. onyx 34 (8% evoh and 92% dmso, onyx 34 ; micro therapeutics, inc., irvine, ca, usa) is chosen for the embolization of distal feeder to achieve higher occlusion speed. the microcatheter was preflushed with dmso, and onyx 34 was injected slowly under fluoroscopic control until occlusion of the distal feeder. the total amount of injected onyx 34 was 0.8 ml. angiography after embolization of the distal feeder showed occlusion of distal feeder and lateral portion of the nidus, but the avm was still supplied by the proximal feeder. therefore, we cannulated the proximal feeder using a marathon flow - directed microcatheter coaxially through a 6 fr. since the angiography of the proximal feeder showed a relative slow - flow vascular malformation, embolization was performed with onyx 18 (6% evoh and 94% dmso, onyx 18 ; micro therapeutics, inc., irvine, ca, usa) to obtain a better penetration into the nidus. the total amount of injected onyx 18 was 0.6 ml. no obvious visible overflow of onyx was seen during the embolization procedure, but subtle overflow of onyx can not be completely excluded under angiography. control angiography after embolization of both feeders demonstrated complete obliteration of the renal avm, with a resultant infarction at the lateral aspect of the middle left kidney (figure 1b, right image). nonenhanced abdominal ct was performed on the 5th postembolization day to follow up the extent of injected onyx. onyx was localized within the expected region of the avm. however, diffuse fine hyperdense linear striations were incidentally found along the course of the left upper renal medulla, which favored the presence of tantalum powder rather than intravascular migration of embolizer (figures 1b, left image). the patient was discharged uneventfully, and follow - up ct 3 months later confirmed the disappearance of the tantalum powder from the renal medulla and the resolution of the renal striation sign (figure 2). both blood test (red blood cell count 4.50 million/l and hemoglobin level 13.4 g / dl) and urinary analysis (color of urine : yellow and red blood cell count 1 /l) were normal during postembolization follow - up. multipanel imaging with comparison of the initial postembolization ct at 5 days after embolization and the subsequent ct at 3 months follow - up revealed the resolution of the striated tantalum powder. (a) post - treatment follow - up (5 days) : axial ct scan at the level of onyx embolizer (left) and at a level below the onyx embolization (right) revealed presence of hyperdense renal striation sign(arrows). (b) post - treatment follow - up (3 months) : axial ct scans at the same levels confirmed disappearance of the tantalum powder. treatment of renal avm is often necessary when complications such as heart failure, uncontrolled hypertension, hemorrhage, or recurrent flank pain occur, as the case of our patient. the optimal treatment of renal avm should include complete eradication for the vascular lesion while preserving the maximal - functioning renal parenchyma. in this study, transarterial embolization became the treatment of choice because the peripelvic location of the nidus posed a high risk of renal parenchymal injury, if surgery was performed. even though several embolic materials are available, the lava - like flow pattern and long solidification time of onyx allow better nidal penetration and more controllable embolization procedure, which is particularly beneficial in treating visceral avm. in addition, onyx injection can be interrupted and continued again until the desired result is achieved. even if the catheter was entrapped by onyx, catheter removal onyx 18 has a lower evoh concentration, resulting in slower occlusion speed and allows better nidal penetration distally, whereas onyx 34 has a higher concentration of evoh and allows a higher occlusion speed, but less nidal penetration ability. the two mixtures can be selected according to the need of embolization speed and distal nidal penetration. as previously mentioned, we employed onyx 34 for the distal feeder to achieve higher occlusion speed and onyx 18 for the proximal one for better nidal penetration. as for the other embolizers, metallic coils are not suitable embolic agents because the delivery catheters can not superselectively enter the nidus for the placement of the coils. the proximal coil embolization may result in development of collateral branches that are difficult to treat. nbca, which is a tissue - adhesive liquid embolic material, has the ability to penetrate and occlude at the level of the nidus of the avm. however, there is a risk that the catheter tip may adhere to the vessel wall. the catheter should be retrieved before nbca polymerizes, which requires great experience and skill. in addition, due to the low viscosity, the embolic material may be washed away before it polymerizes. although beam - hardening artifact can be an image mimicker of the renal hyperdense striation sign, the confinement of the hyperdense striations within the kidney contour, along with the height difference between the peripelvic embolizer and the striations, can allow differentiation of these two entities. this finding is further confirmed by follow - up ct with disappearance of the hyperdense striation sign. we believe the renal hyperdense striation sign is secondary to the presence of tantalum powder in the renal medulla. in one animal experimental study of transarterial embolization of the kidney, with nbca mixed with tantalum powder, and reported by gnther, particles of tantalum powder (average particle size of 75 m) were found to deposit in the arteries and in the glomeruli of kidneys on histologic specimens after the embolization, and tantalum particles smaller than 7 m were found in the pulmonary vascular system. the particle size of onyx tantalum powder ranged from 0.7 m to 22 m, and it is reasonable to suspect that the diffuse linear hyperdensities in the renal parenchyma might be related to the deposition of the tantalum powder. congenital renal avms had tortuous varicous vessels, which are found immediately beneath the urothelium, which is the cause of the hematuria. bypassing of the filtration barrier with subtle overflow may explain for the presence of tantalum powder in the renal medulla. follow - up ct image reveals resolution of the striated tantalum powder, and the renal function remains normal, which probably implied that the phenomenon would not be problematic in patients with normal renal function. however, we do not know if the phenomenon affects the renal function in patients with renal insufficiency. further studies in patients with renal function impairment may be helpful, and the recognition of the renal hyperdense striation sign is important to avoid confusion with embolizer migration and understand its influence on renal function. despite its frequent usage in neural intervention, the application of onyx in avm of visceral organs is gradually graining interest, especially in cases in which delicate nidal penetration of the avm is desired. renal hyperdense striation sign should be recognized to avoid confusion with embolizer migration, and further studies in the effect of tantalum powder in patients with renal function impairment may be helpful to understand its influence of renal function. | abstractonyx is an emerging treatment modality for visceral vascular malformations, especially in cases in which delicate nidal penetration of the arteriovenous malformation (avm) is desired. a computed tomography (ct) image presentation of hyperdense striations along the renal medulla secondary to the tantalum powder has not been previously reported.a 65-year - old woman presented to our institution with intermittent gross hematuria and left flank pain for 10 days. both ct and conventional angiographies confirmed cirsoid - type renal avm, which was successfully treated with onyx. follow - up ct after treatment revealed presence of hyperdense striations along the renal medulla, which resolved during later image follow-up.despite its frequent usage in neural intervention, the application of onyx in visceral avm is gradually gaining interest, especially in cases in which delicate nidal penetration of the avm is desired. renal hyperdense striation sign should be recognized to avoid confusion with embolizer migration, and further studies in patients with renal function impairment may be helpful in understanding its influence of renal function. |
cobalamin, c63h88o14n14pco (figure 1), participates in only two known mammalian enzymatic reactions. yet, these two cbl - dependent enzymes, cytosolic methionine synthase (ms) [ec 2.1.1.13 ], requiring methylcobalamin (mecbl), and mitochondrial methylmalonyl - coa mutase (mu) [ec 5.4.99.2 ], requiring adenosylcobalamin (adocbl) [1, 2 ], are critically involved in key metabolic pathways essential for gene expression and regulation, via formation of s - adenosylmethionine (sam) and methylation, and in protein synthesis and catabolism, cellular respiration, and energy. activation of methionine synthase also ensures key antioxidant defense status, as it triggers concurrent activation of cystathionine -synthase (cs), the pivotal enzyme at the homocysteine junction in the trans - sulfuration pathway to glutathione (gsh). anaemia, and macrocytic or megaloblastic anaemia, as well as for subacute combined degeneration of the spinal cord. however, an increasing body of work suggests that cbl may also play a central role in the regulation of immunity and inflammation (reviewed in). cbl confers significant protection in various animal models of shock, from anaphylaxis to trauma and sepsis [57 ], and has remarkable organ / tissue protective effects when used clinically for the treatment of analogous inflammation in cn poisoning (reviewed in). amongst cbl 's known immunological effects are an augmentation of the cd8+/cd4 + t - lymphocyte ratio and natural killer cell activity [9, 10 ], both significantly reduced in inflammatory pathology, with negative consequences in septic patients. interesting homeostatic links between cbl and pivotal cytokines are also emerging, indicative of complex but still incompletely defined regulatory circuits : mecbl lowers interleukin-6 (il-6) expression in peripheral blood monocytes, whilst cbl deficiency raises circulating il-6 in humans and cbl physiological status regulates il-6 levels in rat cerebrospinal fluid. moreover, in both rodents and humans there appears to be an inverse relation between cbl physiological levels and tumour necrosis factor alpha (tnf-) serum levels. in vitro, a reasonable hypothesis is that such cbl / tnf-/il-6 regulation may be partly effected via cbl indirect regulation of the central immune regulatory transcription factor, nuclear factor kappa b (nf-b). normal physiological levels of cbl in spinal fluid appear to correlate with nf-b quiescence, at least, in a non - inflammatory / non - immune challenge model. recently, a kinetic study reported that cob(ii)alamin reacts with superoxide at rates approaching superoxide dismutase. cncbl protects human aortic endothelial cells, and neuronal cells, in vitro, against superoxide induced injury [19, 20 ]. given that oxidative stress is a major trigger of nf-b activation, this potential antioxidant effect of cbl could theoretically lead to nf-b inhibition. it may also be of critical local importance in vivo, as the phagocytic burst includes release of the cbl carrier, haptocorrin (hc / tc 3), in the immediate vicinity of nadph oxidase [8, 21 ] one of the major biochemical sources of superoxide in immune challenge and inflammation. hc / tc3, moreover, is upregulated by il-1, itself expressed within fifteen minutes of inflammatory challenge. nevertheless, though antioxidant effects of cbls have been observed in vitro and may, indeed, be important in vivo, no systematic analysis of the in vivo mechanisms of cbl conferred protection against inflammation during acute immune challenge has hitherto been done. we wondered if a more comprehensive explanation for cbl effects on inflammation and immunity, and thence beneficial outcomes in sepsis and other forms of shock, may lie in a potential direct / indirect regulation by cbl of one or more of the several actions of nitric oxide (no) as a ubiquitous, cell - signal transduction molecule and second messenger for post - translational modification, whose targets include soluble guanylate cyclase.no is the product of three nitric oxide synthases (nos) : two constitutive, nnos (neuronal nos ; nos i) and enos (endothelial nos ; nos iii), and one inducible, inos (nos ii), at much higher levels of expression, with the potential to produce 1000-fold higher than normal amounts of no, during gestation, growth, and the immune response. cobalamins are known to have effects on no [2729 ], but these have hitherto been thought to be a consequence of cbl / no scavenging effects [7, 3035 ] demonstrable chemically and in vitro [36, 37 ], but biologically unproven in vivo and still controversial [3840 ]. nitrosylcobalamin has not been detected, to date, in vivo or in vitro, amongst naturally occurring intracellular cbls. nevertheless, if the hypothesis that cbl is involved in nos catalysis has any substance [42, 43 ], then it is conceivable that, in analogy to previously observed ferric - heme - no complex formation at the conclusion of nos catalysis, nocbl might be transiently formed, just prior to release of free no by the nos. such a theoretical transience and discrete localisation might account for the failure to detect nocbl in vivo to date. ubiquitous and continuous cbl scavenging of no, on the other hand, may pose biochemical hazards. for no has important antioxidant and cell - signalling actions which might be obstructed by hocbl 's previously proposed, indiscriminate no scavenging, or even just by a recently proposed, cbl structural - based, direct inhibition of the nos tout court and nothing else. there is some evidence that hocbl can discriminate between exogenous no donors and the natural endogenous donor, s - nitrosoglutathione, gsno, actually prolonging only gsno - induced, gastric fundus relaxations. moreover, there are also diverse indications that positive cbl status is allied to beneficial no activity : in diabetic rats, high cobalamin levels correlate with high nos protein levels, no activity, and increased erectile function ; cbl supplementation of vegetarians with low cbl status significantly increases enos no release in the brachial artery ; in the digestive tract of endotoxemic rats, the highest expression of inos is in the ileum, precisely where cbl is internalized, and both cbl and no are known to mediate cell protective effects via erk1/2 and akt [5054 ]. these protective effects of no and cbl include induction and regulation of heme oxygenase-1 (ho-1) [52, 5558 ], which converts biliverdin to the powerful antioxidant bilirubin, and carbon monoxide. (for a more comprehensive list of coincidences of cbl's / no 's positive actions, see table 1 and its related discussion in). thus, in these studies we explored an alternative hypothesis to that of cbl as just an no, or, indeed, superoxide, mop. we posited that the principal mechanism behind cbl 's beneficial, pleiotropic effects in inflammation may involve a biphasic regulation of nos expression and protein translation and the ensuing no synthesis, during the two distinct pro- and anti - inflammatory phases of the immune response. male c57bl/6 mice, weighing 20 to 25 g, were purchased from harlan, uk, and maintained on a standard chow pellet diet, containing standard amounts of cbl (50 g / kg vitamin b12/cncbl), with tap water supplied ad libitum. animals were kept in a 12:00 h light / dark cycle, and all were housed for 7 days prior to experimentation. all experiments were performed in accordance with uk home office regulations (guidance on the operation of animals : scientific procedures act, 1986). the coenzymes, 5-deoxyadenosylcobalamin, and methylcobalamin ; vitamin b12a, cyanocobalamin, and hydroxocobalamin (cas 78091 - 12 - 0) were purchased from sigma - aldrich (uk). glutathionylcobalamin and n - acetyl - cysteinyl - cobalamin were synthesized and supplied by professor nicola brasch (kent state university, ohio, usa). all cbls (and cbl - treated animal samples) were protected from light during storage and handling, and were 98% to 99.5% pure. 5-deoxyadenosylcobalamin (adocbl), methylcobalamin (mecbl), hydroxocobalamin (hocbl), glutathionylcobalamin (gscbl), and n - acetyl - cysteinyl - cobalamin (nac - cbl) were all stored at 20c, and fresh solutions of them were made using sterile, pyrogen - free, phosphate - buffered saline (pbs ; gibco), prior to the experiments. for the in vivo experiments, cobalamins were diluted at 10 ml / kg prior to treatments (with pbs used as vehicle). raw 246.7 macrophage cells, stably transfected with nf-b luciferase reporter construct (stratagene), were maintained in dulbecco 's modified eagle 's medium, supplemented with 10% (v / v) fetal bovine serum, 2 mm l - glutamine, 1 g / ml geneticin, and 50 g / ml g418. cells (2 10 cells) were seeded in 96-well plates and then preincubated for 1 h with increasing concentrations (110100 m) of the five principally occurring, intracellular cbls. thereafter, at time 0 h, cells were stimulated with e. coli lps (0111 : b4 ; 1 g) for 4 h and then processed for measurement of luciferase activity in a luminometer (luminometer td-20/20 ; turner designs instruments). endotoxaemia was induced by the intraperitoneal injection of lps (0.1 mg / kg), alone (non - lethal) or, in the lethal endotoxaemia protocol, in combination with 1 g / kg d - galactosamine (table 1). sample collection in non - lethal endotoxaemia was carried out at both 4 and 24 h after lps challenge. animal survival, in all lethal endotoxaemia experiments, was monitored for a total of 5 days, and all data were analysed using chi - squared or kaplan - meier tests. times shown are in relation to time 0 h, when either lps alone or lps+d - gal was administered by intraperitoneal injection. individual cobalamins were injected into the peritoneum at the doses and times reported in table 1. blood (500 l) was centrifuged (for 5 min, at 2500 rpm), and the plasma then collected for elisa analysis. 500 l of trizol reagent (invitrogen) was added to the remaining fraction. 20 l) was treated with 2 u (1 l) of turbo dnase 1 (ambion, austin, tx), as described by the manufacturer, to remove any contaminating genomic dna. an aliquot of the dna - free rna (7.6 l) was then transferred to a new rnase - free tube and reverse - transcribed into complementary dna (cdna), using superscript iii reverse transcriptase (invitrogen), as described by manufacturer. the following reagents were used : oligo dt primers (invitrogen) ; 1 l, 10 mm dntp (bioline) ; 4 l of 5x first - strand buffer ; 1 l, 0.1 m dtt ; 1 l (40 u) rnaseout ; and 1 l (200 u) of superscript iii reverse transcriptase (invitrogen). after synthesis, cdna was quantified using a nanodrop nd-1000 and diluted (80 ng/l) in molecular biology grade water and then loaded into 384-well plates for real - time pcr. real - time pcr assays were performed on the various samples in order to evaluate the expression of the following genes : gapdh, rpl32, il-1, cox-2, inos, enos, tnf-, and hmgb1 (table 2). for each gene analyzed, reactions were performed using 1 l of the qiagen quantitect primer assay, added to 5 l power sybr green pcr master mix (applied biosystems, warrington, uk) and then diluted with 2 l molecular grade water. a final volume of 8 l was dispensed into each well and 2 l of diluted cdna (160 ng / reaction) was added. each sample was tested in triplicate for each gene, and pcr reactions were performed using abi prism 7900 real - time pcr equipment. the thermal profile consisted of 95c for 15 min, then 40 cycles of 94c for 15 s, 55c for 30 s, and 72c for 30 s. this rest mcs software was utilized for the calculation of the relative difference between the test groups. liver tissues were harvested from (n = 5) animals, after lps endotoxaemia, with or without hocbl treatment and then homogenized in lysis buffer, which contained a cocktail of protease inhibitors. protein concentrations prior to loading were determined using the bradford assay (sigma) : samples were mixed with 6x laemmli sample buffer, and equal protein amounts (100 g) then underwent electrophoresis on a 10% polyacrylamide gel in running buffer (0.3% tris base, 1.44% glycine, and 0.1% sds in distilled water). this was followed by transfer of the proteins onto pvdf membranes in transfer buffer (using 0.3% tris base, 1.44% glycine, and 20% methanol, in distilled water). membranes were blocked for 1 h with 5% nonfat milk solution in tbs containing 0.1% tween 20. inos expression was assessed using a specific monoclonal antibody (1 : 1000 ; santa cruz, usa). the signal was amplified with hrp - linked anti - mouse secondary antibody (1 : 2000) and visualized by ecl (western blotting detection reagent ; amersham biosciences, usa). densitometric analysis was performed using nih imagej software and normalised to tubulin loading controls in the same sample. animals (n = 5) were challenged with lps and treated with cbls as described above. at 4 h and 24 h after lps challenge, lung and liver tissue samples were harvested, homogenized, and processed for determination of nos activity, as measured by nitrate / nitrite end - products of no. the ultrasensitive, nos assay used (oxford biomedical research, oxford, mi, usa : ultrasensitive colorimetric nos assay : cat no. nb78) employs an nadph recycling system nadp, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and the substrate, l - arginine, but not the cofactor, bh4,to ensure that nos operate linearly for up to 6 hours, as no - derived nitrate and nitrite accumulate. the assay kit can accurately measure as little as 1 pmol / millil (~1 millim) no produced in aqueous solution. in these studies, the enzyme nitrate reductase was used to convert all nitrate to nitrite, then griess reagent employed to quantify nitrite levels, with the generation of a nitrite standard, as recommended by the supplier. after collection, blood was centrifuged and the plasma separated, under low lighting conditions, then stored at 80c until performance of the analyses. for determination of circulating tnf- and il-6 levels, using elisa assays, samples were diluted 1 : 10 in the assay diluent, as specified by the manufacturer (r&d, uk). absorbance was plotted in a standard curve, and data expressed as the content of tnf- (ng) or il-6 (pg) per ml of plasma. unless otherwise stated, all reagents were purchased from sigma - aldrich, poole, uk. of 5 animals per group for the analyses in non - lethal endotoxaemia and, initially, 79 per group, then 12 per group, for lethal endotoxaemia survival, series i and ii, respectively. chi - square and kaplan - meier tests were used for the lethality studies. in all cases, a p 18 h and then released extracellularly, hmgb1 can trigger further late release of tnf-, il-1, and inflammatory products from cox-2, inos, and excessive ros and rni species, leading to pathology. it is known that the nervous system can modulate circulating tnf- levels via release of acetylcholine by the vagus nerve. but our studies show that cbl essential for acetylcholine synthesis is the first known endogenous inhibitor of late hmgb1 mrna expression. both nicotine and ethyl - pyruvate have been used to block extracellular release of hmgb1 but, in addition to the fact that hocbl appears to impact on hmgb1 much further upstream, at least in tissues, neither drug is endowed with the safety profile of cbl and, more pertinently, neither is known to exert such a central, endogenous regulation of the immune response. but, on the evidence of the general anti - inflammatory regulation observed in our studies, we predict that extracellular release of hmgb1, from macrophages and pmn, should be negligible with hocbl treatment. the theory that cbl may impact on the nos indirectly, through the contribution of its two known mammalian coenzymatic functions to nos substrate and cofactor assembly and, indeed, to assembly of the nos protein itself, may further explain our findings, including the cbl - promoted high nos protein (figure 10 scheme). furthermore, a deficiency of any of the nos substrates and cofactors (the likely result of cbl functional deficiency in endotoxaemia) is known to result in less tightly coupled nos activity and increased free radical generation [112, 113 ], with a corollary increase in inflammatory mediators and prolonged period of nos activity, indexed by our observed higher lps - only nos nitrite levels. (in a forthcoming study we will also analyse more exactly how cbl may shift the ratio of no / gsno / onoo and related species). it may also be that cbl, as adocbl and its radical, takes a direct, active part in nos catalysis, as a third mammalian cbl cofactor. from this perspective, the high nos protein seen with high cbl administration may be a classical instance of the cofactor promoting coenzyme assembly. such a central, direct cbl / nos, catalytic interaction would further reduce excess production of toxic forms of no, as well as superoxide and other related ros and rni species. the consequent, more precise, pro- and anti - inflammatory signalling should again result in a shorter, more effective period of nos activity, thus lower detectable nitrite levels (as seen) with the beneficial signalling and antioxidant effects of no predominant. however, direct cbl scavenger interactions, in discrete intracellular compartments, with primarily toxic rnis, such as onoo-/onooh / no2, with which cbl interacts ex vivo,, may also play a part and can not be ruled out as contributing to a more complex picture behind our observed results (figure 10). these novel observations on the mechanism behind cobalamin protection in endotoxaemia suggest that we may be looking at the ideal natural, selective and collective regulator of the nos, and thence of cytokines and other pivotal factors, in immune challenge and sepsis. in fact, it is now accepted that anti - inflammatory therapies (based on blocking a specific mediator) fail toutcourt in sepsis and that a more modulatory approach, which regulates the homeostatic inflammatory response, (in itself beneficial), could be successful. thus, our findings may have significant clinical implications, not only for the treatment of sepsis, but also for other analogous inflammation - driven conditions, such as cancer and malaria, where nos / no deregulation, and consequent loss of control over key inflammatory mediators, are equally pathogenic [115, 116 ]. | background. nos/no inhibitors are potential therapeutics for sepsis, yet they increase clinical mortality. however, there has been no in vivo investigation of the (in vitro) no scavenger, cobalamin 's (cbl) endogenous effects on nos/no / inflammatory mediators during the immune response to sepsis. methods. we used quantitative polymerase chain reaction (qpcr), elisa, western blot, and nos griess assays, in a c57bl/6 mouse, acute endotoxaemia model. results. during the immune response, pro - inflammatory phase, parenteral hydroxocobalamin (hocbl) treatment partially inhibits hepatic, but not lung, inos mrna and promotes lung enos mrna, but attenuates the lps hepatic rise in enos mrna, whilst paradoxically promoting high inos / enos protein translation, but relatively moderate no production. hocbl / nos/no regulation is reciprocally associated with lower 4 h expression of tnf-, il-1, cox-2, and lower circulating tnf-, but not il-6. in resolution, 24 h after lps, hocbl completely abrogates a major late mediator of sepsis mortality, high mobility group box 1 (hmgb1) mrna, inhibits inos mrna, and attenuates lps - induced hepatic inhibition of enos mrna, whilst showing increased, but still moderate, nos activity, relative to lps only. experiments (lps+d - galactosamine) hocbl afforded significant, dose - dependent protection in mice conclusions. hocbl produces a complex, time- and organ - dependent, selective regulation of nos/no during endotoxaemia, corollary regulation of downstream inflammatory mediators, and increased survival. this merits clinical evaluation. |
aortic valve stenosis is a quite frequent valvular disease which could require surgical treatment [1, 2 ]. aortic valve replacement by open chest surgery is still considered the gold standard for symptomatic patients, but recently transcatheter aortic valve implantation (tavi) has become an option for patients at high surgical risk. for one third of these patients, not accepted for surgical treatment due to advanced age, comorbidities, and high surgical risk, medical evidence suggests that tavi is feasible and provides a hemodynamic and clinical improvement up to two years, even if long - term durability is still debated. aortic stenosis causes increased left ventricular (lv) afterload, which leads to lv hypertrophy and remodeling towards heart failure, increased morbidity and mortality. surgical treatment, on the one hand, has already been demonstrated to reduce lv mass and improve systolic and diastolic lv parameters, thus prolonging survival. tavi, on the other, seems to improve diastolic function and to favor left ventricular mass regression (lvmr) after six months. the impact of the procedure on lv systolic parameters like peak longitudinal strain have not yet been extensively evaluated. the aim of the present study was to assess lvmr and peak longitudinal strain improvement six months after tavi compared to pre - procedural assessments. moreover, we wanted to investigate whether longitudinal strain parameters before the procedure could predict lvmr. subjects. from january 2011 to august 2011 we consecutively enrolled 23 patients with pure symptomatic severe aortic stenosis and left ventricular ejection fraction > 45% who underwent successful tavi at the cardiologic unit of university civil hospital of brescia, italy. exclusion criteria were : history of ischemic heart disease, high blood pressure, hypertensive cardiomyopathy, dilated cardiomyopathy, and congenital heart defects. patients were treated with tavi if the aortic valve area was 2, if the european system for cardiac operative risk evaluation score (euroscore ; supplementary data online, appendix s1) was > 20% or if one or more of the following criteria was met : contraindication for surgery, severely reduced pulmonary function, liver cirrhosis, or metastatic cancer. all patients underwent the tavi procedure with a third - generation self - expanding corevalve prosthesis (medtronic, minneapolis, mn). the procedure was performed at the catheterization laboratory under local anesthesia and mild sedation with fluoroscopy guidance. procedural success was defined as implantation of a functioning aortic prosthesis valve without intraprocedural mortality and with a paravalvular leak < 2. we state that our study complies with the declaration of helsinki and that all patients gave their informed written consent to access their data for scientific purposes. the arm in which the highest sitting diastolic pressures were found was the arm used for all subsequent readings throughout the study. every effort was made to have the same staff member obtain blood pressure measurements in each individual patient, at the same time of day, using the same equipment. systolic pressure was recorded when the initial sound was heard (phase i of the korotkoff sound), while diastolic pressure was recorded at the disappearance of the sound (phase v of the korotkoff sound). echocardiograms were done using vivid 7 (general electric medical systems, milwaukee, wi, usa) equipment using a 3.5 mhz transducer, with the patients in the left lateral decubitus position, in accordance with the standardization of the american society of echocardiography. digital loops were stored on the hard disk of the echocardiograph for on - line and off - line analyses and transferred to a workstation (echopac ; ge health - care, waukesha, wi, usa) for off - line analysis. the echocardiographic measurements of the left ventricular end - diastolic diameter (lvedd), interventricular septal thicknes (ivst), and posterior wall thickness (pwt) were performed off - line according to recommendations of the american society of echocardiography. left ventricular volume and ejection fraction were obtained by the modified biplane simpson method. lv mass was calculated according to the formula : 0.8 x [1.04 x (lvedd + pwt + ivst)3 - lvedd3 ] + 0.6 (g), and indexed for body surface area. lvmr was defined as the difference between lv mass index for body surface area after six months and before the procedure. end - diastolic relative wall thickness was the ratio of 2 x pwt / lvedd. aortic valve area was calculated by the continuity equation, and the maximum pressure gradient across the restrictive orifice was estimated by the modified bernoulli equation. mean transaortic pressure gradient was calculated by averaging the instantaneous gradients over the ejection period on the continuous - wave doppler recordings. the assessment of longitudinal peak systolic strain was performed offline to the apical two - chamber, four - chamber, and long - axis views of the left ventricle. in brief, the endocardial contour was manually traced at an end - systolic frame. the software then automatically traced a concentric region of interest including the entire myocardial wall. strain analysis was performed by dividing each lv image into six segments per view. with the beginning of the qrs complex and the aortic valve closure time as reference points, systolic global longitudinal strain was calculated by averaging the peak systolic values of the 18 segments [20, 21 ]. all analyses were carried out using ibm spss statistics 20 for windows (spss, inc., continuous variables were tested for normality with the kolmogorov - smirnov test and are represented by mean standard deviation, while categorical variables are represented as frequency (n) and percentage of the sample. paired - samples student s t - test and wilcoxon signed rank test were performed to analyze the difference between means for continuous variables between baseline and 6-month follow - up, and 2 test was performed to assess the difference between proportions for categorical variables (fisher s exact test if dichotomic). bivariate spearman s correlation and linear regression were run between lv strain parameters at baseline and lvmr to investigate their predictive value. mean age was 838 years, ten patients were male (43.5%) and the logistic euroscore was 24.915.1%. one patient was in new york heart association (nyha) class i (4.3%), seven in nyha class ii (30.5%) and 15 in nyha class iii (65.2%). the peak and mean baseline transvalvular gradients were 8821 mmhg and 5616 mmhg, respectively. calculated aortic valve area index at baseline was 0.330.15 cm / m. at six months of follow - up nyha class improved with ten patients in nyha class i (43.5%), 12 in nyha class ii (52.2%), and three in nyha class iii (4.3%) (p<0.001) (table 2), while systolic blood pressure increased from 1159 to 1228 (p=0.024) and diastolic pressure from 786 to 836 (p=0.031). lv = left ventricular ; ivst = interventricular septal thicknes ; pwt = posterior wall thickness ; lvedd = left ventrhicular end - diastolic diameter. a complete study data set was available for all patients : the echocardiographic parameters evaluated at baseline and during follow - up of six months are summarized in table 2 and figure 1. there were significant periprocedural reductions in peak (to 177 mmhg ; p<0.001) and mean (to 105 mmhg ; p=0.005) transvalvular gradients. lv ejection fraction improved from 566% to 5811% without reaching statistical significance (p=0.626), as well as lvedd index (p=0.079). vice versa, ivst and pwt significantly decreased in six months (p < 0.001 for both). the distribution of the lvmr grade is shown in figure 2. left ventricular mass regression six months after tavi. there were no gender - related differences in lvmr, being -2716% in males and -2012% in females (p=0.376). bivariate correlations between lvmr and lv longitudinal strain parameters at baseline are shown in table 3 and figure 3, while linear regression in table 4. scatter plots of lvmr and lv longitudinal strain parameters at baseline (a = peak longitudinal strain 2 chambers ; b = peak longitudinal strain 3 chambers ; c = peak longitudinal strain 4 chambers ; d = global longitudinal strain). lv hypertrophy in aortic stenosis is an adaptive mechanism to ensure a normal relation between systolic wall stress and ejection fraction. nevertheless, it has widely been associated with impaired long - term survival, myocardial infarction, sudden death, heart failure, and cerebrovascular accidents. therefore, lvmr is an important target in this kind of patient, due to the influence of residual lv hypertrophy on long - term survival [27, 28 ]. lvmr ensues after the relief of lv outflow obstruction following aortic valve replacement, which leads to subsequent improved hemodynamics, clinical status, and prognosis. strain imaging is the most appropriate method to assess lv myocardial contractility as it is able to catch subclinical changes in lv performance in aortic stenosis patients [32, 33 ]. in fact, peak longitudinal strain is reduced with increasing severity of aortic stenosis and, in addition, increased lv mass and higher relative wall thickness are associated with reduced lv regional and global myocardial deformation assessed by 2d speckle tracking. as a consequence, lv global longitudinal strain improves after surgical aortic valve replacement [13, 36 ]. recently demonstrated that global longitudinal strain accurately predicts lvmr in patients with pure aortic stenosis undergoing surgical treatment. this study confirms data reported by our group in 2012 regarding lv diastolic function improvement and mass reduction after tavi. moreover, to the best of our knowledge, only a very recent study analyzed lv longitudinal strain 12 months after the cited procedure. moreover, our findings seem to extend the predictive value of baseline global longitudinal strain regarding lvmr after aortic valve replacement even in the tavi context. myocardial fibrosis in an aortic stenosis heart is responsible for lack of lvmr and poor clinical outcome. in this study we confirmed the hypothesis according to which impaired global longitudinal strain before aortic valve replacement (tavi in this case) may be due to a certain amount of fibrosis, thus conditioning future lack of lvmr and prognosis. importantly, our study deals with patients older than cited studies (about 15 years older than spethmann s). this is the reason for a more reduced lv longitudinal strain than cited studies, even if our patients had all preserved lvef and no other cardiological comorbidities. first, the small number of patients and its retrospective nature limit its strength. however, this issue reflects the rarity of patients with severe aortic stenosis who underwent tavi and without any other cardiovascular disease. second, although performed by experienced echocardiographists, lv mass measurement and epicardial / endocardial border tracing for 2d strain analysis might be affected by potential errors. fourth, the small number of patients prevented us from carrying out a multivariate approach. in conclusion, baseline global longitudinal strain seems to predict lvmr in patients with pure aortic stenosis undergoing tavi. the assessment of global longitudinal strain, in addition to other echocardiographic parameters, may be helpful in detecting patients undergoing tavi who are unlikely to benefit from it. | introductiontranscatheter aortic valve implantation is the option of choice for high surgical risk patients suffering from symptomatic aortic stenosis. we aimed to evaluate the influence of baseline global longitudinal strain on left ventricular mass regression after the procedure.methodswe enrolled 23 patients with pure symptomatic severe aortic stenosis who underwent corevalve prosthesis (medtronic, minneapolis, mn) implantation. everyone had echocardiography registration before the procedure and after six months in order to analyze two-, three-, and four - chamber peak longitudinal strain and global longitudinal strain.resultsafter the procedure new york heart association class, peak and mean aortic valve gradients (p<0.001 for all) improved. interventricular septum and posterior wall thicknesses decreased (p<0.001 for both). moreover, left ventricular mass index for body surface area changed from 19044 to 14330 g / m2, (p<0.001). finally, global longitudinal strain significantly increased (from 9.40.9 to 11.50.8% ; p<0.001), as well as its components. baseline global longitudinal strain correlated with left ventricular mass regression (r=0.560 ; p=0.005 ; 2-sided) and predicted it at linear regression analysis (b=23.707 ; p=0.005 ; adjusted r2=0.281).conclusionsglobal longitudinal strain and its components improved six months after the procedure. moreover, baseline global longitudinal strain seemed to predict left ventricular mass regression in patients with pure aortic stenosis undergoing transcatheter aortic valve implantation. this finding could be related to the extent of myocardial fibrosis which is also responsible for lack of left ventricular mass regression and poorer prognosis. |
rhinophyma, exuberant hypertrophic acne, with tumoral aspect of the skin of nasal pyramid is characterized by large, bulbous, erythematous appearance of the nose. there were incriminated several factors such as : the excess of steroid hormones (androgens), the presence of a parasite (demodex folicolorum)[1, 3 ], and vitamin deficiency. it is said that alcoholism was a trigger for rhinophyma, because a lot of alcoholics have erytherma of the face, but alcoholism does not cause rhinophyma. histological, besides sebaceous hyperplasia, it can also be observed an abnormal vascular development (telangiectasis), hypertrophy of subcutaneous tissue and atrophy of dermis. a 56-year - old patient, from urban area, chronic alcohol consumer known with chronic hepatitis presented a giant rhinophyma with a slowly progressive development of approximately 10 years. because of his unaesthetic aspect, the patient was excluded from his group of friends. at the clinical examination it was found : in the glabellar and malar region, the tegument was changed, the skin being fibrous, crossed by red, purple, dilated follicular orifices - fig.1. the nasal tumor was crossed by glandular orifices from which, at pressure, sebum flows. this nasal tumor, because of its size, caused the obstruction of external nasal orifices and difficulty in eating due to the ptosis through lips. the diagnosis of rhinophyma was established on the clinical aspect. in order to confirm it a biopsy was performed fig.2. the surgical treatment consisted in the excision of the nasal tumor, with preservation of the cartilages, perichondrium and periosteum. hemostasis was done by injecting saline and adrenaline peritumoral - fig.3. in the same operatory time, there were covered the skin defect, the cartilage, the perichondrium and the periosteum and then the graft suture and a tie over bandage were realized. the histopathological exam helps to establish a precise diagnosis and can point some modifications bounded on injury progression. in our patients case we encounter the next histopathological features : in rhinophyma, the hyperplasia of sebaceous glands came over so obvious and sebaceous ducts are enlarged and filed with keratin and sebum. was observed a vasodilatation in the upper and the middle part of derma, presenting a perivascular and perifollicular inflammatory infiltrate with lymphocytes and histiocytes fig.6. dermal infiltrates are gathering in little nodular formations and creating granulomas, observing epithelioid histiocytes and, occasionally, can be identified multinucleate giant cells fig.7. he, enlarged sebaceous ducts filed with keratin and sebum, he, x10 lymphocytes and histiocytes perivascular and perifollicular, he, x10 dermal infiltrates formations creating granulomas, he, x10 under pre and post - operative antibiotic therapy the evolution was good and the patient was discharged 12 days after the intervention. after the surgical intervention, the respiratory problems and the discomfort in eating disappeared. furthermore, due to the good aesthetic aspect the patient was reintegrated into society -fig.8. acne rosacea is a chronic or long term facial skin disorder and rhinophyma is the most advanced form of this disease. in the evolution of acne rosacea there are 4 stages : stage i (vascular) it appears around the age of 20 years and it is characterized by sudden reddening of the face and neck, with sensation of intense heat. the factors responsible for these facts are : the change of temperature, ingestion of hot drinks, alcohol or spicy food stage ii it appears around the age of 30 years and it is clinically characterized by permanent facial redness associated with telangiectasia located on the nose, cheek and forehead. stage iii- it appears around the age of 40 years and it is characterized by inflammatory papules and pustules on an erythematous background. the lesions are spread to areas around the eyes and mouth, without scars and the skin is sensitive, with intolerance at application of local cream. stage iv appears around the age of 50 years and it is clinically characterized by thick red skin, with purple striae and nodules located on the nose, ear and zygomatic region the diagnosis is easy to establish even in the first stage. the clinical examination reveals an enlarged nose, with erythematous and thick skin and tumors of different sizes located on the external nose. histopathological changes can differ considerably from case to case, reflecting the clinical aspect on presentation. is observed vasodilatation in upper and middle part of derma, and generally, is present in all cases a perivascular and perifollicular inflammatory infiltrate with lymphocytes and hystiocites and, occasionally, plasmocytes. can be present a dilatation of lymphatic vases. can be observed epithelioid hystiocites in a tuberculoid pattern and, occasional, can be identified multinucleated giant cells. can be constituted unfamiliar body granulomas around necrotic material storages and around materials resulted after lacerating and destructing the follicles. concerning follicular involving can differ also from the infundibular spongiosis and exocytosis, to severely alterations type superficial pustular folliculitis. in rhinophyma, sebaceous glands hyperplasia becomes obvious sand sebaceous ducts are enlarged and filled with keratin and sebum. it can be dermal fibroplasia and the amount of the quantity of conjunctive tissue. the tumor is painless and while pressing it can appear an increased quantity of whitish sebum with fetid smell. in gigantic forms can be associated with a suborbital region lymphedema, having as a result blepharitis, conjunctivitis and keratitis [2, 7 ]. in this situations the differential diagnosis is made with : angiosarcomas, squamous carcinoma, basocellular carcinoma, sebaceous carcinoma, dermal carcinoma, skin metastasis especially in pulmonary neoplasm, eozinophilic facial granuloma, hemangioma, nasal keloid scars, lymphoma,. in this context the therapeutically possibilities in rhinophyma are [7, 9, 10, 11, 12 ] : laser (co2, argon, nd : yag) ; hydrodissection (versajet system) ; mechanical dermabrasion ; laser treatment, hydrodissection and dermabrasion are used in small dimensioned lesions, at the beginning of the disease. the excision is performed in monobloc ; the covering of skin defect can be performed in the same operatory time like in the presented case or after granulation in almost 14 - 16 days from the first intervention. the skin graft is gathered in all its thickness from hairless areas and face skin - like coloration. the principal complication that can appear is represented by the nasal cartilages chondrolysis ; to avoid this complication it must be not used the electrical scalpel and the electrocoagulation that will compromise the perichondral vascularization. to avoid the appearing of postoperatory chondritis must be administrated antistaphylococcal antibiotheraby on systemic as on local way. rhinophyma represent stage iv of rosacea acne, that give an inaesthetic aspect sometimes monstrous of nose and of face too that required a radically surgical treatment in cases where tumoral mass is big, obtaining an satisfactory aesthetic and functional aspect dissipating the respiratory obstruction and also the alimentation discomfort. | the aim of the article is to present an update on the pathophysiology, clinical features and treatment of rhinophyma. a 56 years old patient, living in urban area, presented with a giant rhinophyma which caused him not only upper airways obstruction and difficulty in eating, but also aesthetic and psycho - social disadvantages.the treatment of the patient was a surgical intervention consisting in removal of the nasal tumor and split - thickness skin grafting of the defect. the aesthetic result after surgical intervention was very good, there were no postoperative complications or recurrences.rhinophyma represents the most advanced form of acne rosacea. the diagnosis is easy to establish based on the clinical features of the disease. in advanced forms of rhinophyma, when the tumor is giant, the main method of treatment is surgery. |
hemangiopericytoma is a mesenchymal tumor that occurs most commonly in the soft tissues of the upper and lower extremities, pelvis, and retroperitoneal space and seldom in visceral organs (1). due to its vascular origin transarterial embolization is a procedure which can be performed prior to surgery of highly vascular tumors in order to avoid perioperative bleeding. although there are a few reports (3, 4) of preoperative embolization of hemangiopericytomas, to our knowledge, there has been no report to apply a preoperative embolization of a primary hemangiopericytoma of the rib. a 62-yr - old man was admitted to our department with the complaints of progressive dyspnea, an aching chest pain on the right side, malaise and anorexia with weight loss on 4 december 2001. his medical history was noteworthy for diabetes mellitus type ii, a 10-pack year history of smoking, and a moderate history of alcohol. on physical examination, a chest roentgenogram on admission demonstrated a large, indistinct opacity on the peripheral region of the right lower zone associated with blunting of the right lateral costophrenic sulcus. computed tomographic (ct) scans of thorax revealed a homogeneous mass expanding the right seventh rib and invading into the adjacent lung, and a right sided pleural effusion (fig. histopathologic examination of the pleural biopsy specimens were reported as chronic pleuritis. to clearly identify the mass, a ct - guided, core needle biopsy was performed. the ultrasonography and ct of the abdominopelvic region, magnetic resonance imaging of both upper and lower extremities and ct of the cranium were reported as normal. preoperative angiography showed hypervascularization of the tumor from the right 7th and 8th (fig. 2), and also from 9th intercostal arteries. we performed embolization with polyvinyl alcohol (pva) particles. firstly, we embolized arterioles within the tumor for which we used pva, 250 m in size. then, embolization of the feeding arteries of the tumor was done with pva, 750 - 1,000 m in size. complete resection of the mass with 7th, 8th, and 9th rib with a surgical margin of 5 cm and a wedge resection of the adjacent lung was done. macroscopic evaluation of the resected tumor revealed an encapsulated and lobulated mass located posteriorly to the 6th to 9th ribs. microscopic evaluation of the tumor revealed a solid mesenchymal mass composed of indistinct fusiform to rounded cells arranged around slit - like and branching thin - walled vessels (fig. immunohistochemically tumor cells stained positively for vimentin and focally for cd-34, and negatively for cytokeratin, smooth muscle actin, s-100 and cd-31. hemangiopericytomas, that are believed to be derived from the vascular pericyte, rarely originate from ribs, although they can be found in any region which contains pericytes (5). but, it may be asymptomatic and may be detected as an abnormal shadow on a chest radiography film (3). the radiographic appearance is that of a mass on the lateral chest wall (6). rarely, it may reach a huge size occupying two thirds of one hemithorax (7). in our case, thorax ct revealed that a large indistinct opacity on the peripheral region of the right lower zone on chest radiography was a homogeneous mass expanding the right seventh rib. microscopically the tumor consists of numerous vascular spaces of variable size and shape separated by aggegates of tightly packed oval to spindle - shaped cells with bland nuclei. mitotic figures and nuclear atypia are variable in number and degree (6). in our case, microscopic evaluation of the tumor revealed a solid mesencymal mass composed of indistinct fusiform to rounded cells arranged around slit - like and branching thin - walled vessels. gomori silver impregnation method revealed a dense reticulin meshwork confirming the perivascular distribution of tumoral cells. the results of immunohistochemical studies were consistent with the diagnosis (8). because hemangiopericytomas are highly vascular tumours, because the choice of treatment for primary ones is surgery, and because some authors (3, 5) reported massive bleeding during the operation of hemangiopericytomas, we performed transarterial embolization preoperatively in order to prevent or decrease perioperative bleeding (3). and then, the patient underwent a right thoracotomy during which there was no any significant bleeding. because the recurrence rate was reported as 50% (7, 9), long - term follow - up is necessary for the patient. in our case finally, our case report has two important features : 1) primary hemangiopericytoma of the rib is an extremely rare tumor ; 2) preoperative embolization of a primary hemangiopericytoma of the rib have not been reported heretofore. | primary hemangiopericytoma of the rib is extremely rare and only a few cases have been reported. a 62-yr - old man presented with an aching chest pain and dyspnea. thoracic computed tomography revealed a homogenous mass expanding the right seventh rib. a diagnosis of hemangiopericytoma was established by percutaneous needle biopsy. preoperative embolization of the feeding vessels of the tumor was performed in order to prevent perioperative bleeding. there was no significant bleeding during the surgery, where complete resection of the tumor with 7th to 9th ribs with a surgical margin of 5 cm was performed. postoperative course was uneventful and there has been no recurrence for thirteen months. to our knowledge, there has been no report to apply a preoperative embolization of a primary hemangiopericytoma of the rib. |
influenza is an acute respiratory disease caused by an rna virus belonging to the orthomyxoviridae family. influenza viruses are classified into three types (a, b and c) based on matrix protein (m) or nucleoprotein (np) and are further characterized by both hemagglutinin (h) and neuraminidase (n) proteins. to date, 16 ha and 9 na subtypes have been identified for influenza a. reassortment and mutations occur frequently, contributing to the variation and evolution of influenza viruses, and also influencing the potential of the virus to be the causative agent of epidemics and pandemics. following the emergence of a novel h1n1 influenza a virus strain, later named the pandemic (h1n1) 2009 (ph1n1), in the human population, the initial concern in the general population was with regard to zoonotic potential and humantohuman transmission because of the high mortality rate associated with the virus in early cases. however, in the veterinary field, because this virus was quickly dubbed the swine flu because of its genetic similarity with influenza viruses of swine origin, the need at the veterinary diagnostic community quickly arose to be able to rapidly and accurately detect this organism and differentiate it from endemic influenza a viruses in nonhuman species. many of these,,,,, specifically detect the virus but not other influenza a viruses, which may increase the time and expense of diagnosing endemic influenza. others target either the n or h,,,, genes, which tend to have a high rate of genetic mutation and may lead to false negative results if mismatches occur between the viral sequence and the primer or probe sequence. others are sybr green assays,, which may lack the specificity of a probebased assay as sybr green can intercalate with any doublestranded dna product (including primer dimers or nonspecific products that may be amplified in the reaction) and cause fluorescence, whereas a probebased assay requires complementarity of both primers as well as a probe. the lack of specificity can usually be overcome by performing a melt curve analysis but this adds additional time to the assay. in some instances, the differentiation of primer dimers and specific product is not possible and an additional confirmatory test must be performed. the assay described herein is a probebased assay, detecting ph1n1 as well as endemic, north american swine influenza viruses (sivs) in a differential manner. the m gene is known to be genetically conserved among all influenza a viruses, so this was chosen as the target for a realtime rtpcr assay capable of detecting all influenza a and differentiating ph1n1 from endemic influenza a virus directly from clinical specimens of various species origin. a suitable target region for a realtime rtpcr assay was determined by using clustal w alignment function of lasergene software (dnastar, inc., madison, wi, usa) for the sequences of the matrix gene from six u.s. human ph1n1 cases (genbank accession numbers : epi176471, fj966954, fj966968, fj966972, fj966975, and fj966983) and 16 u.s. sivs (genbank accession numbers : cy031252, cy031253, cy031254, cy031255, cy032930, cy035071, cy036800, cy036864, dq145542, dq889688, eu409951, eu409957, eu409962, eu409966, eu409970 and eu604695). primer express software (applied biosystems, carlsbad, ca, usa) was used for the primer and probe design as summarized in table 1. initially, one forward (m_f) and three reverse primers (m_r1, m_r1a and m_r1b) were selected to amplify all influenza a targets, and 2 mgb probes (m_eapr and m_napr1) were incorporated to differentiate ph1n1 (eurasian or ea) matrix gene from endemic siv (north american or na) matrix gene. sequence information and additional test validation resulted in the incorporation of a second probe (m_napr2) into the assay to improve the detection of endemic influenza a in clinical specimens. primers and probes used in assay extraction of rna from isolates or clinical specimens was performed using the applied biosystems / ambion magmax viral rna isolation kit and a kingfisher semiautomated magnetic particle processing system (thermo scientific, waltham, ma, usa) according to manufacturer s recommendations. realtime rtpcr was carried out with the applied biosystems / ambion pathid multiplex onestep rtpcr kit in a 20 l reaction according to supplier s recommendations, with the addition of 10 u of multiscribe reverse transcriptase (applied biosystems) per reaction. the forward primer (m_f) was added at a final concentration of 0375 m. the three reverse primers (m_r1, m_r1a and m_r1b) were combined in equal molar concentrations and added at a final concentration of 0375 m. each probe (m_ea and m_na1) when an additional probe (m_na2) was incorporated into the assay, each of the na probes was added at a final concentration of 00625 m. reactions were run on an applied biosystems 7500fast instrument with the following cycling conditions in fast mode : 45c for 10 minutes, followed by 95c for 10 minutes, then 45 cycles of 97c for 2 seconds and 60c for 40 seconds. fluorescence curves crossing the threshold at or before 40 cycles were considered positive for the respective genotype. a cycle threshold (ct) between 40 and 45 was considered suspect and retested if appropriate. criteria for retesting include the presence of clinical signs supporting a diagnosis of influenza, compatible lesions or a realtime fluorescence curve of atypical shape. ph1n1 strain (a / california/04/2009), 10 human specimens positive for ph1n1 using the cdc pcr obtained from the university of iowa state hygienic laboratory (uihl) and two siv isolates [a / swine / iowa/73 (h1n1) and a / swine / tx/98 (h3n2), both supplied by the national veterinary services laboratory, ames, ia ]. in addition, eight isolates that had previously been tested with the isuvdl general and subtyping assays and further characterized by sequencing were tested with this assay and were also sent to the uihl in a blind fashion where they were tested by the cdc pcr protocols. these included swine h1 clade (a / swine / iowa/1973 h1n1), swine h1 clade (a / swine / ia/15231/2008 h1n1), swine h1 clade (a / swine / ia/13381/2008 h1n1 ; a / swine / ia/14913/2008 h1n1;a / swine / ia/31256/2006 h1n2), swine h1 clade (a / swine / ia/18755/2008 h1n2), cluster i h3n2 (a / swine / ia/41305/1998) and cluster iv h3n2 (a / swine / ia/18916/2008) viruses. classifications of h1 clades and h3 clusters are described by vincent. and olsen., respectively. phylogenetic relationship between these viruses and contemporary human influenza viruses including ph1n1 based on h gene is illustrated in figure 1. phylogenetic relationship between representative swine influenza viruses and human influenza viruses based on hemagglutinin gene sequence. the viruses that were tested by various pcr protocols (isu, cdc, nahln) are marked with arrow. the assay was then evaluated on specimens from 165 animals representing 32 cases submitted to the iowa state university veterinary diagnostic laboratory (isuvdl). these specimens were from a variety of species, including porcine, feline (domestic and lion), canine, primate (chimpanzee) and rodent (hamster). samples tested included lung tissue, swabs (nasal, pharyngeal or tracheal) and bronchoalveolar lavage. all samples were also tested by isuvdl s inhouse screening pcr, which targets influenza a nucleoprotein (np) gene, isuvdl s h subtyping pcr that identifies and differentiates h1 and h3, nahln siv surveillance pcr and/or sequencing for the confirmation of virus classification. matrix gene sequences from two additional ph1n1positive influenza cases and 122 contemporary h1n1 sivs, all from clinical specimens submitted to the isuvdl, were used to further assess primer and probe suitability. specimens with sequences in which a mismatch was observed between primer / probe sequence and viral sequence were tested with the assay. sequence of samples not giving the expected result were evaluated and utilized to design an additional probe for incorporation into the assay. for sequencing, extraction of rna was performed using magmax viral rna isolation kit (applied biosysems / ambion) in conjunction with the kingfisher magnetic particle processor according to manufacturer s recommendations. rtpcr was performed using the superscriptiii onestep rtpcr system (invitrogen life technologies, carlsbad, ca, usa) with each primer being added at a level of 132 m in the reaction mixture. also, 32 u of rnaseout rnase inhibitor (invitrogen) was added to each reaction. reverse transcription was performed at 50c for 30 minutes, followed by denaturation at 94c for 2 minutes. pcr amplification was executed for 35 cycles of 94c for 20 seconds, 58c for 30 seconds and 68c for 7 minutes. pcr product purification was performed using qiaquick pcr purification kit (qiagen, valencia, ca, usa) or the chargeswitch pcr cleanup kit (invitrogen) in conjunction with the kingfisher magnetic particle processor, according to manufacturer s recommendations. purified pcr product and sequencing primers were submitted to the iowa state university dna sequencing and synthesis facility. a suitable target region for a realtime rtpcr assay was determined by using clustal w alignment function of lasergene software (dnastar, inc., madison, wi, usa) for the sequences of the matrix gene from six u.s. human ph1n1 cases (genbank accession numbers : epi176471, fj966954, fj966968, fj966972, fj966975, and fj966983) and 16 u.s. sivs (genbank accession numbers : cy031252, cy031253, cy031254, cy031255, cy032930, cy035071, cy036800, cy036864, dq145542, dq889688, eu409951, eu409957, eu409962, eu409966, eu409970 and eu604695). primer express software (applied biosystems, carlsbad, ca, usa) was used for the primer and probe design as summarized in table 1. initially, one forward (m_f) and three reverse primers (m_r1, m_r1a and m_r1b) were selected to amplify all influenza a targets, and 2 mgb probes (m_eapr and m_napr1) were incorporated to differentiate ph1n1 (eurasian or ea) matrix gene from endemic siv (north american or na) matrix gene. sequence information and additional test validation resulted in the incorporation of a second probe (m_napr2) into the assay to improve the detection of endemic influenza a in clinical specimens. extraction of rna from isolates or clinical specimens was performed using the applied biosystems / ambion magmax viral rna isolation kit and a kingfisher semiautomated magnetic particle processing system (thermo scientific, waltham, ma, usa) according to manufacturer s recommendations. realtime rtpcr was carried out with the applied biosystems / ambion pathid multiplex onestep rtpcr kit in a 20 l reaction according to supplier s recommendations, with the addition of 10 u of multiscribe reverse transcriptase (applied biosystems) per reaction. the forward primer (m_f) was added at a final concentration of 0375 m. the three reverse primers (m_r1, m_r1a and m_r1b) were combined in equal molar concentrations and added at a final concentration of 0375 m. each probe (m_ea and m_na1) when an additional probe (m_na2) was incorporated into the assay, each of the na probes was added at a final concentration of 00625 m. reactions were run on an applied biosystems 7500fast instrument with the following cycling conditions in fast mode : 45c for 10 minutes, followed by 95c for 10 minutes, then 45 cycles of 97c for 2 seconds and 60c for 40 seconds. fluorescence curves crossing the threshold at or before 40 cycles were considered positive for the respective genotype. a cycle threshold (ct) between 40 and 45 was considered suspect and retested if appropriate. criteria for retesting include the presence of clinical signs supporting a diagnosis of influenza, compatible lesions or a realtime fluorescence curve of atypical shape. ph1n1 strain (a / california/04/2009), 10 human specimens positive for ph1n1 using the cdc pcr obtained from the university of iowa state hygienic laboratory (uihl) and two siv isolates [a / swine / iowa/73 (h1n1) and a / swine / tx/98 (h3n2), both supplied by the national veterinary services laboratory, ames, ia ]. in addition, eight isolates that had previously been tested with the isuvdl general and subtyping assays and further characterized by sequencing were tested with this assay and were also sent to the uihl in a blind fashion where they were tested by the cdc pcr protocols. these included swine h1 clade (a / swine / iowa/1973 h1n1), swine h1 clade (a / swine / ia/15231/2008 h1n1), swine h1 clade (a / swine / ia/13381/2008 h1n1 ; a / swine / ia/14913/2008 h1n1;a / swine / ia/31256/2006 h1n2), swine h1 clade (a / swine / ia/18755/2008 h1n2), cluster i h3n2 (a / swine / ia/41305/1998) and cluster iv h3n2 (a / swine / ia/18916/2008) viruses. classifications of h1 clades and h3 clusters are described by vincent. and olsen., respectively. phylogenetic relationship between these viruses and contemporary human influenza viruses including ph1n1 based on h gene is illustrated in figure 1. phylogenetic relationship between representative swine influenza viruses and human influenza viruses based on hemagglutinin gene sequence. the viruses that were tested by various pcr protocols (isu, cdc, nahln) are marked with arrow. the assay was then evaluated on specimens from 165 animals representing 32 cases submitted to the iowa state university veterinary diagnostic laboratory (isuvdl). these specimens were from a variety of species, including porcine, feline (domestic and lion), canine, primate (chimpanzee) and rodent (hamster). samples tested included lung tissue, swabs (nasal, pharyngeal or tracheal) and bronchoalveolar lavage. all samples were also tested by isuvdl s inhouse screening pcr, which targets influenza a nucleoprotein (np) gene, isuvdl s h subtyping pcr that identifies and differentiates h1 and h3, nahln siv surveillance pcr and/or sequencing for the confirmation of virus classification. matrix gene sequences from two additional ph1n1positive influenza cases and 122 contemporary h1n1 sivs, all from clinical specimens submitted to the isuvdl, were used to further assess primer and probe suitability. specimens with sequences in which a mismatch was observed between primer / probe sequence and viral sequence were tested with the assay. sequence of samples not giving the expected result were evaluated and utilized to design an additional probe for incorporation into the assay. for sequencing, extraction of rna was performed using magmax viral rna isolation kit (applied biosysems / ambion) in conjunction with the kingfisher magnetic particle processor according to manufacturer s recommendations. rtpcr was performed using the superscriptiii onestep rtpcr system (invitrogen life technologies, carlsbad, ca, usa) with each primer being added at a level of 132 m in the reaction mixture. also, 32 u of rnaseout rnase inhibitor (invitrogen) was added to each reaction. reverse transcription was performed at 50c for 30 minutes, followed by denaturation at 94c for 2 minutes. pcr amplification was executed for 35 cycles of 94c for 20 seconds, 58c for 30 seconds and 68c for 7 minutes. pcr product purification was performed using qiaquick pcr purification kit (qiagen, valencia, ca, usa) or the chargeswitch pcr cleanup kit (invitrogen) in conjunction with the kingfisher magnetic particle processor, according to manufacturer s recommendations. purified pcr product and sequencing primers were submitted to the iowa state university dna sequencing and synthesis facility. primers used for amplification and sequencing reactions are listed in table 2. matrix gene sequencing primers the 10 human ph1n1positive specimens provided by uihl were detected by only the vic dye, as expected for the ea gene of the ph1n1 virus. the a / swine / iowa/73 (h1n1, clade) and a / swine / tx/98 (h3n2, cluster i) viruses were detected only by the fam dye, confirming specificity of the assay for the north american matrix gene. classification of the eight siv isolates sent to uihl is summarized in table 3. although all eight isolates were endemic sivs, five of the eight viruses were identified as positive for ph1n1 by cdc pcrs and the remaining three isolates as reassortant between ph1n1 and human seasonal influenza viruses. in contrast, the nahln siv surveillance pcr did not identify any of the eight isolates as ph1n1 as shown in table 3. these isolates all tested na matrix gene positive (i.e., endemic influenza virus) by the assay described herein. comparison of detection and subtyping influenza a realtime rtpcr assays siv, swine influenza virus. a / swine / iowa/1973 (h1n1). a / swine / iowa/15231/2008. a / swine / iowa/13381/2008 (h1n1) ; a / swine / iowa/14913/2008 (h1n1) ; a / swine / iowa/31256/2006 (h1n2). a / swine / iowa/41305/1998 (cluster i) ; a / swine / iowa/18916/2008 (cluster iv). a / california/04/2009. specimens from 165 animals were tested by the differential m gene rtpcr. all results (e.g., positive for influenza a, identification of virus genotype) were confirmed by isuvdl inhouse rtpcr assay (np, h, n genes) or by the nahln surveillance pcr (m and n genes) and/or by sequencing for h and m genes. six samples were positive for endemic (na matrix) influenza a (five porcine and one feline), five samples were positive for pandemic (ea matrix) influenza a (two porcine, two feline and one canine) and the remaining 154 samples were negative for influenza a virus. number of clinical samples tested by matrix differential rrtpcr assay sequencing was performed on 122 clinical porcine samples or virus isolates from samples that had previously tested as h1n1 by the isuvdl assay and on two ph1n1 samples. analysis revealed total homology between the ea matrix primer / probe sequences and that from the ph1n1 matrix sequences. however, minor mismatches in the probe target region were found in six of the endemic h1n1 matrix sequences from clinical specimens. these samples, all confirmed by sequencing as na genotype, were tested with the rtpcr assay described here. three of these samples tested as na matrix positive by the assay but the remaining three samples were either inconclusive or negative for influenza a by the assay. the matrix sequence derived from the latter three specimens (cagaaacttgaagatgt) had a single mismatch to the na genotype probe (cagagactygaagaygt). a probe reflecting this mismatch (m_napr2) the newly developed realtime pcr assay targeting the m gene detects and differentiates ph1n1 and us influenza a viruses in various sample matrices and species. it was used to detect the firstreported humantofeline reverse zoonotic transmission of ph1n1 virus and has also been used to detect this virus in canine and porcine specimens. (for the usda s list of ph1n1 influenza presumptive and confirmed results in nonhuman species, see : http://www.usda.gov/documents/final_results_2009_pandemic_h1n1_influenza_cht.pdf). the assay is rapid (approximately 70 minutes), and results were confirmed by other pcr tests. one of the assays used for comparison was deployed by the national animal health laboratory network (nahln). this organization, established in 2002, is a cooperative effort between the animal and plant health inspection service (aphis) and national institute of food and agriculture (nifa) and the american association of veterinary laboratory diagnosticians (aavld). initially, the network was established with 12 core diagnostic laboratories ; there are currently 62 member laboratories. the mission of nahln is to safeguard animal health by providing standardized protocols and training for detection of animal diseases, coordinating proficiency testing within the member laboratories and overseeing surveillance testing for the early detection of various veterinary agents of concern. more information on this organization is available at http://www.aphis.usda.gov / animal_health / nahln/. the pcr protocol deployed by nahln is a series of two rtpcr tests (one for general detection using m gene and one to differentiate between pandemic and endemic siv using n gene). the m gene assay was originally described by spackman. and was developed for the detection of avian influenza. to update the assay to detect the ph1n1 virus, isuvdl s inhouse ha subtyping pcr is able to differentiate between h1 and h3 subtypes and between n1 and n2 subtypes but can not distinguish between endemic and pandemic h1n1 viruses (table 3). the cdc protocol differentiates ph1n1 from human influenza virus and may be useful in monitoring the emergence of reassortant influenza viruses, but can not differentiate ph1n1 from endemic h1 sivs (table 3), raising a concern on its differential diagnostic utility at public health laboratories as incidence of siv infection in humans has been reported. the differential matrix assay described herein is able to detect and differentiate pandemic from endemic influenza a in a single multiplexed reaction and is currently being used by the isuvdl for monitoring the appearance of ph1n1 in samples submitted to the laboratory. the assay may be a good supplement to the cdc testing protocol and to the nahln pcrs. the study observation emphasizes that primers and/or probes should be designed with good bioinformatics support. like other rtpcr assays, monitoring the variation of sequences in field strains and updating the primers and/or probes is necessary to ensure reliable performance of the assay. this assay will be useful in observing the appearance of ph1n1 in various species and helpful in monitoring the potential for interspecies transmission of this virus. | please cite this paper as : harmon. (2010) a matrix gene based multiplex realtime rtpcr for detection and differentiation of 2009 pandemic h1n1 and other influenza a viruses in north america. influenza and other respiratory viruses 4(6), 405410. background the emergence in humans of pandemic (h1n1) 2009 (ph1n1) with similarities to swine influenza viruses (sivs) caused much concern for both human and animal health as potential for interspecies transmission was initially unknown. objectives the goal of this study was to develop a realtime rtpcr test for the detection and differentiation of 2009 ph1n1 and endemic influenza a viruses in north america. methods matrix (m) gene sequences from u.s. human ph1n1 cases and u.s. sivs were aligned to determine a suitable region for an assay target. primers were selected to amplify all influenza a. two probes were designed to differentiate ph1n1 (ea matrix) from endemic (na matrix) sivs. the assay was validated using the first u.s. ph1n1 strain, 10 human ph1n1positive specimens and nine u.s. siv isolates, then evaluated on 165 specimens of swine and other animal origin submitted to the iowa state university veterinary diagnostic laboratory. results were compared to other influenza a pcr assays. sequences from additional ph1n1 strains and contemporary h1n1 sivs were used to assess robustness of the selected primers and probes for the intended purpose. results the new assay s results from clinical specimens concurred with confirmatory pcr testing. the additional probe designed from sequence analysis improved detection of the na matrix subtype when added to the reaction mixture. conclusion this assay detects and differentiates ph1n1 and us influenza a viruses in various sample matrices and species. good bioinformatics support is critical when designing rtpcr assays and monitoring their performance. |
our work is funded by the medical research council, eu grant qlk6 - 2000 - 530, and by the muscular dystrophy campaign. | gene - expression profiling has yielded important information about simple systems, but complex tissues have not yet been widely profiled. four recent studies of mammalian skeletal muscles have added to the catalogs of their gene expression differences, but have yet to lead to better understanding of the molecular processes underlying their physiological differences. |
postpneumonectomy syndrome (pps) is an ominous complication, with prevalence in children ranging from 1 in 6 to 1 in 5 pneumonectomies. it is caused by mediastinal shift following massive lung resection that results in compression and stretching of the great vessels and airways. it usually occurs after right pneumonectomy, performed for infections, congenital malformations, or metastatic tumours. we report on a patient with oesophageal atresia (oa) who developed a pps following oa repair. a 1-day - old girl was referred to our department for the suspect of oa. she was born by caesarean section at 37 weeks of gestation after an uneventful pregnancy. her birth weight was 2.1 kg and the apgar score was 7/7 at 1/5 min. on admission she was well, with normal oxygen saturation during spontaneous breathing. chest x - rays showed the nasogastric tube folding at the level of the first thoracic vertebra and a distended stomach (fig. echocardiography demonstrated normal left sided aortic arch, a large patent ductus arteriosus and hypoplastic right pulmonary artery. preoperative fiberoptic tracheoscopy was performed to exclude the presence of a tracheo - oesophageal cleft, and to measure the oesophageal gap. the tip of the bronchoscope was placed at the level of the fistula and a contrasted naso - gastric tube was inserted in the upper pouch. she underwent uneventful surgical ligation and section of the tof and primary oesophageal anastomosis. on post - operative day one, she had progressive clinical worsening with marked hypoxia despite mechanical ventilation. chest x - rays showed total collapse of the right lung and right mediastinal shift (fig. a fiberoptic tracheobronchoscopy performed by an experienced otolaryngologist revealed a long malacic trachea, extending to the left main bronchus. the suspect of a right oesophageal bronchus associated with the oa was confirmed by oesophagogram showing the origin of the right main bronchus from the oesophagus, caudal to the patent anastomosis (fig. the right lung collapse caused massive mediastinal shift and rotation, clinically mimicking a pps. a high resolution ct scan with 3d images reconstruction showed a collapsed hypoplastic right lung with a single, small, right pulmonary artery and an associated left pulmonary artery sling (fig. 2a), not visible during the pre - operative echocardiography focused to the side of the aortic arch. to push the mediastinum in the correct position, a 12 ch foley catheter was inserted in the right hemithorax and sterile normal saline (50 ml) was infused (fig. d), and serial saline infusions were performed in the next days. in the occasion of surgical correction of the left pulmonary artery sling (day 22 of life) a tissue expander was inserted in the right hemithorax to stabilize the mediastinum. on post - operative day 25, a right pneumonectomy was performed and the tissue expander was replaced with a larger one. post - operative course was uneventful ; she was extubated on post - operative day 2, and discharged home on post - operative day 24. she is alive and well at 24 months of follow - up with moderate thoracic asymmetry and good respiratory function. we present a case of acute pps following oa repair in a patient with associated right oesophageal lung. the association of oa with an abnormal bronchial implantation on the oesophagus is extremely rare and falls in the so - called communicating bronchopulmonary foregut malformations (cbpfm). in our series of 256 patients with oa / tof consecutively treated from 1995 we found only one such patient and gou. first used the term bronchopulmonary foregut malformations in 1968 to describe a spectrum of congenital malformations including pulmonary sequestration with or without connections to the alimentary tract. srikanth. later suggested the term cbpfm to describe a segment of lung tissue connected to the foregut, and introduced a classification. the first group of this classification is represented by sequestered lung tissue associated with oa / tof and connected to the distal oesophagus. in our patient, surgical repair of ea suddenly impeded the ventilation to the lung fed by the oesophageal bronchus, thereby leading to total right lung collapse and contralateral single lung ventilation. the pathogenetic cascade is confirmed by the only reported case that did not have surgical ligation of the fistula after detection of the anomaly. that patient s respiratory conditions remained stable because the fistula allowed airflow to the right lung. in our patient, the limitation of the preoperative endoscopic study to exclude a tracheo - oesophageal cleft and to define the oesophageal gap misled our intraoperative management and delayed the diagnosis of cbpfm. a detailed tracheobronchoscopy would probably have allowed the diagnosis of cbpfm before oa repair, as suggested by the subsequent tracheobronchoscopy that showed the absence of the right main bronchus. in any case, in the event of acute worsening of clinical conditions during oa / tof repair or shortly thereafter, with unilateral total lung collapse, the presence of an associated cbpfm should always be born in mind and actively investigated. oesophagogram is the gold - standard diagnostic test as it shows the aberrant origin of the oesophageal bronchus. the study should be performed in lateral decubitus as the bronchus originates from the side of the oesophagus, usually the right one. oesophagoscopy should be avoided in the acute setting as it can cause disruption of the fresh anastomosis. in our patient, lung resection is generally well tolerated, with good long - term outcomes. however, there are anatomic characteristics specific to children that are thought to predispose them to specific post - operative complications. moreover, during childhood, lungs are more compliant due to the higher content of elastic tissue. this relative flexibility of the mediastinal structures could lead to contralateral pulmonary distension after a pneumonectomy. mediastinal shift and rotation causes compression and stretching of the great vessels and airways that in association with overdistension of the remaining lung can result in critical worsening of pulmonary function leading to major respiratory failure, the so called pps. this condition is most frequently encountered in children than in adults for the anatomical characteristics mentioned above. pps can occur almost immediately after pneumonectomy but is more common in the progressive and delayed form. in our patient, oa / tof repair was followed by acute complete right lung collapse, simulating a right pneumonectomy, with massive right - sided mediastinal shift, clinically mimicking a pps. the left pulmonary artery sling that caused left main bronchus stenosis further worsened the clinical picture. as a consequence, both air and blood flow in the remaining left lung was acutely reduced by the mediastinal shift. when such a clinical picture occurs in patients with group 1 cbpfm, the term pseudo - pps seems more appropriate as they do not undergo lung resection surgery. classically, treatment of cbpfm is to resect the aberrant lung tissue with lobectomy or pneumonectomy and mediastinal repositioning using rigid prostheses (e.g. breast prostheses) or tissue expanders in the empty hemithorax. reimplantation of the anomalous bronchus is the ideal management but was attempted in only one patient with cbpfm associated with oa, which was successful. this option should be considered in patients with normal pulmonary vasculature and without lung tissue damage due to aspiration or prolonged consolidation. however, in some cases pps may be life - threatening, and its treatment requires emergency repositioning of the mediastinum in axis. in our patient, we performed an iatrogenic hydrothorax by infusing sterile normal saline in the right hemithorax, which was followed by immediate improvement of her clinical conditions. serial infusions were performed to maintain the mediastinum in axis, which allowed the patient to sustain clinical stability and to undergo cardiac surgery. in our patient, bronchial reconstruction was not possible as definitive treatment because the right bronchus was too short, not allowing a good anastomosis with the trachea, and the lung remained collapsed too long to allow its re - expansion. the association of oa with an abnormal bronchial implantation on the oesophagus is extremely rare and falls in the so - called communicating bronchopulmonary foregut malformations (cbpfm). in our series of 256 patients with oa / tof consecutively treated from 1995 we found only one such patient and gou. first used the term bronchopulmonary foregut malformations in 1968 to describe a spectrum of congenital malformations including pulmonary sequestration with or without connections to the alimentary tract. srikanth. later suggested the term cbpfm to describe a segment of lung tissue connected to the foregut, and introduced a classification. the first group of this classification is represented by sequestered lung tissue associated with oa / tof and connected to the distal oesophagus. surgical repair of ea suddenly impeded the ventilation to the lung fed by the oesophageal bronchus, thereby leading to total right lung collapse and contralateral single lung ventilation. the pathogenetic cascade is confirmed by the only reported case that did not have surgical ligation of the fistula after detection of the anomaly. that patient s respiratory conditions remained stable because the fistula allowed airflow to the right lung. in our patient, the limitation of the preoperative endoscopic study to exclude a tracheo - oesophageal cleft and to define the oesophageal gap misled our intraoperative management and delayed the diagnosis of cbpfm. a detailed tracheobronchoscopy would probably have allowed the diagnosis of cbpfm before oa repair, as suggested by the subsequent tracheobronchoscopy that showed the absence of the right main bronchus. in any case, in the event of acute worsening of clinical conditions during oa / tof repair or shortly thereafter, with unilateral total lung collapse, the presence of an associated cbpfm should always be born in mind and actively investigated. oesophagogram is the gold - standard diagnostic test as it shows the aberrant origin of the oesophageal bronchus. the study should be performed in lateral decubitus as the bronchus originates from the side of the oesophagus, usually the right one. oesophagoscopy should be avoided in the acute setting as it can cause disruption of the fresh anastomosis. in our patient, lung resection is generally well tolerated, with good long - term outcomes. however, there are anatomic characteristics specific to children that are thought to predispose them to specific post - operative complications. moreover, during childhood, lungs are more compliant due to the higher content of elastic tissue. this relative flexibility of the mediastinal structures could lead to contralateral pulmonary distension after a pneumonectomy. mediastinal shift and rotation causes compression and stretching of the great vessels and airways that in association with overdistension of the remaining lung can result in critical worsening of pulmonary function leading to major respiratory failure, the so called pps. this condition is most frequently encountered in children than in adults for the anatomical characteristics mentioned above. pps can occur almost immediately after pneumonectomy but is more common in the progressive and delayed form. in our patient, oa / tof repair was followed by acute complete right lung collapse, simulating a right pneumonectomy, with massive right - sided mediastinal shift, clinically mimicking a pps. the left pulmonary artery sling that caused left main bronchus stenosis further worsened the clinical picture. as a consequence, both air and blood flow in the remaining left lung was acutely reduced by the mediastinal shift. when such a clinical picture occurs in patients with group 1 cbpfm, the term pseudo - pps seems more appropriate as they do not undergo lung resection surgery. classically, treatment of cbpfm is to resect the aberrant lung tissue with lobectomy or pneumonectomy and mediastinal repositioning using rigid prostheses (e.g. breast prostheses) or tissue expanders in the empty hemithorax. reimplantation of the anomalous bronchus is the ideal management but was attempted in only one patient with cbpfm associated with oa, which was successful. this option should be considered in patients with normal pulmonary vasculature and without lung tissue damage due to aspiration or prolonged consolidation. however, in some cases pps may be life - threatening, and its treatment requires emergency repositioning of the mediastinum in axis. in our patient, we performed an iatrogenic hydrothorax by infusing sterile normal saline in the right hemithorax, which was followed by immediate improvement of her clinical conditions. serial infusions were performed to maintain the mediastinum in axis, which allowed the patient to sustain clinical stability and to undergo cardiac surgery. in our patient, bronchial reconstruction was not possible as definitive treatment because the right bronchus was too short, not allowing a good anastomosis with the trachea, and the lung remained collapsed too long to allow its re - expansion. in patients with oa, the development of post - operative severe respiratory distress and unilateral lung collapse must lead to the suspect of an associated cbpfm. neonatologists and paediatric surgeons should be aware of this rare association that may cause acute life threatening worsening of patient s clinical conditions. in the case of pps development, infusion of sterile normal saline in the affected hemithorax is a simple, fast and effective method that allows pushing the mediastinum in the correct position, thereby leading to dramatic improvement of the clinical conditions. although no identifying details are reported, parents of the patient have given their informed consent. | highlightsafter esophageal atresia repair, if postpneumonectomy syndrome develops, an associated esophageal lung must be considered.contrast esophagography, with the patient lying on the side of collapsed lung, is the optimal diagnostic study.in infants with postpneumonectomy syndrome, infusion of normal saline in the hemithorax on the side of collapsed lung is a simple, rapid, and effective method to obtain mediastinum realignment in emergency with immediate improvement of clinical conditions. |
fifty to eighty - five percent of the population in a modern industrial society experience low back pain more than once in their lifetime. low back pain is one of the common symptoms having a prevalence rate of 40 - 67.6% and a point prevalence of 20% (1 - 4). approximately 50% of patients who experience low back pain show improvement within seven days and almost 90% are in remission within one month. nevertheless, 4 - 15% of patients can not return to their job for more than a month, and 1% will suffer chronic or recurring low back pain (5). in many cases, the symptoms may not coincide with the histologic and radiologic findings, resulting in a great deal of diagnostic and therapeutic medical expenses as well as leading to complementary and alternative medical therapies. therefore, low back pain has become one of the greatest causes of manpower loss and escalating medical expenditure (6). extensive research has been carried out on the factors relevant to low back pain worldwide. many of these studies have examined age, obesity, exercise, smoking, education and stress as associated factors. in this study, a questionnaire was used to analyze the correlation between these factors and low back pain among 772 hospital - visiting male patients. the patients with a symptom or test result suspicious of a specific illness and those who were already diagnosed as having a vertebral disease were excluded. this study also examined which factors in ordinary life are most helpful in reducing and preventing low back pain. of a total of 28,100 patients who underwent medical examination in this center, patients 19 yr old or younger, females, and people with symptoms or test results suspicious of a particular disease were excluded. seven hundred and seventy two patients, who filled out the precision medical questionnaire, were used as subjects for retrospective analysis. the precision medical questionnaire consisted of 13 general items and 9 questions for each system, as well as questions related to the level of stress. general items included the presence of any particular motivation for health and medical examination, a history of previous medical examinations, marital status, extent of smoking, alcohol consumption, educational level, monthly income, previous medical history, previous surgical history, previous drug - taking history and previous familial medical history. the survey questions for each system included detailed items such as the general body state, digestive, respiratory, cardiovascular, endocrine, renal - urethral, nervous and dental questions. of these questions, the item of the general body state included a question " have you recently suffered low back pain ? " included in this study were the low back pain - related survey questions such as age, obesity, exercise, smoking, level of education and smoking. for each item, the number of people who had low lumbar pain and those that did not were compared and analyzed. using these survey questions, the following cases were excluded : 1) cases where there were other factors relevant to low back pain in addition to a simple description of " i have low back pain, " under the item " recently, i felt something wrong regarding my health. " 2) cases where patients underwent any one of the following : gastric, duodenal, gallbladder, colonic or kidney surgery with respect to the item " have you ever had surgery ? " 3) regarding the item " up to now, have you ever been diagnosed by a doctor as having any of the following diseases ? " any patient who checked any one of the following : cerebral infarct / paralysis, angina pectoris / myocaridal infarct, acute hepatitis, chronic hepatitis / liver cirrhosis, renal disease, bladder disease, malignancy or trauma. 4) in the item, " presently, are you continuously taking any drug twice or more weekly ? ", a patient who marked on any one or more of the the following : aspirin, nsaids, calcium, anti - anxiety drugs, sleeping pills, any other drugs which can modify back pain including herbal medicines or restorative drugs. 5) finally, cases with the following symptoms : " i lost body weight without any particular reason,""i bruise easily or often have a nose bleed,""i have difficulty swallowing and feel pain while swallowing,""i often feel nausea or vomit, and the vomitus has a coffee or green color,""i once had black - colored feces, " " i once had a tight chest and pain radiating to the arm or the back,""when i exercise, i feel tightness in the chest and become quite breathless,""my heart suddenly throbs fast or irregularly,""my face, hand or foot swells easily,""i feel chest tightness and shortness of breath while lying down or sleeping but feel comfortable sitting down,""i cough continuously for a long time,""i have blood in my sputum or expectorated blood,""i have cold sweats during sleep,""i have a hoarse breathing sound,""i feel breathless even after slight activity,""recently, my urine volume has increased,""i recently lost my appetite,""my fingernails or toenails break easily,""i have difficulty urinating and unsatisfactory feeling after urination,""i can not stand the urge of urination,""i have red or cocoa colored urine,""i have severe pain in the flank or lower abdomen,""i often wake up to pass urine,""i once passed urine without knowing,""i often feel dizziness,""i have an experience of passing out,""i once had paralysis in the body,""i have a symptom of slow movement or hand shaking " or"i have joint pain or swelling. " " i lost body weight without any particular reason, " " i bruise easily or often have a nose bleed, " " i have difficulty swallowing and feel pain while swallowing, " " i often feel nausea or vomit, and the vomitus has a coffee or green color, " " i once had black - colored feces, " " i once had a tight chest and pain radiating to the arm or the back, " " when i exercise, i feel tightness in the chest and become quite breathless, " " my heart suddenly throbs fast or irregularly, " " my face, hand or foot swells easily, " " i feel chest tightness and shortness of breath while lying down or sleeping but feel comfortable sitting down, " " i cough continuously for a long time, " " i have blood in my sputum or expectorated blood, " " i have cold sweats during sleep, " " i have a hoarse breathing sound, " " i feel breathless even after slight activity, " " recently, my urine volume has increased, " " i recently lost my appetite, " " my fingernails or toenails break easily, " " i have difficulty urinating and unsatisfactory feeling after urination, " " i can not stand the urge of urination, " " i have red or cocoa colored urine, " " i have severe pain in the flank or lower abdomen, " " i often wake up to pass urine, " " i once passed urine without knowing, " " i often feel dizziness, " " i have an experience of passing out, " " i once had paralysis in the body, " " i have a symptom of slow movement or hand shaking " or " i have joint pain or swelling. " a total of 772 survey participants were divided into 4 groups according to age : 20 - 40 yr old, 40 - 50 yr old, 50 - 60 yr old and 60 yr or older. the body mass index (bmi, kg / m) was used to indicate obesity. the participants were grouped according to the classification for asians into normal (bmi less than 23), over weight (bmi 23 - 25) and obesity (25 or higher) (7). the extent of smoking in pack years (py) was obtained by multiplying the average number of cigarrettes smoked by the number of years smoked. they were classified into 5 groups : individuals who never smoked, a smoker who stopped smoking in the past, smoker with 10 py or less, smoker with 10 - 20 py, smoker with 20 py or more. with respect to the level of exercises, the respondents were grouped into 5 groups : never exercised, exercised 2 - 3 times monthly, exercised 1 - 2 times weekly, exercised 3 - 4 times weekly and regularly exercised more than 5 - 6 times weekly. the participants were divided into 3 groups according to their educational levels : less than high school graduation, high school graduation to college graduation, and higher than college education. regarding the level of stress, the respondents were grouped into 4 groups : no stress at all, slight stress, moderate stress and a great deal of stress. a chi - square test was used to examine each item for determining a correlation with the development of low back pain. a logistic regression test with a bonferroni 's correction was used for multivariant analysis. a p - value < 0.05 was considered significant. a total of 772 patients were included in this survey. of these 772 patients, 80 (10.4%) responded as having low back pain (table 1). among the six factors examined, age, obesity, smoking, level of exercise, educational level and level of stress, there was a statistically - significant correlation between low back pain and the educational level (p=0.0029, chi - square test), as well as level of exercise (p=0.0409, chi - square test). when the other variables were constant, multivariate analysis indicated that the development of low back pain is not dependent on age, obesity, smoking and level of stress. nevertheless, when the other variables were constant, the development of low back pain was found to be associated with the level of exercise. the patients who exercised regularly 3 - 4 times a week (p=0.028, logistic regression test with bonferroni ' correction) and 5 - 6 times or more a week (p=0.040, logistic regression test with bonferroni ' correction) had a lower chance of developing low back pain than those who did not exercise at all. when the other variables were constant, the educational level was found to be associated with the development of low back pain. the group with high school to college education (p=0.006, logistic regression test with bonferroni 's correction) had a higher chance of experiencing low back pain than the group with a higher than college education (table 2). statistically, there are many issues being considered as risk factors for low back pain. there are largely work - related factors, such as type of work and attitude to life, and individual factors such as gender, age, obesity, level of back muscle development, level of exercise, educational level, economic status, smoking and level of stress (8, 9). this is a retrospective study that analyzed results of a questionnaire at the health & physical examination center. therefore, it was difficult to examine the cause and effect of the various risk factors on low back pain when only a correlation was observed. a sufficient number of samples were used to achieve good accuracy. this study excluded females in whom low back pain might be associated with various gynecologic conditions. by analyzing only male data, analytical errors, which could be brought on by gender differences, was minimized. this study also excluded the survey results with a check mark on various items that forecasted other disease, malignancies and metastatic disorders, and eliminating the causes of other disorders, which would have made it difficult to make differential diagnosis. in this investigation, after excluding the other variables, smoking, age and level of stress did not significantly increase the development of low back pain. there have been many studies in which smoking had been associated with low back pain (10, 11). intervertebral disc degeneration was the cause of low back pain in most of these studies. although direct mechanical pressure following repeated coughing or osteoporosis can cause intervertebral disc degeneration, a blood circulation disorder due to vascular contraction, carboxyhemoglobin generation, atheroma formation and cellulose dissolution problems can also lead to intervertebral disc degeneration (12). intervertebral discs receive their blood supply directly at a young age, but as people age, these discs instead receive their blood supply through diffusion from the adjacent cartilagenous end plate surrounding these discs (13). according to several animal experiments, a blood circulation impediment in the adjacent vasculature can occur, which affects the cellular uptake and solute exchange capacity. this can lead to a reduction in the collagen and proteoglycan level, which are the main constituents of the disc (12, 14, 15). however, many other studies argued that this is not be relevant (16). nevertheless, in this study, there was no statistically significant correlation between smoking and the development of low back pain. previous retrospective studies reported that the incidence of low back pain increased as people aged toward their 50 - 60s, with the incidence of low back pain decreased gradually thereafter (8, 17, 18). several prospective studies have revealed that age is not a significant factor for low back pain (10, 19). although many studies on how mental stress induces low back pain have been carried out (2, 9), most stopped at verifying their correlation using retrospective studies (2, 9). however, considering that mental symptoms such as depression concur with chronic diseases, there are some doubts that mental stress is a cause of low back pain. on the other hand, several retrospective studies demonstrated a correlation between the level of mental stress and low back pain (10, 20). in this study, after excluding other variables, subjects with an educational level of high school graduation and up to less than college graduation had higher chance of developing low back pain than those with college graduation or higher educational levels. the proportion of blue collar workers having a lower socioeconomic status increases with decreasing education level. as the level of education increases, the proportion of white collar workers having to do less difficult work and physical labor increases (2, 21, 22). a correlation between the type of work and low back pain has been reported (23, 24). in these studies, hard manual work, heavy weight lifting, pushing and pulling jobs, jobs at which people are exposed to continuous whole body vibration (i.e., truck drivers) and work that requires sitting for long periods of time have been considered relevant risk factors (2, 4, 25, 26). there are many reports asserting that physical labor causes low back pain (4, 23). however, there was a report showing that such hard labor did not specifically induce low back pain (20). nevertheless, many studies concurred with the fact that hard physical labor leads to degenerative disc changes to the extent that they could be discovered radiographically (27). furthermore, even without having done hard physical work, bending down or twisting the waist frequently while sitting for a long time increases the chance of experiencing low back pain (28). however, in this study, subjects with less than high school graduation did not have a high rate of low back pain compared with the college graduates. most of the twenty - seven subjects with a level of education less than high school graduation were actually high income earners of 4 million won (about $ 4,000) per month or higher (13). the reason behind the low rate of low back pain might be due to the fact many of them do not perform hard physical labour. the mechanisms by which exercises may prevent low back pain are believed to be : 1) they strengthen the back muscles and increase trunk flexibility ; 2) they increase blood supply to the spine muscles and joints and intervertebral disks, minimizing injury and enhancing repair ; and 3) they improve mood and therby alter the perception of pain (5, 29 - 31). however, it has not clearly been determined what type and how much exercises one should carry out (31). moreover, it is unclear if a reduction in body weight to a normal level is essential for its effectiveness or if long exercise is truly effective. in this study, excluding other variables, patients who exercised 3 - 4 times weekly or 5 - 6 times regularly every week were able to decrease the low back pain significantly compared to patients who did not exercise at all. many studies have examined the correlations between obesity and low back pain. many have insisted that obesity might have been the cause of low back pain (10, 22, 32). however, recent studies carried out by excluding many variables showed otherwise (33, 34). indeed, a regular exercise prorgram performed 3 - 4 times weekly not to the extent of the reducing body mass index was found to be most effective in reducing low back pain. | many factors are associated with the development of low back pain. among them, exercise, obesity, smoking, age, educational level and stress are the most common. this study examined the association of these factors with low back pain. an additional aim was to determine a procedure for preventing low back pain. this study analyzed the responses to a questionnaire sent to 772 individuals who had undergone a medical examination at this hospital in 2003 and excluded the individuals who had shown symptoms or their test results indicated a particular disease. assuming that there were no variables, individuals who exercised regularly 3 - 4 times per week would have a lower chance of having low back pain than those who did not exercise regularly. the analysis revealed that individuals with a college degree or higher education have a lower chance of experiencing low back pain than those with only a high school education or even college drop - outs. when the other variables were constant, age, extent of obesity (body mass index), smoking and level of stress were not found to affect the development of low back pain. the level of education was associated with the development of low back pain. however, regular exercise 3 - 4 times per week or more would be most effective in reducing the incidence and duration of low back pain. |
cell - to - cell and interorgan exchange of nutrients and information is essential for growth, development, successful reproduction, and environmental adaptation of multicellular organisms. higher land plants have evolved a plant - specific symplastic network connected via plasmodesmata (pd), cytoplasmic connections between plant cells, which allow a locally restricted exchange of information. in addition, a rapid transfer of information over longer distances is enabled by the phloem. pd and the phloem thus provide a continuous symplastic connection between even the most distant plant organs. the phloem of higher land plants is a channel system mainly responsible for the allocation of organic nutrients that are produced during photosynthesis (photoassimilates) from source organs (those with a net production of photoassimilates, such as green leaves) to sink organs (those with a net consumption, such as roots or fruits). together with the xylem (dead tubes that transport water from root to shoot), the phloem is located in vascular bundles. the transport tubes of the phloem, the so - called sieve elements (ses), are living but partially degenerated cells that lose important cellular components such as vacuoles, the nucleus, or ribosomes during maturation. as a consequence, ses have presumably lost the capacity for transcription and translation, resulting in their inability to synthesise rnas or proteins. the specific maturation and their interconnection with other adjacent ses make these tubes suited for efficient and quick substance transport, but hardly viable. for survival, ses are therefore always intimately associated with their neighbouring companion cells (ccs), and both emerge from the division of a single mother cell. ccs remain fully equipped and develop high metabolic activity in order to provide ses with all the vitally important compounds. to allow a steady transfer of molecules of different molecular classes and sizes, ccs and ses are connected by special pd, namely the pore plasmodesmal units, which are formed by multiple channels on the cc side that lead into a single pore on the se side. as a result, the size of molecules allowed to pass these pd, the so - called size exclusion limit (sel), is quite high (> 67 kda) as compared with that of other pd (1 kda). in addition, the capacity of many phloem polypeptide compounds to further increase the sel should, in principle, also enable macromolecules to pass through. it is well known that many plant viruses (macromolecular structures consisting of nucleic acids and proteins) exploit this symplastic pathway to spread from cell to cell and systemically through the entire plant. it was only recently discovered that a high number of plant endogenous macromolecules, such as proteins and rnas, are also present within the phloem, which suggested that they might act as information - transmitting molecules in a diverse range of physiological processes. however, only recent advances have confirmed that some of these endogenous macromolecules can indeed be transported in living plants to influence developmental processes, as well as stress and defence responses. in addition, advances mainly from plant virus research allow new insights into the mechanisms that underlie the systemic translocation of macromolecules. long - distance transport of macromolecules was first seen by observing the spread of viruses that misuse the phloem for their passive translocation through the plant, leading to the spread of the infection, especially into growing areas. after infection, viruses normally spread locally from cell to cell through pd until they reach the phloem. viruses contain special proteins, namely movement proteins (mps), that can bind and unfold single - stranded rnas and facilitate their intercellular translocation by building protein - rna transport complexes [ribonucleoprotein (rnp) complexes ]. several studies indicate that cell - to - cell and systemic virus transport follow different mechanisms [5 - 7 ], and there seem to be different viral and endogenous plant components involved [7 - 10 ]. in addition to mp, an important viral factor for phloem movement is the coat protein that seems to be strictly required for long - distance translocation of several groups of viruses. with regard to endogenous plant factors, a cell wall glycine - rich protein and a pectin methylesterase have been shown to influence viral spread, probably by modulating the permeability of pd. an interesting recent finding is that the interaction of the groundnut rosette virus orf3 protein with subnuclear cajal bodies and the nucleolus is required to capture the plant protein fibrillarin to form an rnp complex capable of long - distance phloem movement. also, endogenous phloem proteins, namely different phloem lectins and the phloem protein cmpp16 (a protein with properties resembling those of virus mps), have been found to interact with viral rnas and endogenous mrnas and thus have been suggested to be involved in virus import and/or translocation as rnp complexes, but a conclusive demonstration of their importance for viral spread is, as yet, missing. the occurrence of unspecified rna species was described nearly 40 years ago, but it was long regarded as an artefact caused by sampling. the first evidence that endogenous plant mrnas are present in ses came from the in situ localisation of the mrna of a sucrose transporter in ses and the pd connecting ses to ccs. meanwhile, a range of different mrnas from phloem samples of various species, obtained using different sampling techniques, have been detected (reviewed in) and they are nowadays regarded as being authentic components of the phloem stream. at the same time, heterografting studies performed by different research groups could show that specific transcripts can indeed move and, moreover, induce phenotypic alterations in their target tissues [20 - 22 ]. for example, in potato, overexpression of the transcription factor bel1 mrna induced a marked increase of tubers per plant, and this phenomenon could cross graft junctions, thus indicating phloem mobility of this mrna. recently, small rnas of fewer than 30 nucleotides [short interfering rnas (sirnas) and micrornas (mirnas) ] were found to occur in the phloem stream [24 - 26 ]. sirnas are involved in a process called post - transcriptional gene silencing, an innate plant defence mechanism against transposable elements and viruses that can spread systemically throughout the plant. while sirnas are generally assumed to be mobile between cells and also systemic, most mirnas, involved mainly in controlling developmental processes, seem to act cell - autonomously under normal growth conditions in adult tissues, as demonstrated, for example, by mir171. however, it was recently shown that specific nutrient starvation - responsive mirnas (such as mir395 that responds to sulphur, mir398 to copper, and mir399 to phosphate stress) can accumulate in the phloem at high levels when plants are grown under the respective nutrient - deprived condition. moreover, different results suggested that during phosphate deprivation systemic signalling is required, and recently mir399 was shown to be indeed translocated across graft junctions in the model species arabidopsis, indicating that phloem movement of this specific mirna could be involved in regulating the response toward phosphate starvation systemically. this observation, however, does not answer the question of whether mirnas are generally mobile or immobile from cell to cell and over long distances. there might exist a small subset of specific mobile mirnas, or mirna mobility might be restricted to particular developmental stages and/or specific environmental conditions. also, the occurrence of proteins in phloem sap was noticed a long time ago and since then different studies that comprehensively identified phloem proteins from various species have been published [36 - 38 ]. however, the capability of the polypeptides to be transported in vivo has been demonstrated for only a few of them (for example, the pumpkin phloem protein cmpp16 that, as mentioned above, displays similarities to viral mps). moreover, the physiological functions of most of the phloem polypeptides remain unknown. for many of them, roles in signal transduction and stress responses, the solanaceae - specific peptide systemin served as the paradigm for a phloem - mobile protein involved in long - distance signalling, but according to more recent results, it seems rather likely that it is jasmonic acid and not systemin that constitutes the phloem - translocated signal during wound responses. to date, the only convincing example of a phloem protein involved in signalling is the flower - promoting protein flowering locus t (ft), which was recently shown to be present in the phloem at detectable concentrations when plants were sampled at the onset of flowering. subsequently, several independent studies have provided compelling evidence that ft long - distance transport can indeed contribute to flower induction in different plant species [42 - 46 ], and the ft protein could thus be a component of the long - sought florigen. further studies are required to find out whether any of the other phloem sap proteins can act in the information transfer during other developmental processes or stress responses. although the transport mechanisms of endogenous macromolecules are not well understood, it is assumed that phloem import and transport of both proteins and rnas are mediated by specific binding proteins acting as molecular chaperones. it was proposed that phloem heat shock proteins might participate in delivering polypeptides to the phloem, but other components with functions related to protein folding (for example, the abundant phloem cyclophilins) could also be involved. several phloem - sap proteins have also been suggested to transport rnas in the form of rnp complexes since they are rna - binding, are translocatable across graft unions, and can increase the sel of pd. in particular, the pumpkin phloem sap protein cmpp16 possesses properties similar to those of viral mps and mediates the transport of its own and foreign mrnas from cell to cell and over long distances through the phloem, as shown by microinjection studies and grafting experiments. small rna transport in pumpkin seems to follow a similar route mediated by the small rna - binding phloem protein cmpsrp1 that, however, seems to be restricted to this one plant species. in other plants, one or more of the various rna - binding phloem proteins that have been described to occur in phloem sap could possibly do the job. also, the obvious similarities between the translocation of viruses and endogenous macromolecules suggest that the underlying mechanisms are the same or at least highly similar, but both will need to be demonstrated in future experiments. long - distance transport of macromolecules was first seen by observing the spread of viruses that misuse the phloem for their passive translocation through the plant, leading to the spread of the infection, especially into growing areas. after infection, viruses normally spread locally from cell to cell through pd until they reach the phloem. viruses contain special proteins, namely movement proteins (mps), that can bind and unfold single - stranded rnas and facilitate their intercellular translocation by building protein - rna transport complexes [ribonucleoprotein (rnp) complexes ]. several studies indicate that cell - to - cell and systemic virus transport follow different mechanisms [5 - 7 ], and there seem to be different viral and endogenous plant components involved [7 - 10 ]. in addition to mp, an important viral factor for phloem movement is the coat protein that seems to be strictly required for long - distance translocation of several groups of viruses. with regard to endogenous plant factors, a cell wall glycine - rich protein and a pectin methylesterase have been shown to influence viral spread, probably by modulating the permeability of pd. an interesting recent finding is that the interaction of the groundnut rosette virus orf3 protein with subnuclear cajal bodies and the nucleolus is required to capture the plant protein fibrillarin to form an rnp complex capable of long - distance phloem movement. also, endogenous phloem proteins, namely different phloem lectins and the phloem protein cmpp16 (a protein with properties resembling those of virus mps), have been found to interact with viral rnas and endogenous mrnas and thus have been suggested to be involved in virus import and/or translocation as rnp complexes, but a conclusive demonstration of their importance for viral spread is, as yet, missing. the occurrence of unspecified rna species was described nearly 40 years ago, but it was long regarded as an artefact caused by sampling. the first evidence that endogenous plant mrnas are present in ses came from the in situ localisation of the mrna of a sucrose transporter in ses and the pd connecting ses to ccs. meanwhile, a range of different mrnas from phloem samples of various species, obtained using different sampling techniques, have been detected (reviewed in) and they are nowadays regarded as being authentic components of the phloem stream. at the same time, heterografting studies performed by different research groups could show that specific transcripts can indeed move and, moreover, induce phenotypic alterations in their target tissues [20 - 22 ]. for example, in potato, overexpression of the transcription factor bel1 mrna induced a marked increase of tubers per plant, and this phenomenon could cross graft junctions, thus indicating phloem mobility of this mrna. recently, small rnas of fewer than 30 nucleotides [short interfering rnas (sirnas) and micrornas (mirnas) ] were found to occur in the phloem stream [24 - 26 ]. sirnas are involved in a process called post - transcriptional gene silencing, an innate plant defence mechanism against transposable elements and viruses that can spread systemically throughout the plant. while sirnas are generally assumed to be mobile between cells and also systemic, most mirnas, involved mainly in controlling developmental processes, seem to act cell - autonomously under normal growth conditions in adult tissues, as demonstrated, for example, by mir171. however, it was recently shown that specific nutrient starvation - responsive mirnas (such as mir395 that responds to sulphur, mir398 to copper, and mir399 to phosphate stress) can accumulate in the phloem at high levels when plants are grown under the respective nutrient - deprived condition. moreover, different results suggested that during phosphate deprivation systemic signalling is required, and recently mir399 was shown to be indeed translocated across graft junctions in the model species arabidopsis, indicating that phloem movement of this specific mirna could be involved in regulating the response toward phosphate starvation systemically. this observation, however, does not answer the question of whether mirnas are generally mobile or immobile from cell to cell and over long distances. there might exist a small subset of specific mobile mirnas, or mirna mobility might be restricted to particular developmental stages and/or specific environmental conditions. also, the occurrence of proteins in phloem sap was noticed a long time ago and since then different studies that comprehensively identified phloem proteins from various species have been published [36 - 38 ]. however, the capability of the polypeptides to be transported in vivo has been demonstrated for only a few of them (for example, the pumpkin phloem protein cmpp16 that, as mentioned above, displays similarities to viral mps). moreover, the physiological functions of most of the phloem polypeptides remain unknown. for many of them, roles in signal transduction and stress responses the solanaceae - specific peptide systemin served as the paradigm for a phloem - mobile protein involved in long - distance signalling, but according to more recent results, it seems rather likely that it is jasmonic acid and not systemin that constitutes the phloem - translocated signal during wound responses. to date, the only convincing example of a phloem protein involved in signalling is the flower - promoting protein flowering locus t (ft), which was recently shown to be present in the phloem at detectable concentrations when plants were sampled at the onset of flowering. subsequently, several independent studies have provided compelling evidence that ft long - distance transport can indeed contribute to flower induction in different plant species [42 - 46 ], and the ft protein could thus be a component of the long - sought florigen. further studies are required to find out whether any of the other phloem sap proteins can act in the information transfer during other developmental processes or stress responses. although the transport mechanisms of endogenous macromolecules are not well understood, it is assumed that phloem import and transport of both proteins and rnas are mediated by specific binding proteins acting as molecular chaperones. it was proposed that phloem heat shock proteins might participate in delivering polypeptides to the phloem, but other components with functions related to protein folding (for example, the abundant phloem cyclophilins) could also be involved. several phloem - sap proteins have also been suggested to transport rnas in the form of rnp complexes since they are rna - binding, are translocatable across graft unions, and can increase the sel of pd. in particular, the pumpkin phloem sap protein cmpp16 possesses properties similar to those of viral mps and mediates the transport of its own and foreign mrnas from cell to cell and over long distances through the phloem, as shown by microinjection studies and grafting experiments. small rna transport in pumpkin seems to follow a similar route mediated by the small rna - binding phloem protein cmpsrp1 that, however, seems to be restricted to this one plant species. in other plants, one or more of the various rna - binding phloem proteins that have been described to occur in phloem sap could possibly do the job. also, the obvious similarities between the translocation of viruses and endogenous macromolecules suggest that the underlying mechanisms are the same or at least highly similar, but both will need to be demonstrated in future experiments. the results from recent publications enhance our understanding of why, when, and how viruses and endogenous macromolecules can travel long distances through the phloem. on one hand, they have begun to shed light on the complex mechanisms of the long - observed systemic spread of viruses within plants. on the other hand, they have established that specific endogenous phloem proteins and rnas can be transported long distances in vivo following certain triggers. while the translocation of viruses clearly serves to spread infection, the transport of endogenous macromolecules might contribute to communicate essential information during developmental processes or stress responses as, for example, for the flower - promoting ft protein or the phosphate - deprivation - induced mir399. whether this holds true for only a small subset of endogenous macromolecules, or whether there are much more important physiological decisions influenced by the wide range of molecules found in the phloem, requires further investigation. to this end, it will be interesting to comprehensively identify the (sub)set of endogenous macromolecules that can really move long distances in vivo and to analyse the direction of movement and the triggers (developmental and environmental) that initiate transport. it will also be important to identify additional factors and interaction partners required for the movement of different viruses and endogenous macromolecules in order to establish the prerequisites for phloem mobility and import and transport selectivity. this knowledge will be the basis for creating plants with, for example, increased virus or stress resistance, or for influencing important developmental processes by effecting which macromolecules can ride on the phloem information highway. | long - distance phloem transport of small metabolites has long been the subject of many different studies concentrating on resource allocation and signalling between plant organs. also, phloem movement of viruses has long been examined as the route for systemic infection of the plant. only recently, the transport of macromolecules, such as proteins and nucleic acids, has received increasing attention because they are regarded as being a new class of potential information - transmitter. a set of recent publications allows the first insights into the important roles that phloem - mobile macromolecules might play in the regulation of development and the responses to stress. furthermore, they start to shed light on the mechanisms involved in systemic macromolecule transport. |
in 1980 the health care financing administration (hcfa), in cooperation with the health policy center of brandeis university, began planning a demonstration program to provide and finance long - term care services. this demonstration program uses a social health maintenance organization (s / hmo) to combine acute and long - term care services under a single prepaid health care plan for the elderly. s / hmo benefits include all basic acute and ambulatory health care services covered by medicare as well as selected chronic care services. these services are paid for by medicare on a capitated basis, along with a premium paid by the enrollee, or when applicable, medicaid. the demonstration was designed to address the health care and health insurance needs of both a severely impaired and a well group of elderly. beneficiaries meeting their state 's nursing - home - certifiable criteria at enrollment receive an adjusted medicare capitated rate, which is higher than the adjusted average per capita cost (aapcc) rate established for the well enrollees. although all enrollees are eligible for basic medicare benefits, only those assessed as nursing home certifiable or " at risk " of nursing home placement are eligible for chronic care benefits. the availability of long - term chronic care benefits, and the associated case management processes, distinguish an s / hmo from a health maintenance organization (hmo) ; these features plus the capitation payment distinguish an s / hmo from fee - for - service health care. the demonstrations began in early 1985 and will continue for 7 years under the current terms of the program. the four sites involved include : elderplan in brooklyn, new york, sponsored by metropolitan jewish geriatric center ; medicare plus ii in portland, oregon, sponsored by kaiser permanente center for health research ; seniors plus in minneapolis, minnesota, sponsored by group health inc. and ebenezer society ; and scan health plan in long beach, california, sponsored by senior care action network. although under different organizational auspices, all s / hmo 's share the common purpose of testing the ability of these health plans to attract members and to provide an expanded array of acute and chronic care benefits within a capitated budget. hcfa has commissioned a comprehensive evaluation of the s / hmo 's by a consortium of researchers. the prime contractor is the institute for health & aging at the university of california ; others include berkeley planning associates, the center for demographic studies at duke university, and westat, inc. it explores the cost effectiveness of each s / hmo demonstration model as well as internal operations and environmental factors affecting program outcomes. the evaluation investigates nine major issues : selection bias in initial enrollment and in attrition. public, third party, and out - of - pocket expenditures for acute and chronic care services. marketing efforts, the market area, and environmental conditions affecting each site 's operations. patterns of change in the organizational form, management, and financing at each site as the programs evolve. the effectiveness of case management in controlling chronic care service use and cost and assuring access to appropriate levels of care. an interim evaluation report covers organizational and operational issues (health care financing administration, 1988). current field work is gathering health status, health service utilization, and out - of - pocket expenditure data on s / hmo enrollees and the fee - for - service comparison group. the focus of this article is on case management roles and activities across the four s / hmo demonstration sites. the information presented is drawn from data gathered during site visits in 1986 and from progress reports during the first 2 years of operation of the s / hmo 's, which began enrollment in 1985. throughout this article, emphasis is placed on the 1986 data, which represent a more stable program period and are less susceptible to variations experienced during the initial startup period. it is important to note that the s / hmo is an evolving form, and as such, there have been some important changes in case management and its activities since this article was prepared. the information provided here describes s / hmo case management as it functioned at the close of the second year of the demonstration and does not reflect changes made as the plans assumed full financial risk. at each s / hmo demonstration site, the case management component was given responsibility for managing the non - acute, long - term care services. in the s / hmo, the role and authority of the case manager was envisioned as much broader than in earlier long - term care demonstrations (leutz., 1985). in most earlier demonstrations, the case manager role focused primarily on screening and assessment and the coordination and/or authorization of community - based care (austin, 1983 ; berkeley planning associates, 1985 ; health care financing administration, 1988 ; zawadski, 1984). in the s / hmo, the case manager was to have primary responsibility for authorizing all long - term care services, responsibility for monitoring the chronic care services and budget, and final authority over chronic care resource allocation. beyond this, it was hoped that the case management component would also be able to establish new norms of practice regarding linkages with other components of the health care system. it was also hoped that these norms would lead to care of a consistently high quality that would simultaneously maintain chronic care costs at budgeted levels. within these broad goals and objectives, each demonstration plan was permitted flexibility in developing its case management model. as implemented, no two case management models were alike. there were no restrictions placed on the plans by hcfa as to which members should be eligible to receive the expanded long - term care benefits. during the planning phase, there was debate about the advisability of limiting services to the severely impaired versus providing services to less impaired members as a preventive measure. in addition, because the plans were reimbursed at a higher capitated rate for members assessed as meeting their state 's nursing home certification (nhc) criteria, it was decided to link eligibility for the chronic care services, at least in part, to nursing home status. although each plan paralleled its state nhc form and guidelines to qualify a member for chronic care benefits (and a higher reimbursement rate), the nhc criteria were different among the four demonstrations. (forty - five variables were used for certification and only 11 were common to all plans.) each plan also expanded eligibility criteria beyond nhc, allowing at least some services to members who were not nursing home certifiable (henceforth referred to as certifiable), but who were considered to be at risk of future institutionalization by case management staff. the final determination of eligibility for chronic care services and/or case management was determined by interrelated factors : the stringency of the state 's nursing home criteria and the plan 's application of these criteria ; the extent to which the plan permitted provision of chronic care services to less impaired members ; and the extent to which the plan provided case management services to members who did not qualify for chronic care services. the eligibility criteria of the four plans can be placed on a continuum ranging from the most restrictive to the least restrictive. seniors plus and scan health plan were much less restrictive, electing to provide preventive services to moderately impaired members. at all of the sites, the case management department was the single entry point into the chronic care service component of the s / hmo. initially, every new enrollee received by mail a baseline health status form (hsf). if this form, or a followup telephone screening to review hsf responses, indicated that the person might be impaired, a case manager conducted an in - person comprehensive health assessment. telephone screenings and comprehensive assessments could also be triggered by referrals from s / hmo medical staff and other service providers. following the comprehensive assessment, a final determination was made about eligibility for chronic care services. given the differences in the eligibility criteria and processes at the four sites, it was not surprising to find that different proportions of the total membership were being permitted access to chronic care services. in table 1 one can see the numbers and proportions of the s / hmo membership who were certifiable, of the members who were receiving chronic care services, and of the members who were receiving case management services only during the fourth quarter of 1986. also shown, where available, are plan projections of case mix made during the planning stages of each s / hmo. medicare plus ii projected that only 5 percent of its membership would be certifiable and only those enrollees would receive chronic care services. by the fourth quarter of 1986, the s / hmo had 4,300 members ; 6.7 percent were certifiable, but only 4.5 percent (67 percent of the certifiable group) were receiving chronic care services. another 4 percent of members received case management services only (i.e., periodic monitoring). the proportion of certifiable members was larger than the proportion of members receiving chronic care services for two reasons. second, during the first 24 months of the demonstration, there was an anomaly in the state of oregon 's nhc criteria that resulted in several incontinent medicare plus ii members being evaluated as certifiable, although they did not actually need services. at elderplan, the actual number of functionally impaired persons receiving chronic care benefits was considerably lower than that projected in its demonstration protocol. elderplan projected that 13.8 percent of its members would be impaired and using chronic care services at any given time. of the four plans, elderplan had the highest projected use of chronic care benefits. by the fourth quarter 1986, this s / hmo had 2,502 members ; 4.1 percent were certifiable, and only 2.9 percent were receiving chronic care services (i.e., approximately 71 percent of certifiable members were receiving chronic care services). seniors plus projected that 4.3 percent of its members would be certifiable, but that 8.5 percent of enrollees would receive chronic care benefits. this reflected this plan 's orientation toward using chronic care services as a form of third - level safeguard to forestall further deterioration of the condition of moderately or severely impaired members at risk of becoming certifiable. by the fourth quarter of 1986, the s / hmo had 1,688 members ; 7.2 percent of enrollees were certifiable ; 11 percent of the members were receiving chronic care services ; and an additional 8.2 percent of members received case management services only. scan health plan also followed the seniors plus strategy of preventive use of chronic care services and projected that a larger proportion of its membership would receive those services (10.0 percent) than would be certifiable (4.0 percent). as was the case for seniors plus, the actual membership was more impaired than was anticipated. by the fourth quarter of 1986, scan health plan had 2,061 members ; 5.5 percent were certifiable ; 12.1 percent were receiving chronic care services ; and 7.9 percent received case management monitoring only. these data indicate that at the end of the second year of the demonstration, two of the plans were providing chronic care benefits (either case management or chronic care services) to a large proportion of the membership who were assessed by case managers as " at risk " of institutionalization but who did not meet their state 's nhc criteria. at scan health plan, only 28 percent of those receiving chronic care benefits were certifiable ; at seniors plus, the percentage was only slightly higher35 percent. in sharp contrast, at elderplan and medicare plan ii, at least three - fourths of the chronic care recipients were certifiable. these differences are noteworthy because as the plans assumed full financial risk in year three, scan health plan, citing fiscal reasons, petitioned hcfa and changed its eligibility criteria, offering chronic care services only to members who were certifiable. in large part, the roles, responsibilities, and authority given to the case management component of the demonstration plans were determined by their organizational model whether the plan 's principal sponsor was an established hmo or a long - term care provider that created a newly formed hmo. the organizational model was also a key determinant of case management department linkages to the larger s / hmo delivery system. indeed, the organizational model often determined whether the case managers had direct control over a service or whether the case manager 's role was primarily that of coordinating with another health care provider. the major difference in case management practices between the established hmo and long - term care organization s / hmo models is found in the degree of involvement of s / hmo case managers in monitoring acute care utilization. the two s / hmo 's affiliated with established hmo 's (medicare plus ii and seniors plus) chose to leave primary responsibility and control over acute care utilization (i.e., hospital and post - hospital, skilled nursing home, and home health care) to their respective hmo utilization review and discharge planning staff. the s / hmo 's founded on preexisting long - term care organizations (scan health plan and elderplan) attempted to gain control over acute care utilization by assigning part of the utilization review and discharge planning responsibilities to the case management component of the s / hmo. both plans experienced considerable difficulty with the original providers when they attempted to place case managers in these new roles. for example, at one site the case manager role was a source of contention with the regular hospital utilization review and discharge planning staff, who maintained that the job could be done more efficiently and effectively in - house. a more detailed discussion of organizational factors influencing hospital and nursing home utilization is presented in the interim congressional report (health care financing administration, 1988). other case managers ' roles were similar across all plans, reflecting traditional case management functions (e.g., assessment, care planning, and service arrangement). at all sites, the case managers were responsible for coordinating the fairly comprehensive array of institutional and community - based long - term care services that constituted the chronic care benefit package. in addition, case managers routinely contacted non - s / hmo service providers to obtain information or refer members for services not covered by the s / hmo, such as legal help, social security assistance, shared housing, friendly visitors, and senior centers. face - to - face meetings or telephone calls were conducted with caregivers to negotiate care plans, explain benefits and copayments, clarify client needs, help the family accept client disabilities, facilitate family interaction, identify tasks family members could reasonably perform, and support family caregiving efforts. all plans had written guidelines specifying that no chronic care services could be authorized without first exploring the availability of potential help from informal caregivers. at each of the sites, in most cases this required the development of a long - term chronic care service utilization plan to prevent clients from exhausting their benefits. additionally, the plans had somewhat different benefit periods and cost - sharing arrangements (health care financing administration, 1988). it is important to place in perspective the actual dollar amount available for a case manager 's use in developing a chronic care plan. for example, scan health plan offered a fairly generous $ 7,500 annual benefit, yielding an average monthly benefit of $ 625. if an elderly person with alzheimer 's disease needed day care 3 days a week (at $ 27 per day) and a home health aide to assist the family with the member 's personal care needs for 2 hours (at $ 8.75 per hour) on the days when day care was not attended, the weekly cost would be $ 151, which would exceed the budget. for members with short - term or time - limited chronic care service needs, benefit limits usually posed no problem. for a highly impaired person requiring services on an ongoing basis, services had to be carefully allocated to maximize member benefits. to date, it appears that the case managers have been able to monitor and maximize benefits with considerable success. for example, during the fourth quarter of 1986, the plans reported that only a small number of members had exhausted their benefits by the end of the second year of the demonstration, plans varied considerably in the extent to which the acute and long - term services had been integrated to provide an effectively coordinated continuum of care for impaired elderly. in general, at medicare plus ii, s / hmo case mangement was a fairly insular unit functioning with a reasonable, although not a high, degree of coordination with the larger kaiser system. most physicians and providers generally did not know whether a member was in the medicare plus ii program. the larger kaiser system remained responsible for acute hospital care, medical care, and medicare - covered home or nursing home care. the s / hmo case managers were only responsible for the expanded chronic care services and budget. at elderplan, there was little evidence of an integrated service system. it appeared that the s / hmo could have benefited from stronger ties and coordination between the medical / hospital service components and the case management component responsible for long - term care. a large number of medical specialists operating independently of the s / hmo were responsible for acute hospital care. the primary care physician group was under contract to the s / hmo but was not closely linked to the case management component. in an attempt to gain control over hospital utilization, a case manager was assigned the utilization review and discharge planning functions, but did not have the power or authority to influence practice patterns. further, working relationships between key personnel in the principal acute care hospital and the case managers were not strong. long - term institutional and in - home care were the sole responsibility of the case managers, with little input from the medical component. in contrast to elderplan, seniors plus appeared to effectively integrate the s / hmo case management department with two strong preexisting service providers group health, inc. and ebenezer society. at each level of health care acute hospital, outpatient clinic, nursing home, community - based services, and in - home services there were well defined, closely coordinated working relationships between the case managers and the other service providers. in turn, the other service providers recognized that when a s / hmo client required chronic care services, the case managers then had control over chronic care service use and cost. continued coordination between the acute care and long - term care service teams was evidenced in the case conference meetings attended by the medical director, select physicians, the home care service director from ebenezer, the geriatric nurse practitioner who managed nursing home care, and the s / hmo case managers. in the scan health plan, a large number of health care providers could be involved in a member 's plan of care. even though the scan program had a number of years of experience working with many of the providers in the multipurpose senior services program (mssp), the s / hmo case management department faced a tremendous challenge as it attempted to coordinate this diverse group of service providers (more than 100) into a comprehensive continuum of care for the impaired s / hmo members. each type of health care was provided by a different vendor under contract. with the assistance of the scan health plan medical director, the role of the case management department was to coordinate this array of service providers. at the end of the second year of the demonstration, based on service provider interviews, it appeared that some of the linkages needed to be strengthened and better coordinated especially between the s / hmo case managers and the primary care physicians, the medical specialists, and the hospital discharge planning unit. a detailed description of the acute and long - term care service delivery systems at each site at the end of the second year of the demonstration is provided in the congressional report (health care financing administration, 1988). this report also provides information on the health care environment and the market in which each of the plans was implemented. this preliminary review of the case management component of the s / hmo demonstrations has identified a number of issues and questions to be examined in subsequent analyses. in addition, the ongoing research agenda contains specific questions about s / hmo members who are eligible for and are receiving chronic care benefits. how do the health and functional limitations of members receiving chronic care services and case management vary across plans ? to what extent do plans provide case management to persons determined not to be nursing home certifiable ? what are the implications of this for state nursing home criteria, plan resource allocation, and beneficiary satisfaction ? how do services differ between members assessed as nursing home certifiable and members assessed as " at risk " of institutionalization ? what is the potential impact on client case mix and program revenues of standardizing nursing home certification criteria ? what are the quantitative and qualitative differences in each plan 's approach to case management for members with the same primary diagnosis ? what out - of - pocket costs are incurred for noncovered long - term care services ? can case managers remain within chronic care budgets as the membership ages and frailty advances ? should case managers participate in hospital utilization review and discharge planning ? what impact will multiple vendor agencies have on case management work load and member satisfaction ? will case managers be able to develop formal chronic care standards and protocols for issues such as normal usage, equity of service allocation, and quality of care ? what similarities and differences are evolving between case management in the s / hmo 's and earlier long - term care demonstrations ? what aspects of the s / hmo case management approach are transferable to other prepaid health care settings ? | in this article, case management departments and roles during the early years of the social health maintenance organization (s / hmo) demonstrations are compared. these organizations provide acute and chronic care services under a prepaid plan for the elderly. eligibility criteria for case management and chronic care services at each site are compared, followed by a description of the resultant case mix of members receiving chronic care benefits. case managers principal activities are described, and a preliminary assessment is made about the strength of the linkages that have been developed between the case management component of these plans and the larger health care system. |
the majority are benign ; however, inflammatory symptoms (pain, reddened skin) and facial asymmetry are characteristics that strongly suggest a malignant lesion. the duration of complaints in such a histologically diverse group of benign tumors and malignancies can vary greatly. facial nerve weakness alarms the patient, family, and general practitioner and prompts an earlier referral and consultation. preoperative facial nerve palsy is a manifestation of malignant infiltration and so far, the only preoperative indicator for planning total parotidectomy with nerve resection and facial reanimation [3, 6, 15 ]. when the facial nerve function is intact, early involvement with tumor can not be definitively ruled out. preoperative imaging (ultrasonography, computed tomography, and magnetic resonance) do not have the resolution for nerve structure visualization. therefore, intraoperative nerve assessment is crucial in the decision making process as to whether the nerve should be resected. the aims of the study are to (1) assess the frequency of facial paralysis and undiagnosed nerve infiltration in patients with parotid malignancies ; (2) analyze the duration of symptoms before presentation ; and (3) define the risk factors for nerve impairment and the impact of facial nerve involvement on treatment methods, outcomes, and survival. one hundred and three patients had surgery for parotid gland malignancies in a single institution, the tertiary center for ent as the university department in pozna between 1996 and 2006. brackmann 6-score scale (h - b), was identified at the initial presentation in 32 patients : 28 had total facial palsy (h - b 6) and 4 had partial facial palsy (h - b 2 - 5). in five other patients, intra - operative features of facial nerve infiltration were found although the facial mobility was normal preoperatively (h - b 1). the study group included 53 women and 50 men ranging in age from 12 to 87 years, with a mean age of 55 years. the tumor histology was assessed according to who1991 classification, modified in 2003 : adenoid cystic carcinoma27, acinic cell carcinoma 9, undifferentiated carcinoma8, mucoepidermoid carcinoma8, squamous cell carcinoma18, anaplastic carcinoma3, adenocarcinoma7, lymphoma malignum5, ex - pleomorphic carcinoma6, and metastases12. twelve cases assessed on the base of histology as metastases had primaries in : skin (two malignant melanoma), kidney (two clarocellular cancer), prostatic gland (two adenocarcinoma), and upper aerodigestive tract (six squamous cell carcinoma). in preoperative oligobiopsy atypical cell or malignant cell were diagnosed in all cases from metastases group. thus, these patients were primarily graded with t stage. finally, 2 weeks after the procedure, the pathological examination revealed metastases and the further diagnostics of the primary was carried up in further analysis, this group would be distinguished and its outcomes compared with other histological entities. the stage of the primary tumor in the examined group of 103 patients is as follows : 20t1, 31t2, 20t3, 32t4a. the diameter of the tumors in preoperative imaging (ultrasonography, computed tomography, or magnetic resonance imaging) ranged from 1 to 9 cm, with a mean 3 cm. the duration of symptoms before the initial presentation ranged between 1 and 120 months, with a mean of 12 months. the mean duration of complaints in patients with facial palsy was 16.8 months and the mean duration of complaints in patients without facial palsy was 14.1 months. the duration of complaints for a particular histologic diagnosis was as follows : adenoid cystic carcinoma15 months, acinic cell carcinoma7 months, undifferentiated carcinoma7 months, mucoepidermoid carcinoma15 months, squamous cell carcinoma6 months, and adenocarcinoma34 months. taking into consideration the cancer histology, facial nerve paresis was noted in : 11 out of 27 with adenoid cystic, 5 out of 9 with acinic cell, 4 out of 8 with undifferentiated, 3 out of 8 with mucoepidermoid, 6 out of 18 with squamous cell, 4 out of 6 with ex - pleomorphic cancer, 1 out of 7 with adenocarcinoma, and in 0 out of 20 patients from the rest of the group (12 with metastases in parotid, 5 with lymphoma malignum, and 3 with anaplastic carcinoma). all patients were treated surgically with the intent for complete resection. in most cases, the facial nerve was identified by anterograde dissection, but in some it was identified by the retrograde technique. the data were analyzed statistically by chi - square test to compare categorical variables between groups or non - parametric kruskal the overall survival results were calculated using the kaplan meier method and differences were analyzed by the log - rank test. the correlation between facial nerve function before treatment and patients characteristics, including the treatment methods, were analyzed. facial paresis at the initial presentation was found in 32 patients (28 total, 4 partial), equally distributed in male and female patients. the duration of symptoms in both groups : with and without facial nerve paresis is shown in table 1. the average duration of complaints before the initial presentation was 16.8 months in the paresis group, and 14.1 months for the non - paresis group. however, the difference did not achieve a statistical significance (mann whitney test : p = 0.121).table 1correlation between duration of symptoms and paresis of the facial nerve at the initial presentationparesis of the facial nervenduration of symptoms (months)meanstandard deviationminimummaximumyes3216.817.72.072.0no7114.120.11.0120.0 correlation between duration of symptoms and paresis of the facial nerve at the initial presentation taking into consideration tumor histology, the shortest duration of symptoms to presentation was found in squamous cell cancers6.2 months, followed by metastasis to the parotid7.8 months, then adenoid cystic carcinoma15.3 months, and acinic cell cancer after 55 months. the results were statistically significant (kruskal wallis test : p 4 cm (table 2). the correlation between tumor size and paresis is statistically evident (chi - quadrate test p 4 cmyes4 (12.5%)7 (21.9%)21 (65.7%)32 (100.0%)no15 (21.1%)46 (64.8%)10 (14.1%)71 (100.0%)all19 (18.4%)53 (51.5%)31 (30.1%)103 (100.0%) correlation between tumor size and facial nerve paresis at the initial presentation during the study period, 778 parotidectomies were performed in our institution. of this group, there were 103 malignancies. partial lateral parotidectomy was performed in 37 patients, although the majority of the cases treated by this technique took place before the year 2000. between 2001 and 2006, the analysis of surgical treatment of parotid malignancies included the comparison of three different surgical techniques : tumor removal with a margin of healthy tissue, lateral parotidectomy, and total parotidectomy. in table 3, the choices of surgical procedures are presented. in patients with positive nodes (n+), neck dissection was performed, depending on the age of the patient, advanced stage of the cancer (t&n), and the presence of facial nerve palsy. importantly, this study compared the selection of three operating techniques : partial lateral parotidectomy, lateral parotidectomy, and total parotidectomy. table 3 analyzes the factors of age, tumor size, nodal status, and facial nerve status compared to the choice of operation with or without neck dissection. there was no difference between the age groups when analyzes by treatment method, t stage, n stage, and facial nerve palsy. 27 neck dissections (15 selective level ii and iii, and 12 radical) were performed on the 103 patients with parotid malignancies. the positive nodal disease was not identified by pathologist in the primary specimen in any patient. the presence of positive lymph nodes was not a statistically significant risk factor for facial nerve involvement. logline analysis performed for the following factors : the type of operation, t stage, n stage, nerve palsy, and age (categorized in two groups) revealed that the greatest influence on the chosen type of operation was t stage and facial nerve palsy. with higher t stage, the number of total parotidectomies performed increased and lateral parotidectomies decreased ; however, this is noticed most clearly in the group of patients with facial nerve palsy.table 3treatment methodstreatment of primary tumorpartial lateral parotidectomy 37 patientslateral parotidectomy 34 patientstotal parotidectomy 32 patientsneckn0 31neck dissection 6n0 28neck dissection 6n0 17neck dissection 15age > 57 years1751021210 80, reconstruction was not performed because of poor general status.table 4occurance of facial nerve paresis and treatment methodtreatmentfacial nerve palsyallyesnopartial lateral parotidectomy0 (0.0%)37 (52.1%)37 (35.9%)lateral parotidectomy4 (12.5%)30/1 (42.3%)34 (33.0%)total parotidectomy28 (87.5%)4/4 (5.6%)32 (31.1%)all32 (100.0%)71 (100.0%)103 (100.0%)facial nerve palsy including only a marginal branchfacial nerve resection with immediate reconstruction occurance of facial nerve paresis and treatment method facial nerve palsy including only a marginal branch facial nerve resection with immediate reconstruction in all patients, pathology was performed and the margins were assessed. histological analysis revealed that in 18 out of 103 cases, free margins were not obtained (resection margins incomplete or close). this included 8 (25%) patients with facial nerve paresis and 10 (14%) patients without paresis. local - regional control and disease - free survival were studied in the all of patients with parotid malignancies in a 3-year follow - up. this included six (18.8%) patients with facial paresis and in five (7%) patients without. this was not statistically significant (chi - quadrate test ; p = 0.075). the data are shown in table 5.table 5comparison of recurrence frequency in patients with and without facial nerve paresislocal recurrencefacial nerve paresisallyesnono26 (81.3%)66 (93.0%)92 (89.3%)yes6 (18.8%)5 (7.0%)11 (10.7%)all32 (100.0%)71 (100.0%)103 (100.0%) comparison of recurrence frequency in patients with and without facial nerve paresis neck node control was 90% for non - paresis patients and 92% for patients with nerve paresis. the analysis of factors influencing the survival in patients with parotid malignant tumors revealed a statistical significance for shorter survival in the high tumor stages (p = 0.014), in high grade histology (p = 0.008), and in facial paresis before treatment (p = 0.001). percentage disease - free 3-year survival differed dramatically for advancing tumor (t stage) and was 85, 90, 70, and 32% for t1, t2, t3, and t4, respectively (fig. 1). the percentage disease - free 3-year survival was 45 and 88% for patients with and without facial paresis (fig. 2).fig. 1curve of survival probability depending on tumor stage according to kaplan meierfig. 2curve of survival probability depending on facial nerve paresis according to kaplan meier curve of survival probability depending on tumor stage according to kaplan meier curve of survival probability depending on facial nerve paresis according to kaplan the present study describes one center s 10-year experience with parotid malignancy treatment with a focus on facial nerve status. in this retrospective analysis, the authors have taken into consideration patients age, gender, the duration of complaints (lump and facial paresis), preoperative clinical features of the primary (diameter, skin involvement, total or partial nerve paresis), histologic data, postoperative assessment of specimens, and outcome measures (time until relapse and survival). in the examined group however, of the patients with parotid tumors of advanced stage, over 50% had facial nerve palsy as the presenting and dominant symptom. facial nerve palsy was the clinical symptom in 60% of patients with adenoid cystic carcinoma and in 66% with squamous cell cancer. taking into consideration metastases, obrien and co - authors proposed a new staging system for patients with metastatic squamous carcinoma (scc) to the parotid irrespective of cervical nodal status. in a series of 87 patients, metastases were staged according to the clinical extent of disease in three groups : p1, tumor up to the 3 cm in diameter ; p2, tumor greater than 3 cm up to 6 cm in diameter or multiple metastatis parotid nodes ; p3, tumor greater than 6 cm in diameter, vii nerve palsy or skull invasion. author recommends that the clinical staging system for cutaneous scc of the head and neck should separate parotid and neck disease. the results of this study demonstrated, that patients with metastatic cutaneous scc in both the parotid gland and neck had a significantly worse prognosis than those with in the parotid alone. these observations were confirmed in group of 232 patients by bron. and by andruchow. in multicenter study of 322 patients from six different institutions. for metastases to the parotid, none out of our 12 (100%) patients had a facial palsy., who revealed facial nerve palsy in 33% of primary malignant tumors and in only 6% of metastasis to the parotid8. in terhaard.s analysis describing 324 malignant tumors of the parotid, 27% of the patients were found with no facial paresis, 14% were observed with partial paralysis, and 7% had total paralysis. they found 8 patients with facial paralysis and in 16 others the infiltration was diagnosed intra - operatively. the assessment of risk factors for facial nerve palsy, in cases of malignant tumors, was presented by terhaard.. there was a statistically significant correlation between facial nerve paralysis and localization in deep lobe, old age, pain, and infiltration of perineurium. huang., based on 60 patients with post - operative weakness of the facial nerve function, showed a significantly higher risk of paresis in operations for cancers greater than 4 cm. post - operative paresis was also higher in patients with intra - operative signs of nerve infiltration (95.5%) versus no infiltration (51.3%). the authors mentioned above did not demonstrate a connection between the histology of the tumor and the presence of palsy. however, tumor size, presence in the deep lobe, and nerve infiltration were highly significant. this is confirmed by preis., who recognized the location in the deep lobe and the size of the tumor as important factors. they did not show a correlation with age, sex, pain, and metastases to lymph nodes. our research demonstrated that facial nerve palsy occurs significantly more in tumors greater than 4 cm. it is well known that malignant infiltration of the facial nerve happens more frequently with a longer duration of symptoms and an advanced stage. a longer duration of symptoms and a delayed initiation of treatment increase the risk of facial palsy, and worsen the outcome. this was confirmed in our research. despite the fact that the results were not statistically significant, the duration of symptoms in patients with facial nerve palsy was nearly 3 months longer than in patients whose only symptom was a tumor. non - radical surgery (positive surgical margins) was more prevalent in the paretic group (25%) versus the non - paretic group (14%). they demonstrated that facial nerve palsy correlated with a significantly lower percentage of survival, 13% for total and 37% for partial paralysis, in comparison to 69% for patients without paresis. our study clearly proved a correlation of both t stage and facial nerve palsy at presentation and the duration of survival of patients with parotid cancer. the quality of life of the patients after radical removal of the cancer depends on the extent of resection, facial nerve preservation, and the preservation of neighboring structures such as the great auricular nerve, hypoglossal nerve, mandibular nerve, and the lateral wall of the pharynx [4, 11 ]. removal of one or several of the facial nerve branches leads to long - term functional deficits, and facial muscle atrophy cable graft techniques and neural repair are vital in the reconstruction and recovery of the resected facial nerve. muscle rehabilitation is also important to preserve the mass of muscles while they are temporarily deprived of motor innervation [7, 14 ]. in the study from holland, among 332 patients, the facial nerve was sacrificed in 21% of patients, and a reconstruction was performed in only 1/3 of those with sacrificed nerves (19/60). final results of treatment showed a partial paresis in 22% of patients and total paresis in 17%. in our analysis, overall, 32 out of 103 were left with permanent facial paralysis, with 28 cases involving all three branches. in our study, the incidence of facial nerve palsy due to malignant tumors of the parotid was not correlated with age, sex, duration of symptoms, and histological type of tumors. however, tumor size had a strong statistical correlation with paresis and emphasizes the importance of early diagnosis and treatment to optimize outcome. facial nerve paresis had a very strong prognostic correlation with a poor outcome in the treatment of malignant neoplasm of parotid. similarly, a high t stage, and a highly malignant histology, both have a directly negative influence on the patients survival. in conclusion, on the basis of analyzed data, prognostic factors concerning pre - treatment risk of facial paresis did not show a correlation with gender, age, duration of history, and tumor histology. however, a longer duration of symptoms and a higher grade histology was observed in paresis group. increased tumor diameter was the single factor that was statistically correlated with a risk of facial palsy. pre - treatment facial nerve status was prognostically significant. facial nerve impairment before resection was very strongly predictive for a shorter disease - free survival. facial nerve impairment was correlated with larger tumors and a higher histologic grade of malignancy. | the objectives of this article are to assess the frequency and significance of facial paralysis and undiagnosed nerve infiltration in patients with parotid malignancies. 103 patients with parotid gland malignancies were treated in a single institution, the tertiary center for ent at the university department in poznan between 1996 and 2006. facial palsy at the initial presentation was found in 32 patients. the stage of the primary tumor in the examined group of 103 patients is as follows : 20t1, 31t2, 20t3, 32t4. the correlation between facial nerve function before treatment and patients characteristics, including the treatment methods, were analyzed. intact facial nerve function at patient presentation was a very strong prognostic factor determining the treatment and final outcome for malignant neoplasms of parotid gland. similarly, t stage and a high - grade malignant histology had a direct influence on the duration of patients survival. |
catatonia as a syndrome was described first in the year 1874 by the german psychiatrist karl karrl kahlbaum as cardinal features noticed are not communicating or repeating what others speak or gesturing what others gesture, extreme negativism or automatic obedience without interpretation of the command, which is supposed to be due to inability to disobey commands. they may maintain peculiar postures for prolonged periods of time as well as have features of purposeless stereotypies and mannerisms. catatonia can complicate mental illness such as schizophrenia, bipolar disorders, depression, and posttraumatic syndromes. medical causes known to be associated are autoimmune disorders, paraneoplastic syndromes, seizures, encephalitis, metabolic disorders, use of steroids, neuroleptics, phencyclidine, inhaled abuse substances and abrupt withdrawal of benzodiazepines. diagnostic and statistical manual of mental disorders - fifth edition (dsm - v) separates catatonia from schizophrenia resulting in diagnostic and therapeutic changes in the approach. catatonia is categorized as follows : catatonia associated with general medical problems (293.89)catatonia as a subtype of schizophrenia (295.20),catatonia as an episode specifier for depressive and bipolar disorders (without any code),neuroleptic malignant syndrome considered as malignant catatonia. catatonia associated with general medical problems (293.89) catatonia as a subtype of schizophrenia (295.20), catatonia as an episode specifier for depressive and bipolar disorders (without any code), neuroleptic malignant syndrome considered as malignant catatonia. irrespective of the context, it is a very debilitating disease in view of the difficulty in communicating verbally, motor dysregulation clubbed with pronounced distress. when confounded in the background of an organic neurologic illness, it poses great problem in getting an insight into the diagnosis. most often it is mistaken as sequelae of the primary insult resulting in under diagnosis more so with reference to children. targeted diagnostic approach is indicated when there is the slightest suspicion as treatment options are different and outcome is good if treated early at least in a small percentage of the cases. criteria for catatonia in dsm - v includes the presence of three or more of the following : catalepsy (i.e., passive induction of a posture held against gravity),waxy flexibility (i.e., slight and even resistance to positioning by examiner),stupor (no psychomotor activity ; not actively relating to environment),agitation, not influenced by external stimuli, mutism (i.e., no, or very little, verbal response [note : not applicable if there is an established aphasia]),negativism (i.e., opposing or not responding to instructions or external stimuli),posturing (i.e., spontaneous and active maintenance of a posture against gravity),mannerisms (i.e., odd caricature of normal actions)stereotypies (i.e., repetitive, abnormally frequent, nongoal directed movements),grimacing, echolalia (i.e., mimicking another 's speech),echopraxia (i.e., mimicking another 's movements). catalepsy (i.e., passive induction of a posture held against gravity), waxy flexibility (i.e., slight and even resistance to positioning by examiner), stupor (no psychomotor activity ; not actively relating to environment), agitation, not influenced by external stimuli, mutism (i.e., no, or very little, verbal response [note : not applicable if there is an established aphasia ]), negativism (i.e., opposing or not responding to instructions or external stimuli), posturing (i.e., spontaneous and active maintenance of a posture against gravity), mannerisms (i.e., odd caricature of normal actions) stereotypies (i.e., repetitive, abnormally frequent, nongoal directed movements), echolalia (i.e., mimicking another 's speech), echopraxia (i.e., mimicking another 's movements). catatonia is a treatable disorder which carries 60 fold increased risk of premature death compared to general population if not treated properly. there is very scanty literature in catatonia in children and it is said that children present with motor and verbal slowness, difficulty initiating and completing action, increased dependence on cues, passivity, parkinsonian features, reversal of circadian rhythm, agitation and ritualistic behavior more than adults. in this study we report four cases of pediatric catatonia seen by us in the last 1-year which posed great diagnostic challenge before the correct diagnosis was made. children admitted to the pediatric neurology ward under our team in the last 1-year with features of psychomotor dysfunction following systemic illness and referred to neurology for diagnosis and later turned out to be catatonia are discussed. inclusion criteria - patients satisfying dsm - v criteria for catatonia were included in the study. they were screened and severity was graded using bush - francis catatonia screening instrument (bfcsi) [table 1 ]. case detection was done by the presence of the following signs : excitement, immobility and stupor, mutism, staring, posturing, grimacing, echopraxia, stereotypy, mannerisms, verbigeration, rigidity, negativism, waxy flexibility and withdrawal. their severity was rated for items 1 - 23 using a 0 - 3 point scale for each parameter. once the suspicion was raised all the patients were given slow intravenous (iv) lorazepam 2 mg over 2 min and relief of the catatonic symptoms were looked for. patients were investigated for central nervous system infections, acute and chronic, hepatic, renal and hematological assessment, electroencephalography (eeg) and magnetic resonance imaging. they all underwent ultrasound abdomen and chest x - ray for any detectable mass lesions. paraneoplastic antibody screening, autoantibody screening, iron and ferritin estimation could not be done in all cases due to financial constraints. rheumatoid factor, antinuclear antibodies, thyroid functions, hiv / venereal disease research laboratory (vdrl) screening was done in all the cases. screening and severity grading scores using bfcsi a 14-year - old girl who had features of viral meningoencephalitis in the form of fever and convulsions which was diagnosed as herpes encephalitis and received the full course of antiviral agents and anticonvulsants. during convalescence after a brief period of recovery from the illness, which lasted for about a week, patient became noncommunicative, irritable in between, confused, agitated and stiff. all investigations turned out to be negative. in view of the clinical suspicion of postinfective catatonia this response to lorazepam and absence of evidence for any other illness, possibility of postencephalitic catatonia was considered. she was maintained on 1 mg of lorazepam iv for 3 days, followed by oral lorazepam 2 mg 3 times daily. slowly, patient improved and at the time of discharge (15 days after admission) patient was taking oral feeds, ambulant with support and at 1-month follow - up child was independent for activities of daily living. a 6-year - old child who was admitted with fevers with rashes of 3 days duration and claimed to have been punished for poor obedience at school following which child went into withdrawal state with occasional eye blinking, not communicating, paucity of movement in all four limbs, occasional involuntary tongue protrusion. all her investigations were negative except for low total iron and transferrin saturation values [table 2 ]. she showed very good response to iv lorazepam, which was later followed - up with nutritional care and lorazepam 2 mg 4 times a day iv and later shifted over to oral. she was discharged after 2 weeks of admission at request. at the time of discharge child was walking without support, was communicating and independently feeding. a 16-year - old female was admitted with history of complex seizures with fever and altered sensorium, which persisted for more than 3 weeks and therefore referred to our center. at the time of examination, patient was not opening eyes and was resisting attempt to open. she was occasionally asking mother whether her experiences are real or false and there were occasional features of lack of inhibition. therefore, possibility of limbic encephalitis, infective versus noninfective was considered and her investigations were noncontributory. therefore, a trial with iv lorazepam was given to which she showed significant response. a 12-year - old girl born to nonconsanguineous parents had been symptomatic for the last 6 months. 6 months ago she had a febrile illness, which was treated as viral meningitis and following recovery from the same to almost independent activities of daily living, child started showing signs of deterioration. she was given a trial with 2 mg iv lorazepam following which she became extremely hyper dynamic, was tossing in the bed violently, occasionally trying to get up, and making frightening gestures with protrusion of the tongue, which stopped after some time. she also had autonomic features in the form of shiny skin, which was severely blanching on mild pressure in addition to coldness of the limbs. patient showed fluctuating response to treatment and at the time of discharge she was occasionally vocalizing, taking oral feeds, showing bizarre postures, moving limbs in sleep, gesturing to consultant in the form of waving but she was going back to episodes of motor and verbal inactivity. patient was discharged with oral lorazepam 2 mg 4 times daily, oral iron and syndopa [figures 1 and 2, video 1 ]. except one child all the three children belonged to higher socioeconomic status and all four of them had fever preceding the illness with an average gap of 2 weeks to 2 months from fever to onset of catatonic symptoms. all of them satisfied dsm - v criteria for the diagnosis of catatonia and their scores as per bfcsi was 10 - 14 in screening and the rating score varied from 33 to 63. the highest score was seen in the patient with longest period of symptoms which was 7 months. herpes simplex virus titers in cerebrospinal fluid (csf) were 1:125 in two cases which is not significant. neuropsychological assessment could not be done in any of the patients at the time of admission and three of the patients were active and responding at the time of discharge. detailed neuropsychological workup was not done in any of the patients as they were children [tables 18 ]. a 14-year - old girl who had features of viral meningoencephalitis in the form of fever and convulsions which was diagnosed as herpes encephalitis and received the full course of antiviral agents and anticonvulsants. during convalescence after a brief period of recovery from the illness, which lasted for about a week, patient became noncommunicative, irritable in between, confused, agitated and stiff. all investigations turned out to be negative. in view of the clinical suspicion of postinfective catatonia this response to lorazepam and absence of evidence for any other illness, possibility of postencephalitic catatonia was considered. she was maintained on 1 mg of lorazepam iv for 3 days, followed by oral lorazepam 2 mg 3 times daily. slowly, patient improved and at the time of discharge (15 days after admission) patient was taking oral feeds, ambulant with support and at 1-month follow - up child was independent for activities of daily living. a 6-year - old child who was admitted with fevers with rashes of 3 days duration and claimed to have been punished for poor obedience at school following which child went into withdrawal state with occasional eye blinking, not communicating, paucity of movement in all four limbs, occasional involuntary tongue protrusion. all her investigations were negative except for low total iron and transferrin saturation values [table 2 ]. she showed very good response to iv lorazepam, which was later followed - up with nutritional care and lorazepam 2 mg 4 times a day iv and later shifted over to oral. she was discharged after 2 weeks of admission at request. at the time of discharge child was walking without support, was communicating and independently feeding. a 16-year - old female was admitted with history of complex seizures with fever and altered sensorium, which persisted for more than 3 weeks and therefore referred to our center. at the time of examination she was occasionally asking mother whether her experiences are real or false and there were occasional features of lack of inhibition. therefore, possibility of limbic encephalitis, infective versus noninfective was considered and her investigations were noncontributory. therefore, a trial with iv lorazepam was given to which she showed significant response. a 12-year - old girl born to nonconsanguineous parents had been symptomatic for the last 6 months. 6 months ago she had a febrile illness, which was treated as viral meningitis and following recovery from the same to almost independent activities of daily living, child started showing signs of deterioration. she was drooling and had a constant silly smile on her face. occasionally during sleep she was given a trial with 2 mg iv lorazepam following which she became extremely hyper dynamic, was tossing in the bed violently, occasionally trying to get up, and making frightening gestures with protrusion of the tongue, which stopped after some time. she also had autonomic features in the form of shiny skin, which was severely blanching on mild pressure in addition to coldness of the limbs. patient showed fluctuating response to treatment and at the time of discharge she was occasionally vocalizing, taking oral feeds, showing bizarre postures, moving limbs in sleep, gesturing to consultant in the form of waving but she was going back to episodes of motor and verbal inactivity. patient was discharged with oral lorazepam 2 mg 4 times daily, oral iron and syndopa [figures 1 and 2, video 1 ]. except one child all the three children belonged to higher socioeconomic status and all four of them had fever preceding the illness with an average gap of 2 weeks to 2 months from fever to onset of catatonic symptoms. all of them satisfied dsm - v criteria for the diagnosis of catatonia and their scores as per bfcsi was 10 - 14 in screening and the rating score varied from 33 to 63. the highest score was seen in the patient with longest period of symptoms which was 7 months. herpes simplex virus titers in cerebrospinal fluid (csf) were 1:125 in two cases which is not significant. neuropsychological assessment could not be done in any of the patients at the time of admission and three of the patients were active and responding at the time of discharge. detailed neuropsychological workup was not done in any of the patients as they were children [tables 18 ]. they often occur in the background of minor and major systemic illness and therefore get masked under the title of some organic disorder. whether organic catatonia and purely psychogenic catatonia, catatonia in adults and catatonia in children differ pathophysiologically and in long term prognosis is not well known. however, benzodiazepines, specially lorazepam and antiparkinsonian drugs are very useful in the management of these patients irrespective of the underlying pathology. electroconvulsive therapy (ect) is also useful in resistant cases. clinical features of psychological verbal and motor withdrawal unsupported by laboratory data and lack of response to conventional methods of treatment in those circumstances should raise the possibility of catatonia. shorter the duration of illness, better is the outcome as evidenced in our own observations. females are more affected than males and probably occur in clusters suggesting a common underlying etiology in the environment like some unknown neurotropic viruses. irrespective of the cause, patient show good response to lorazepam challenge as well as follow - up treatment with lorazepam. none of our patients needed ect and 75% of our patients became completely normal at 1-month of follow - up. the only patient who had a partial response had duration of illness more than 6 months. hence it is important to be conscious about the possibility of catatonia in unresponsive children following minor or major systemic disease longer follow - up and larger number of patients needs to be studied to know the long - term outcome of these patients. 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 can not 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 can not be guaranteed. | background : the term catatonia was first introduced in 1874 and several etiologies, both organic and psychiatric have been attributed to the clinical phenotype of catatonia. the interesting feature is their response to lorazepam irrespective of their etiology.patients and methods : four patients admitted with verbal and motor unresponsiveness following febrile illness were evaluated for infective and metabolic causes. those who qualified for catatonia as per diagnostic and statistical manual of mental disorders - fifth edition criteria and bush - francis catatonia screening instrument screening tool and rating scale were evaluated in detail and reported. observations : catatonia occurs in clusters, females are more affected than males. electroencephalogram can be abnormal based on the precipitating symptom. minor changes in serum total iron and transferrin saturation and nonspecific elevation of viral antibody titers are seen in some patients. lorazepam challenge always gives the diagnosis.result:all patients where females and had preceeding systemic or cns infection. three out of the four patients where independent at the end of one month.conclusion:catatonia should be considered as a differential diagnosis in all children with verbal and motor unresponsiveness, which have no other explanation. early initiation of treatment is very rewarding at least during short term follow - up. |
adrenal cyst thus a rare disease entity, lacks common clinical features, making it difficult to diagnose preoperatively, especially before the introduction of modern radiological devices, computerized tomograms and sonograms. although several hundred cases have been repoted in world literature, the majority of them were discovered upon autopsy or as incidental findings at operation for other disease, the remainder of the cases were diagnosed after surgery due to gastrointestinal or renal symptoms caused by pressure or displacement of the adjacent organs by the cyst s large size. furthermore, in order to classify it a wide variety of descriptive terms has been introduced to denote the same or different types of adrenal cysts. abeshous proposed the following simple and inclusive classification : 1) parasitic cysts, 2) congenital glandular cysts, 3) cystic adenoma, 4) endothelial cysts, and 5) pseudocysts. the pseudocysts were further subdivided into 1) hemorrhagic cysts originating in hematoma and 2) hemorrhagic cysts originating in benign or malignant tumors. she had been relatively well until several weeks previous, when she suffered from a headache coupled with palpitations and facial flushing. on admission, she was known to have mild hypertension, which had not been monitored and treated regularly. there was no history of pulmonary disease, diabtes, fever, or arthralgia, and she did not take alcohol, anorexigenic agents, or contraceptives. on examination, the lung sound was clear and no heart murmur was audible. on the abdomen, no mass was palpated and no bruit was heard, and the abdomen was not rigid. the complete blood count, urinalysis, and liver function test were within normal limits. the electrolyte counts were sodium 142 meq / l, potassium 2.55 meq / l, chloride 103 meq / l, and total calcium 8.8 mg%. blood urea nitrogen and creatinine were 14.2 mg / dl and 0.9 mg / dl, respectively. the patient was given a large dose of antihypertensive medication (cateolol 5 mg per orally bid, prazocin 8 mg per orally bid, and nifedipine 10 mg per orally bid), but her blood pressure was refractory to these medications. in order to exclude secondary hypertension, a hormonal assay was begun and an abdominal sonogram taken. the sonogram revealed a slightly irregular tabulated low echogenic mass in the left adrenal fossa, and there was no calcification and no posterior enhancement. serum acth was 19.8 pg / ml, ketosteroid 3.8 ng / day, and 17 ohcs 2.45 ng / day. serum aldosterone (at supine) was 4.4 ng / dl, and plasma cortisol was 13.0 ug / dl. plasma renin activity was within the normal limit, but urine vma and metanephrine was elevated above the upper normal limit, 12.0 mg / day and 1.4 mg / day, respectively. a computerized tomogram of the abdomen showed a well - defined non - enhancing low - density oval - shaped mass in the left adrenal gland. the patient was transferred to the department of surgery and underwent a laparotomy on the 14th hospital day under the impression of cystic pheochromocytoma. through a left flank incision, the retroperitoneum was exposed, and a child fist - sized (454 cm) brown - gold colored adrenal tumor was noted. the tumor was removed while preserving the ipsilateral kidney. during manupulation of the tumor, the blood pressure rapidly increased to 260/150, so nitroprusside was infused to control it. grossly, the adrenal gland was cystic in nature with a smooth outer surface that measured 543 cm and weighed 23.5 grams. the cut surface showed a large cyst, which had mainly replaced the adrenal gland. a portion of the remaining adrenal tissue contained serous fluid with various sized numerous cysts. this large cyst was located at the medullary portion, and its wall was composed of loose fibrous tissue with focal calcification. these small remaining cysts were lined with flattened single endothelial cells and contained pale eosinophilic fluid with a few lymphocytes., the patient s blood pressure dropped sharply to 60/40 without nitroprusside, and dopamine was infused until the next day to increase the blood pressure. from the 3rd postoperative day, her blood pressure was stabilized without medication, and urine catecholamine metabolites checked on the 7th postoperative day were reported to be within the normal limit. the vma was 2.4 mg / day, and metanephrine was 1.1 mg / day. after her discharge and throughout the follow - up period until 14 months later, the patient remained normotensive with intermittent minimal doses of antihypertensive medication and was reported free of previous symptoms. lymphangiectatic cyst is the most common type of adrenal cyst, but there is no general agreement regarding the exact pathogenesis of this adrenal lesion. some authors attribute this variety of cyst formation to an ectasia of the lymphatic vessels, normally present in the capsule and medulla but absent in the cortex. consequently, it is possible that faulty development or incomplete linkage of these minute lymph spaces with larger lymphatic channels may lead to the development of multiple lobules or cystic areas but rarely to the formation of a large lymphangiomatous cyst. others suggest that is the end result of a cystic regeneration of hamartoma, though unlike a pseudocyst, which is caused by absorbed hamartoma in the normal or pathological adrenal gland. the lymphangiectatic cyst, occasionally referred to as cystic lymphangioma, could be regarded as a true benign tumor - like overgrowth of the adrenal cells. from a pathological view point, the distinguishing features between the two most common types of cyst, the lymphangiectatic and hemorrhagic pseudocyst, appear to be : 1) a layer of flattened endothelium which usually lines the former but is absent in the latter. 2) the contents, if clear, give no help, which may be milky if of lymphatic origin or bloody or reddish - brown if of hemorrhagic origin, and 3) lymphangiomatous cysts, which are commonly multilocular with many loculi separated by thin septa of fibrous tissue and until pseudocysts, which are usually unilocular and frequently have blood clots or deposits of fibrin on the wall. however, in spite of these criteria, it may be impossible in any given case to differentiate between the two, because, as we mentioned earlier, most adrenal cysts are small, asymptomatic, and lacking characteristic signs and symptoms. so the most important clinical problem is still finding out how this cyst attains its large size to produce pressural symptoms. however, some cases have been reported regarding adrenal cortical hypertension, cushing syndrome, or virilism. geelhead and budd dc presented two cases of adrenal cyst with refractory hypertension which was corrected by surgical removal of the cyst, but they could not verify the endocrine dysfunctions biochemically. likewise, in our case, we think it is noteworthy that we diagnosed an adrenal lymphangiectatic cyst while in search of an endocrine dysfunction as the cause of hypertension. our patient s severe hypertension was normalized through the surgical removal of the adrenal cyst, while the catecholamine metabolites, which had increased before surgery, became normal. although an adrenal vascular study was not performed in our case, we believe that the size of the cyst was not large enough to compromise the renal blood flow, considering the fact that catecholamine levels are not measurably higher in patients with unilateral renovascular hypertension. we suggest that the overproduction of catecholamine, caused by an adrenal medullary lymphangiectatic cyst, played an important role and may not have been the sole cause in the elevation of blood pressure in our patient. in conclusion, whatever mechanism activates the sympathochromaffin system, we think that the adrenal medullary lymphangiectatic cyst could be regarded as a functioning tumor in some cases rather than a simple space - occupying lesion. | adrenal lymphangiectatic cyst is a very rare pathological and clinical disease entity, and its clinical silence and lack of characteristic symptoms and signs makes it difficult to diagnose preoperatively. we experienced a case of adrenal lymphangiectatic cyst, accompanied by severe refractory hypertension, which was corrected by surgical removal of the cyst.we reprot it with a review of the literature. |
the renin - angiotensin system (ras) plays a key role in several target organs, such as heart, blood vessels, and lungs, exerting a powerful control in the maintenance of the homeostasis [14 ]. this system is activated by the conversion of the angiotensinogen to the inactive peptide angiotensin (ang) i through the renin action. subsequently, ang i is cleaved by the angiotensin - converting enzyme (ace) generating ang ii, the main angiotensin peptide, whose actions are mediated by two g protein - coupled receptors (gpcr), at1 and at2 [7, 8 ] (figure 1). the major physiological functions of ang ii are mediated by at1 receptor [9, 10 ]. in pathological conditions, activation of this receptor induces deleterious effects, such as vasoconstriction, fibrosis, cellular growth and migration, and fluid retention [11, 12 ]. on the other hand, ang ii binding to the at2 receptor generally causes opposite effects when compared with those actions mediated by the at1 receptor [13, 14 ]. recently, it has been proposed that, in addition to the ace / ang ii / at1 receptor axis, the ras possesses a counter regulatory axis composed by ace2, ang-(17), and mas receptor (figure 1). ang-(17) is a biologically active component of the ras which binds to mas inducing many beneficial actions, such as vasodilatation, antifibrosis, and antihypertrophic and antiproliferative effects [1523 ]. this peptide is produced mainly through the action of ace2, which has approximately 400-fold less affinity to ang i than to ang ii [2426 ] ; thereby, ang ii is the major substrate for ang-(17) synthesis. in fact, the conversion of ang ii to ang-(17) by ace2 is important to regulate the ras activity since ang-(17) induces opposite effects to those elicited by ang ii [1624 ]. additionally, ace2 can form ang-(17) less efficiently through hydrolysis of ang i to ang-(19) with subsequent ang-(17) formation. the relevance of the ras is highlighted by the success obtained in therapeutic strategies based on the pharmacological inhibition of this system in cardiovascular and respiratory diseases [2732 ]. blockade of the ras with ace inhibitors (acei) or at1 receptor antagonists (arbs) improves the outcomes of patients with hypertension, acute myocardial infarction, and chronic systolic heart failure [3335 ]. furthermore, based on the involvement of the ace / ang ii / at1 axis in respiratory diseases and the crucial role of the lungs in the ras metabolism, several studies have reported the contribution of the ras in lung pathophysiology [28, 30, 31, 3640 ]. importantly, it has been shown that administration of acei and arbs causes substantial increases in plasma ang-(17) levels, leading to the assumption that part of their clinical effects might be mediated by this heptapeptide [4143 ]. indeed, some effects of acei and arbs can be blocked or attenuated by a-779, a mas antagonist, confirming the role of ang-(17) in the actions of these compounds. the beneficial effects of ang-(17), as well as its likely participation in the effects of the acei and arbs, represent evidences for the potential of the ace2/ang-(1 - 7)/mas axis as a therapeutic target. in this review, we will focus on the recent findings related to the pathophysiology actions of the ace2/ang-(17)/mas axis in the cardiovascular and respiratory system. also, we will discuss the promising initiatives to develop new therapeutic strategies based on this axis to treat pathological conditions. the heart is one of the most important targets for the actions of the ace2/ang-(17)/mas axis. in the heart, ace2 is expressed in the endothelium, myofibroblasts, cardiomyocytes, and fibroblasts [47, 48 ]. classical pharmacotherapeutic agents used to treat heart failure, including acei, arbs, and aldosterone receptor blockers, increase ace2 activity and/or expression, indicating its importance in the cardiac diseases establishment and progression [4951 ]. additionally, pharmacological and genetic (transgenic animals and gene transfer) approaches have evidenced the significance of ace2 in cardiac pathologies. despite some controversies concerning the consequences of the ace2 deficiency, in general, evidences indicate a protective role of ace2 in the heart [48, 5257 ]. crackower and colleagues were the first to demonstrate that genetic ablation of ace2 results in severe blood - pressure - independent systolic impairment. also, disruption of ace2 was able to accelerate cardiac hypertrophy and shortened the transition period to heart failure in response to pressure overload by increasing local ang ii. recently, it has been demonstrated that loss of ace2 enhances the susceptibility to myocardial infarction, with increased mortality, infarct expansion and adverse ventricular remodeling. in keeping with these genetic findings, on the other hand, cardiac overexpression of ace2 prevented hypertension - induced cardiac hypertrophy and fibrosis in spontaneously hypertensive rats (shr) and in ang - ii - infused rats [59, 60 ]. indeed, transfection of lenti - ace2 (lentivirus containing ace2 cdna) or ad - ace2 (recombinant adenovirus carrying the murine ace2) into the surrounding area of the infarcted myocardium was protective against pathological remodeling and cardiac systolic dysfunction in a rat model of myocardial infarction [61, 62 ]. this effect was associated with decreased expression of ace and ang ii and increased expression of ang-(17). collectively, these observations reveal that ace2 effectively plays a protective role in the cardiac structure and function. since the discovery of ang-(17) in the late 1980s [63, 64 ], several studies have demonstrated important effects of this peptide in hearts. the presence of ang-(17) and its receptor mas in the heart [65, 66 ] and the ability of this organ to produce ang-(17) [55, 67 ] are evidences of the role of this peptide in cardiac tissues. functionally, ang-(17) induces an antiarrhythmogenic effect against ischemia / reperfusion injuries in rats [17, 68 ] as well as prevents atrial tachycardia and fibrillation in rats and dogs [69, 70 ]. treatment with ang-(17) improved the coronary perfusion and cardiac function in rats after myocardial infarction and after ischemia / reperfusion injury. increases in circulating ang-(17) levels in transgenic rats reduced the cardiac hypertrophy and fibrosis [20, 22 ] induced by isoproterenol administration. these effects are apparently independent of changes in blood pressure since grobe and colleagues have demonstrated that the antifibrotic and antihypertrophic actions of ang-(17) are still observed in ang - ii - infused hypertensive rats. local overexpression of ang-(17) in hearts of mice and rats improved the myocardial contractility and prevented the isoproterenol- and hypertension - induced cardiac remodeling [19, 21 ]. altogether, these findings support a direct effect of ang-(1 - 7) in the heart. further evidence for the role of ang-(17)/mas in the pathophysiology of the heart came from experimental protocols utilizing mice with genetic deficiency of mas. they revealed that the cardiac function is impaired in mas knockout mice likely due to the increased extracellular matrix proteins deposition in the heart [66, 73 ]. this profibrotic phenotype may be related to changes in matrix metalloproteinases (mmps) and tissue inhibitors of metalloproteinases (timps) levels and/or activities [74, 75 ]. although further elucidations regarding the signaling pathways involved in mas activation are necessary, some mechanisms have been proposed. overexpression of ang-(17) in hearts of rats causes an improvement in the [ca ] handling in cardiomyocytes and increases the expression of serca2a. in keeping with these results, cardiomyocytes from mas - deficient mice present slower [ca]i transients accompanied by a lower ca atpase expression in the sarcoplasmic reticulum [66, 76 ]. although acute ang-(17) treatment failed to alter ca handling in ventricular myocytes of rats, these findings suggest an important role of the ang-(17)/mas in the long - term maintenance of the ca homeostasis in the heart. one of the mechanisms by which ang-(17) plays its effects in the heart is stimulating the nitric oxide (no) production. indeed, it has been demonstrated that ang-(17) via mas increases the synthesis of no through a mechanism involving the activation of the endothelial no synthase (enos). these effects were abolished by a-779 and are absent in cardiomyocytes from mas - deficient mice. recently, gomes. found that the treatment of isolated cardiomyocytes of rats with ang-(17) efficiently prevents the ang - ii - induced hypertrophy by modulating the calcineurin / nfat signaling cascade. these effects were blocked by no synthase inhibition and by guanylyl cyclase inhibitors, indicating that these effects are mediated by the no / cgmp pathway. also, ang-(17) inhibits serum - stimulated mitogen - activated protein kinase (mapk) activation in cardiac myocytes and prevents the ang - ii - mediated phosphorylation of erk1/2 and rho kinase in hearts in a dose - dependent manner. in line with these data, activation of endogenous ace2 significantly reduced the phosphorylation of erk1/2 in hearts of hypertensive rats (shrs). however, mercure. reported that overexpression of ang-(17) in hearts of rats decreases the ang - ii - induced phosphorylation of c - src and p38 kinase, whereas the increase in erk1/2 phosphorylation was unaffected by the expression of the transgene, thereby suggesting a selective effect of ang-(17) on intracellular signaling pathways related to cardiac remodeling. overall, these data reveal a key role of the ace2/ang-(17)/mas axis in the pathophysiology of the cardiac structure and function. activation of this axis might be an important strategy to develop a new generation of cardiovascular therapeutic agents against cardiac dysfunction and pathological remodeling of the heart. early studies have reported the endothelium as the major site for generation and metabolism of ang-(17). in addition to ang-(17), endothelial cells also express ace2 and mas [80, 81 ]. thus, now it is recognized that the ace2/ang-(17)/mas axis is present in vascular endothelial cells and modulates its function promoting vasorelaxation, reduction of the oxidative stress [83, 84 ], and antiproliferative effects [85, 86 ]. the vasodilatory actions of ang-(17) have been reported in many studies in several vascular beds and preparations, including mouse [16, 23 ] and rat aortic rings, canine and porcine coronary arteries, canine middle cerebral artery, porcine piglet pial arterioles, feline mesenteric vascular bed, rabbit renal afferent arterioles, and mesenteric microvessels of normotensive and hypertensive rats. for example, it has been shown that ang-(17) causes vasodilation in forearm circulation of normotensive subjects and patients with essential hypertension while other studies were unable to report any significant effect of ang-(17) in the same vascular territory in acei - treated patients. in fact, many of these actions are completely abolished by a-779 or partially blocked by this antagonist [3, 86, 96 ]. importantly, the endothelium - dependent relaxation induced by ang-(17) in mouse aortic rings is absent in vessels derived from mas - knockout mice. however, other studies have shown that ang-(17) also interacts with ace, at1, and at2-like receptors, suggesting the existence of additional sites of interaction for ang-(17) [3, 97, 98 ]. indeed, silva. reported evidence for the presence of a distinct subtype of ang-(17) receptor sensible to d - pro - ang-(17), a second mas antagonist, but not to a-779 in aortas of sprague - dawley rats. the vascular effects of ang-(17) are endothelium dependent and involve the production of vasodilator products, such as prostanoids, no, and endothelium - derived hyperpolarizing factor (edhf) [16, 81, 100 ]. pinheiro and coworkers found that ang-(17) promotes an increase in no release in mas - transfected chinese hamster ovary (cho) cells. furthermore, short - term infusion of ang-(17) improved the endothelial function by a mechanism involving no release in rats. mas deletion resulted in endothelial dysfunction associated with an unbalance between no and oxidative stress. also, mas activation by ang-(17) in human endothelial cells stimulated enos phosphorylation / activation via the akt - dependent pathway. roks. have shown that ang-(17) inhibits the vasoconstriction induced by ang ii in human internal mammary arteries, thereby suggesting that ang-(17) can regulate the ang ii effects. in fact, ang-(17) negatively modulates the ang ii type 1 receptor - mediated activation of c - src, and its downstream targets erk1/2 and nad(p)h oxidase. the counterregulatory action of ang-(17) on ang ii signaling has been also observed in cardiomyocytes, vascular smooth muscle cells, and fibroblasts. additionally, an interaction between mas and bradykinin (bk) type 2 (b2) receptors may modulate some of the ang-(17) effects in blood vessels. indeed, it has been demonstrated that ang-(17) potentiates the vasodilator and hypotensive effects of bk in several vascular beds [93, 108110 ]. as the major enzyme involved in ang-(17) lovren. have demonstrated that ace2 ameliorates the endothelial homeostasis via a mechanism involving reduction of the reactive oxygen species production. of note moreover, overexpression of ace2 in vessels of hypertensive rats resulted in reduction in the arterial blood pressure and improvement of the endothelial function associated with increased circulating ang-(17) levels. overall, these data indicate that the beneficial effects of ace2 are, at least in part, mediated by ang-(17). recently, we have demonstrated that activation of endogenous ace2 causes a dose - dependent hypotensive effect in normotensive and hypertensive rats. also, the response to bk administration was augmented in rats chronically treated with xnt, an ace2 activator. however, we were unable to demonstrate any significant effect of xnt on blood pressure in response to the administration of ang ii or losartan in normotensive and hypertensive rats (figure 2). in the past few years, the participation of the ace2/ang-(17)/mas axis in the establishment and progression of pulmonary diseases has become evident. indeed, the important role of the ras in the lung pathophysiology and the side effects and pulmonary toxicity induced by the acei raised the interest to evaluate the activation of the ace2/ang-(17)/mas axis as an alternative target to treat pulmonary pathologies. thus, it has been reported beneficial outcomes induced by the activation of this axis in animal models of acute respiratory distress syndrome (ards), pulmonary hypertension (ph), fibrosis, and lung cancer [31, 37, 114117 ]. these studies pointed out that the imbalance between the ace / ang ii / at1 and the ace2/ang-(17)/mas axes of the ras might be relevant in lung diseases. taking into account that systemic hypotension is an important limitation to the use of acei and arbs in pulmonary patients, therapies based on the ace2/ang-(17)/mas axis emerge as a safe and efficient approach since studies using the ace2 activator xnt or ace2 gene transfer have shown that these strategies induce beneficial pulmonary outcome without changes in systemic blood pressure in rats and mice [39, 117, 118 ]. imai and colleagues demonstrated the role of ace2 in ards pathogenesis. they found that a more severe ards was reached in ace2 knockout mice, and this phenotype was reversed by double genetic deletion of the ace2 and ace genes or by the treatment with recombinant human ace2 (rhace2). furthermore, ang ii levels were related to the severity of the lung injury. of note, ace2 is widely expressed in the pulmonary endothelium, vasculature, and pneumocytes [119, 120 ]. also, rhace2 inhibited the increase of ang ii and tnf- levels, attenuated the arterial hypoxemia and ph, and ameliorated the distribution of the pulmonary blood flow in lipopolysaccharide - induced lung injury in piglets. therefore, these studies suggest that ace2 is a suitable target to arrest the development of ards in patients at risk. the stimulation of the ace2/ang-(17)/mas axis has been successful used to prevent and reverse ph and fibrosis in animals. ace2 activation using the compound xnt or induction of ace2 overexpression by gene transfer efficiently prevented and, more importantly, reversed the increase of the right systolic ventricular pressure (rsvp), pulmonary fibrosis, imbalance of the ras, and inflammation in animals (rats and mice) with ph induced by monocrotaline (mct) or in rats with pulmonary fibrosis caused by bleomycin treatment [39, 117, 118 ]. in keeping with these findings, ang-(17) in addition, ang-(17) via mas prevented the apoptosis of alveolar epithelial cells and the jun n - terminal kinase (jnk) activation induced by bleomycin. the involvement of the ang-(17)/mas in ph was further evidenced by the observation that the xnt effects are blocked by a-779. furthermore, in both lung specimens from patients with idiopathic pulmonary fibrosis and from animals with bleomycin - induced pulmonary fibrosis were reported a reduction in mrna, protein, and activity of ace2 with a reciprocal increase in ang ii level. a growing body of studies has focused on the relevance of the ace2/ang-(17)/mas axis in the pulmonary cancer pathophysiology. the protein expression of ace2 is reduced in non - small - cell lung carcinoma (nsclc) along with an increase in ang ii levels. moreover, overexpression of ace2 in cultured a549 lung cancer cells and in human lung cancer xenografs inhibited the cell growth and the vascular endothelial growth factor - a (vegfa) expression induced by ang ii [123, 124 ]. gallagher and tallant evaluated the effects of several angiotensin peptides [ang i, ang ii, ang-(28), ang-(38), and ang-(37) ] in sk - lu-1 cancer cells growth, and only ang-(17) showed significant attenuation of the dna synthesis and proliferation. the antiproliferative effect of ang-(17) was mediated by its receptor mas and inhibition of the erk1/2 pathway. neither the blockage of at1 nor at2 succeeded in inhibiting the action of ang-(17). in keeping with these data, the antiproliferative effect of ang-(17) was observed in human a549 lung tumor xenograft growth along with a marked decrease in the vessel density in mice through a mechanism involving cyclooxygenase-2 (cox-2) [126, 127 ]. of note, in a nonrandomized phase i clinical trial conducted by petty and colleagues, subcutaneous injections of ang-(17) were administered in 18 patients with advanced solid tumors refractory to standard therapy. despite the mild adverse effects observed with the ang-(17) treatment, altogether, these studies provide insights into the involvement of the ace2/ang-(17)/mas axis in lung cancer. current therapies based on the modulation of the ras include the acei, arbs, and renin inhibitors. in general, these drugs prevent or reverse endothelial dysfunction and atherosclerosis, reduce cardiovascular mortality and morbidity of patients with coronary artery disease, and hold antihypertensive effects. classically, the mechanisms of action of the acei and arbs involve the blockade of the synthesis and actions of ang ii, respectively. however, the ras is a complex hormonal system and, consequently, other mechanisms are likely implicated in the actions of these drugs [42, 86, 129 ]. they cause substantial increase in plasma levels of ang-(17), leading to the assumption that their clinical effects might be partly mediated by this heptapeptide [42, 130 ]. indeed, a variety of effects of the acei and arbs can be abolished or attenuated by mas antagonism, confirming the role of ang-(17) in the actions of these compounds [129, 131 ]. the beneficial effects of ang-(17) as well as its likely involvement in the effects of the acei and arbs represent a strong evidence for the therapeutic potential of the activation of the ace2/ang-(17)/mas axis (figure 3). the beneficial effects of ang-(17) are well known ; however, the therapeutic utilization of this peptide is limited due to its unfavorable pharmacokinetic properties. ang-(17) has a short half - life (approximately 10 seconds) since it is rapidly cleaved by peptidases. furthermore, ang-(17) is degraded during its passage through the gastrointestinal tract when orally administrated. thus, new strategies are crucial to make feasible the clinical application of ang-(17). recently, a formulation based on the ang-(17) included into hydroxypropyl -cyclodextrin [hpcd / ang-(17) ] was developed by lula and colleagues. cyclodextrins are pharmaceutical tools used for design and evaluation of drug formulations, and they enhance the drug stability and absorption across biological barriers and offer gastric protection. the amphiphilic character of cyclodextrins allows the possibility of formation of supramolecular inclusion complexes stabilized by noncovalent interactions with a variety of guest molecules [133, 134 ]. in this regard, the formulation hpcd / ang-(17) pharmacokinetic and functional studies showed that oral hpcd / ang-(17) administration significantly increases plasma ang-(17) levels and promotes an antithrombotic effect that was blunted in mas deficient mice. marques and colleagues have found that chronic oral administration of hpcd / ang-(17) significantly attenuates the heart function impairment and cardiac remodeling induced by isoproterenol treatment and myocardial infarction in rats. administration of liposomes containing ang-(17) in rats led to prolonged hypotensive effect for several days in contrast to the response observed when the free peptide was used [137, 138 ]. a strategy used to protect the ang-(17) against proteolytic degradation was proposed by kluskens and coworkers. using the ability of prokaryotes to cyclize peptides, they synthesized a cyclic ang-(17) derivative [thioether - bridged ang-(17) ] which presented an increased stability in homogenates of different organs and plasma and enhanced the ang-(17) bioavailability in rats. furthermore, cyclized ang-(17) induced a relaxation in precontracted aorta rings of rats which was blocked by the ang-(17) receptor antagonist d - pro - ang-(17), providing evidence that cyclized ang-(17) also interacts with mas. ave 0991 was the first nonpeptide synthetic compound developed with the intention of stimulating the mas receptor. this compound mimics the ang-(17) effects in several organs such as vessels [140, 141 ], kidney, and heart [142, 143 ]. similar to ang-(17), ave 0991 induced a vasodilation effect which was absent in aortic rings of mas - deficient mice. moreover, its effects in aortic rings were blocked by the two ang-(17) receptor antagonists, a-779 and d - pro - ang-(17). ave 0991 potentiated the acetylcholine - induced vasodilation in conscious normotensive rats, and this effect was abolished by a-779 and l - name. similarly, it was able to increase the hypotensive effect of bk in normotensive rats, and a-779 also blocked this effect. ferreira. [142, 143 ] reported that ave 0991 protects the heart against cardiac dysfunction and remodeling caused by isoproterenol treatment or by myocardial infarction in rats [142, 143 ]. in mas - transfected cells, ave 0991 induced no release which was blunted by a-779 and not by at2 or at1 antagonists. all these data support the concept that ave 0991 is an ang-(17) mimetic and that its actions are mediated by the interaction with mas. using a computational discovery platform for predicting novel naturally occurring peptides that may activate gpcr, two novel peptides, designated as cgen-856 and cgen-857, with amino acid sequence unrelated to angiotensin peptides, these peptides elicited ca influx in cho cells overexpressing mas without any activity in at1 or at2 receptors. cgen-856s, a derivative of the cgen-856 peptide, induced beneficial cardiovascular effects similar to those caused by ang-(17). this compound competes with ang-(17) for the same bind site in mas - transfected cells. furthermore, similar to ang-(17), cgen-856s produced a vasodilation effect which was absence in mas - deficient mice, indicating that this compound also acts via mas. this was confirmed by the inhibition of the cgen-856s effects by the mas antagonist a-779.. showed that cgen-856s promotes antiarrhythmogenic effects and produces a small dose - dependent decrease in arterial pressure of conscious shr. a new approach addressing the therapeutic potential of the activation of the ace2/ang-(17)/mas axis based on the crystal structure of ace2 and using a virtual screening strategy, it was identified small molecules that may interact with this enzyme leading to changes in its conformation and, consequently, enhancing its activity. thus, the ace2 activator, namely xnt, was identified and its administration in shr decreased blood pressure, induced an improvement in cardiac function, and reversed the myocardial and perivascular fibrosis observed in these animals [48, 113 ]. the beneficial effects of xnt were also observed in rats with ph induced by mct. furthermore, this compound attenuated the thrombus formation and reduced the platelet attachment to vessels in hypertensive rats. it appears that the pharmacological activation of ace2 promotes its beneficial effects due to an increased ang-(17) production with concomitant degradation of ang ii. in fact, coadministration of a-779 abolished the protective effects of xnt on ph. in addition, the antifibrotic effect of xnt observed in hearts of shr was associated with increases in cardiac ang-(17) expression. however, it is also pertinent to point out that off - target effects of xnt on these beneficial outcomes can not be ruled out at the present time. there is growing evidence that changes in the novel components of the ras [ang-(17), ace2, and mas ] may take part of the establishment and progression of cardiovascular and respiratory diseases. importantly, these new components of the ras, due to their counter regulatory actions, are candidates to serve as a concept to develop new cardiovascular and respiratory drugs. | angiotensin (ang)-(17) is now recognized as a biologically active component of the renin - angiotensin system (ras). the discovery of the angiotensin - converting enzyme homologue ace2 revealed important metabolic pathways involved in the ang-(17) synthesis. this enzyme can form ang-(17) from ang ii or less efficiently through hydrolysis of ang i to ang-(19) with subsequent ang-(17) formation. additionally, it is well established that the g protein - coupled receptor mas is a functional ligand site for ang-(17). the axis formed by ace2/ang-(17)/mas represents an endogenous counter regulatory pathway within the ras whose actions are opposite to the vasoconstrictor / proliferative arm of the ras constituted by ace / ang ii / at1 receptor. in this review we will discuss recent findings concerning the biological role of the ace2/ang-(17)/mas arm in the cardiovascular and pulmonary system. also, we will highlight the initiatives to develop potential therapeutic strategies based on this axis. |
diosmin is a member of the citrus flavonoid family and is a modified form of hesperidin [1 - 4 ]. it is a semisynthetic drug that is used as an oral phlebotropic drug in the treatment of venous disease, i.e., chronic venous insufficiency and hemorrhoidal disease. in some countries, daflon 500 mg (which contains 90% diosmin+10% hesperidin) is used as a prescription medication. diosmin prolongs the vasoconstrictor effect of norepinephrine on the vein wall, increases the venous tone, and therefore reduces venous capacitance, distensibility, and stasis. furthermore, it improves lymphatic drainage by increasing the frequency and intensity of lymphatic contractions and at the same time decreases the diameter of the lymphatic capillaries [1 - 4 ]. in addition to its use in vascular disease, diosmin has been found to be effective in mitigating hyperglycemia in diabetic rats. it is also speculated that diosmin may have potential in the treatment of neurodegenerative diseases, such as alzheimer 's disease. moreover, its anti - inflammatory and anti - apoptotic activity has been demonstrated in neuronal cells. it has been mentioned that the anticancer, cardiovascular, and anti - inflammatory activities are the properties of citrus flavonoids. until now, however, clinical studies of the effects of diosmin have been inconclusive. furthermore, only a limited number of articles have been published and few articles are available on the use of diosmin in renal diseases. kidney stones (nephrolithiasis) are a solid concretion or crystal aggregation formed in the kidneys from dietary minerals existing in the urine. the africo - asian stone - forming belt stretches from sudan, the arab republic of egypt, saudi arabia, the united arab emirates, the islamic republic of iran, pakistan, india, myanmar, thailand, and indonesia to the philippines. in this area of the world, the disease affects all age groups from less than 1 year old to more than 70 years old. because nephrolithiasis is a widespread disease, blocking the process of stone formation and finding new therapeutic options is an important area of study. the present study was therefore conducted to investigate the effects of diosmin in a rat model of calcium oxalate (caox) nephrolithiasis by use of stereological techniques. the quantitative data obtained showed the total volume of the deposited caox and structural changes to the kidney, including the total volume of the glomeruli, proximal and distal convoluted tubules (pct and dct), loop of henle, collecting duct, interstitial tissue, and vessels. a rapid and simple stereological method was used to obtain the total quantities on one microscopic slide without the need for serial sectioning of the renal tissue. the animals were treated according to the standard directive as recommended and approved by the research authorities of shiraz university of medical sciences (license no. the rats were randomly divided into three groups of 10 animals each, which is acceptable for stereological studies. the first group (control) did not receive any treatments. in groups two and three the second group received distilled water by gavage, whereas the third group received diosmin (80 mg / kg / d) dissolved in a distilled water vehicle given by gavage for 21 days. to induce an experimental model of caox nephrolithiasis, the rats were orally administered 2.5% (v / v) ethylene glycol+2.5% (w / v) ammonium chloride (2 ml / d) by gavage. in addition, the rats experienced a restriction on their intake of drinking water (20 ml / d) for 21 days. after 21 days the primary volume of the kidney, vprimary, was measured by using the immersion method of sherle. estimation of the shrinkage and the length of the tubular structure of the kidney requires isotropic, uniform, random sections [13 - 15 ]. the estimated shrinkage was also used for the estimation of the final kidney volume to avoid the consecutive sectioning that is required for the cavalieri method. the final volume of the kidney (the reference space) was corrected by using the following formula : vfinal = vprimary(1-volume shrinkage). each sampled section was analyzed by using a video microscopy system that was made up of a microscope (e-200, nikon, tokyo, japan) linked to a video camera, a computer, and a monitor to determine the parameters. between 8 and 12 microscopic fields were selected in a systematic random manner. afterwards, the stereological grids were superimposed on the images by means of the stereology software designed at histomorphometry & stereological research center, shiraz university of medical sciences, shiraz, iran. however, the volume density of each structure (its volume per unit volume of the kidney) was estimated by using the point - counting method [13 - 15 ]. 3. the volume density (vv) of the renal cortex, the medulla, the glomeruli, the pct, the dct, the collecting ducts, the henle 's loop, the vessels, the connective tissue, and the caox deposition was obtained at the final magnification of 1,800. the point - counting method used the following formula : vv (structure, reference)=p(structure)/p(total) where p(structure) and p(total) represent the sum of the number of the points hitting the structure and all sampled fields of the kidney, respectively. finally, the total volume of each structure was estimated by multiplying the fractional volume by the final volume of the kidney to prevent the " reference trap " [13 - 15 ]. the diameter of the capillaries and vessels sampled by the unbiased counting frame was measured. the diameter was measured perpendicular to the long axis where the vessel was widest [13 - 15 ]. the statistical comparisons between the group means were performed by analysis of variance and also mann - whitney u - test. the animals were treated according to the standard directive as recommended and approved by the research authorities of shiraz university of medical sciences (license no. the rats were randomly divided into three groups of 10 animals each, which is acceptable for stereological studies. the first group (control) did not receive any treatments. in groups two and three the second group received distilled water by gavage, whereas the third group received diosmin (80 mg / kg / d) dissolved in a distilled water vehicle given by gavage for 21 days. to induce an experimental model of caox nephrolithiasis, the rats were orally administered 2.5% (v / v) ethylene glycol+2.5% (w / v) ammonium chloride (2 ml / d) by gavage. in addition, the rats experienced a restriction on their intake of drinking water (20 ml / d) for 21 days. the primary volume of the kidney, vprimary, was measured by using the immersion method of sherle. 1. the kidney was fixed in neutral buffered formaldehyde for 2 days. then the tissue shrinkage produced by fixation, processing, and staining was estimated. estimation of the shrinkage and the length of the tubular structure of the kidney requires isotropic, uniform, random sections [13 - 15 ]. the estimated shrinkage was also used for the estimation of the final kidney volume to avoid the consecutive sectioning that is required for the cavalieri method. the final volume of the kidney (the reference space) was corrected by using the following formula : vfinal = vprimary(1-volume shrinkage). each sampled section was analyzed by using a video microscopy system that was made up of a microscope (e-200, nikon, tokyo, japan) linked to a video camera, a computer, and a monitor to determine the parameters. between 8 and 12 microscopic fields were selected in a systematic random manner. afterwards, the stereological grids were superimposed on the images by means of the stereology software designed at histomorphometry & stereological research center, shiraz university of medical sciences, shiraz, iran. however, the volume density of each structure (its volume per unit volume of the kidney) was estimated by using the point - counting method [13 - 15 ]. 3. the volume density (vv) of the renal cortex, the medulla, the glomeruli, the pct, the dct, the collecting ducts, the henle 's loop, the vessels, the connective tissue, and the caox deposition was obtained at the final magnification of 1,800. the point - counting method used the following formula : vv (structure, reference)=p(structure)/p(total) where p(structure) and p(total) represent the sum of the number of the points hitting the structure and all sampled fields of the kidney, respectively. finally, the total volume of each structure was estimated by multiplying the fractional volume by the final volume of the kidney to prevent the " reference trap " [13 - 15 ]. the diameter of the capillaries and vessels sampled by the unbiased counting frame was measured. the diameter was measured perpendicular to the long axis where the vessel was widest [13 - 15 ]. the statistical comparisons between the group means were performed by analysis of variance and also mann - whitney u - test. the mean and the standard deviation of the kidney volume and the volume of the cortex and medulla are presented in table 1. the total volume of the glomeruli, pct, dct, henle 's loop, collecting duct, cortical and medullary degeneration, and fibrotic tissue in the different groups is shown in table 2. however, after the induction of nephrolithiasis, the volume of the glomeruli, pct, dct, henle 's loop, collecting ducts, and vessels was reduced -57%, 34%, 35%, 58%, 54%, and 32%, respectively, in the rats (p<0.001). after treating the nephrolithiasis with diosmin, on average, -70% to 96% of the glomeruli, pct, of course, the degeneration of the cortical tissue was 5-fold that of the medulla. after treating the rats with nephrolithiasis with diosmin, the volume of caox deposition was -63% less in the rats with nephrolithiasis treated with diosmin (p<0.01). in the nephrolithiasis+diosmin rats, degeneration in the renal cortical tissue and medulla was reduced -70% and 44% compared with that in the rats with untreated nephrolithiasis (p<0.01). the diameter of the capillaries and vessels in the cortex was reduced -7% and -12%, respectively, in the rats with nephrolithiasis (p<0.001). after treating the nephrolithiasis with diosmin, the reduction of the diameter of the cortical capillaries and vessels was recovered (table 3). the mean and the standard deviation of the kidney volume and the volume of the cortex and medulla are presented in table 1. the total volume of the glomeruli, pct, dct, henle 's loop, collecting duct, cortical and medullary degeneration, and fibrotic tissue in the different groups is shown in table 2. however, after the induction of nephrolithiasis, the volume of the glomeruli, pct, dct, henle 's loop, collecting ducts, and vessels was reduced -57%, 34%, 35%, 58%, 54%, and 32%, respectively, in the rats (p<0.001). after treating the nephrolithiasis with diosmin, on average, -70% to 96% of the glomeruli, pct, henle 's loop, collecting ducts, and vessels remained intact (p<0.01). of course, the degeneration of the cortical tissue was 5-fold that of the medulla. after treating the rats with nephrolithiasis with diosmin, the volume of caox deposition was -63% less in the rats with nephrolithiasis treated with diosmin (p<0.01). in the nephrolithiasis+diosmin rats, degeneration in the renal cortical tissue and medulla was reduced -70% and 44% compared with that in the rats with untreated nephrolithiasis (p<0.01). the diameter of the capillaries and vessels in the cortex was reduced -7% and -12%, respectively, in the rats with nephrolithiasis (p<0.001). after treating the nephrolithiasis with diosmin, the reduction of the diameter of the cortical capillaries and vessels was recovered (table 3). the results of the present investigation revealed that diosmin massively reduces caox formation and also protects the renal tissue from damage in a rat model of nephrolithiasis. formation of caox stones starts from the attachment of a crystal formed in the lumen of the renal tubules to the surface of the epithelial cells [16 - 23 ]. most of the crystals formed in the lumen of tubules are discharged in the urine. in healthy individuals, the crystals that attach to the surface of the epithelial cells are digested by macrophages or cellular lysosomes. however, in patients with hyperoxaluria or crystal urine, the renal tubular cells are injured, thus allowing crystals to easily attach to them. then, the crystals aggregate and grow, and finally a stone is formed [16 - 23 ]. several studies have provided evidence that oxalate - induced toxicity is related to superoxide and lipid peroxidation. the oxalate - induced increase in free radical production appears to be related to cell death in a concentration - dependent manner [16 - 23 ]. finding a therapy for preventing the caox deposition seems to be possible by controlling the above - mentioned mechanism, in particular controlling the tubular cell injury and the inflammation process. concluded that urinary glycosaminoglycans might play a critical role in preventing the adhesion of crystals by inhibiting the renal tubular cell injury. reported that the antioxidative effect of green tea decreases caox stone formation, osteopontin expression, and apoptosis and increases superoxide dismutase activity in the kidney tissues of rats. also, sarica. reported a significant decrease in the mean level of apoptosis in the animals receiving vitamin e and allopurinol. in the present study, the protective role of diosmin on the renal structure diosmin has been used to prolong the vasoconstrictor effect of norepinephrine on the venous and lymphatic vessels. as a flavonoid, however, it also exhibits anti - inflammatory, free - radical scavenging, and antimutagenic properties. in the present research capillary hyperpermeability is one of the primary processes of the inflammation. at the microcirculation level, diosmin reduces capillary hyperpermeability and increases capillary resistance by protecting the microcirculation from the damaging processes [1 - 4 ]. in addition, diosmin reduces the expression of endothelial adhesion molecules and inhibits the adhesion, migration, and activation of leukocytes at the capillary level [1 - 4 ]. it has been reported that these effects lead to a reduction in the release of inflammatory mediators, principally oxygen free radicals and some prostaglandins. in a recent article, srinivasan and pari showed the ameliorative effect of diosmin against the oxidative stress in the kidney of diabetic rats. reduction of the degenerative tissue as a result of using diosmin was another finding of the study. this finding might be explained by the reduction in tubular cell injury and inflammation after the treatment with diosmin. in addition, observation of more intact tubules after diosmin treatment in the present survey is an indicator of less tubular cell injury. another aspect of the present research was the reduction of the diameter of the capillaries and vessels in the cortex after nephrolithiasis and the recovery of this after diosmin treatment. as stated previously, diosmin prolongs the vasoconstrictor effect of norepinephrine on the vein. note the diameter reduction of the histological sections is a structural change that might be due to tissue fibrosis, because a significant reduction was seen in the cortex and not the medulla, which contains less fibrous tissue. diosmin reduces caox deposition and the degeneration of glomeruli and tubules in a rat model of nephrolithiasis. | purposekidney stones (nephrolithiasis) are a widespread disease. thus, blocking stone formation and finding new therapeutic methods is an important area of study. diosmin (a major component of the bile) is known to have antioxidant as well as renoprotective effects. the present investigation aimed to evaluate the effect of diosmin on renal tissue protection in rats with ethylene glycol - induced nephrolithiasis.materials and methodsthe rats were randomly divided into three groups. group one (control) did not receive any treatments. in groups two and three, nephrolithiasis was induced by 2.5% (v / v) ethylene glycol + 2.5% (w / v) ammonium chloride (2 ml / d). the second and the third groups received distilled water or diosmin (80 mg / kg / d) by gavage for 21 days.resultsstereological estimation of the renal structures revealed that the average volume of calcium oxalate (caox) in the nephrolithiasis+diosmin rats was -63% less than in the rats with untreated nephrolithiasis (p<0.01). the volume of the glomeruli, proximal and distal convoluted tubules, henle 's loop, collecting ducts, and vessels was reduced -32% to 58% after the induction of nephrolithiasis (p<0.001). in the nephrolithiasis+diosmin rats, on average, -70% to 96% of the glomeruli, proximal convoluted tubules, henle 's loop collecting ducts, and vessels remained intact (p<0.01). degeneration of the cortical tissue was 5-fold that of the medulla. in the nephrolithiasis+diosmin rats, degeneration in the renal cortical tissue and medulla was reduced -70% and 44%, respectively, compared with that in the untreated nephrolithiasis group (p<0.01).conclusionsdiosmin reduces caox deposition and the degeneration of glomeruli and tubules in a rat model of nephrolithiasis. |
invasive candidiasis (ic) is a serious infection associated with a high mortality rate and limited definitive diagnostic signs. colonization is a key component of ic pathogenesis, yet the significance of gastrointestinal candida colonization as a predictor of ic remains controversial. although yeast are normal bowel flora and recovery of low numbers in stool cultures is common, some hospitalized patients exhibit large numbers of yeast in stools obtained for enteric pathogen screening. hospitalized patients undergoing evaluation for a possible inflammatory etiology of diarrhea may heighten concern that heavy yeast colonization of the gastrointestinal tract could progress to ic. clinicians question whether prophylaxis of ic is warranted in such cases, and clinical microbiologists question whether the reporting of heavy yeast carriage is necessary. the study objective was to determine if a relationship exists between ic and recovery of large numbers of yeast from stool cultured for enteric pathogens. all inpatients at a 689-bed academic medical center from january 2004july 2008 with an enteric pathogen stool culture revealing yeast were eligible for inclusion. only stools with moderate to many yeast detected (growth in two or more quadrants of an isolation streak from the original stool on sheep blood agar) were evaluated. sheep blood agar, incubated for 72 hours, was included as a primary medium to screen for aeromonas hydrophila and recognize patients that lack stool flora. each case patient, included only once, was matched by age, gender, and admission date in a 1:3 ratio to subjects with enteric pathogen stool cultures negative for yeast in a retrospective matched case - control study. subjects with ic prior to the index stool and those who received systemic antifungals within 30 days after the index stool were excluded. the primary outcome was development of ic, identified from an institutional database of patients diagnosed with ic and defined by a positive culture from blood, intra - abdominal abscess, peritoneal fluid, or cerebrospinal fluid indicative of ic. a retrospective chart review was done to collect the following : demographics ; admission/ discharge dates ; culture / susceptibility results ; presence and date of ic ; antimicrobial, immunosuppressant, and chemotherapy regimens ; mechanical ventilation ; catheterizations (intravascular and urinary) ; hemodialysis ; and parenteral nutrition received during hospitalization, as well as internal prosthetic devices and surgical procedures performed within 30 days prior to index stool. wilcoxon rank sum and chi - square or fisher s exact tests were used for continuous and categorical variables, respectively. 115 unique subjects had a stool culture that revealed moderate to many yeast out of 2373 total unique stool cultures performed among hospitalized patients during the study period. fifty - seven subjects were excluded [developed ic prior to index stool (n=1), received systemic antifungals after index stool (n=40), and outpatient status at time of culture (n=16) ]. the remaining cases (n=58) were matched to 174 controls in a 1:3 ratio. no significant differences were detected between cases and controls for age, gender, and ethnicity, (data not shown) but antimicrobial use was significantly more common in cases versus controls (95% vs. 73%, p=0.0005). the length of hospitalization was significantly longer (median, 2.5 days longer) for cases (p=0.002). table 1patient characteristics comparing patients with gastrointestinal yeast colonization to those without colonization.patient characteristicyeast in stool cultureppositive(n=58)n (%) negative(n=174)n (%) received antimicrobials55 (94.8)127 (72.9)0.0005received immunosuppressants24 (41.4)63 (36.2)0.481received chemotherapy0 (0)2 (1.1)0.99medical prosthetic devices12 (20.7)20 (11.5)0.08surgical procedures19 (32.8)41 (23.6)0.166mechanical ventilation11 (19)20 (11.5)0.148intravascular catheterization44 (75.9)113 (65)0.12hemodialysis4 (6.9)12 (7.5)0.99parenteral nutrition8 (13.8)28 (16.1)0.68total length of hospitalization [median (range)]8.5 (1, 116)6.0 (1, 231)0, 002aduring hospitalization;bwithin 30 days of index stool culture. during hospitalization ; within 30 days of index stool culture. one control subject developed ic. other studies investigating candidal colonization and systemic disease have neither focused on incidental demonstration of heavy gastrointestinal colonization from an enteric pathogen culture nor firmly concluded a correlation between colonization and subsequent ic. magill. investigated whether colonization site impacted the likelihood of developing ic in 182 surgical intensive care unit (sicu) patients. no sites (urine, oropharynx, trachea, gastric aspirate, or rectum / ostomy) were associated with a high positive predictive value for ic. conversely, all patients with negative rectum / ostomy, urine, and tracheal aspirate cultures remained without ic. the high negative predictive values for these cultures suggest a limited strategy to identify patients who are unlikely to benefit from antifungal prophylaxis. blumberg. assessed risk factors for candidemia in a multicenter, prospective cohort involving sicu patients (n=4276). recovery of candida from rectum and urine samples was not associated with a significant risk of developing candidemia. the authors concluded urinary or rectal colonization alone did not predict patient populations for antifungal prophylaxis. while these findings, in addition to the present study, suggest that rectal / gastrointestinal colonization alone does not predict ic, another study suggested that gastrointestinal colonization was the source for c. albicans candidemia. however, surveillance cultures were obtained after candidemia documentation, and thus, whether gastrointestinal tract colonization preceded candidemia was not elucidated. other investigators have incorporated colonization as one of many risk factors to be included in rules to help predict whether a given patient is likely to develop ic. these studies have focused on patients at high risk for ic and have included colonization sites other than the gastrointestinal tract. additionally, the ability to include colonization with yeast in such a clinical prediction rule requires routine surveillance cultures, which are not performed at our institution. these studies demonstrate that the predictive value of colonization for invasive candidiasis is variable. in our study, the only case of ic occurred in a control subject who did not have heavy gastrointestinal colonization with yeast. thus, if a negative gastrointestinal culture had been used to decide upon prophylactic therapy, this patient would not have received prophylaxis. this matched case - control study was intended to investigate the significance of incidental demonstration of substantial gastrointestinal yeast colonization as a predictor of ic. although conducted in a general population not selected as high risk for ic, many risk factors were present in both cases and controls (table 1). since cultures collected for detection of enteric pathogens were used, these observations may not be applicable to other situations. furthermore, only patients who had an enteric stool specimen submitted for analysis were included in the study, and thus, these results may not be generalizable to the majority of patients who do not have stool specimens collected. other limitations include the retrospective, single - centered study design, small sample size, and lack of data regarding intensive care unit days. additionally, a significant difference was found in length of hospital stay between cases and controls, suggesting more time in the hospital may increase the probability of colonization. | a matched case - control study was conducted to investigate gastrointestinal colonization with yeast as a predictor of invasive candidiasis (ic) in patients who underwent an enteric pathogen test. no significant association was detected between gastrointestinal colonization and ic. however, gastrointestinal colonization with yeast was associated with increased antimicrobial exposure and median length of hospitalization. |
cellular and viral oncogenes, such as ras and adenovirus e1a, trigger p53 activation by inducing the expression of arf 9, which inhibits mdm2 mediated p53 degradation. arf is lost in 58% of cancers9, which had previously been invoked as the critical factor that prevents p53 activation in e1b-55k infected tumor cells 23. using a u2os stable cell - line (p53 wild - type, arf negative) in which arf expression is induced by isopropyl--d - thiogalactopyranoside (iptg), we show that arf stabilizes p53 and activates p21 transcription in mock infection. nevertheless, although arf expression increases basal p53 activity, the induction of p21 is repressed in both wild - type (wt) and e1b-55k infected cells (fig 1b and supp. furthermore, endogenous arf induction also fails to activate p53 targets ine1b-55k infected saecs (fig 1a). thus, p53 is inactivated, irrespective of e1b-55k and arf expression in adenovirus infected cells. dna damage signals also play a critical role in activating p53, triggering p53 phosphorylation and protein stabilization 8,24. in clinical trials, e1b-55k (onyx-015), was used in combination with genotoxic chemotherapies, such as 5-fluorouracil (5-fu) 19,20. we reasoned that the induction of p53 levels alone may not be sufficient to activate p53 in infected cells, and that dna damage is also required. therefore, we also analyzed e1b-55k infected saecs and show that p53 transcriptional targets can not be activated by irradiation (fig 1c and supp. fig 6), uv irradiation (supp. fig 7) or doxorubicin (dox, fig 1e). the activation of p53 in response to dna damage is mediated via kinases, such as atm, atr, dna - pkcs, chk1 and chk2, which phosphorylate p53 8 at key residues, stabilizing p53 and potentiating p53 dna binding 24. a possible explanation for the failure of dna damage to activate high p53 levels in e1b-55k infected cells is that p53 phosphorylation is inhibited by viral infection. however, even without the introduction of exogenous genotoxic stress, p53 is already highly phosphorylated at multiple sites targeted by dna damage kinases in e1b-55k infected saecs (fig 1d). thus, although oncogenes and dna damage trigger p53 stabilization and phosphorylation ine1b-55k infected cells, p53 fails to activate the transcription of downstream effectors. we next examined if in the absence of e1b-55k, mdm2 binds and inactivates p53 in adenovirus infected cells. in contrast to mock, nutlin fails to stabilize p53 further or induce p21 in e1b-55k infected saecs (fig 1e). the histone deacetylase (hdac) inhibitor, trichostatin a (tsa), induces the expression of p21 independently of p53 stabilization or phosphorylation (fig 1e). we conclude that p53 transcriptional targets are dominantly suppressed in adenovirus infected cells, irrespective of e1b-55k, and can not be activated in response to radiation, genotoxic drugs, arf, mdm2 antagonists or hdac inhibitors. our data strongly suggest that there is a previously undiscovered adenoviral protein that inactivates p53 independently of p53 degradation. to test this, we screened for p53 activation in primary cells infected with adenoviruses that have compound mutations in e1b-55k and other early viral genes (supp. in addition to deleting e1b-55k, the loss of either e1a-13s or e4-orf3 is required to activate p53 in infected cells (fig 2a). this is surprising, especially since e1a is a potent oncogene that triggers p53 activation in cellular transformation 7,14. in adenovirus infection, the e1a-13s splice form is required for the transactivation of other viral genes 17, including e4-orf3 (fig 2a). consistent with this, we show that in contrast to gfp, the ectopic expression of e4-orf3 rescues p53 inactivation in both e1b-55k/e4-orf3 and e1b-55k/e1a-13s infected cells (fig 2b). the slight reduction of p21 by ad - gfp ine1b-55k/e4-orf3 co - infection is due to the partial activation of e4-orf3 transcription (in trans) by e1a-13s, which does not occur ine1b-55k/e1a-13s co - infection (supp. hence, e1a-13s induces the expression of e4-orf3 which then inactivates p53 via an e1b-55k independent mechanism. moreover, the expression of e4-orf3 alone is also sufficient to inhibit p53 activation (supp. these data reveal e4-orf3 as a novel adenoviral protein that inactivates the p53 tumor suppressor pathway. the proposed p53 tumor selectivity of the e1b-55k oncolytic therapy, onyx-015, is based on p53 stabilization being the sole critical event that determines p53 activated transcription. although there is some basal p53 activity in e1b-55k infected cells compared to wild - type virus, the additional deletion of e4-orf3 is necessary for p53 to activate downstream effectors over the course of infection (fig 2c - d). in contrast to p53 transcriptional targets, the mrna levels of p53 and the housekeeping gene, gusb, are not impacted by e4-orf3 (supp. p53 stabilization is required to activate p53 transcriptional targets, and does not occur in e4-orf3 infection where p53 is degraded by e1b-55k / e4-orf6. fig 12), we show that the induction of p21 and mdm2 in e1b-55k/e4-orf3 infection is p53-dependent. thus, the deletion of both e1b-55k and e4-orf3 is necessary to activate p53 in adenovirus infection. we conclude that e4-orf3 has a critical and novel role in inactivating p53 independently of e1b-55k and p53 degradation. dna tumor virus proteins, such as e1b-55k, sv40 lt and hpv e6 inactivate p53 via direct high affinity protein - protein interactions 1. however, contrary to this established paradigm, e4-orf3 does not co - localize with p53 (supp. the induction of p53 levels and phosphorylation induces p53 conformational changes that drive sequence - specific dna binding and the recruitment of transcription co - factors 25. a p53 dna binding domain that is competent to bind to dna can be distinguished by immunoprecipitation with pab 1620 versus pab 240 26. p53 is immunoprecipitated selectively by pab 1620 in bothe1b-55k and e1b-55k/e4-orf3 infected cells (supp. fig 14), demonstrating that the p53 dna binding domain is in a protein conformation that should be capable of binding to dna 26 in both cases. to functionally determine if e4-orf3 prevents p53 dna binding, we transfected u2os cells with a p53 luciferase plasmid (p53-luc), where p53 binding to consensus dna sequences activates luciferase transcription 27. a control pgl3-luciferase reporter (non - p53 promoter) is activated to similar levels in all viral infections (supp. infected cells, p53 activated transcription of luciferase is inhibited after 24 hours (fig 3a), which is expected due to p53 degradation. in contrast, p53-luciferase is activated in both e1b-55k and e1b-55k/e4-orf3 infection (fig 3a). the induction of luciferase requires p53 dna binding, since a mutated p53 response element (p53-mutant) abolishes luciferase activity. these experiments demonstrate that e4-orf3 does not compete with p53 for binding to consensus dna target sequences or prevent p53 transcriptional activation of promoters in ectopic reporter plasmids. the ability of e4-orf3 to prevent p53 activated transcription of endogenous targets but not ectopic p53-luciferase plasmids is at first difficult to reconcile. plasmid dna is not subject to the same architectural and packing constraints as dna in cellular chromatin. therefore, we performed p53 chromatin immunoprecipitations (chips) to determine if e4-orf3 specifically prevents p53 dna binding in the context of cellular chromatin. p53 binding to target sites in the p21 (5 and 3 site) and mdm2 promoters 25 is induced upon doxorubicin treatment and e1b-55k/e4-orf3 infection, where it activates the transcription of p21 and mdm2 rnas (fig 3b - c). in contrast, although p53 is induced to similar levels, e4-orf3 prevents p53 dna binding to the p21 and mdm2 promoters in e1b-55k infected cells (fig 3b c and supp. thus, e4-orf3 inactivates p53 by preventing p53 binding to dna target sites specifically in the context of cellular chromatin. we reasoned that p53 dna binding depends not only on the protein conformation of p53 but also the accessibility of target promoters in the cellular genome. we hypothesized that e4-orf3 could inactivate p53 by inducing heterochromatin at endogenous target promoters, preventing the access of p53 to dna. heterochromatin compaction is specified by the loss of histone acetylation and induction of repressive histone methylation 28. tsa fails to induce p21 in e1b-55k infected saecs (fig 1e) suggesting that e4-orf3 inactivates p53 targets via a mechanism that is dominant to the inhibition of histone deacetylation. in cancer, the aberrant epigenetic silencing of tumor suppressor genes, such as p16, is initiated by the methylation of histone h3 at lysine 9 (h3k9) 29. however, in e1b-55k infected cells, where p53 is inactive, dense regions of h3k9me3 repressive heterochromatin are induced at the periphery of the nucleus (fig 3d and supp., we show that suv39h1 and suv39h2 (which share 59% sequence identity and have redundant functions) 30,31, but not setdb1 32 or g9a 33, are specifically associated with the formation of de novo h3k9me3 heterochromatin domains in e1b-55k infected nuclei (fig 3e). the formation of these domains requires e4-orf3 and does not occur in either mock or e1b-55k/e4-orf3 infected cells (supp. these data demonstrate that e4-orf3 induces novel h3k9me3 heterochromatin, which could deny p53 access to endogenous target promoters. to test this, we performed p53 and h3k9me3 chips. the induction of repressive heterochromatin by e4-orf3 is not associated with a global upregulation of either total histone h3 or h3k9me3, which are at similar levels in all infections (fig 4a). in e1b-55k/e4-orf3 infected cells, p53 binding is induced at p21 and mdm2 promoter sites (consistent with fig 3c) while h3k9me3 is at a similar level to an igg negative control (fig 4a and supp. in contrast, in e1b-55k infected cells, h3k9me3 is enriched at the p21 and mdm2 promoters where p53 binding is prevented. h3k9me3 is also induced at the -5 kb region of the p21 promoter and is not restricted to p53 binding sites (supp. thus, in cells expressing e4-orf3, there is an inverse correlation between p53 and h3k9me3 at p53 regulated promoters. the same conclusions were reached for additional p53 targets, including gadd45a, fas, puma and pig3 (supp. in contrast, at non - p53 regulated promoters, such as actin and polr2, h3k9me3 is not induced in e1b-55k infected cells relative to mock (supp. basal h3k9me3 is decreased at these promoters ine1b-55k/e4-orf3 infected cells, suggesting that e4-orf3 may also restrain global demethylase activity. we conclude that e4-orf3 inactivates p53 by inducing de novo h3k9me3 heterochromatin silencing at p53 target promoters. with access denied thus, a major question is how is e4-orf3 directly involved in inducing repressive h3k9me3 heterochromatin at p53 target promoters ? e4-orf3 does not co - localize with p53 and forms a distinctive web - like structure in the nucleus (supp. fig 13). we show that e4-orf3 demarcates the formation of de novo h3k9me3 heterochromatin domains ine1b-55k infected cells. e4-orf3 is, for the most part, adjacent to h3k9me3, suggesting it acts as a novel platform that catalyses heterochromatin formation through transient or long - range interactions (supp., we show that e4-orf3 forms a continuous scaffold that specifies de novo heterochromatin assembly as it weaves through the nucleus (fig 4b). these data demonstrate a direct role for e4-orf3 in orchestrating h3k9me3 heterochromatin silencing at p53 target promoters. furthermore, they reveal an extraordinary nuclear scaffold that either builds on existing architectural features that organize cellular dna or is a novel viral construction that targets heterochromatin assembly at p53 target promoters. these data beg the question as to the specificity of e4-orf3 in silencing p53 targets. to determine the global consequences on cellular transcription, we performed genome - wide expression analyses on infected saecs (supp. these studies demonstrate that e4-orf3 is an exclusive player in the global transcriptional changes induced upon viral infection. there are 1,730 overlapping genes that are similarly up or downregulated by a log fold change (fc) greater than two in bothe1b-55k and e1b-55k/e4-orf3 versus mock, which reflect a common transcriptional program (fig 5a). these global changes are associated with the cell cycle and e2f activation (supplementary table i and ii). this is consistent with e1a mediated inactivation of rb 34 and recruitment of p300 and pcaf to induce active histone acetylation marks at the promoters of genes involved in cell growth, division and dna synthesis 35,36. thus, e4-orf3 induced heterochromatin silencing, as well as the scaffold it forms throughout the nucleus, does not affect the global activation of cellular transcripts induced by viral infection. to define the genes specifically targeted by e4-orf3 e4-orf3 prevents the transcriptional activation of 265 genes by a log fold change of two or more in e1b-55k infected cells. to determine how many of these genes are likely to be regulated by p53, we used two criteria : the presence of consensus p53 dna binding sites in their promoters and their induction upon treatment with the mdm2 antagonist, nutlin. a heat map of top transcripts differentially upregulated in response to e1b-55k/e4-orf3 and nutlin includes well known p53 targets (mdm2, fas, pig3, tp53inp1, btg2, lrdd) associated with growth inhibition and apoptosis, as well as novel targets (hrh1, rnase7, jmjd1c) (fig 5b and supp. 73% are induced in response to nutlin and/or have predicted p53 binding sites (fig 5c and supplementary table iii a pathway analysis of e4-orf3 regulated transcripts indicates that in addition to the p53 pathway, there is a significant overrepresentation of genes associated with immune modulation as well as tissue / vascular remodeling (supplementary table v). these data suggest that e4-orf3 may target p53 promoters as part of a general anti - viral transcriptional silencing program, which is consistent with the highly defective replication of e1b-55k/e4-orf3 in primary cells (supp. the conclusions of our study challenge the general assumption that p53 induction and phosphorylation is tantamount to p53 activity, which is the premise for several cancer therapies 1113. our data reveal a novel and dominant mechanism of p53 inactivation that acts via the targeted epigenetic silencing of p53 target promoters. we identify a viral protein, e4-orf3, which appears to form a novel scaffold that weaves through the nucleus, directing suv39h1/2 h3k9me3 heterochromatin assembly at p53 target promoters to silence p53 activated transcription in response to genotoxic and oncogenic stress (fig 5d). remarkably, this suppressive nuclear web selectively ensnares p53 and anti - viral genes while operating in the backdrop of global transcriptional changes that drive pathological cellular and viral replication. thus, a major question is if e4-orf3 reflects or exhorts an existing cellular mechanism and nuclear structure that censors p53 transcriptional activity. strikingly, all of the known targets of e4-orf3, pml 38, the mre11/rad50/nbs1 (mrn) dna damage / repair complex 39 and tif1 40 are subverted by tumor mutations. it is intriguing to speculate that e4-orf3 physically integrates the inhibitory effects of several cancer pathway mutations, both known and yet to be discovered, which together have emergent functions 4 in silencing p53 activity. similar to the discovery of p53 with a viral protein 2,3, e4-orf3 provides a powerful dynamic probe with which to define critical cellular factors that induce de novo epigenetic silencing of p53 target promoters in somatic cells. this has important implications for understanding how high levels of wild - type p53 might also be inactivated in cancer as well as the dynamic mechanisms that induce aberrant epigenetic silencing of tumor suppressor gene loci. finally, our identification of e4-orf3 changes the fundamental definition of how p53 is inactivated in adenovirus infected cells, which is a critical mechanistic insight that could now enable the rational development of true p53 tumor selective adenoviral therapies. real - time quantitative pcr (rt - qpcr) was used to quantify p53 targets 21, and normalized relative to 18s. for luciferase assays, 100 um d - luciferin was added 4 hours post infection (h.p.i.) and luminescence quantified every hour. global gene expression was determined using affymetrix human exon 1.0st arrays and analyzed with partek and genomatix software. | the transcription factor p53 guards against tumor and virus replication and is inactivated in almost all cancers. p53 activated transcription of target genes is thought to be synonymous with the stabilization of p53 in response to oncogenes and dna damage. during adenovirus replication, the degradation of p53 by e1b-55k is considered essential for p53 inactivation, and is the basis for p53 selective viral cancer therapies. here we reveal a dominant epigenetic mechanism that silences p53-activated transcription, irrespective of p53 phosphorylation and stabilization. we show that another adenoviral protein, e4-orf3, inactivates p53 independently of e1b-55k by forming a nuclear structure that induces de novo h3k9me3 heterochromatin formation at p53 target promoters, preventing p53 dna - binding. this suppressive nuclear web is highly selective in silencing p53 promoters and operates in the backdrop of global transcriptional changes that drive oncogenic replication. these findings are important for understanding how high levels of wild - type p53 might also be inactivated in cancer as well as the mechanisms that induce aberrant epigenetic silencing of tumor suppressor loci. our study changes the longstanding definition of how p53 is inactivated in adenovirus infection and provides key insights that could enable the development of true p53 selective oncolytic viral therapies. |
hypokalemic periodic paralysis is a rare disorder characterized by transient attacks of flaccid paralysis of varying intensity and frequency. reported incidence of hypokalemic paralysis in patients with type 1 renal tubular acidosis (rta) type 1 rta or distal rta (drta) is a disorder of renal tubular acidification characterized by hyperchloremic metabolic acidosis with a normal serum anion gap. inherited forms include autosomal - dominant, autosomal - recessive, or x - linked recessive, of which autosomal - dominant form causing mutations in the basolateral chloride bicarbonate exchanger (ae1) has been identified as the most common form of inheritance. acquired causes include hypergammaglobulinemic states, such as hyperglobulinemic purpura, cryoglobulinemia, fibrosing alveolitis, sjogren syndrome, lupus, chronic active hepatitis, thyroiditis, graves disease, primary biliary cirrhosis ; disorders of calcium metabolism, e.g. primary hyperparathyroidism, vitamin d intoxication, idiopathic hypercalciuria, familial absorptive hypercalciuria, medullary sponge kidney. renal involvement in systemic lupus erythematous is mainly glomerulonephritis and isolated tubular dysfunction is rare. this unusual presentation of systemic lupus erythematosus (sle) was first presented by fortenberry and kenney in 1991 and infrequent cases have been reported in the literature ever since. we present the case of a woman who was admitted with acute onset hypokalemic quadriparesis. six months after the initial presentation, the patient fulfilled 5 out of 11 american college of rheumatology (acr) criteria for diagnosis of sle and patient was managed successfully with oral and intravenous potassium chloride, sodium bicarbonates and oral hydroxychloroquine and a short course of steroid. a 30-year - old woman presented with gradually increasing weakness of both upper and lower limbs. she reported having had a similar episode twice previously, most recently 2 months ago ; and at all of these occasions, she had documented hypokalemia and she was managed by a local physician with parenteral and oral administration of potassium chloride that improves her condition on these occasions. resting after exercise or eating patient also had complaints of recurrent oral ulceration, photosensitivity, hair loss, loss of appetite, easy fatigability, pain in multiple joints and she experienced this pain almost throughout the day, but it was worse in the morning. she had also off and on low grade fever over 3 months before admission but she had no burning in the urine, cough and abdominal pain or vomiting, pruritus, dryness of mouth, difficulty in swallowing, gritty or sandy sensation under eyelids and reynaud 's phenomenon. on the day of admission, the patient also complained of difficulty in swallowing solid food and inability to speak loudly but there was no history of visual blurring, diplopia, facial asymmetry, sensory, bladder, or bowel complaints. she had a clear sensorium with no history of seizures, involuntary movements, root pains, or band - like sensation over the trunk. she did not give a history of prior drug intake, colicky abdominal pain and recent vaccination. six months before her current presentation, the patient had progressive weakness in all four limbs ; it started in one limb, but progressed to all four limbs within a few hours and her past medical record and history given by her husband revealed that she was conscious and oriented at the onset of weakness. her eye movements were normal and there was no facial asymmetry and seizure at the onset of weakness. at current admission to our hospital in physical examination, blood pressure was 130/80 mm hg ; pallor was present. musculoskeletal and neurological examination revealed muscle strength was poor (flicker 1/5) in all the muscle groups. laboratory investigations done revealed blood urea 28 mg / dl, serum creatinine 1.3 mg / dl, hypokalemia 2.2 mmol / l, hyperchloremic metabolic acidosis (chloride 112 meq / l, arterial blood ph 7.279) and alkaline urine (urinary ph 7.5). ultrasonography of the abdomen revealed right nephrolithiasis with bilateral normal size kidney and normal liver size and echotexture. in view of the normal anion gap acidosis (12.4 mmol / l), alkaline urine (urinary ph 7.5), low serum potassium (2.2 mmol / l), absence of fanconi 's syndrome, positive urinary anion gap (46 mmol / l), the diagnosis of drta was made. type 2 rta was excluded, as her 24 h urinary excretion of phosphate and uric acid was normal with no glycosuria. renal biopsy was deferred in view of normal renal function and no active sediment in urine routine microscopy. her biochemical parameters corrected with treatment and she attained blood urea of 24 mg / dl, serum creatinine 1.0 mg / dl, potassium 3.9 meq / l, chloride 95 meq / l and arterial blood ph of 7.332. she had continued on the oral supplements with sodium bicarbonate (500 mg twice daily) and oral hydroxychloroquine 200 mg twice a day and had been asymptomatic on regular follow - up [table 1 ]. our patient initially presented with generalized weakness and was diagnosed drta due to sle on evaluation. generalized weakness is a very subjective feeling and is an uncommon objective finding in outpatients and emergencies. there is a large differential diagnosis for muscle weakness, but only a few medical conditions develop rapid onset generalized muscle weakness. on initial presentation, we kept the possibility of inflammatory myopathy, thyroid disorder, channelopathy and neuropathy and electrolyte disturbance as the cause of our patient symptoms. investigations revealed that patient did not have inflammatory myopathy as she had normal creatine phosphokinase and serum lactate dehydrogenase. she was diagnosed sle based on acr criteria for diagnosis of sle and drta on the basis of hyperchloremic metabolic acidosis, hypokalemia with a positive urinary anion gap and the absence of fanconi 's syndrome. our case is considered to be an acquired form of drta with predominant tubular involvement in sle. in drta, the kidney is not able to adequately excrete an acid (h+) load and loses its normal acid - base balance and characterized by hyperchloremic metabolic acidosis, a normal serum anion gap, a urinary ph 5.5, hypokalemia, a positive urinary anion gap, nephrolithiasis and daily replacement of sodium bicarbonate 4 mmol / kg generally improve the conditions. previous studies have also shown the association of sle with various tubular defects, drta being the most common type of tubular involvement in sle, although type 4 rta has also been recently reported. tubular dysfunction is well - described in sle and coincides with active proliferative glomerulonephritis, although patients usually show manifestations of lupus rather than features of rta. pasternack and linder found the deposition of immunoglobulin with complement as well as immunoglobulin - producing mononuclear cells around the tubuli, which was highly suggestive of an immunological process affecting the tubuli, eventually leading to the destruction of some of them. to conclude, tubular function should be assessed carefully in lupus patients and a work - up for the presence of sle should be done in all rta cases. | we report recurrent hypokalemic periodic quadriparesis in a 30-year - old woman. patient had also symptoms of multiple large and small joint pain, recurrent oral ulceration, photosensitivity and hair loss that were persisting since last 6 months and investigations revealed systemic lupus erythematosus (sle) with distal tubular acidosis. our patient was successfully treated with oral potassium chloride, sodium bicarbonate, hydroxychloroquine and a short course of steroids. thus, tubular dysfunction should be carefully assessed in patients with sle. |
to start the discussion, the relevant results from a survey of the isds community (3) were presented. there were 165 responders from 28 countries. when asked whether they collaborated regularly with workers from a sector different from theirs, 93% answered affirmatively. seventy - two percent of the respondents were from the human health sector, and among those stating collaboration with another health sector, 60% pointed to collaborations with the animal sector. however, at the 2013 isds conference, only five out of the 180 abstracts presented work related to animal health (three dealing exclusively with animal health and two dealing with both animal health and public health). it became apparent during the roundtable discussion that most collaboration results from efforts to control a zoonotic disease. public health workers know of veterinarians working at many health departments in what is called veterinary public health. however, they are not aware of, or have little collaboration themselves, with animal health agencies and epidemiologists. the question raised was, should they be more aware?. the audience presented many different opinions. one participant noted that it should not be relevant whether public health epidemiologists are familiar with animal health and/or collaborate with those agencies and individuals responsible for animal health per se (i.e. aimed at diseases exclusively affecting animals) ; instead, they should focus efforts on zoonotic diseases and areas where multi - disciplinary work is more effective and needed. other participants, however, pointed out that animal and human disciplines have largely overlapping methodologies, and more collaboration would speed development as each discipline learns from the other 's experience. one example mentioned was salmonella outbreak investigations, which are often closed once their source has been identified as an animal product. stronger collaborations with the animal health sector would allow continuing investigations to better understand the entire contamination pathway and result in control measures to prevent further outbreaks in both humans and animals. a very practical comment reminded the audience that this is not only a question of should we do it? but also, how could we do it?. a participant said, we should care about animal health for the sake of it.. it may be difficult to foster interest in animal health by the human health sector if we leave zoonoses out of the equation. a former ohio department of health worker reported that the veterinary college, the department of health, usda wildlife services, and state agriculture partners used to routinely hold joint meetings to discuss surveillance coordination. we did it very easily and we kept individual goals in mind, but the important thing is that it opened doors for so many other collaborations and activities : education of veterinary students and public health students, a rabies baiting program, coordinating efforts and logistics, etc.. this case highlighted many ways agencies can benefit from multi - disciplinary collaborations. it also led to a discussion on how building and routinely fostering / nurturing personal connections among the agencies can greatly improve the coordination of efforts when rapid action is needed, such as during an outbreak control situation. a success story was reported from singapore, where a true one health governmental initiative was built by joining animal health, human health, and environmental health interests. the initiative 's aim was to develop joint activities in four areas : capability development, protocol development, risk communication, and information sharing. indicator was created for animal and human health workers to routinely look at cases together. in her view, the threat of zoonoses in the state, such as tuberculosis and west nile virus, caused public health and animal health agencies to understand the need to collaborate and work together. she discussed how collaborations built to fight specific diseases opened doors for joint work that strengthened disease control efforts against new emerging threats in the state. a participant from the us department of defense shared his experience meeting three times a year with others from institutions involved in human, animal (including wildlife), plant, and environmental health. most participants enthusiastically agreed that the bridges must be built in a time of peace, but also pointed out that in reality, most fruitful collaborations were established after a zoonotic disease outbreak forced animal and public health workers to connect and work together. overall, most participants recognized that such collaborations are often built on a personal, rather than institutional, level, and each worker must actively build networks of public and animal health workers to guarantee fast communication and crisis response. participants discussed whether different goals and strategies among the human and animal disciplines could hinder collaboration. some differences included the economic value of animals (livestock in particular), different epidemiological units (individual in humans versus herds in animals), and different concerns regarding data sharing and protection, which can be less strict in animal health. for example, new methods develop much faster in human health due to more resources, and animal health can benefit from these developments. however, in other fields, animal health scientists have more available research subjects and individual data, and in those cases they could lead the development of new methods. another participant disagreed, stating that animal owners economic considerations do not necessarily mean that animal and public health workers have different missions. veterinary health agencies also cite maintaining the health and welfare of animals as their primary institutional goal. while their missions may be similar, health workers often have different work cultures and ways of handling their target populations. increased collaboration needs to be based on educating workers from both disciplines on issues they will face when working together with animal and human populations. the importance of a joint mission statement and joint communication strategy to the public should not be underestimated. communication failures resulting in different messages from human and animal agencies often result in mistrust by the public. health workers from both disciplines expressed concern about whether one side could appropriately handle epidemiological knowledge / data from the other side. others strongly defended their right not to share such data, as agencies must guarantee confidentiality to access the information in the first place. the group mentioned examples of public overreaction to common animal health issues, such as anthrax. but the audience was positive that trust can be built when both sides are committed. participants touched on the need for animal and public health institutions to integrate their functions around the one health framework, rather than building informal networks of specific individuals. judy akkina described the one health coordination office established by usda 's animal and plant health inspection service (aphis), veterinary services, whose goal is to form a culture of internationally recognized experts to meet the need of animal health integration with public health ; to build and to strengthen collaborations. this office 's focus areas are pandemic and animal disease preparedness, zoonoses engagement and response, global health security, pre - harvest food safety, and antimicrobial resistance. cline dupuy mentioned the example of the triple - s (syndromic surveillance systems in europe), a 3-year project that provided a platform for animal and public health scientists to discuss how to implement syndromic surveillance systems and build guidelines. the audience agreed that conferences intended for both public and animal health scientists and stakeholders would be good venues to establish relationships, and the isds annual meeting was cited as one such opportunity. an invitation was extended to the discussion group to participate in the international conference on animal health surveillance in havana, cuba, may 79, 2014. of interest is a comment from the audience regarding the need to extend this discussion to those tasked with surveillance design and implementation. gray literature resources on one health are still scattered and hard to make sense of, and that is a limitation to reach many health stakeholders. the roundtable leaders felt strongly that concrete opportunities need to follow these proposals, starting with writing this document. other actions should include the promotion of one health webinars and utilizing the support offered by the isds. since this roundtable, isds support of one health activities has resulted in the organization of the first isds animal health literature review. discussions among participants identified possible topics that could interest both human and animal health sectors, such as evaluation of surveillance systems cost effectiveness and performance (e.g. data quality, sensitivity, and specificity). these literature reviews will also provide a good opportunity to identify future speakers for one health webinars. the roundtable showed interest in one health not only from animal health scientists but also many public health stakeholders who realize the importance of building common programs and establishing closer connections. even though collaborations between human and animal health sectors are often built to respond to zoonotic outbreaks (during crisis time) and are mainly based on personal networks, this roundtable highlighted the interest in maintaining such collaborations after the crisis. ways to maintain and strengthen these links should be investigated and documented, and some examples include joint surveillance planning, tabletop exercises for emergency response, laboratory capacity and testing cooperation, and regular one health stakeholder meetings and trainings. the support of the isds by promoting the roundtable, joint literature reviews, and webinars has been very important to stimulate connections. this interest needs to result in concrete actions aimed at permanently integrating the animal health and one health theme into the society 's program. the authors have not received any funding or benefits from industry or elsewhere to conduct this study. | motivated by the perception that human and veterinary medicines can cooperate in more ways than just fighting zoonoses, the authors organized a roundtable during the 2013 annual meeting of the international society for disease surveillance (isds). collaborations between human and animal health sectors were reported to often rise in response to zoonotic outbreaks (during crisis time) and be mainly based on personal networks. ways to maintain and strengthen these links were discussed. |
empathy is one of the most important aspects in the medical profession (110) and when it is employed in the right capacity it can be very effective in the relationship between doctor and patient (11). empathy is a very important tool in understanding the concerns and suffering of the patient from their perspective, so that the patient feels understood and has been given assistance (1215). some studies (16, 19) suggest that empathy in physicians would lead to better patient care because (a) it increases satisfaction (2, 19), (b) it increases indulgency (20), (c) it enhances the ability of physicians to diagnose and treat (20), and (d) it could lead to a significant reduction in medical malpractice (21). the jefferson scale of empathy (jse) is an instrument that has been designed to assess empathy in the medical profession (hp version) and related students (s - version) (22). in iran, several studies have been carried out in the field on medical students (2, 22), but there is no study regarding one of the most important components of the patient - physician relationship which is among ras and their potential as future professionals. the aim of this study was to measure empathy score among ras in mashhad, iran, during a period from march to september, 2015. similar studies were used to determine the sample size (3). in iran, the iranian student version of the jpse - s was distributed to 120 clinical ras from five large governmental hospitals in mashhad (iran) in september 2015 and they were give twenty minutes to independently complete the questionnaire. a total of 98 volunteers completed the jspe - s. participation was completely voluntary and ras received no remuneration for their part in the study. jspe - health professional version is an instrument to measure patient care among physicians (17). jspe is a self - report tool and includes 20 likert - type items answered on a seven point scale (from strongly disagree to strongly agree). furthermore, a factor structure of jse has been evaluated using exploratory factor analysis (efa) and a three - factor structure has been used, including perspective taking, compassionate care, and ability to stand in patients shoes (14). the data of this study were analyzed using spss version 17 (spss inc., chicago, illinois, usa) descriptive statistics including tests for assessing the normality of the data and independent - samples t - test to assess the difference of empathy between the two sexes. independent sample t - test and multivariate analysis of variance (manova) were used to assess the effect of gender on scores of jse and its three factors. in order to investigate the underlying components of the jspe, data were subjected to principal component analysis (pca) by using varimax rotation method. similar studies were used to determine the sample size (3). in iran, the iranian student version of the jpse - s was distributed to 120 clinical ras from five large governmental hospitals in mashhad (iran) in september 2015 and they were give twenty minutes to independently complete the questionnaire. a total of 98 volunteers completed the jspe - s. participation was completely voluntary and ras received no remuneration for their part in the study. jspe - health professional version is an instrument to measure patient care among physicians (17). jspe is a self - report tool and includes 20 likert - type items answered on a seven point scale (from strongly disagree to strongly agree). furthermore, a factor structure of jse has been evaluated using exploratory factor analysis (efa) and a three - factor structure has been used, including perspective taking, compassionate care, and ability to stand in patients shoes (14)., chicago, illinois, usa) descriptive statistics including tests for assessing the normality of the data and independent - samples t - test to assess the difference of empathy between the two sexes. independent sample t - test and multivariate analysis of variance (manova) were used to assess the effect of gender on scores of jse and its three factors. in order to investigate the underlying components of the jspe, data were subjected to principal component analysis (pca) by using varimax rotation method. of the 120 ras who received the jspe - s, 98 completed and returned the survey. seventy four of the 98 who completed the survey were effective, giving an overall effective response rate of 83.5%. the average age was 31.49 4.49 and the majority were male (58%). there were three specialties : 15 (20.3%) in surgery, 30 (40.5%) in internal medicine, and 29 (39.2%) in psychiatry. there were no statistically significant differences between the groups regarding gender and age (p0.05). mean score of jse in female was 83.87(15.68) and in male was 89.37(14.48). the mean scores for each of the three factors of the scale were also calculated. the mean scores for perspective taking were 38.90 (13.11) and compassionate care were 39.27 (7.94), standing in the patients shoes was 8.89 (2.80). table 1 shows the rotated factor loadings for the jspe, based on the responses of the 70 ras. subsequent tests of between - subjects effects showed that the female group scored significantly higher on perspective taking (p=0.044) (mean of perspective taking in female vs. male : 39.7/35.7). also, an inverse significant relationship was found between the ras age and mean empathy score (pearson correlation= 0.204, p= 0.08). statistical analysis showed that perspective taking between the two age groups was statistically significant (p= 0.027 ; confidence interval ci = 1.3913.35). spearman s rho test also showed a significant negative correlation between age and average score of empathy (correlation coefficient : 0.257 ; p= 0.02). among the three specialties : psychiatric, internal medicine, and surgery, statistical analysis showed that there was a significant difference in total empathy score and perspective taking. post hoc tests also showed that empathy score in psychiatric ras is more than surgery ras (table 2, figure 1). in the fourth year of education, students had higher perspective taking scores than students in second year (p=0.04). in compassionate care between groups, there was a significant difference (p 0.001) and the average score of empathy extended further by increasing the interest in their field. the aim of this study was to examine the levels of patient empathy in ras. the mean empathy score of this study (mean = 87.67) was lower than in the japanese (mean = 104.3), kuwaiti (mean = 104.6), iranian medical students (mean = 105.1 and 110.41), and chinese (mean = 109.60) (2224) studies respectively. this may be due to the differences in cultural factors, custom, ethnicity, spiritual belief, educational system, variation of selection of ras, and availability of appropriate role model. average score of empathy was higher in women than in men, which is similar to other studies (25) (2629). also in dental students, it was shown that females scored significantly higher on the jspe than males (29, 30) (31). women show a greater understanding of the emotional support, which is important to develop interpersonal relationships with patients (32). also, several studies have shown that female physicians (2) and female medical students (1, 23) gain higher empathy scores in the care of patients. the results of this study are aligned with previous studies. gender also has shown the difference of empathy in the three factors of the scale and observed that, difference exists only in perspective taking, while in the study of van ryn. (33), in both domains of perspective taking and compassionate care, there were significant differences. in the study of prabhu. (34), the mean empathy score of male students is greater than that of female students. in this study, regarding empathy among different ages, there was a decrease with increase in age, which is similar with the study of khademalhosseini. but in a deliang wen study, there were no significant differences in the age groups. these studies are similar to those of chinese (25), japanese (23), korean (33), portuguese (34), and kuwaiti (24) medical students, but were different with american (35, 36) medical students. empathy score among ras was found to increase by the advance in their educational years. ras should be educated in a way that they learn both scientific concepts of medicine and communication with patients and also learn how to empathize with them. ras must learn how to treat patients, not just to treat their diseases (29, 37). a cohort study conducted on internal residency students showed that the amount of empathy with patients was much higher in the first year as compared to the last year of specialty (38), which was contradictory to our study. this study was cross - sectional in nature so causal inference can not be mentioned and also a prospective study is needed to follow ras annually from the beginning of first year until graduation, also larger study populations covering different ras are required to validate the results of this study this study shows significant differences in total empathy score and perspective taking in three specialties. practical significance of these findings is that there is need for a curriculum regarding iranian student residence assistants. with an increased sample size, a cohort study could be a possible route for future research on this topic. | introductionempathy, an essential component of the physician patient relationship, may be linked to positive patient outcomes. this study aimed to determine the empathy score among student residence assistants (ras).methodsin this descriptive design (cross - sectional study), 102 iranian ras participated in the study during 2015, completing the jefferson scale of empathy (jspe). data collection was analyzed using spss version 17. manova, independent - samples t - test, spearman correlation and confirmatory factor analysis (cfa) were used for data analysis.resultsmean score of jse in the sample was 87.06 (15.14). the mean scores for perspective taking, compassionate care, and standing in the patients shoes were 38.90 (13.11), 39.27 (7.94), and 8.89 (2.80) respectively. among the three specialties, (psychiatric, internal medicine, surgery) results showed significant differences in total empathy score (p=0.001) and perspective taking score (p= 0.008).conclusionsthis study showed significant differences in total empathy score and perspective taking in three specialties. we suggest that the curriculum in iranian ras include more teaching on empathy and communicational skills. |
increased recognition and improved understanding of the neuropathic component of low back pain raises the potential for the development of mechanismbased therapies. open and retrospective studies suggest that agents like tapentadol and topical analgesics such as the capsaicin 8% patch and the lidocaine 5% medicated plaster may be effective options for the treatment of neuropathic low back pain in defined patient groups. low back pain (lbp) defined as pain and discomfort localized below the costal margins and above the inferior gluteal folds, with or without referred leg pain (airaksinen., 2006) is one of the most common chronic pain conditions encountered in worldwide clinical practice. lifetime prevalence of lbp is estimated to be > 70% in industrialized countries, with a 1year prevalence of 1545% (kaplan., 2013), it is generally accepted that only a minority of patients report persistent pain after an acute episode. however, a recent systematic review of prospective cohort studies, set in primary care suggests that as many as twothirds of patients go on to develop chronic lbp (itz., 2013). results of the 2010 global burden of disease study show lbp to be the most common cause of years lived with disability (ylds) and the sixth leading cause of disabilityadjusted lifeyears (dalys) worldwide (murray., 2012 ; vos., 2012). lbp was estimated to be responsible for 58.2 million ylds in 1990, increasing to 83.1 million in 2010 (vos., 2012). lbp is frequently associated with comorbid conditions, most notably depression, panic and anxiety disorders, and sleep disturbances (freynhagen., 2006b ; hagen., 2006 ; freynhagen and baron, 2009). chronic lbp is a complex, heterogeneous condition, where both nociceptive and neuropathic pain mechanisms may be involved. in lbp, nociceptive pain results from activation of nociceptors that innervate ligaments, joints, muscles, fascia and tendons as a response to tissue injury or inflammation and biomechanical stress. neuropathic back pain describes pain arising from injury or disease directly affecting the nerve roots that innervate the spine and lower limbs, and pathological invasive innervation of the damaged lumbar discs. chronic lbp is increasingly considered to be a mixed pain syndrome consisting of both nociceptive and neuropathic components (treede., 2008 ; freynhagen and baron, 2009), and it has been suggested that neuropathic components in chronic lbp may be underrecognized and therefore undertreated. this paper reviews the role of neuropathic mechanisms in chronic lbp and discusses implications for clinical management, with particular focus on currently available pharmacological treatment options. clinical practice guidelines typically suggest that the prevalence of neuropathic pain in lbp is approximately 5% ; however, some reports suggest that as many as 1655% of patients with chronic lbp have possible neuropathic pain components (hassan., 2004 ; kaki., 2005 ; freynhagen., 2006a, b ; freynhagen and baron, 2009 ; beith., 2011 ; the wide variation in the reported prevalence of neuropathic pain in lbp is most likely due to differences in methodology between studies, particularly in terms of the definition of neuropathic pain, pain assessment tools and the body area assessed. in a study investigating the neuropathic component of lbp in patients with or without leg pain using the douleur neuropathique en 4 questions (dn4), the relative contribution of neuropathic mechanisms was found to increase with the degree of distal pain radiation (attal., 2011). the proportion of patients with neuropathic pain as a component ranged from 8% in patients with pain restricted to the lumbar area, to 15% in patients with pain radiating proximally, 39% in patients with pain radiating below the knee without neurological signs and 80% in patients with pain radiating towards the foot in a dermatomal distribution with neurological signs corresponding to typical radiculopathy. neuropathic lbp is associated with increased likelihood and severity of medical comorbidities (freynhagen., 2006a ; beith., 2011 ; mehra., 2012), reduced quality of life (qol) (beith., 2011) and higher health care costs (berger., 2004 ; schmidt., 2009 ; mehra., 2012), when compared with low back pain without a neuropathic component. in a study in germany, health care costs in patients with chronic lbp were 67% higher in those with neuropathic pain than in those with nociceptive pain alone, and approximately 16% of the total costs associated with lbp were estimated to be attributable to neuropathic pain (schmidt., 2009). furthermore, an analysis of a us claims database found that 90% of patients with chronic lbp have a neuropathic component (mehra., 2012). total annual direct costs of chronic lbprelated health care resource use were approximately us$96 million. chronic lbp with a neuropathic component accounted for 96% of these total costs, with a mean annual perpatient cost of care approximately 160% higher in patients with neuropathic lbp than in those without neuropathic pain (us$2577 vs. us$1007, respectively ; p < 0.0001). results from the 2010 global burden of disease study found that in germany between 1990 and 2010, lbp caused the loss of 2.1 million dalys, with only ischaemic heart disease accounting for a greater loss in dalys. moreover, the absolute number of dalys lost as a result of lbp rose by 11% during the study period (plass., 2014). the disproportionately high health care costs in patients with neuropathic lbp suggest a need for more targeted therapeutic interventions to improve patient outcomes and reduce the burden on health care systems. low back pain is classified on the basis of both the clinical characteristics of a patient and the underlying pathophysiology of the condition (quebec task force on spinal disorders, 1987 ; task force on taxonomy of the international association for the study of pain, 1994 ; bogduk, 2009). the quebec task force on spinal pain suggested classifying patients with lbp into 11 subgroups, of which the first four were based on pain location and the presence or absence of neurological signs : (i) lbp only ; (ii) lbp and pain above the knee ; (iii) lbp and pain below the knee and (iv) lbp with pain above and below the knee and signs of nerve root involvement (quebec task force on spinal disorders, 1987 ; kongsted., 2013). using this classification, patients with lbp and leg pain and signs of nerve root involvement have been shown to be more severely affected and have a worse prognosis than those with lbp alone (kongsted. the oswestry disability index is an important tool that researchers and physicians use to classify functional disability as a result of lbp (fairbank and pynsent, 2000), and is considered the gold standard of low back functional outcome tools, but does not differentiate between nociceptive and neuropathic components. nociceptive lbp is understood to be pain arising from the vertebral column or its adnexa, evoked by noxious stimulation of structures in the lumbar spine, or from the deep soft tissues of the back (muscles and thoracolumbar fascia) (hoheisel., 2013). noxious stimulation of structures in the lumbar spine can also produce referred pain in addition to back pain. in clinical terms, referred pain is defined as pain perceived as occurring in a region of the body topographically distinct from the region in which the actual source of pain is located. referred pain arises from central processing of afferent activity in intact nerves ; it does not imply an underlying neuropathic mechanism. the mechanism of referred pain (convergenceprojection model) consists of convergence of inputs from two tissues onto the same spinal neuron, and projection of the resulting pain sensation into the wrong tissue (i.e. not the one where the injury is located) (arendtnielsen and svensson, 2001). as the source of spinal referred pain lies in the somatic tissues of the lumbar spine, it is often called somatic referred pain (bogduk, 2009). somatic referred pain is generally perceived in regions that share the same segmental innervation as the source. nociceptive lbp and somatic referred pain do not involve injury or disease of nerves and/or nerve roots. radiculopathy is defined as objective loss of sensory and/or motor function as a result of damage to the nerve root and can occur with or without associated pain (task force on taxonomy of the international association for the study of pain, 1994). when radiculopathy is associated with pain, this is referred to as painful radiculopathy. according to the proposed neuropathic pain grading system developed by the special interest group on neuropathic pain (neupsig), painful radiculopathy fulfils the criteria for definite neuropathic pain when the diagnosis is also based on sensory signs, and the criteria for probable neuropathic pain if it is only based on motor signs (treede., 2008 ; painful radiculopathy perceived as arising in a limb or the trunk, with a distribution that is consistent with one or more dermatomes, fulfils the criteria for possible neuropathic pain according to the same grading system. painful radiculopathy can qualify as being neuropathic, when the underlying neurological lesion or disease is demonstrated by confirmatory tests (as detailed below), when there are sensory signs within the pain distribution, or when both elements are present. although radiculopathy and radicular pain often coexist, and may be caused by the same lesion, they may also exist in isolation. typically, painful radiculopathy is associated with direct damage to nerve roots ; however, it can occur independently from this, for example, as a result of inflammation affecting the spinal nerves. sciatica is a common term used by both doctors and patients to describe a specific pattern of pain in the back of the thigh and sometimes the calf and foot that has radiated along the sciatic nerve.. failed back surgery syndrome (fbss) is the term used to describe chronic back and/or leg pain that persists or occurs after spinal surgery, usually laminectomy. fbss may or may not also include a neuropathic component (hussain and erdek, 2014) ; and as in other postsurgical pain syndromes, the neuropathic component is likely to contribute to the pain being chronic (kehlet., 2006). although radicular pain and radiculopathy are distinct diagnostic entities, a recent systematic review undertaken to assess how radiating leg pain is defined in randomized controlled trials of conservative treatments in primary care found the two terms to be used inconsistently and interchangeably, highlighting the need for further consensus on the classification and definitions of neuropathic back pain (lin., a number of pathophysiological mechanisms have been implicated in neuropathic lbp (fig. 1). in chronic lbp, neuropathic pain may be caused by lesions of nociceptive sprouts within a degenerated disc (local neuropathic pain), by mechanical compression of the nerve root (mechanical neuropathic root pain), or by the effects of inflammatory mediators arising from a degenerative disc that results in inflammation and damage to the nerve roots (freynhagen and baron, 2009 ; cohen and mao, 2014). (a) anatomy of a spinal nerve emerging from the spinal cord. the spinal nerve branches into a dorsal ramus innervating the skin of the lower back and a ventral ramus innervating the leg (via the lumbosacral plexus) ; (b) proposed pathophysiological mechanisms in neuropathic back pain (freynhagen and baron, 2009). with kind permission from springer science+business media : curr pain headache rep 2009;13:185190, freynhagen r, baron r, figure 1. various preclinical models have been developed that attempt to mimic aspects of pathophysiological mechanisms that contribute to chronic lbp. these include application of nucleus pulposus material near the lumbar dorsal root ganglia (drg), chronic compression of the drg or localized inflammation of the drg, and nerve growth factor injections into the multifidus muscle (hoheisel., 2013 ; strong., 2013). these models, which are primarily developed in rats, have many common features including behavioural hypersensitivity of the hind paw, enhanced excitability and spontaneous activity of sensory neurons, and locally elevated levels of inflammatory mediators including cytokines. however, some drugs shown to be effective in preclinical models of neuropathic pain fail in clinical studies, either due to lack of tolerability or testing in heterogeneous groups of patients, highlighting the need for careful selection of patient subgroups in trials of potential neuropathic pain drug therapies. these components require different pain management strategies directed at peripheral and central processes, but there is currently no goldstandard approach for the diagnosis of neuropathic lbp (freynhagen and baron, 2009 ; haanp., 2011). a focused clinical examination, following a full patient history, should be the first step in the differential diagnosis of any suspected neuropathic pain condition in order to document the distribution of the pain, any associated sensory or motor signs within that distribution, as well as any evidence for an underlying neurological lesion or disease (treede., 2008 ; haanp., 2011 ; nijs., however, one recent study revealed that as many as 43% of patient visits for lbp did not involve any form of direct physical examination and nearly 20% did not even involve palpation (i.e. no sensory examination) (press., the substantial lack of a routine approach to the diagnosis of pain is further highlighted by the results of a recent study undertaken in a rehabilitation setting, where lbp is a major clinical problem (casale., 2012). painful signs and symptoms arising in an area of altered sensation are the hallmarks of neuropathic pain ; however, signs and symptoms of neuropathic pain can vary between patients and even within individual patients over time. cardinal features include spontaneous pain (i.e. arising without stimulus), abnormal response to nonpainful stimuli such as light touch and moderate heat or cold (allodynia), or an exaggerated response to painful stimuli (hyperalgesia). spontaneous pain can be paroxysmal (e.g. shooting, stabbing or electric shocklike), dysaesthetic (unpleasant abnormal sensations of touch, for example prickling, pins and needles or crawling) or associated with abnormal thermal sensations (e.g. burning or ice cold). these signs and symptoms can coexist in an area with a loss of afferent sensations (numbness). signs of neuropathic pain can be assessed using bedside sensory tests when they are due to root compression or inflammation, but not when they result from a lesion that affects nerve sprouts that are pathologically innervating the spinal disc. clinical examination of a patient with lbp in which a neuropathic component is suspected should focus on identifying possible sites of an underlying somatosensory lesion, which is consistent with the anatomical distribution and type of symptoms described by the patient (cohen., 2008 ; treede., 2008 ; careful assessment of the patient 's sensory, motor and autonomic systems should be done, in conjunction with musculoskeletal examination and palpation of their spine, in order to identify any neurological dysfunction or structural abnormality. because clinical examination of these patients is rarely, if ever, definitive in isolation it will often be used to guide further laboratory investigation, and rule out other potentially causative pathologies as part of a differential diagnosis (haanp., 2011). several screening tools have been developed to facilitate identification of a neuropathic pain component in patients with chronic lbp (bennett., 2007 ; cruccu and truini, 2009 ; haanp., 2011). these tools are generally based on elicitation of verbal pain descriptors, although some also include bedside testing ; sensitivity and specificity typically range from 80% to 90% (table s1). however, these tools are not a substitute for the clinical examination of the patient. douleur neuropathique en 4 questions, paindetect (pdq) and the standardized evaluation of pain (step) are the only screening tools to have been specifically validated in patients with lbp (freynhagen. the dn4 comprises both interview questions and physical tests, and has been shown to have high sensitivity and specificity for the detection of neuropathic pain components in patients with lbp (attal., 2011). the pdq questionnaire screens for typical signs and symptoms of neuropathic pain, pain course pattern and the presence of radiating pain (freynhagen., 2006a). it is easy to administer, even in primary care settings, and has demonstrated high sensitivity, specificity and accuracy in patients with chronic lbp. a score of 19 is considered strongly suggestive of a neuropathic pain component, with a score of 1318 indicating that a neuropathic pain component may be present. however, it appears that further research is required to establish whether the pdq score can be used to predict treatment response (mors., step includes six interview questions and ten physical tests, and has been shown to distinguish between radicular pain and nonneuropathic low back pain with high sensitivity and specificity (scholz., 2009). more detailed radiological and neurological assessments may be indicated in some patients, including quantitative sensory testing (qst). qst is used to reveal pathological signs of neuropathic pain and is recognized to be a useful additional diagnostic tool (freynhagen and baron, 2009 ; schfer., 2014). additionally, neurophysiological investigations utilizing electroneuromyography (i.e. nerve conduction studies) may be useful in helping to differentiate peripheral lesions from suspected lbp with a neuropathic component, but only when considered in conjunction with a detailed patient history and careful clinical examination (cruccu and truini, 2009 ; haanp., 2011). conventional electrophysiological techniques can also be used to document radiculopathy, albeit not painful radiculopathy, while nociceptive sensory deficit can be documented objectively using laser evoked potentials (quante., radiological imaging studies, primarily mri, may help when conducting a differential diagnosis in patients with neuropathic lbp, but need to be interpreted with caution as there is a high prevalence of asymptomatic degenerative disorders in older adults and areas of abnormal mri signal do not necessarily imply tissue damage or dysfunction (cohen. currently, there is an unmet need for a dedicated diagnostic algorithm for the clinical assessment of patients with lbp with a suspected neuropathic component. the goal of treatment of chronic lbp is to reduce pain, maintain function and prevent future exacerbation. numerous evidencebased clinical practice guidelines for the management of chronic lbp have been published (chou., 2007 ; national institute for health and clinical excellence, 2009 ; koes., 2010 ; german medical association, national association of statutory health insurance physicians, and association of scientific medical societies, 2013). available guidelines typically advise a multimodal approach to the management of chronic lbp, combining pharmacological therapies for symptomatic relief with nonpharmacological approaches, such as physical activity and psychosocial / behavioural interventions. choice of treatment should be individualized according to the nature and severity of symptoms, the presence of comorbid conditions (e.g. depression or sleep disorders), potential for adverse effects and drug interactions, risks of misuse and abuse, and cost. however, these guidelines typically do not include specific recommendations for the treatment of neuropathic components of chronic lbp. clinical practice guidelines are also available for the treatment of neuropathic pain (attal., 2010 ; dworkin., 2013 ; national institute for health and clinical excellence, 2013 ; finnerup, attal., 2015). however, the definitions used by these guidelines do not typically include all forms of neuropathic lbp, for example, the most recent update of the neupsig guidelines only covers back pain with radiculopathy (finnerup, attal., most randomized controlled trials of drug therapies for neuropathic pain have been undertaken in patients with postherpetic neuralgia (phn) or painful diabetic peripheral neuropathy (pdn), and the extent to which results of these studies can be extrapolated to other neuropathic conditions, such as chronic lbp, is unknown. typically, no more than half of patients experience clinically meaningful pain relief with currently available oral pharmacotherapy, and all oral agents are associated with a risk of significant adverse effects which can have a serious impact on patients quality of life. furthermore, studies undertaken to date are typically short term (<3 months duration) and evidence of effectiveness and risks associated with longterm treatment is limited. in addition, few headtohead trials comparing different treatments have been undertaken, so direct comparisons of efficacy and tolerability are generally not possible. in one recent study undertaken to assess adherence of french general practitioners to chronic neuropathic pain clinical guidelines, typical radicular pain was correctly identified in most cases (90.7%). in contrast, very few respondents (5.2%) were able to identify all the recommended firstline drugs (pregabalin, gabapentin, tricyclic antidepressants and duloxetine), and only 44.3% would have prescribed one of these agents (martinez., 2014). nonpharmacological options for the management of chronic lbp are often applied in the context of multimodal and multidisciplinary pain therapy, with specialist physiotherapy input and cognitivebehavioural therapies making important contributions. other options may also include noninvasive approaches, such as transcutaneous electrical nerve stimulation (tens), and invasive procedures, including epidural steroid injections (esis) and spinal cord stimulation (scs). it is a relatively safe, noninvasive and easytouse modality that can be conveniently selfadministered by patients at home, making it an attractive treatment option. however, a cochrane review found conflicting evidence regarding the benefits of tens for chronic lbp (khadilkar., 2008). esi is a common approach in patients with radiculopathy ; however, recent systematic reviews suggest only modest evidence of shortterm benefits (3 months) (cohen., 2013 ; dworkin., 2013) a number of studies support the efficacy and costeffectiveness of scs for the treatment of fbss (kumar., 2012 ; kumar and rizvi, 2013 ; hussain and erdek, 2014 ; kapural, 2014). pharmacological agents available for the management of chronic lbp include paracetamol (acetaminophen), nonsteroidal antiinflammatory drugs (nsaids), antidepressants, anticonvulsants, opioids, tapentadol and topical treatments (table 1). paracetamol and nsaids target the nociceptive component of lbp and have no effect against neuropathic pain components, while currently available neuropathic pain medications generally show only modest evidence of efficacy in patients with chronic lbp. this may be because studies undertaken to assess these agents in this setting generally have not specifically selected patients with a significant neuropathic component. response rate with neuropathic pain medications is typically only around 3050% in patients with classical neuropathic conditions, and may be lower in patients with chronic lbp. overview of pharmacological agents that can be used for the treatment of chronic lbp lbp, low back pain ; snri, serotonin noradrenaline reuptake inhibitor ; ssri, selective serotonin reuptake inhibitor ; trpv1, transient receptor potential vanilloid 1. firstline and longterm treatment with opioids a recent study in the uk found high use of opioid analgesics as firstline treatment (either as monotherapy or in combination with other therapies) in 64% of patients with neuropathic lbp (hall., 2013). a recent systematic review found evidence of moderate shortterm efficacy for opioids in chronic lbp compared with placebo ; however, the few trials that compared opioids with nsaids or antidepressants did not show any differences in treatment outcome (chaparro., 2013). results of another metaanalysis also fail to support the use of opioids alone for the treatment of chronic noncancer pain (reinecke., 2015). tramadol is a weak opioid receptor agonist, which also appears to inhibit serotonin and noradrenaline reuptake. it is generally considered to have a lower sedative effect and risk of abuse compared with other opioids. however, there are only limited data to support the use of tramadol in this setting (vorsanger., 2008). tapentadol, a dual opioid receptor agonist and noradrenaline reuptake inhibitor, has been shown to be as effective as oxycodone for the treatment of chronic lbp. it was effective in patients with nociceptive and neuropathic low back pain (steigerwald., 2012 ; glvez., 2013), with better gastrointestinal tolerability and improved treatment adherence compared with oxycodone alone (pergolizzi., 2011). in a recent phase iiib study, tapentadol monotherapy was found to be as effective as combination therapy with tapentadol and pregabalin in patients with severe, chronic lbp with a neuropathic component (baron., 2015). neuropathic pain and qol measures improved significantly in both groups ; however, the incidence of dizziness and/or somnolence was significantly lower in patients who received tapentadol alone. antidepressants are often used in patients with neuropathic pain, particularly those with comorbid depression or anxiety ; their analgesic properties are mediated through their effects on noradrenergic and serotoninergic neurotransmission. systematic reviews show tricyclic antidepressants, for example, amitriptyline, and dual serotonin, and norepinephrine reuptake inhibitors, for example, duloxetine and venlafaxine, to have efficacy for the treatment of neuropathic pain (saarto and wiffen, 2007 ; dharmshaktu., 2012 ; finnerup, attal., 2015). in contrast, the analgesic effects of selective serotonin reuptake inhibitors such as fluoxetine, paroxetine and citalopram appear limited and inconsistent (saarto and wiffen, 2007 ; dharmshaktu., 2012). however, robust data to support the use of antidepressants for the treatment of neuropathic lbp are lacking. indeed, a cochrane review of randomized, controlled trials comparing antidepressants with placebo in patients with nonspecific lbp, which included patients with neuropathic pain components, failed to reveal clear evidence of efficacy for antidepressants in this setting (urquhart. the anticonvulsants gabapentin and pregabalin are also frequently used in the treatment of neuropathic pain ; these agents are calcium channel alpha2delta ligands. for other types of neuropathic pain, such as spinal cord injury, it was found that these agents reduce pain as well as comorbid depression, anxiety and sleep disturbances, and improve qol (mehta., 2014). however, robust data are lacking to support the use of these agents for the treatment of neuropathic lbp (chung., 2013). in one study specifically undertaken to assess the efficacy and safety of pregabalin for the treatment of neuropathic pain in patients with chronic lumbosacral radiculopathy, most patients responded to pregabalin therapy ; however, time to loss of response (the primary study endpoint) did not significantly differ between pregabalin and placebo (baron., 2010). results of another small, prospective randomized study in patients with chronic lbp suggest that pregabalin may be most effective when used in combination with celecoxib (roman.,, placebocontrolled study has recently been initiated to assess the efficacy of pregabalin in addition to usual care for the treatment of sciatica (mathieson., 2013). future studies of potential drug therapies for use in this setting should aim to carefully select patients with welldefined neuropathic pain components using appropriate screening and diagnostic tools. two topical analgesics the capsaicin 8% patch and the lidocaine 5% medicated plaster are available for the treatment of peripheral neuropathic pain. currently the 8% capsaicin patch is licensed for use in the treatment of peripheral neuropathic pain in adults, while the lidocaine 5% medicated plaster is only indicated for use in phn (astellas pharma europe b.v., 2015). emerging data suggest that these agents may also be effective for the treatment of patients with chronic neuropathic lbp. although both treatments are topical they are applied in different ways, the capsaicin 8% patch is applied once every 3 months, under physician supervision, for either 30 or 60 min, whereas the lidocaine 5% medicated plaster is applied by the patient and worn daily, for up to 12 h a day. capsaicin is a selective agonist of the transient receptor potential vanilloid 1 (trpv1) channel, which is highly expressed on nociceptors. the capsaicin 8% patch was found to be well tolerated and effective for the treatment of peripheral neuropathic pain (backonja., 2008 ; simpson., 2008 ; maihfner and heskamp, 2013), in a prospective, noninterventional study involving over 1000 patients with a variety of neuropathic pain conditions, including patients with radiculopathy, the proportions of patients achieving a 30% and 50% decrease in pain at 3 months were 43% and 24%, respectively, following a single treatment (maihfner and heskamp, 2013). highest treatment response rates were observed in patients with preexisting pain for < 6 months when compared with patients whose duration of pain was 6 months to 2 years (30% and 50% decreases in pain score in 62% and 39% of patients, respectively, in the former group, vs. 41% and 23% of patients in the latter group), suggesting that early initiation of treatment may be beneficial (maihfner and heskamp, 2014). a retrospective analysis of patients with peripheral neuropathic pain of varying aetiologies, including radiculopathy and fbss, treated in a clinical setting found that the capsaicin 8% patch provided rapid and sustained pain relief, with a significant reduction in prescribed concomitant pain medications (wagner., 2013 fbss achieved reductions in pain score of 30% and 50%, respectively ; however, these results should be interpreted with caution due to the small number of patients studied (n = 6). lidocaine blocks voltagegated sodiumchannels and hence action potential conduction of nociceptors at the level at which it is applied (mick and correaillanes, 2012). in two uncontrolled, openlabel studies that included patients with moderatetosevere lbp, treatment with the lidocaine 5% plaster for 6 weeks significantly reduced both the intensity of the pain and its impact on the patients qol (galer., 2004 ; gimbel., 2005). a retrospective case series also reported marked reductions in pain intensity in patients with neuropathic pain after disc herniation during longterm treatment with the lidocaine 5% plaster (mean treatment duration 7.6 months) (likar., 2012). in a more recent study, addon therapy with the lidocaine 5% plaster was associated with a clinically meaningful reduction in pain scores after 3 months of treatment in 24 patients experiencing lbp with a neuropathic component (casale., 2014). both types of topical treatment are applied directly to the most painful skin area, either on the back or more peripherally in the corresponding dermatome and multiple patches / plasters may be used to cover the affected region if needed. these topical approaches are generally well tolerated ; applicationsite reactions are the most common adverse event. risks of systemic adverse events and pharmacokinetic interactions with concomitant oral medications are minimal owing to low systemic exposure, making them attractive options for use in combination with other pharmacological approaches for chronic lbp. nonpharmacological options for the management of chronic lbp are often applied in the context of multimodal and multidisciplinary pain therapy, with specialist physiotherapy input and cognitivebehavioural therapies making important contributions. other options may also include noninvasive approaches, such as transcutaneous electrical nerve stimulation (tens), and invasive procedures, including epidural steroid injections (esis) and spinal cord stimulation (scs). these approaches have been reviewed in detail elsewhere (kumar., 2012 ; morlion, 2013). it is a relatively safe, noninvasive and easytouse modality that can be conveniently selfadministered by patients at home, making it an attractive treatment option. however, a cochrane review found conflicting evidence regarding the benefits of tens for chronic lbp (khadilkar., 2008). esi is a common approach in patients with radiculopathy ; however, recent systematic reviews suggest only modest evidence of shortterm benefits (3 months) (cohen., 2013 ; dworkin., 2013) a number of studies support the efficacy and costeffectiveness of scs for the treatment of fbss (kumar., 2012 ; kumar and rizvi, 2013 ; hussain and erdek, 2014 ; kapural, 2014). pharmacological agents available for the management of chronic lbp include paracetamol (acetaminophen), nonsteroidal antiinflammatory drugs (nsaids), antidepressants, anticonvulsants, opioids, tapentadol and topical treatments (table 1). paracetamol and nsaids target the nociceptive component of lbp and have no effect against neuropathic pain components, while currently available neuropathic pain medications generally show only modest evidence of efficacy in patients with chronic lbp. this may be because studies undertaken to assess these agents in this setting generally have not specifically selected patients with a significant neuropathic component. response rate with neuropathic pain medications is typically only around 3050% in patients with classical neuropathic conditions, and may be lower in patients with chronic lbp. overview of pharmacological agents that can be used for the treatment of chronic lbp lbp, low back pain ; snri, serotonin noradrenaline reuptake inhibitor ; ssri, selective serotonin reuptake inhibitor ; trpv1, transient receptor potential vanilloid 1. firstline and longterm treatment with opioids is generally not recommended due to concerns regarding tolerability and dependence. despite this, a recent study in the uk found high use of opioid analgesics as firstline treatment (either as monotherapy or in combination with other therapies) in 64% of patients with neuropathic lbp (hall., 2013). a recent systematic review found evidence of moderate shortterm efficacy for opioids in chronic lbp compared with placebo ; however, the few trials that compared opioids with nsaids or antidepressants did not show any differences in treatment outcome (chaparro., 2013). results of another metaanalysis also fail to support the use of opioids alone for the treatment of chronic noncancer pain (reinecke., 2015). tramadol is a weak opioid receptor agonist, which also appears to inhibit serotonin and noradrenaline reuptake. it is generally considered to have a lower sedative effect and risk of abuse compared with other opioids. however, there are only limited data to support the use of tramadol in this setting (vorsanger., 2008). tapentadol, a dual opioid receptor agonist and noradrenaline reuptake inhibitor, has been shown to be as effective as oxycodone for the treatment of chronic lbp. it was effective in patients with nociceptive and neuropathic low back pain (steigerwald., 2012 ; glvez., 2013), with better gastrointestinal tolerability and improved treatment adherence compared with oxycodone alone (pergolizzi., 2011). in a recent phase iiib study, tapentadol monotherapy was found to be as effective as combination therapy with tapentadol and pregabalin in patients with severe, chronic lbp with a neuropathic component (baron., 2015). neuropathic pain and qol measures improved significantly in both groups ; however, the incidence of dizziness and/or somnolence was significantly lower in patients who received tapentadol alone. antidepressants are often used in patients with neuropathic pain, particularly those with comorbid depression or anxiety ; their analgesic properties are mediated through their effects on noradrenergic and serotoninergic neurotransmission. systematic reviews show tricyclic antidepressants, for example, amitriptyline, and dual serotonin, and norepinephrine reuptake inhibitors, for example, duloxetine and venlafaxine, to have efficacy for the treatment of neuropathic pain (saarto and wiffen, 2007 ; dharmshaktu., 2012 ; finnerup, attal., 2015). in contrast, the analgesic effects of selective serotonin reuptake inhibitors such as fluoxetine, paroxetine and citalopram appear limited and inconsistent (saarto and wiffen, 2007 ; dharmshaktu., 2012). however, robust data to support the use of antidepressants for the treatment of neuropathic lbp are lacking. indeed, a cochrane review of randomized, controlled trials comparing antidepressants with placebo in patients with nonspecific lbp, which included patients with neuropathic pain components, failed to reveal clear evidence of efficacy for antidepressants in this setting (urquhart., 2008). the anticonvulsants gabapentin and pregabalin are also frequently used in the treatment of neuropathic pain ; these agents are calcium channel alpha2delta ligands. for other types of neuropathic pain, such as spinal cord injury, it was found that these agents reduce pain as well as comorbid depression, anxiety and sleep disturbances, and improve qol (mehta., 2014). however, robust data are lacking to support the use of these agents for the treatment of neuropathic lbp (chung., 2013). in one study specifically undertaken to assess the efficacy and safety of pregabalin for the treatment of neuropathic pain in patients with chronic lumbosacral radiculopathy, most patients responded to pregabalin therapy ; however, time to loss of response (the primary study endpoint) did not significantly differ between pregabalin and placebo (baron., 2010). results of another small, prospective randomized study in patients with chronic lbp suggest that pregabalin may be most effective when used in combination with celecoxib (roman., 2009). a large doubleblind, randomized, placebocontrolled study has recently been initiated to assess the efficacy of pregabalin in addition to usual care for the treatment of sciatica (mathieson., 2013). future studies of potential drug therapies for use in this setting should aim to carefully select patients with welldefined neuropathic pain components using appropriate screening and diagnostic tools. two topical analgesics the capsaicin 8% patch and the lidocaine 5% medicated plaster are available for the treatment of peripheral neuropathic pain. currently the 8% capsaicin patch is licensed for use in the treatment of peripheral neuropathic pain in adults, while the lidocaine 5% medicated plaster is only indicated for use in phn (astellas pharma europe b.v., 2015). emerging data suggest that these agents may also be effective for the treatment of patients with chronic neuropathic lbp. although both treatments are topical they are applied in different ways, the capsaicin 8% patch is applied once every 3 months, under physician supervision, for either 30 or 60 min, whereas the lidocaine 5% medicated plaster is applied by the patient and worn daily, for up to 12 h a day. capsaicin is a selective agonist of the transient receptor potential vanilloid 1 (trpv1) channel, which is highly expressed on nociceptors. the capsaicin 8% patch was found to be well tolerated and effective for the treatment of peripheral neuropathic pain (backonja., 2008 ; simpson., 2008 ; maihfner and heskamp, 2013), in a prospective, noninterventional study involving over 1000 patients with a variety of neuropathic pain conditions, including patients with radiculopathy, the proportions of patients achieving a 30% and 50% decrease in pain at 3 months were 43% and 24%, respectively, following a single treatment (maihfner and heskamp, 2013). highest treatment response rates were observed in patients with preexisting pain for < 6 months when compared with patients whose duration of pain was 6 months to 2 years (30% and 50% decreases in pain score in 62% and 39% of patients, respectively, in the former group, vs. 41% and 23% of patients in the latter group), suggesting that early initiation of treatment may be beneficial (maihfner and heskamp, 2014). a retrospective analysis of patients with peripheral neuropathic pain of varying aetiologies, including radiculopathy and fbss, treated in a clinical setting found that the capsaicin 8% patch provided rapid and sustained pain relief, with a significant reduction in prescribed concomitant pain medications (wagner., 2013). in this study, 67% and 33% of patients with radiculopathy and fbss achieved reductions in pain score of 30% and 50%, respectively ; however, these results should be interpreted with caution due to the small number of patients studied (n = 6). lidocaine blocks voltagegated sodiumchannels and hence action potential conduction of nociceptors at the level at which it is applied (mick and correaillanes, 2012). in two uncontrolled, openlabel studies that included patients with moderatetosevere lbp, treatment with the lidocaine 5% plaster for 6 weeks significantly reduced both the intensity of the pain and its impact on the patients qol (galer., 2004 ; a retrospective case series also reported marked reductions in pain intensity in patients with neuropathic pain after disc herniation during longterm treatment with the lidocaine 5% plaster (mean treatment duration 7.6 months) (likar., 2012). in a more recent study, addon therapy with the lidocaine 5% plaster was associated with a clinically meaningful reduction in pain scores after 3 months of treatment in 24 patients experiencing lbp with a neuropathic component (casale., 2014). both types of topical treatment are applied directly to the most painful skin area, either on the back or more peripherally in the corresponding dermatome and multiple patches / plasters may be used to cover the affected region if needed. these topical approaches are generally well tolerated ; applicationsite reactions are the most common adverse event. risks of systemic adverse events and pharmacokinetic interactions with concomitant oral medications are minimal owing to low systemic exposure, making them attractive options for use in combination with other pharmacological approaches for chronic lbp. neuropathic pain is challenging to manage, and many patients with chronic lbp have pain that is refractory to existing treatments. there remains a clear need for improved treatment options for the management of the neuropathic component of chronic lbp. as chronic lbp is often characterized by both nociceptive and neuropathic components, combination therapy with drugs with different mechanisms of action would appear to be an attractive treatment option ; however, clinical studies to support this approach are limited (roman., 2012). combining oral agents also raises the potential for drug drug interactions and increased adverse effects. the positive results from trials with tapentadol may reflect the benefit of a single molecule that possesses two mechanisms of action, thereby modulating both nociceptive and neuropathic elements. emerging data suggest that it may be possible to profile patients with chronic lbp according to the sensory abnormalities they experience, possibly reflecting differences in underlying pathophysiological mechanisms (mahn., 2011 ; frster., analysis of epidemiological and clinical data for 2094 patients with painful radiculopathy showed touchevoked allodynia and thermal hyperalgesia to be relatively uncommon in radiculopathy compared with classical neuropathic pain syndromes, such as pdn and phn (mahn., 2011). this difference may be related to the fact that the site of the nerve lesion in radiculopathy is often located proximally to the dorsal root ganglion. a distinct sensory profile was identified in patients with radiculopathy, namely severe painful attacks and pressureinduced pain in combination with mild spontaneous pain, mild mechanical allodynia and thermal hyperalgesia. the painful attacks in these patients might be explained by compressioninduced ectopic discharges from a dorsal root and not necessarily by nerve damage. such differences in sensory phenotype between different neuropathic pain conditions may explain, at least in part, why many therapeutic approaches shown to be effective in pdn and phn have failed to demonstrate efficacy in chronic lbp. sensory phenotyping of patients with chronic lbp is a promising technique that may enable individualized treatment, potentially leading to improved patient outcomes, and could assist in the development of more targeted drug therapies. it represents a serious burden both in terms of the health of the individual patients and the costs to society as a whole. treatment recommendations in current guidelines for lbp and for neuropathic pain differ substantially, which can leave clinicians at a loss as to which guidelines to follow when a patient has lbp with an associated neuropathic component. to resolve this issue, increased recognition and improved understanding of the neuropathic component of lbp is needed, together with the development of dedicated evidencebased diagnostic and therapeutic algorithms. this may lead to the development of individualized mechanismbased treatment regimens, which can be expected to result in improved patient outcomes. table s1. overview of screening tools for detection of neuropathic components in chronic lbp (bennett. | abstractbackground and objectivelow back pain (lbp) is one of the most common chronic pain conditions. this paper reviews the available literature on the role of neuropathic mechanisms in chronic lbp and discusses implications for its clinical management, with a particular focus on pharmacological treatments.databases and data treatmentliterature searches were performed in pubmed, key pain congresses and proquest dialog to identify published evidence on neuropathic back pain and its management. all titles were assessed for relevant literature.resultschronic lbp comprises both nociceptive and neuropathic components, however, the neuropathic component appears underrecognized and undertreated. neuropathic pain (np) is challenging to manage. many patients with chronic lbp have pain that is refractory to existing treatments. typically, less than half of patients experience clinically meaningful analgesia with oral pharmacotherapies ; these are also associated with risks of adverse effects. paracetamol and nsaids, although widely used for lbp, are unlikely to ameliorate the neuropathic component and data on the use of np medications such as antidepressants and gabapentin / pregabalin are limited. while there is an unmet need for improved treatment options, recent data have shown tapentadol to have efficacy in the neuropathic component of lbp, and studies suggest that the capsaicin 8% patch and lidocaine 5% medicated plaster, topical analgesics available for the treatment of peripheral np, may be a valuable additional approach for the management of neuropathic lbp.conclusionschronic lbp often has an underrecognized neuropathic component, which can be challenging to manage, and requires improved understanding and better diagnosis and treatment.what does this review add?increased recognition and improved understanding of the neuropathic component of low back pain raises the potential for the development of mechanismbased therapies.open and retrospective studies suggest that agents like tapentadol and topical analgesics such as the capsaicin 8% patch and the lidocaine 5% medicated plaster may be effective options for the treatment of neuropathic low back pain in defined patient groups. |
teaching methods with advanced technology have significantly increased in clinical nursing skills training over the past decade. today, due to constant technological changes in clinical environments, working with patients, especially in adverse conditions, has become more complicated for nurses. therefore, nursing students need better training methods in order to be prepared for entering the clinical environment. however, factors such as fear of mistakes and uncertainty make their clinical experience difficult. in order to overcome these problems, using high - precision simulators in nursing education has gradually expanded and become important. in the clinical skills laboratory, a safe environment for learning, students practice skills on human patient simulation manikins (hpsms) and simulators with less stress. with the help of teaching aids and educational software in these centers, real opportunities are provided for learning and developing clinical skills, and it is also possible to objectively evaluate students. using simulators is the most widely used strategy in nursing education. simulator manikins promote science, critical thinking, and students satisfaction with education. with simulators, using simulators for training clinical skills is an approach with international value and it is widely used in nursing education today. results of different studies show that students are anxious in their initial encounters with patients, and if they do not have enough practice, fear of failure and mistake impedes their proper function. thus, universities seeking modern and efficient methods for clinical skills learning have been encouraged to advance training through this method. using simulators enables to learn skills without compromising patients health. by getting trained through using simulators, learners can better understand the real situation by manipulating the simulator. in using simulators results of a study in the united states by harlow and sportsman indicated that in 34 nursing schools, simulators were used in internal surgery education (56%) and advanced medical surgical nursing education (64%). in this regard, blood pressure is one of the most important clinical skills to assess health ; nursing students must have sufficient proficiency in it, and the necessary conditions for exact measurements should be provided for them. blood pressure measurement is a complex skill that requires considerable practice in order to gain competence in the recognition of korotkoff sounds. for example, the systolic pressure provides basic information about the condition of the heart and large arteries. the method currently used for teaching measurement of blood pressure is by the teacher measuring the blood pressure values of students. it is taught by occluding the cuff of a barometer on the arm of a student. however, since new methods should be used to teach and assess students, educational simulators can be used in teaching students to take blood pressure measurement. results of the study by karadag. showed that students in the experimental group who had used the simulator to learn to measure blood pressure heard korotkoff sounds more effectively. the aim of the present study was to compare two methods of blood pressure measurement training on nursing students performance, so that the best and most appropriate method would be used to teach students. after obtaining approval of study from the ethics committee of isfahan university of medical sciences, the study purpose was explained to the participants and informed consent was obtained from them. in this quasi - experimental study, 36 first - term nursing students, studying in the clinical skills laboratory of the school of nursing and midwifery in isfahan university of medical sciences, iran, were enrolled by census sampling method. information was collected by direct observation, performance skills checklist of blood pressure measurements, and a questionnaire to assess students knowledge on blood pressure measurements (including 12 open questions) prepared using scientific sources. the checklist of blood pressure measurement included 21 items such as the right position of the patient and his / her hand and arm, palpating the brachial pulse, occluding the cuff around the arm, placing a manometer on the surface of the eye, radial and brachial pulse palpation, obtaining baseline number for blood pressure, correct use of the stethoscope, filling the cuff, emptying the cuff using control valves, and determining the systolic and diastolic pressures. the prepared checklist was handed over to some of the nursing faculty members, and based on their opinions, the validity of the questionnaire was assessed and confirmed. to assess the reliability of the checklist, test - retest was conducted. the students (n = 36, 12 males and 24 females) were divided into six groups of six people each for clinical skills training. the 12 male students were divided into two groups by random draw and the teaching method for blood pressure measurement was randomly determined for the two groups. the 24 female students were also randomly divided into four groups by random draw and the teaching method for blood pressure measurement was randomly determined for the four groups. the students knowledge about the blood pressure measurement methods before training was obtained using a questionnaire in order to compare the results with those obtained after the training. from among the groups, three were trained to measure blood pressure on the right arm of a simulator and three were trained to measure blood pressure on the right arm of a student, both conducted by two professors during 2 h. then, in the next week, during a 2-h session, the students practice was conducted in the presence of the professor separately for each group, and the students problems were resolved. this means that the simulator group practiced blood pressure measurement on the simulator and the group experimenting with human subjects practiced on the students. the evaluation of the students was done the next week using observation of blood pressure measurement on human subjects and according to the performance on the measuring blood pressure checklist, and using a double educational stethoscope by the professor. the duration of 5 min was considered for examining each student. to analyze the data, the methods of descriptive statistics (mean) and analytical statistics (independent and paired t - tests) were used with spss for windows (version 16 ; spss inc., table 1 shows that on determining and comparing the mean scores of the students performance in the two methods of blood pressure measurements (simulator and human subjects), there was no significant difference between the two groups. comparison of the mean scores of students performance in the two studied groups according to table 2, there was a significant difference between the two study groups (simulator and human subjects) in the mean score of knowledge about the blood pressure measurement before and after training (p < 0.05). comparison of the mean knowledge scores of the method of blood pressure measurement before and after training in the two studied groups determination and comparison of measured mean systolic blood pressure by the two groups and the measured blood pressure by the professor (standard), based on student 's independent t - test, showed that there was no significant difference between the simulator and human subjects groups and also between the groups and the standard. based on student 's independent t - test, there was no significant difference between the two groups regarding diastolic blood pressure considering the measured blood pressure by the professor (standard). the findings showed that there was no significant difference between the two groups in terms of mean performance scores. thus, the two study groups were similar in terms of performance of blood pressure measurement after training. in the study by lee., simulator arm and human subjects were used for measuring blood pressure and it was found that blood pressure measurements using these two methods were the same ; there was no difference in students performance regarding the accuracy of the measurement technique. in the present study, there was a statistically significant difference before and after training with regard to the mean score of knowledge of blood pressure measurement. this means that the two teaching methods were effective in increasing knowledge about blood pressure measurement. results of the study by hosseini - nasab. indicated that mean and standard deviation of pre- and post - test scores of the two groups had increased after training. pre- and post - test scores showed significant differences. according to the results of the present study, there was no statistically significant difference between the simulator group and the standard regarding the measured mean systolic and diastolic blood pressure levels. this means that training students in blood pressure measurement using a simulator is a good method and, by this method, students acquired the ability to measure systolic and diastolic blood pressure correctly. in recent decades, with the teaching process becoming more practical, development of educational technology, and increased importance given to assumed values in medical ethics (including respect for the patient and causing him / her less damage during the training), the traditional methods are questioned and clinical skills laboratories, which provide a simulator and secure learning environment, are used for training and managing skills. moreover, in this environment, clinical interventions can be performed using a simulator or manikin and students can be trained in the skills with less stress. using a high - precision simulator is effective in learning psychomotor skills because students are able to repeat the skills safely without being concerned about compromising the patients health. based on the results of the study by harlow and sportsman, 75% of educational simulators tend to result in learners satisfaction and give very good educational results. according to the present results, there was no significant difference regarding systolic and diastolic blood pressure measurements by students on human subjects and blood pressure measurement by the professor (standard). this means that students gained correct knowledge on the ability of measuring blood pressure and that blood pressure measurement training given to students using human subjects is a good method to teach them. compared computer - assisted instruction and academic views on learning vital signs measurement in nursing students and found no significant differences between the two teaching methods. according to the results, the two training methods (on human subjects and simulator) provide the necessary ability for the student to measure the systolic and diastolic blood pressures correctly. a comparison with previous studies shows that using simulators is an effective teaching strategy and facilitates learning and development of students knowledge because simulators provide secure clinical opportunities and assist in the development of clinical skills. using a simulator arm is an acceptable method for teaching students the skills for measuring blood pressure. the simulator arm allows the students to practice blood pressure measurement in circumstances similar to a living example. the educational simulator is like a tolerant patient that the student can use repeatedly for hearing and distinguishing korotkoff sounds. in addition, as an independent educational method, it results in improvement of learning for the students. therefore, to enrich the educational programs of students, using the simulator training is recommended. | background : new teaching methods are essential in the promotion of competence in nursing students. measuring blood pressure, which is one of the most essential skills, is the foundation of nursing care and clinical decision - making, and students should be trained to master this skill. this study aimed to determine the impact of two teaching methods on students performance in blood pressure measurements.materials and methods : in this semi - experimental study, 36 first - term nursing students from isfahan university of medical sciences, iran, were divided into six groups of six people each. the type of training for each group was determined by random draw. blood pressure measurement training was given to three groups on a simulator and to the other three groups on human samples. pre- and post - test evaluation of knowledge of the methods of measuring blood pressure was conducted using a researcher - made checklist and by observation of the students actions. the knowledge and skill scores were compared using paired and independent t-tests.results:the mean performance scores of students in simulator (19.14 1.60) and human (19.64 1.08) groups were not significantly different. in both methods, there was no significant difference between systolic and diastolic blood pressures measured by students and by teachers (standard). mean knowledge scores of both groups increased significantly after training compared to before training.conclusions:according to the results of the study, the two training methods (on human subjects and simulator) provide the students with the necessary ability to measure the systolic and diastolic blood pressures correctly. therefore, to enrich the educational program of nursing students, using simulators is recommended as an effective teaching strategy to facilitate learning and for the development of students knowledge. |
conventional esophagogastroduodenoscopy (egd) is limited by the requirement for sedation and its associated recovery time, endoscopy suite resources, and anesthesia monitoring. these limitations likely decrease the effectiveness of current screening strategies for esophageal precancerous lesions 1. unsedated small - caliber transnasal endoscopy (tne) has been investigated as a less expensive, efficient, office - based alternative to egd for screening for gastroesophageal reflux complications. tne is a well tolerated procedure with an excellent safety profile 2 3 4 5. the acceptability rate of tne was 85.2 % in a recent meta - analysis including 1597 patients ; furthermore, 63 80 % of individuals preferred tne to egd for future procedures 6 7 8. technical success rates of tne and egd were comparable (particularly when tne scope diameter was 95 % of the gej was visualized in nearly all cases. the overall quality scores for tne and egd were excellent without statistically significant differences (p = 0.30). there was no significant difference in any quality assessment score between mtne and htne groups, which were different only in procedural location. inter - rater agreement of endoscopy quality was excellent (kappa = 0.88). in this brief report, we found that the quality of esophageal assessment with tne (conducted in a gastrointestinal endoscopy suite (htne) or in a mobile research van (mtne)) was comparable to egd in terms of overall quality and gej visualization. tne received higher esophageal intubation scores, likely due to passage through the nasal cavity into the superior oropharynx, which provided superior visualization of the laryngeal and oropharyngeal structures. egd scored higher in tubular esophagus passage metrics likely due to greater insufflation capabilities compared to the ultrathin tne endoscope. tne quality was not affected by procedure setting as there was no difference in quality scores between the mtne and htne groups. these results indicate that tne using the endosheath technology is a feasible efficient option for endoscopic assessment of reflux complications in a population - based setting. the scoring tool employed in this study to assess endoscopic quality was validated in a large multicenter study performed in the uk that included over 4000 subjects with a diverse group of endoscopists 12. the scoring tool did not include assessments to evaluate the endoscopist s diagnostic yield or esophageal lesion identification. in our previously published study, sami. reported no difference in suspected or confirmed be rates between the three arms (egd, mtne, or htne) of the study 7. these results are akin to multiple studies showing that tne is comparable to egd at detecting be 9 10 11. the scoring tool also did not assess the endoscopist s level of comfort or perceived difficulty of the procedure. these additional personal viewpoints could be incorporated into future investigations of tne used in primary care settings with endoscopists less experienced with tne. it was also difficult to completely blind reviewers from the potentially different appearances of tne and egd videos. nevertheless, by applying a clear scoring system, it was hoped that videos were scored on their merit rather than as a comparison. as we have previously reported, there was no difference in the rate of successful intubation, complete evaluation, or be screening yield between the three arms (egd, mtne, or htne) of this prospective population - based study 7. comparing tne and egd examinations, the absolute difference in the mean procedure length was minimal in this study, likely due to the research protocol driven acquisition of gej and esophageal biopsies in all three arms. however, recovery time (extubation to discharge) was substantially longer by approximately 50 minutes for subjects who received sedated egd compared to tne 7. this decreased recovery time associated with tne is beneficial for the patient as well as the endoscopist. by including a disposable sheath given that tne examination quality did not differ by procedure setting, tne could likely be employed effectively in an office - based setting, community center or even a mobile research unit as investigated in this study for assessment of complications from gastroesophageal reflux. furthermore, a majority of primary care providers may be more willing to refer patients for screening with tne if it was readily available (62 %) and to perform tne in their office if trained (52 %) 13. these time - saving, mobile features associated with tne are ideal for increasing case volume and accessibility which would be necessary in community - based be screening programs. current limitations for tne include the lack of readily available tne training opportunities and current endoscopists with tne experience. recently reported that physician assistants and nurse practitioners can be trained to conduct be screening with tne and gain proficiency in 50 procedures 14. reasonable, plausible tne training strategies using available providers and resources need to be further delineated. in conclusion, the overall quality of esophageal assessment and gej visualization was comparable for tne performed using the endosheath technology and egd in a community cohort. tne quality was not affected by procedure location and thus could be used effectively in mobile or office - based settings. these results indicate that tne is a feasible option for endoscopic assessment of reflux complications, and can be used in a community setting. video i d : scorer : date: time, you began scoring : hr min sec this section is to evaluate dexterity and safety of performance of ogd. this includes instrument entry, passage, and manipulation through the mouth, throat, and oesophagus. pause the video each time before scoring and rewind as many times as needed to ensure accurate scoring. 1. oesophageal intubation passage under direct vision all the time following centre of tongue visualizing epiglottis visualize the cricoarytenoid folds and vocal cords insertion posterior to the larynx between the pyriform sinuses 2. passage through oesophagus insertion under direct vision all the time no mucosal red or white outs adequate air insufflation no mucosal wall collisions suctioning any secretions 3. examination of og junction (z - line or squamocolumnar junction in particular) on insertion on withdrawal > 95 % visibility 66 95 % visibility 36 65 % visibility 5 35 % visibility < 5 % visibility 4. overall score of oesophageal exam complete examination and no concerns over technique or content probably complete examination with minor concern over technique or content incomplete examination with moderate concern over technique or content incomplete examination with major concern over technique or content incomplete examination and totally unacceptable technique or content time, when you finish scoring : hr min sec this includes instrument entry, passage, and manipulation through the mouth, throat, and oesophagus. pause the video each time before scoring and rewind as many times as needed to ensure accurate scoring. | background unsedated transnasal esophagoscopy (tne) may offer a less expensive, mobile alternative to sedated esophagogastroduodenoscopy (egd) for evaluations of reflux related complications. comparisons of imaging quality by these methods are lacking. methods two reviewers evaluated videos of tne and egd procedures, performed during a community randomized study comparing endoscopic techniques. subjects were randomized to egd, tne in endoscopy suite, or tne in mobile research unit. endoscopic quality was assessed using a validated scoring tool. results in total, 115 videos (58 egd, 28 endoscopy suite tne, and 29 mobile tne) were reviewed. overall quality scores for tne and egd were excellent without a statistically significant difference (p = 0.30). there were no differences in gastroesophageal junction (gej) visualization scores, though egd scored higher in esophageal passage (p < 0.05) and tne scored higher in esophageal intubation (p < 0.05). there was no significant difference in any quality score between mobile tne and gastrointestinal suite tne. conclusion esophageal assessment with tne or egd was comparable in overall quality and gej visualization. tne quality was not affected by procedure location. tne is a feasible option for endoscopic assessment of reflux complications. |
the introduction and widespread use of antenatal ultrasonography has increased the diagnosis of fetal hydronephrosis in utero [1 - 3 ]. one of the most common causes of antenatally diagnosed hydronephrosis is ureteropelvic junction obstruction (upjo). many reports deal with the unilateral idiopathic upjo with a functionally, ultrasonographically, and renographically normal contralateral renal unit and many researchers manage their patients by initially conservative approaches with subsequent ultrasonographic or renographic follow - up [5 - 7 ]. compared with unilateral upjo, however, few published papers deal with the management protocol for neonatal patients with prenatally diagnosed bilateral hydronephrosis that leads to the postnatal diagnosis of severe bilateral upjo. factors to be considered in planning the surgical treatment of neonatal severe bilateral upjo are as follows. how long can we wait to correct the later - operated renal unit after the initial pyeloplasty ? is it necessary to place percutaneous nephrostomy (pcn) tube drainage to the later - operated renal unit for the preservation of renal function during the waiting period ? these questions have been on the minds of many pediatric urologists for a long time. but it is true that there is a paucity of clear recommendations regarding the management of severe bilateral upjo. therefore, we evaluated the clinical outcome of treatment in a prenatally diagnosed group with severe bilateral upjo at our institution to offer suggestions for the management of these clinical situations. from january 2000 to march 2009, 12 boys and 1 girl (26 renal units) with antenatally diagnosed grade 3 or higher bilateral hydronephrosis that led to the diagnosis of severe bilateral upjo on postnatal ultrasonography at 2 days after birth were selected. infants with grade 2 or less hydronephrosis in both renal units and those who had other urinary tract anomalies were excluded. the medical records of the selected 13 patients were reviewed for their hospital course and their postoperative follow - up imaging studies by using ultrasonographic and renographic assessment were reviewed on the picture archiving & communications system (pacs). all neonates underwent voiding cystourethrography (vcug) at 2 weeks after birth to rule out reflux diseases, but no reflux was found on vcug. within 4 weeks after birth, ultrasonography was repeated for the reassessment of hydronephrosis and a technetium mercaptoacetyltriglycine (tc - mag3) renal scan using the standard intravenous furosemide and 20-minute delay technique was performed for evaluating preoperative relative renal function (rrf) and diuretic drainage characteristics. the hydronephrosis of each renal unit with upjo was graded according to the society for fetal urology (sfu) grading guidelines from 1 to 4. among the renal units with hydronephrosis of sfu grade 4 in particular, we defined sfu grade 5 as hydronephrosis with ' paper thin ' renal parenchyme. all neonates had sfu grade 3 or higher bilateral hydronephrosis and sfu grade 4 or 5 hydronephrosis was involved in all of the cases. all cases of dismembered anderson - hynes open pyeloplasty through a flank incision were performed by a single surgeon (k.s.k.). subsequent ultrasonography and tc - mag3 renal scans were performed at 1 month postoperatively and we then decided whether delayed pyeloplasty in the opposite renal unit was necessary according to the findings of the ultrasonography and tc - mag3 renal scan. of 26 renal units, 4 renal units had hydronephrosis of sfu grade 3 and 22 had hydronephrosis of sfu grade 4 or more on postnatal ultrasonography (table 1). the hydronephrosis grade and rrf for both renal units in each neonate at the preoperative, the postoperative, and the most recent follow - up are given in table 1. a total of 13 patients were managed surgically by initial unilateral dismembered open pyeloplasty, and the mean age of the patients at the time of surgery was 3 months (range, 1 - 11 months). the mean follow - up time after the initial pyeloplasty was 37 months (range, 5 - 84 months). of 13 patients, 9 (cases 1 - 9) had bilateral upjo with different degrees of hydronephrosis of both renal units on ultrasonography at 2 weeks after birth. these 9 patients underwent initial unilateral dismembered open pyeloplasty on the renal unit with a higher sfu hydronephrosis grade on ultrasonography. two patients (cases 10 and 11) had bilateral upjo with the same degree of hydronephrosis of both renal units on ultrasonography and their tc - mag3 renal scan demonstrated that the difference in rrf between each renal unit was not significant. these 2 patients underwent initial unilateral dismembered open pyeloplasty on the renal unit with a lower rrf on the tc - mag3 renal scan. the remaining 2 patients (cases 12 and 13) had bilateral upjo with the same degree of hydronephrosis of both renal units on ultrasonography but the difference in rrf on the tc - mag3 renal scan between the renal units was 12% and 28%, respectively. these 2 patients underwent pcn under general anesthesia on the worse renal unit with a significantly lower rrf and simultaneous initial unilateral dismembered open pyeloplasty on the better renal unit with a higher rrf. at 1 month after the initial pyeloplasty, the hydronephrosis of the non - operated renal unit resolved or improved spontaneously in 5 patients (cases 1 - 5 ; 38.5%). in the remaining 8 patients (cases 6 - 13 ; 61.5%), the high - grade hydronephrosis on ultrasonography and the obstructive drainage pattern on the tc - mag3 renal scan persisted. at 2 months after the initial pyeloplasty, six patients showed improvement of the hydronephrosis on ultrasonography and a good drainage pattern on the tc - mag3 renal scan after consecutive bilateral pyeloplasty. two patients (cases 8 and 13) required redo - pyeloplasty because of persistent grade 5 hydronephrosis and a decrease in rrf. the management of neonates with unilateral upjo has remained a controversial issue for a long time, and this issue has stayed on the minds of many pediatric urologists. king reported that early correction of upjo is advocated as soon as the diagnosis is established and that young infants show more rapid improvement in rrf than do older children. tapia and gonzalez reported that pyeloplasty in children younger than 1 year with grades 3 or 4 hydronephrosis secondary to upjo is effective at improving renal function and they recommended early pyeloplasty for children with reduced function of the involved kidney. ransley reported that deterioration of renal function occurred in only 23% of renal units with good function as ascertained by renography initially, and they proposed that conservative observation could be possible in infants who demonstrate relatively good renal function postnatally. cartwright proposed that in the kidney with apparent upjo and good function, an initial conservative approach with sequential renal scan follow - up and delayed pyeloplasty as needed is a reasonable management. recently, many institutes around the world have followed these trends in reserving pyeloplasty with conservative observation in the management of unilateral hydronephrosis with apparent upjo. compared with unilateral upjo, however, few published management protocols exist for neonates with prenatally diagnosed bilateral hydronephrosis that leads to the postnatal diagnosis of severe bilateral upjo. theoretically, there are two possible approaches to these patients with severe bilateral upjo : (1) initial conservative management followed by intervention when renal function deteriorates or progression of hydronephrosis occurs, and (2) early intervention independent of initial renal function. there are many reports to suggest delayed surgical intervention followed by close, conservative observation focused on surveillance. onen reported that only 35% of total renal units with prenatally diagnosed primary grade 3 to 4 bilateral hydronephrosis required unilateral or metachronous bilateral pyeloplasty, and the remaining 65% of renal units that were followed nonoperatively showed resolution or improvement of the hydronephrosis for a mean 54 month follow - up. those authors thus suggested conservative management with close follow - up during the first 2 years to be a safe and recommended approach for neonates with primary bilateral ureteropelvic junction type hydronephrosis. kim reported that patients who underwent early surgical correction were expected to show faster and reliable improvements in hydronephrosis, although not only the patients who underwent early surgical correction but also those treated by the conservative method could yield acceptable improvements. they concluded that early surgical intervention should be considered in patients with bilateral upjo. in many studies, patients with bilateral upjo had hydronephrosis of sfu grade 3 or less, which is a mild form and not an emergent case requiring surgery. however, all of the cases recruited for this study had a single or two renal units with hydronephrosis of sfu grade 4 or more, which is a severe grade and thus an emergent case requiring surgery. eckstein and drake reported the feasibility of concurrent bilateral open pyeloplasties, and schwab and casale performed successful concurrent bilateral laparoscopic pyeloplasties with favorable results. but traditionally staged pyeloplasties have been recommended as a safe surgical modality. therefore, we performed early unilateral pyeloplasty in all cases without initial conservative management. the non - operated renal units with mild hydronephrosis of sfu grade 3 (cases 1 - 4) showed resolution of the hydronephrosis (sfu grade 0 or 1) or improvement of the hydronephrosis (sfu grade 2 or 3) after unilateral pyeloplasty for the worse renal unit at 1 month postoperatively however, the non - operated renal units with severe hydronephrosis of sfu grade 4 (cases 5 - 10) showed improvement of the hydronephrosis after unilateral pyeloplasty in only one case (case 5). the remaining 4 cases (cases 6 - 10) showed persistent hydronephrosis following the initial pyeloplasty and consequently delayed pyeloplasty was performed after 1 month. after delayed pyeloplasty, one patient (case 6) showed resolution of the hydronephrosis and 3 patients (cases 7, 9, and 10) showed improvement of the hydronephrosis at the last follow - up. we observed that all of the renal units with sfu grade 3 hydronephrosis and some of the renal units with sfu grade 4 hydronephrosis improved or resolved after unilateral pyeloplasty. therefore, we suggest that delayed pyeloplasty for the renal unit that has relatively mild hydronephrosis should be considered at 1 month following the initial unilateral pyeloplasty for the worse renal unit. preservation of the optimal renal function of both units is a main goal of follow - up of a patient with bilateral upjo. previously published papers reported that although there was no consensus regarding such a cutoff value for the recommendation of early surgical correction, an initial rrf < 30 - 40% can serve as an indication for early pyeloplasty. however, these well - organized studies included mild hydronephrosis such as sfu grade 1 or 2 and enrolled cases of unilateral upjo. in our series, we planned the initial pyeloplasty depending on the severity of hydronephrosis on renal ultrasound rather than the rrf on the tc - mag3 renal scan. we performed initial pyeloplasty of the renal unit with a higher grade of hydronephrosis on renal ultrasound irrespective of the difference in rrf on the tc - mag3 between the two renal units in one patient. if two renal units had the same degree of hydronephrosis on renal ultrasound, our indication for initial pyeloplasty depended on the rrf on the tc - mag3 renal scan. two patients (cases 12 and 13) had the same degree of hydronephrosis of both renal units on ultrasonography but the difference in rrf on the tc - mag3 renal scan between the renal units was more than 10%. we thought pcn could protect against deterioration of renal function, and these 2 patients therefore underwent pcn on the worse renal unit with a significantly lower rrf and simultaneous initial pyeloplasty on the better renal unit with a higher rrf. however, pcn tube placement followed by delayed pyeloplasty did not preserve the renal function as we expected. although the number of cases was small, our results suggest that preoperative pcn tube placement before delayed pyeloplasty is not a feasible management protocol. the number of cases was very small and the follow - up period was uneven in some patients. strictly speaking, our results could be specific to our patients only and may not be generalizable to all bilateral upjo cases. in children younger than 1 year of age with severe bilateral upjo, improvement or resolution of the hydronephrosis following unilateral pyeloplasty for the worse renal unit did occur in the non - operated renal units with sfu grade 3 hydronephrosis and some with sfu grade 4 hydronephrosis. therefore, delayed pyeloplasty for the opposite renal unit should be considered at 1 month following initial unilateral pyeloplasty for the worse renal unit.. a greater number of cases and a longer follow - up in the setting of a prospective and randomized study could help to clarify the optimal management guidelines for severe bilateral upjo. | purposethe management of prenatally detected bilateral ureteropelvic junction obstruction (upjo) remains controversial. we attempted to develop a treatment plan for patients with severe bilateral upjo.materials and methodswe evaluated the records of 13 patients with prenatally diagnosed grade 3 or more bilateral hydronephrosis that led to the postnatal diagnosis of severe bilateral upjo. ultrasonography and 99mtechnetium mercaptoacetyltriglycine (99mtc - mag3) renal scans were performed within 1 month. four renal units had grade 3 and 22 had grade 4 hydronephrosis. all 13 patients were managed by unilateral pyeloplasty, and the patients ' mean age was 3 months. at 1 month postoperatively, we decided whether delayed surgery in the opposite renal unit was necessary according to the findings of ultrasonography and 99mtc - mag3 scans.resultsof 13 patients, 11 underwent initial pyeloplasty on renal units with more severe hydronephrosis or lower relative renal function (rrf) on 99mtc - mag3 scans. the remaining 2 patients simultaneously underwent percutaneous nephrostomy on renal units with a lower rrf and initial pyeloplasty on renal units with a higher rrf. in 5 patients, contralateral hydronephrosis had spontaneously improved at 1 month postoperatively, and 8 patients underwent delayed contralateral pyeloplasty at 2 months postoperatively.conclusionsin children with severe bilateral upjo, the non - operated renal units with grade 3 and some with grade 4 hydronephrosis improved spontaneously after unilateral pyeloplasty. therefore, delayed pyeloplasty of the opposite side should be considered at 1 month following initial pyeloplasty. |
insulin receptor is a tetramer protein composed by two extracellular subunits and two transmembrane subunits. the subunits have a binding site to insulin while the subunits contain an intrinsic tyrosine kinase activity towards intracellular side. insulin binding to the subunit leads to a conformational change and activation of the subunit, resulting in tyrosyl autophosphorylation of the insulin receptor. after being activated and phosphorylated, several intracellular docking proteins bind to the insulin receptor and are also tyrosyl phosphorylated, including insulin receptor substrates 1 and 2 (irs-1 and irs-2) [1, 2 ], src homology collagen (shc), and associated protein substrate (aps). these proteins activate several signaling pathways involved in the regulation of important cellular events such as glucose uptake and metabolism, protein synthesis, gene expression, cell survival, growth, development, and differentiation [46 ]. irs proteins are phosphorylated on various tyrosine residues of the c - terminal region, generating specific sites for binding of proteins containing src homoly-2 (sh2) domains, including phosphatidylinositol-3 kinase (pi-3k), nck, and grb-2. pi-3k is composed by a catalytic subunit (p110) and a regulatory subunit (p85). this kinase is an important signaling molecule, mediating metabolic effects of the insulin. binding of p85 subunit to phosphorylated tyrosine residues of irs proteins leads to activation of the catalytic activity of p110 subunit and subsequent increase in the generation of phosphatidylinositol 3,4-bisphosphate (pip2) and phosphatidylinositol 3,4,5-trisphosphate (pip3) content. downstream proteins from pi3 k pathway comprehend several serine / threonine kinases, for example, phosphoinositide - dependent protein kinase-1 (pdk-1), protein kinase b (pkb, also known as akt), protein kinase c (pkc), p70 s6 kinase (p70s6 k), and glycogen synthase kinase-3 (gsk-3). these kinases are involved in the most important biological effects induced by insulin, such as translocation of glucose transporter-4 (glut-4) from intracellular vesicles to plasma membrane, glycogen and protein synthesis, antiapoptotic effects, and gene expression (figure 1) [711 ]. other signaling pathways involved in the glucose uptake induced by insulin start with the recruitment of aps to the activated insulin receptor and subsequent association and tyrosine phosphorylation of cbl, which interacts with cbl associated protein (cap) through an sh3 domain and with flotillin, a constituent of lipid raft, through a sorbin domain. the complex crkii / c3 g then binds to the phosphorylated tyrosine residues of cbl, activating the c3 g activity that exchanges gdp for gtp of tc10, a small g - protein that belongs to the rho family. after being activated, mitogen - activated protein kinase (mapk) cascade starts with the association of shc to insulin receptor, binding of grb-2 to shc or to irs-1, and formation of the grb-2/sos (son of sevenless) in the plasma membrane [1719 ]. this complex leads to the activation of c - ras and raf, starting the mapk cascade. mapk pathway is involved in the differentiation, cell growth, and development induced by insulin, as well as some metabolic effects, as glycogen synthesis and glut-4 translocation to plasma membrane (figure 1) [2224 ]. however, this cascade is not enough or even required to this later effect. disturbances in several proteins involved in the insulin signaling pathways have been found in different conditions of insulin resistance, including obesity, type 2 diabetes mellitus, metabolic syndrome, cardiovascular diseases, inflammatory disorders, and cancer [2628 ]. here, we will discuss possible mechanisms involved in the development of insulin resistance related to inflammatory processes. insulin resistance occurs when the insulin - sensitive tissues, mainly skeletal muscle, adipose tissue, and liver, lose the ability to respond properly to the hormone [29, 30 ]. it is associated with several chronic diseases, especially those linked to obesity, such as type 2 diabetes mellitus, metabolic syndrome, dyslipidemias, cardiovascular diseases, cancer, and neurodegenerative diseases [3133 ]. however, the precise mechanisms involved in insulin resistance are not fully understood yet [3437 ]. several factors have been proposed to participate in the development of insulin resistance, including increased plasma - free fatty acid level, subclinical chronic inflammation, oxidative and nitrative stress, altered gene expression, and mitochondrial dysfunction [29, 37 ]. since free fatty acids are elevated in obesity and related diseases, these metabolites have been proposed to be responsible for the impairment in the insulin action, but the mechanisms are not completely known yet [38, 39 ]. high availability of fatty acids, specially long - chain saturated fatty acids, results in the establishment of insulin resistance in liver, skeletal muscle, and adipose tissue [40, 41 ]. various hypothesis have been proposed to explain the insulin resistance induced by saturated fatty acids, including randle cycle, oxidative stress, modulation of gene transcription, inflammation, accumulation of intracellular lipid derivatives (diacylglycerol and ceramides), and mitochondrial dysfunction [4247 ] (for review, see martins.). a chronic state of inflammation in the insulin responsive tissues is a major contributor to insulin resistance in obesity and related diseases. thus, a crosstalking between inflammation and insulin resistance has been suggested by several authors. however, the precise mechanism as well as the mediators involved in this interaction is not completely defined yet. in this paper, we discuss how inflammatory signaling pathways impair insulin signaling (see below). intracellular redox balance is a finely regulated process that involves several generating pathways and degrading systems. physiologically, ros participate in important biological responses, but accumulation of these molecules leads to oxidative stress condition. ros are highly oxidant molecules that can oxidize various intracellular components, including membrane phospholipids, proteins, and dna [49, 50 ]. usually, these reactions cause cellular damage, reducing the function of oxidized biomolecules. in insulin resistance, increased ros production and/or decreased ros degradation is observed, leading to an oxidative stress condition and activation of signaling pathways related to stress. there is evidence that oxidative stress is also involved in muscle disorders, contributing to the insulin resistance process. transgenic mice expressing human ubiquitin protein e3 ligase, a protein that binds and promotes degradation of superoxide dismutase-1, resulting in reduced superoxide degradation and consequently oxidative stress, present muscle dysfunction (atrophy and sclerosis). activation of signaling pathways to stress has been suggested to participate in the development of insulin resistance by impairing the signaling by this hormone. several serine / threonine kinases activated by oxidative stress pathways, including jnk, pkc, gsk-3, nf - kb, and p38 mapk, have been suggested to impair insulin signaling pathways [53, 54 ], as described below. for example, expression of genes involved in lipid and glucose metabolism, insulin signaling, inflammation, redox balance, and mitochondrial function is modified, suggesting that these processes participate in the pathophysiology of insulin resistance [5557 ]. disturbed mitochondrial function has been suggested to have a central role in these alterations, since this organelle participates in all these processes (for review, see martins.). inflammation is a coordinated process evoked by the tissues in response to noxious stimuli or conditions including the presence of infection, tissue injury, or malfunction. the inflammatory response is activated by molecules released by microorganisms including microbial associated molecular patterns (mamps) such as lipopolysaccharide, flagellin, and peptidoglycans or produced / released by host cells including intracellular components, the so - called damage - associated molecular patterns (damps), of which, hmgb1, dna, and nucleotides are part. these inducers of inflammation bind to their respective receptors and activate biological responses by the resident cells, mainly, macrophages and mast cells. these cells act directly or indirectly on the vasculature and on leukocytes to induce, among other effects, the migration of leukocytes and extravasations of plasma proteins to the tissues. examples of proteins with this function include the receptors of the toll - like (tlrs) family, the c - type lectin receptors, the purinergic and advanced glycation endproductsreceptors (rage), and the intracellular nucleotide oligomerization domain (nod), and retinoic acid - inducible gene (rig)-i - like receptors (rlrs). after recognizing their ligands, several downstream pathways including c - jun nh(2)-terminal kinase (jnk) and ib kinase complex (ikk) are activated, resulting in changes on transcription factors activity and expression of proteins such as cytokines, enzymes, chemokines, adhesion molecules, and amplification of the inflammatory response. the activation of the inflammatory pathways described above is a hallmark of obesity, and it has been associated with the development of insulin resistance, atherosclerosis, and other tissue dysfunctions that are secondary to fat accumulation. an increased production of inflammatory mediators and activation of inflammatory pathways in several tissues including adipose tissue (at), liver, pancreas, skeletal muscle, and hypothalamus are present in obese individuals and define a subclinical inflammatory process also known as meta - inflammation, little information is available regarding the inducers and sensors involved in obesity - associated inflammation. in this respect, some hypotheses to explain the initial activation / recruitment of leukocyte to the tissues (mainly, at) have been proposed. the release of damps by necrotic adipose cells, the increase in the flux of nonesterified fatty acids (increased rates of lipolysis), the reduction in the oxygen tension (hypoxia) leading to activation of hypoxia - induced factor (hif)-1, which controls the expression of proinflammatory proteins, and the production of chemokines by adipose cells have been suggested to play a role in the initiation of inflammatory process. despite several advances in the field, the initial events involved in the beginning of inflammation in the at and the complex interactions between them are not clearly understood, and new components are continuously described and added to the puzzle such as leukotrienes and the apoptosis inhibitor of macrophage (aim) [62, 63 ]. the release of fatty acids, through interaction with tlr4, induces the expression of chemokines that lead to macrophage recruitment. the obesity - induced inflammation is associated with activation of resident cells such as kupffer cells in the liver and leukocyte infiltration and shift in the polarized state of tissue / recruited leukocytes. these changes are better characterized in the adipose tissue, in which an increased infiltration by proinflammatory m1-type macrophages (classical macrophages), th1, th17 and cd8 t cells and a reduction in the content of less inflammatory cells such as m2-macrophage, t reg, and th2 cells were demonstrated [6568 ]. the overproduction of inflammatory mediators by the infiltrating cells together with changes in adipokines production and nefa release by at contribute to the tissue dysfunctions observed in obesity, as discussed below. several chronic diseases are characterized by increased inflammatory process and insulin resistance, such as obesity, type 2 diabetes mellitus, metabolic syndrome, cardiovascular diseases, and cancer. for example, various studies suggest the involvement of some inflammatory factors in the development of insulin resistance, including cytokines (tnf-, il-1, il-6), ros, and rns (figure 2). these factors lead to the activation of signaling pathways that ultimately impair insulin signaling (see below). in addition, other factors involved in inflammatory processes also can impair insulin sensitivity, particularly lipopolysaccharides (lpss) and environmental stress (hypoxia, nutrients, and ph) (figure 2). it has been demonstrated that fatty acids, specially saturated fatty acids, are able to activate tlr-4 in skeletal muscle cells, resulting in increased activity of ikk and jnk. the first kinase degrades the inhibitor of b (ib), resulting in the nuclear factor-b (nfb) release and migration to the cell nucleus, where it induces the transcription of proinflammatory genes. the second kinase activates members of the signal transducers and activators of transcription (stat) family, which are involved in several biological effects, such as expression of genes related to inflammation, apoptosis, differentiation, growth, morphogenesis, migration, and proliferation. both jnk and ikk have been proposed to be the mediators of insulin resistance induced by saturated fatty acids (figure 2). it has been shown that these kinases phosphorylate serine residues on irs proteins, blocking irs phosphorylation on tyrosine residues by the activated insulin receptor and consequently inhibiting insulin effects. moreover, phosphorylation on serine / threonine residues also increases irs protein degradation, contributing to the establishment of insulin resistance [7173 ]. nonfunctional tlr-4 expression protects mice from insulin resistance and inflammation induced by high - fat diet, and tlr-4 gene silencing by small interference of rna reduces inflammation in acute lung injury induced by lipopolysaccharide. obese and type 2 diabetic animals are prevented from insulin resistance and inflammation by specific inhibitors or gene mutation (knockout or nonfunctional gene) of ikk or jnk [7678 ]. these metabolites have been linked to the establishment of inflammatory process, by modulating several signaling pathways related to inflammation. for example, fatty acids can directly activate toll - like receptors (tlrs), g - protein coupled receptors (gpcrs), and tumor necrosis factor- (tnf-) receptor as well as modulate inflammatory signaling pathways involved in the increase in cytokine secretion (tnf-, il-1, and il-6) [60, 79, 80 ], oxidative and nitrative stress, mitochondrial dysfunction, endoplasmic reticulum (er) stress, and proinflammatory gene expression. alterations in the expression of genes and proteins involved in the inflammatory process are clearly associated with insulin resistance and several metabolic abnormalities, including mitochondrial dysfunction, decreased fat oxidation, increased ectopic lipid storage, and impaired insulin signaling pathways (figure 2). proinflammatory cytokines were also involved in the reduction of mitochondrial function [79, 82 ]. palmitate - stimulated il-1 production in macrophages occurs via nlrp3-asc inflammasome and participates in the mitochondrial dysfunction induced by this fatty acid. this dysfunction is also observed when the cells are exposed to other cytokines, such as tnf- or il-6 [83, 84 ]. therapies aimed at neutralizing proinflammatory cytokines such as tnf- and il-1, such as the monoclonal antibody infliximab and canakinumab, respectively, have been under investigation in the treatment of type 2 diabetic patients. considering anti - tnf antibodies, the results are disappointing as many clinical trials in type 2 diabetic patients have failed to demonstrate an effect of tnf neutralization on insulin sensitivity [8589 ]. on the other hand, in patients with high grade inflammatory diseases such as rheumatoid arthritis and ankylosing spondylitis, anti - tnf therapy has been successfully associated with reduction in insulin resistance and metabolic syndrome components [9095 ]. the molecular mechanisms of tnf- blockade on insulin signaling were related to reduction in irs-1 serine phosphorylation and increase in akt phosphorylation in peripheral mononuclear cells from rheumatoid arthritis patients. the long - term effects of anti - il-1 therapy are now examined in the large phase iii clinical trial cantos (canakinumab anti - inflammatory thrombosis outcomes study). the study included and treated 17,200 patients with various doses of anti - il-1 antibody every 3 months and followed up over 4 years. the primary endpoint of the cantos study will be cardiovascular events, and secondary endpoints include new onset type 2 diabetes and diabetes - specific markers. such large and long - term trials could provide a novel cytokine - based therapy for the secondary prevention of new - onset diabetes as well as confirm the autoinflammatory nature of metabolic disorders. however, identification of pharmacologically potent and selective small molecule jnk inhibitors has been limited. compound a, a reversible atp - competitive aminopyridine inhibitor of jnk, was developed by pfizer. testing this compound in obese mice decreased their body weight as well as blood glucose and triglyceride concentrations and increased insulin sensitivity to levels comparable to those in lean control mice. a substrate competitive inhibitor of jnk, bi-78d3, has also been shown to restore insulin sensitivity in a murine model of type 2 diabetes after a single dose. a more recent drug discovery is the compound 19, a potent and selective dual substrate and atp - competitive jnk bidentate inhibitor. glucose intolerant noncnzo10/ltj mice were injected intraperitoneally daily for four days with 25 mg / kg 19, and this compound was remarkably effective in restoring normoglycemia without inducing hypoglycemia compared to the vehicle control. these studies demonstrate that inhibition of jnk is an effective strategy to ameliorate insulin resistance. however clinical trials are needed to test these compounds in humans and show their efficacy and long - term toxicity. g - protein coupled receptors (gpcrs) constitute a family of membrane proteins characterized by a common motif, the seven transmembrane domains. ligand binding to these receptors leads to conformational changes in the receptor and activation of intracellular guanine nucleotide - binding proteins (g - proteins). several enzymes including adenylyl and guanylyl cyclases, phospholipases a2 and c, phosphodiesterases and phosphatidylinositol 3-kinases (pi3ks) and other intracellular transduction cascades are triggered downstream to these receptors. these pathways affect both the insulin signaling and the inflammatory proteins expression (figure 3). several of these receptors have been shown to regulate insulin secretion and tissue sensibility to this hormone, becoming potential targets for intervention in conditions of insulin resistance / deficiency. in this paper, we will briefly discuss a class of gpcrs that share in common the fact of being activated by fatty acids (fas - gpcrs). other gpcrs implicated in the glucose metabolism and regulation of inflammation such as glp-1, glucose - dependent insulinotropic polypeptide (gip), the bile acid (tgr5), cholecystokinin, the cannabinoid receptors (cbs), and muscarinic receptors are beyond the focus of the present paper and are discussed elsewhere. proteins that regulate gpcrs signaling such as gpcr kinases and arrestins, which are implicated in the control of food intake, regulation of insulin action, inflammation, adipogenesis, and other processes that are associated with weight gain and development of insulin resistance, are the focus of recent reviews [100, 101 ], so they are not discussed here. the fas - gpcrs receptors, which include the gprs 40, 41, 43, 84, 119, and 120, present distinct ligand specificity and tissue distribution. regarding their participation in the glucose metabolism, it has been demonstrated that their activation (at least, gpr40 and gpr119) directly stimulates insulin secretion by -cells and protects these cells from gluco- and lipotoxicity (gpr40) [103, 104 ]. activation of fas - gpcrs induces also the release of gut - derived hormones including glucagon - like peptide-1 (glp-1) and glucose - dependent insulinotropic polypeptide (gip) [105109 ]. these latter two - gut - derived hormones not only modulate the gastrointestinal functions such as motility, but also the insulin secretion and food intake. in addition to these effects, fas - gpcrs, mainly, gpr43, 84, and 120, present relevant effects in the inflammatory cell activation. in this sense, activation of gpr120 and -arrestin 2 by n-3 fatty acids (docosahexaenoic and -linolenic acids) attenuates the production of tnf-, il-6, and macrophage chemoattractant protein-1 (mcp-1, also known as ccl2). this anti - inflammatory effect seems to be important for the beneficial action of these fatty acids in the model of obesity induced by high - fat diet, preventing development of glucose intolerance, insulin resistance, and obesity. altogether the findings herein discussed highlight the importance of these receptors for glucose homeostasis, control of inflammatory cells activation, and food intake, processes that are linked through complexes interaction, which are not completely understood. histone deacetylase (hdac) is a family of enzymes that together with the histone acetyltransferases (hats) controls the degree of protein acetylation. inhibition of hdac activity by different compounds (e.g., short chain fatty acids such as butyrate, valproic acid, trichostatin a, and other compounds) increases the acetylation of histone and nonhistone proteins including nfb, myod, p53, and n - fat and, consequently, affects gene expression and proteins activities leading to changes in different aspects of cell biology including cell motility, proliferation, differentiation, and apoptosis. in addition to their well - known anti - inflammatory effects, other recent evidence has been obtained, which together strongly indicates hdac as a target for novel therapies in insulin resistance and diabetes.histone hyperacetylation has been associated with an increase in insulin expression and protection of -cells against cytokine - induced apoptosis, as reviewed by christensen..the isoforms 4, 5, and 9 of hdac are associated with the development of and cells of the pancreas.oral administration of hdaci (itf2357) reduced -cells toxicity associated with streptozotocin administration. additionally, the authors also showed that this hdaci protected islets from cytokine - induced toxicity and reduced production of no and chemokines in islets ; administration of sodium butyrate (diet supplementation or oral tributyrin (a prodrug of butyrate)) to high - fat fed mice attenuated body weight gain, improved lipid and glucose metabolism parameters, and inhibited the development of obesity - associated changes including activation of inflammatory pathways and hepatic steatosis [115, 116 ]. recently, it has been suggested that this effect of butyrate involves inhibition of hdac 3 activity, an effect that leads to activation of ppar- and expression of fgf21, which stimulates lipid oxidation, triglyceride clearance, and energy expenditure ; hdac 6 knockout mice are protected from hyperglycemia, glucose intolerance, and insulin resistance secondary to chronic corticoid administration ; hdaci increases the number of t regulatory cells and their suppressive function, an effect that may be important in the context of adipose tissue inflammation [119, 120 ]. histone hyperacetylation has been associated with an increase in insulin expression and protection of -cells against cytokine - induced apoptosis, as reviewed by christensen.. the isoforms 4, 5, and 9 of hdac are associated with the development of and cells of the pancreas. additionally, the authors also showed that this hdaci protected islets from cytokine - induced toxicity and reduced production of no and chemokines in islets ; administration of sodium butyrate (diet supplementation or oral tributyrin (a prodrug of butyrate)) to high - fat fed mice attenuated body weight gain, improved lipid and glucose metabolism parameters, and inhibited the development of obesity - associated changes including activation of inflammatory pathways and hepatic steatosis [115, 116 ]. recently, it has been suggested that this effect of butyrate involves inhibition of hdac 3 activity, an effect that leads to activation of ppar- and expression of fgf21, which stimulates lipid oxidation, triglyceride clearance, and energy expenditure ; hdac 6 knockout mice are protected from hyperglycemia, glucose intolerance, and insulin resistance secondary to chronic corticoid administration ; hdaci increases the number of t regulatory cells and their suppressive function, an effect that may be important in the context of adipose tissue inflammation [119, 120 ]. ppar- receptor activation has been shown to have significant effects on increasing insulin sensitivity in fat and muscle cells. it improves glucose metabolism and reduces inflammation (figure 3) and has a crucial role in adipocyte differentiation. ppar- is a nuclear receptor that acts as a transcription factor upon activation, by regulating the transcription and expression of specific genes such as adipokines. several studies have demonstrated the anti - inflammatory activities of ppar-. inhibition of ppar- function by inflammatory cytokines may contribute to pathogenesis of many diseases such as insulin resistance, atherosclerosis, inflammation, and cancer cachexia [122124 ]. activation of serine kinases including ikk, erk, jnk, and p38 may be involved in the tnf regulation of ppar- (figure 3) (reviewed in). ikk acts through at least two mechanisms : inhibition of ppar- expression and activation of ppar- corepressor. in macrophages, where ppar gamma is also expressed, it inhibits tlr and ifn- mediated inflammatory responses. in obesity, macrophages invade adipose tissue promoting the inflammation characteristic of insulin resistance. therefore, macrophage ppar- function gained considerable pharmacological interest. diet - induced obesity influences the state of adipose tissue macrophages from an m2-polarized state (that protects adipocytes from inflammation) to an m1 proinflammatory state. studies have demonstrated that this obesity - induced phenotypic alteration of macrophage polarization is orchestrated by ppar-. these researchers demonstrated that ppar- is required for the maturation of alternatively activated macrophages (m2 macrophages) by using mice with specific macrophage deletion of ppar-. prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue and other tissues, leading to metabolic abnormalities such as insulin resistance.. showed that ppar- is involved with adipose tissue - specific lymphocyte accumulation and activation, leading to cell differentiation. t regulatory cells (tregs) are a small subset of t lymphocytes, normally constituting only 520% of the cd4 compartment. these cells are thought to be one of the most critical defenses against excessive immune responses, avoiding autoimmunity, allergy, inflammation, infection, and tumorigenesis [132, 133 ]. typically, tregs control other t cell populations, but can also influence the activities of innate immune system cells. treg cells are characterized by high - level expression of the forkhead / winged - helix transcription factor, foxp3. cipolletta. have demonstrated that ppar- collaborates with foxp3 to impose on naive cd4 t cells the characteristic of visceral adipose tissue treg cells. in fact, feuerer. demonstrated that treg cells with a unique phenotype were highly enriched in the abdominal fat of normal mice, but were specifically reduced at this tissue in an insulin - resistant model of obesity. a recent study highlighted the negative effect of leptin on the proliferative capacity of treg. in fact, it is well demonstrated that obese subjects present low treg cell number and adiponectin production and high leptin secretion. tlrs are transmembrane receptors that play a critical role in the detection of microbial infection and in the induction of inflammatory and immune responses against conserved microbial structures, called pathogen - associated molecular patterns. each member of tlr family recognizes a specific pathogen component which, upon activation, triggers a signaling cascade leading to cytokine production and adaptive immune response. among the tlrs, tlr2 and tlr4 play a critical role in the pathogenesis of insulin resistance, diabetes, and atherosclerosis in both clinical and experimental conditions [138, 139 ]. c3h / hej mice with a mutation in tlr4 are protected against the development of high fat diet - induced obesity. in addition, these mice demonstrate decreased adiposity, increased oxygen consumption, a decreased respiratory exchange ratio, improved insulin sensitivity, and enhanced insulin - signaling capacity in adipose tissue, muscle, and liver. moreover, in all these tissues, control mice fed a high - fat diet showed an increase in ikappab kinase complex and c - jun nh(2)-terminal kinase activity, which is prevented in c3h / hej mice. studies in mice demonstrate that tlr-2 and tlr-4 activation and cytokine production stimulated by these receptors lead to the development of diabetes (figure 3) [140, 141 ]. more recently, tlr4 has been indicated as a molecular link between free fatty acids, inflammation, and the innate immune system. studied tlr2 and tlr4 mrna and protein expression, their ligands, and intracellular signaling in monocytes of recently diagnosed type 2 diabetic patients and observed that there is significant elevation of tlr2 and tlr4 protein, mrna, endogenous ligands, and cofactors which, together with hyperglycemia, contribute to the proinflammatory state of type 2 diabetes. koopet. observed that tlr activation promotes upregulation of il-6 and mcp-1 release in isolated human adipocytes via specific activation of erk. tlr-4 deficient mice had also markedly lower circulating concentrations of mcp-1 and much less nf - kb protein in nuclear extracts prepared from adipose tissue. in contrast, tlr-4 deficiency did not attenuate the induction of tumor necrosis factor - alpha (tnf-) or interleukin-6 (il-6) expression in adipose tissue promoted by diet with high saturated fatty acids. nowadays, based on several studies it is clear that tlr4 inhibition is a pharmacologic tool to avoid inflammation in insulin resistance patients. arachidonic acid (n-6 fatty acid) serves as precursor of immune - active lipid mediators known as eicosanoids. classes of eicosanoids include lipoxins, leukotrienes, and pgs, and their effects on the immune system have been extensively explored and reviewed., resolvins are new mediators generated from n-3 fatty acids docosahexaenoic acid (dha, c22:6n-3) and eicosapentaenoic (epa, c20 : 5n-3) identified first in resolving inflammatory exudates and in tissues enriched with dha. the names resolvin (resolution phase interaction products) and docosatrienes were given because these bioactive compounds demonstrate potent anti - inflammatory and immunoregulatory actions (figure 3). these mediators prevented neutrophil entry to inflammation sites and cytokine production and reduced exudates in rats with experimental peritonitis. the compounds derived from epa carrying potent biological actions are named the e series and are denoted as resolvins of the e series. those synthesized from dha are resolvins of the 17s - d series that have immunoregulatory and neuroprotective actions. in addition, aspirin treatment triggers the formation of 15-epimeric lipoxins, termed aspirin - triggered lipoxins, that also play a role in resolution of inflammation through inhibition of neutrophil tissue infiltration and stimulation of macrophage phagocytosis of apoptotic neutrophils. studies demonstrate that n-3 fatty acid feeding promotes endogenous production of resolving d1 and 17-hydroxy - dha, a marker of resolvin biosynthesis, in adipose tissue of obese - diabetic mice. transgenic overexpression of fat-1, which encodes a desaturase enzyme that is able to convert -6 to -3 fatty acids, partially protects against obesity - induced insulin resistance in mice and is associated with an increase in the resolvin biosynthetic pathway marker 17-hydroxy - dha. these studies demonstrated that high - fat feeding results in a deficient endogenous resolvin and protectin biosynthesis and that these compounds are restored in fat-1 transgenic mice. provided evidence that adipose tissue expresses all the enzymes necessary for the formation of bioactive lipid mediators derived from both omega-6 and omega-3-pufas. in ob / ob mice dha significantly increased adipose tissue levels of adiponectin, which alleviated hepatic steatosis and insulin resistance. recent findings indicate also that dha (at micromolar concentrations) and resolvin d1 (at nanomolar concentrations) consistently decrease m1 macrophage activation in adipose tissue and increase m2 cells. these effects are related to stimulation of arginase 1 expression and attenuation of ifn/lps - induced th1 cytokine secretion. hellmann. suggested that stimulation of the inflammation resolution with the endogenous proresolving mediator resolving d1 provides a novel therapeutic strategy for treating obesity - induced diabetes. the authors observed that in leptin - receptor deficient mouse resolvin d1 prevents the accumulation of macrophages in adipose tissue and restores systemic insulin sensitivity. notably, these inflammation - resolving factors are important tools to decrease adipose tissue inflammation that is common in insulin resistance. micrornas (mirnas or mirs) are short noncoding rnas that have been demonstrated to be master regulators of the cellular transcriptome and proteome [155157 ]. regulatory mirnas bind to the complementary segments within 3-untranslated region (3utr) of target transcripts through watson - crick base pairing, causing translational inhibition or mrna cleavage and suppression of gene expression. over 1000 mirnas have been identified in the human genome, which are estimated to regulate thousands of protein - coding genes [158, 159 ]. there is also increasing experimental evidence that mirnas are involved in the control of several critical biological processes such as metabolism, cell proliferation, apoptosis, and disease development and progression [160163 ]. single - stranded mature mirnas with 2024 nucleotides in length are derived from genomically encoded sequences through transcription and complex mrna processing. change in gene expression or processing in dysfunctional or abnormal cells or tissues leads to an altered mirna expression level. profile of these molecules may be used as biomarkers for classifying human diseases and disease status [162, 164, 165 ]. recent studies have observed an association between specific mirnas and insulin resistance [166, 167 ], supporting the fact that mirnas may play a role in the pathological development of type 2 diabetes mellitus and leading also to the hypothesis that mirnas may represent a new class of glucose metabolism regulators with therapeutic potential for improving insulin sensitivity in peripheral tissues. supporting this idea, frost and olson have demonstrated that global and pancreas - specific overexpression of the mirna let-7 in mice results in impaired glucose tolerance and reduced glucose - induced pancreatic insulin secretion. pharmacological inhibition of the mirnalet-7family with specific anti - mir was sufficient to prevent and treat impaired glucose tolerance in diet - induced obesity mice, at least in part, by improving insulin sensitivity in liver and muscle. in addition, mirna let-7was able to block glucose - induced insulin secretion from the pancreas, suggesting that knockdown of this mirnaalso might improve pancreatic -cell function. a role of the heart in systemic metabolic control and the involvement of the heart - specific microrna, mir-208a, as potential therapeutic target for metabolic disorder have been proposed. impaired metabolism of energy - providing substrates and myocardial lipid accumulation are early abnormalities in obese and insulin - resistant individuals. grueter. have shown that med13, a subunit of the mediator complex, controls transcription by thyroid hormone andother functions of nuclear hormone receptors in heart, controlling metabolic homeostasis and energy expenditure in mice. they have also shown that mir-208a, a heart - specific mirna encoded by an intron ofthe cardiac - specific -myosin heavy - chain (mhc) gene, negatively regulates med13 expression. elevated cardiac expression of med13 or pharmacologic inhibition of mir-208a in mice led to resistance to high - fat diet - induced obesity and improved systemic insulin sensitivity and glucose tolerance in mice. conversely, genetic deletion of med13 specifically in cardiomyocytes enhanced obesity in response to high - fat diet and exacerbated metabolic syndrome. as discussed previously, inflammation and oxidative stress participate in the propagation and development of obesity and associated metabolic disorders associated to insulin resistance. maladaptive production of various adipokines (e.g., adiponectin, resistin, visfatin, and leptin), migration of monocytes, and subsequent transformation into macrophages within affected tissues are key factors in the self - perpetuating inflammation associated with metabolic disorders [171, 172 ]. in particular, plasma levels of adiponectin are significantly lower in obese individuals and have been associated with inflammation, insulin resistance, and cardiovascular disease. hulsmans. identified mir-146b-5p as a downregulated mir in monocytes of obese subjects with targets in the irak / nfkb - related gene cluster. they identified obesity - associated low levels of globular adiponectin as cause of the decrease in mir-146b-5p. the role of mir-146b-5p in the protection against inflammation was further supported by the finding that, after sequestration of mir-146b-5p, the cells lost their potency to raise their anti - inflammatory action in response to high levels of adiponectin. a possible role of mir-107 in regulating the inflammatory process that might lead to type 2 diabetes mellitus has been proposed by foley and o'neill. because the tlr4 (lps receptor) has been shown to downregulate mir-107 in activated macrophages located in adipose tissue, and mir-107 has been demonstrated to be dysregulated in murine and rodent models of obesity and insulin resistance, respectively [176178 ], it has been suggested that mir-107 may be the link between obesity, inflammation, and insulin resistance. the authors hypothesize that decreased mir-107 by tlr4 is required to limit proinflammatory signaling pathways, since this effect will stabilize caveolin-1, blocking the tlr4 pathway by displacement of myd88. decreased mir-107 is an attempt to increase insulin sensitivity in the resolution phase of inflammation. a defect in this process, particularly in the ability of tlr4 to decrease mir-107, an interesting model would be transgenic mice, in which we could manipulate mir-107 in vivo and pharmacologically inhibit mir-107. the discovery and identification of new biomarkers involved in the pathogenesis of chronic inflammation and insulin resistance have been fundamental to understanding how these processes work and to direct further studies for the prevention or treatment of related disorders. particularly, these discoveries have aided to identify new target genes, lipids, proteins, and other metabolites involved in the development or severity of chronic diseases, for example, obesity, type 2 diabetes mellitus, metabolic syndrome, cardiovascular diseases, cancer, dyslipidemia, and cancer. probably, with the help of new tools and advanced methodologies, we will have a better characterization and understanding of the mechanisms and mediators involved in the different inflammatory and insulin resistance conditions, addressing further biotechnological studies for the development of new potential clinical therapies and interventions. | inflammation and insulin resistance are common in several chronic diseases, such as obesity, type 2 diabetes mellitus, metabolic syndrome, cancer, and cardiovascular diseases. various studies show a relationship between these two factors, although the mechanisms involved are not completely understood yet. here, we discuss the molecular basis of insulin resistance and inflammation and the molecular aspects on inflammatory pathways interfering in insulin action. moreover, we explore interventions based on molecular targets for preventing or treating correlated disorders, advances for a better characterization, and understanding of the mechanisms and mediators involved in the different inflammatory and insulin resistance conditions. finally, we address biotechnological studies for the development of new potential therapies and interventions. |
urinary incontinence (ui) after radical prostatectomy (rp) is not uncommon and one of a major postoperative complication, which has been reported in 2% to 87% of patients, depending on the definitions, types of surgery, and diagnostic methods. the cause of persistent postprostatectomy incontinence (ppi) is multifactorial, but can be sphincteric deficiency, bladder abnormalities such as detrusor overactivity (do), reduced bladder compliance, and impaired detrusor contractility. an urodynamic study is considered as the best method that can definitively ascertain the cause of ui and has been used by many previous studies on the cause of ppi. in clinical practice, urodynamic study may be performed only when patients have unresolved ui or new - onset lower urinary tract symptoms (luts) after rp, hence it is not easy to obtain large - scale data on the of bladder and urethral function following rp through performing an urodynamic study. therefore, functional alteration of bladder and urethra after surgery has not been extensively studied. although the various studies reported the urodynamic findings on the bladder and urethral function following rp, most of them performed an urodynamic study only aimed at incontinent patients. in the present study, using a large - scale database from snu - experts - of - urodynamics - leading (seoul) study group, we analyzed the urodynamic parameters to compare bladder and urethral function following rp between men who had luts with ui and those who had luts without ui. between july 2004 and june 2013, we have prospectively collected data on the urodynamics of 555 patients who had undergone rp or other prostatic surgery at three affiliated hospitals of the seoul study group. of these, 303 patients with luts determined by the subjective reports following rp were evaluated for this study. we excluded the patients with neurogenic abnormalities before rp (14 patients), with pelvic irradiation (11 patients), or who had a history of surgery due to bladder neck contracture or urethral stricture (10 patients) after rp. the institutional review board (irb) of the seoul national university bundang hospital (irb no. : b-1506/304 - 107) approved the study protocol based on the declaration of helsinki and the acquisition of the informed consent was waived due to the retrospective analysis of the database. the methods for rp were open retropubic, pure - laparoscopic or robot - assisted approach based on a decision by the surgeon and the patient. before the introduction of robotic surgery, most surgeries had been performed by open approach in three affiliated hospitals and laparoscopic approach had accounted for less than 5% of the whole cases. after the introduction of robotic surgery, most surgeries have been performed by robot approach. regular follow - up after surgery consisted of physical examination, urinalysis, prostate - specific antigen, digital rectal examination, and relevant questionnaires assessing the status of urinary or erectile function, and were performed every 3 months for the first 1 or 2 years, every 6 months in the next 3 or 4 years, and yearly thereafter. the status of ui and the number of pads per day required for urinary control were assessed serially at the follow - up by a physician or a research assistant. usually, all patients with ppi were advised to conduct kegel exercises without participation in a specific rehabilitation program. the evaluation of luts including ui after surgery included a complete history taking of clinical symptoms, physical examination, questionnaires evaluating status of urinary function and symptom bother, noninvasive uroflowmetry, cystoscopic inspection, and urodynamic study. cystoscopy and urodynamic study were performed when luts or ui was persistent beyond 1 year after surgery. ui was defined as regularly wearing one pad or more per day due to any involuntary leakage of urine in daily living, based on the patient interview. all terms and procedures for urodynamic evaluation were based on the guidelines of the international continence society. also, as seoul study group urodynamic database has been obtained from three affiliated hospitals, we have tried to maintain the consistency of data by standardizing the urodynamic procedure and interpretation of the results between the hospitals through the periodic audit and quality control. conventional multichannel urodynamic study (ud-2000, medical measures system, enschede, the netherlands), including a pressure - flow study, was performed after discontinuation of all drugs possibly affecting micturition function for at least 3 - 7 days, based on the predetermined protocol. first, urethral pressure profiles were measured at rest while the catheter was withdrawn at a speed of 2 mm / s with saline perfused at 2 ml / min. an aseptic 6-fr double - lumen catheter and a 9-fr balloon catheter were used to assess the intravesical and abdominal pressures during a filling cystometry. pelvic floor electromyography was performed with surface electrodes attached at 3 and 9 o'clock positions near the anus. intravesical pressure was measured under conditions of room - temperature saline infusion at 50 ml / min. however, in individuals with severe storage symptoms or lower functional bladder capacity in bladder diary, the filling rate was decreased to 20 ml / min. abdominal leak point pressure (alpp) was assessed in the sitting or standing position at the amount of half maximum cystometric capacity (mcc). for patients who failed to demonstrate urine leakage on intubated urodynamic alpp test patients were instructed to void in a standing or sitting position under quiet and relaxed circumstances during a pressure - flow study. considering the possibility of cortical inhibition, an additional voiding trial was performed if the first voiding trial failed. in cases of severe leakage in the sitting or standing position before the command for voiding, manual compression was applied on the penile urethra to identify the mcc and the presence of detrusor pressure during voiding trial. reduced bladder sensation was defined as a diminished bladder sensation or not having a strong desire to void during filling cystometry. do was regarded as positive if spontaneous or provoked involuntary detrusor contraction (idc) was observed. detrusor underactivity (du) was considered as present if the bladder contractility index (bci) was less than 100 and bladder outlet obstruction (boo) was defined as an abrams - griffith number of 40 or greater. patients were divided into individuals with ui (ppi group) and those without ui. the type of ui, such as stress or urgency, was not concerned in the statistical analyses because ui, itself, may be more relevant to patients regardless of the type of ui after surgery. comparisons between groups were assessed by a student t - test or a chi - square test depending on the types of variables. logistic regression analysis was performed to identify the possible factors related with ppi among the variables from patient demographics, uroflowmetry, urethral pressure profilometry, and filling cystometry. the significance level of two - tailed p - value<0.05 in the univariate analyses was a screening criterion for entrance into the multivariate logistic regression analyses. 19.0 (ibm co., armonk, ny, usa) was used, and a two - tailed p - value<0.05 was determined to demonstrate statistical significance. the mean age of 268 patients included in the analysis was 66.8 years at the surgery and 68.2 years when receiving an urodynamic study. patients with ui were significantly older ; however, other demographics were not different between patients with and without ui (table 1). as for other luts except for ui, frequency, nocturia, and urgency were the most common. fourteen patients (9.3%) among the ppi group did not demonstrate urine leakage on urodynamic alpp test, despite the presence of subjective symptoms of ui in daily living. also, 33.9% in the non - ppi group showed a positive result on the test, though they did not report the subjective symptoms on ui. actual value of alpp among the patients with a positive test was lower in the ppi group (table 2). overall, reduced bladder compliance was found in 27.2% of the patients ; and 31.3% had idc. during voiding phase, 63.1% and compared to patients of the non - ppi group, those in the ppi group had a lower maximum urethral closure pressure (mucp) and lower mcc (table 3). as for voiding parameters, maximum flow rate during a pressure - flow study was higher and the detrusor pressures recorded at the onset of urine flow and at maximum measured flow rate were lower in the ppi group. concerning du, incidences between both groups were not dif ferent, while the occurrence of boo was higher in the non - ppi group (table 3). in the multivariate logistic regression models with variables from patient demographics, uroflowmetry, urethral pressure profilometry, and filling cystometry, only mucp and mcc were identified as the related factors with the presence of ppi (table 4). to date, various studies reported the urodynamic findings on the bladder and urethral function following rp. however, these studies only considered small patient number less than 100 cases and some had a heterogenic cohort of both rp for prostate cancer and transurethral resection for benign prostatic hyperplasia. furthermore, most of them performed an urodynamic study only aimed at incontinent patients. in the present study, we analyzed urodynamic parameters to evaluate the bladder and urethral function following rp, not only in the ppi group, but also in the patients with luts other than ui using a large - scale database. a reduced bladder compliance and idc were shown in 27.2% and 31.3% of the patients enrolled. the ppi group patients were older when receiving surgery and urodynamic study, had lower levels of mucp, mcc, and detrusor pressure during voiding, as compared with those in the non - ppi group. finally, only mucp and mcc were identified as the related factors with the presence of ppi in the logistic regression models. therefore, we assume that both impairment of the urethral closure pressure and lower bladder capacity during the storage phase are deeply associated with the presence of ppi. in the previous studies with urodynamic assessment, sphincteric deficiency has been investigated as the main cause of ppi. in our study, mucp in the ppi group was about 10 cmh2o lower than in the non - ppi group and was identified as the related factor with the presence of ppi, supporting the findings of previous researches on the relationship between sphincteric deficiency and ppi. furthermore, cameron. reported no differences in mucp at rest, rather, did observe a large difference in the ability to increase closure pressure during a kegel maneuver between men with and without ppi. as for functional urethral length, no statistical difference was found between men with and without ppi in our study, similar to the study by cameron.. one systematic review revealed that functional urethral length did not show any significant difference between men with and without ppi in three of five studies, whereas mucp was significantly different between these men in five out of seven studies. sphincteric deficiency is a predominant factor associated with ppi, but the bladder components may also contribute to this problem. in the present study, lower mcc was another related factor with the presence of ppi. similar to our findings, cameron. found mcc was 95 ml smaller in men with ppi. the clinical significance of mcc, however, has not been clearly determined in men with ppi until now, and may be difficult to interpret. in women with overactive bladder, it has been reported that mcc is smaller in women with do than in those without do. considering this relation, our findings could be explained by the assumption that lower mcc in the ppi group may be related with higher frequency of idc in this group, albeit not statistically significant (32.7% vs. 29.7%) or is perhaps a new finding needing further study. similar to our findings, kadono. also reported the significant relation of postoperative mcc with the status of ui, measured by urine loss ratio, in men with rp. more well - designed studies will clarify the clinical evidence to support mcc as a relevant factor of ppi. another parameter of bladder dysfunction that can be related with ppi may be a reduced bladder compliance and idc. de novo reduced compliance has been observed in up to 32% of men receiving rp, with persistence in 28% after 36 months and idc has been found in 30%-40% among patients with ppi. however, as previously reported, these parameters are rarely the sole cause of ppi and less than 10%-15% of patients with ppi have only bladder dysfunction without sphincteric deficiency. in our study, the percent of patients with these parameters was not statistically different between both groups though the actual rates were relatively higher in the ppi group. in contrast to men with a prostate, those receiving rp may more readily initiate and maintain urinary flow with relatively less use of detrusor power, and this can particularly apply to the patient with ppi who has a weak sphincteric function. have reported significant decreases in detrusor pressure at the maximum flow, opening urethral pressure, and an increase in the maximum flow rate after rp. therefore, it is not surprising that the ppi patients had the higher maximum flow rate and lower detrusor pressures during the voiding phase in the present study. although we defined du as the bci less than 100, the specific method of assessment for detrusor contractility has not been standardized and there are no acceptable normal levels of contractility for men receiving rp. unlike men with an intact prostate, men receiving rp may have the lower outflow resistance. therefore, the simple use of the bci derived from men with benign prostatic hyperplasia can be inappropriate for patients receiving rp. previous study insisted that the best method for the assessment of contraction strength was the isometric detrusor contraction pressure independent of urinary flow in men receiving rp and, also, found that the use of common methods, such as the bci, was the imprecise way for the diagnosis of du in this population. therefore, further studies on more proper measures of du independent of outlet resistance are needed in men devoid of a prostate. about 10% (14 patients) of the ppi group did not demonstrate urine leakage during the alpp test, despite their clinical symptoms. in these patients, do, du with increased postvoid residual volume, and lower detrusor compliance were identif ied in 3, 4, and 2 patients, respectively. for these five patients, we 're not sure whether ui in daily living arose from the bladder or sphincteric dysfunction. one possible assumption is that the urodynamic test could not replicate the clinical symptoms in these patients with possible sphincteric dysfunction. in contrast, 33.9% of the non - ppi group showed a positive alpp result during the test. as the mean value of alpp in this population did reach nearly 150 cmh2o, it is possible that ui may be insignificant and ignored in daily living in this population. first, we did not determine the type of incontinence, such as stress or urgency, in the statistical analyses and medications such as anticholinergics were not concerned when defining the presence of ppi. however, published data have shown that only 2% of patients with ppi complain of urgency incontinence alone and over 90% of our ppi group showed the positive result during the alpp test. therefore, ui symptoms might be ascribed to the impairment of the urinary sphincter in most of our ppi group. second, we only analyzed the status of ui, but did not assess the qualitative status of various urinary symptoms. we used validated questionnaires, but they could not be unified, in part, for the three affiliated hospitals of the study group and the response rate was not high for a statistical analysis. in some previous studies, do, bladder compliance, and low mucp evaluated preoperatively have been shown to adversely affect continence status after rp. however, there are some evidences on the contrary. to assess the urodynamic change between pre and postoperative period will be essential to identify the predictor for the development of ppi. however, to date, such studies have only involved a small sample size, around 50 patients. our main objective was to identify the urodynamical differences of bladder and urethral function, in other words, the possible cause of ppi among men with luts including ui after rp. therefore, the lack of preoperative urodynamic study would have little impact on our results. forth, because of retrospective nature of the study, we could not analyze the detailed data about the modification of the operative procedure, prostate size, and tumor extent. last, interval between the surgery and an urodynamic study varied (around 18 months), even though the bladder and urethral function following rp could change with time. nevertheless, our findings confirm the urodynamical differences of bladder and urethral function among men with luts including ui after rp and should improve understanding about the pathophysiology of ppi which is one of major postoperative complications experienced by patients undergoing rp. in our study, significant number of patients with luts following rp showed a reduced bladder compliance and do. ppi is associated with both impairment of the urethral closuring mechanism and bladder storage dysfunction. both urethral and bladder factors should be considered in the evaluation and management of the patients with ppi. some effort not to attenuate mucp such as precise identification of the sphincter complex and meticulous dissection to prevent tissue damage would be beneficial to prevent the development of ppi. further studies including symptom assessment using unified form of questionnaire are necessary to elucidate the influence of other bladder abnormalities on the ppi. | purposewe compared bladder and urethral functions following radical prostatectomy (rp) between men with and without urinary incontinence (ui), using a large - scale database from snu - experts - of - urodynamics - leading (seoul) study group.materials and methodssince july 2004, we have prospectively collected data on urodynamics from 303 patients with lower urinary tract symptoms (luts) following rp at three affiliated hospitals of seoul study group. after excluding 35 patients with neurogenic abnormality, pelvic irradiation after surgery, or a history of surgery on the lower urinary tract, 268 men were evaluated. we compared the urodynamic findings between men who had luts with ui (postprostatectomy incontinence [ppi ] group) and those who had luts without ui (non - ppi group).resultsthe mean age at an urodynamic study was 68.2 years. overall, a reduced bladder compliance (20 ml / cmh2o) was shown in 27.2% of patients ; and 31.3% patients had idiopathic detrusor overactivity. the patients in the ppi group were older (p=0.001) at an urodynamic study and had a lower maximum urethral closure pressure (mucp) (p<0.001), as compared with those in the non - ppi group. bladder capacity and detrusor pressure during voiding were also significantly lower in the ppi group. in the logistic regression, only mucp and maximum cystometric capacity were identified as the related factor with the presence of ppi.conclusionsin our study, significant number of patients with luts following rp showed a reduced bladder compliance and detrusor overactivity. ppi is associated with both impairment of the urethral closuring mechanism and bladder storage dysfunction. |
autoantibodies are a hallmark in the diagnosis of many systemic autoimmune rheumatic diseases (sard) including idiopathic inflammatory myopathies (iim) (reviewed in [1, 2 ]). most of those autoantibodies are directed to intracellular proteins, including nuclear and cytoplasmic antigens, and based on their specificity, autoantibodies in iim can be grouped into myositis specific autoantibodies (msa) and myositis associated autoantibodies (maa) (reviewed in [13 ]). the presence of msa and maa has become a key feature for classification and diagnosis of iim and they are increasingly used to define clinically distinguishable iim subsets. among the msa, autoantibodies against aminoacyl - trna synthetases (ars) were detected in 2535% of iim patients. other autoantibodies in iim are directed to the signal recognition particle (srp), chromodomain helicase dna binding protein 4 (mi-2), sae / small ubiquitin - related modifier (sumo-1), mj / nuclear matrix protein 2 (nxp2), melanoma differentiation - associated gene 5 (mda5)/clinically amyopathic dermatomyositis p140 (cadm-140), and transcription intermediary factor (tif1-) gamma (p155/140). anti - jo-1 antibodies are the most common, predominantly found in 1530% of patients with polymyositis (pm) and in 6070% of those with interstitial lung disease (ild). autoantibodies directed towards other ars are less common, each reaching less than 5% prevalence in iim. msa and maa are commonly detected using immunoprecipitation (ip) or line immunoassays (lia). muscle pain and weakness are common side effects of statins which are commonly used to reduce cholesterol levels. about 5% of statin users experience muscle pain and weakness during statin treatment. in 2010, antibodies to 3-hydroxy-3-methylglutaryl coenzyme a reductase (hmgcr) have been identified in patients with autoimmune necrotizing myopathies associated with statin use [57 ]. recently, a significant difference between statin - exposed and statin - unexposed anti - hmgcr positive patients has been found. therefore, diagnostic tests are needed to aid in the diagnosis of this severe clinical condition [9, 10 ]. this study aimed to compare different technologies for the detection of anti - hmgcr antibodies and analyze the clinical phenotype and autoantibody profile of the patients and to investigate the epitope specificity of anti - hmgcr antibodies. a total of 20 samples from myositis patients positive for anti - hmgcr antibodies (see table 1) using a research addressable laser bead assay (albia, rouen, france) identified in a previous study and 20 negative controls (age and sex matched) were collected and tested using various methods. to verify the specificity of the quanta lite hmgcr elisa a total of 824 controls diagnoses of the patients were established based on the respective disease classification criteria and as previously described. patient data was anonymously used in keeping with the latest version of the helsinki declaration of human research ethics. the first antigen was obtained from a commercial source (sigma) and consists of the hmg - coa reductase catalytic domain expressed in e. coli and fused to gst protein with a final molecular weight of 76 kda (including the fusion protein). the hmgcr dna was cloned into the piex / bac-3 vector using homo sapiens hmgcr and transcript variant 1 (nm_000859.2) amino acids 427888. the clone is n - terminal 10x histidine tagged and expressed in sf9 cells with a molecular weight of 51 kda. the cells were grown to 24 10 cells / ml in sf900 ii sfm medium and infected using 20 ml of hmgcr baculovirus per 1 they were incubated at 27c for 108 hrs while rotating at 140 rpm. the cells were harvested by centrifuging at 8000 rpm for 15 minutes using an slc-6000 rotor. the cell pellets were washed with pbs, centrifuged at 4000 rpm and the pellets were stored at 80c prior to extraction. the hmgcr antigen was extracted using 1 m nacl, 20 mm tris, 0.25% chaps, and 10 mm imidazole buffer, ph 8.0. the sonicated mixture was centrifuged at 30,000 rpm for 30 minutes using a 50.2ti rotor. the supernatant was collected and run over a ni++ nta imac column equilibrated on the extraction buffer. the column was washed using 1 m nacl, 20 mm tris, 0.25% chaps, and 140 mm imidazole buffer, ph 8.0 and eluted using 1 m nacl, 20 mm tris, 0.25% chaps, and 400 mm imidazole buffer, ph 8.0. the elution was collected and buffer exchanged using a g25 sec equilibrated using 1 m nacl, 10 mm tris, 0.25% chaps, and 0.09% nan3 buffer, ph 8.0. the hmgcr antigen was quantified using the calculated extinction coefficient and a280 absorbance measured using a uv / visible spectrophotometer and stored at 80c. in - house antigen (lot # alo38) was compared to sigma antigen (hmg - coa reductase h7039 059k4055) via sds page / western blot. both antigens were loaded to a 15-well 412% bis - tris prepact polyacrylamide gel (life technologies, carlsbad, california), at 0.5 g per well. a seeblue plus2 prestained mes ladder (life technologies) was run in lane 1 for molecular weight determination. electrophoresis was performed using a mini blot gel box and mes running buffer (life technologies). proteins were run at 200 volts for 45 minutes using a biorad model 200/2.0 power supply. the remaining samples were then transferred to a nitrocellulose membrane using a life technologies iblot transfer unit. the membrane was then cut into 8 strips with each containing 1 lane of each antigen. strips were then incubated in hrp sample diluent (inova 508551) for 30mins followed by incubation with the appropriate patient samples at a 1 : 100 dilution for 1 hr. the strips were then washed with hrp wash (inova 508552) 4 5 min and incubated with a goat anti - human secondary antibody diluted 1 : 3000 (jackson immuno research) in hrp sample diluent for 1 hr. strips were washed 4 5 min in di water then developed with bcip / nbt (moss, inc.). the quanta flash hmgcr (research use only) assay is a novel cia that is currently used for research purposes only and utilizes the bio - flash instrument (biokit s.a., barcelona, spain), fitted with a luminometer, as well as all the hardware and liquid handling accessories necessary to fully automate the assay. the quanta flash assay for this study prior to use, the reagent pack containing all the necessary assay reagents is gently inverted thirty times and the sealed reagent tubes are then pierced with the reagent pack lid. small amounts of the diluted patient serum, the beads, and the assay buffer are all combined into a second cuvette, mixed, and then incubated for 9.5 minutes at 37c. the magnetized beads are sedimented using a strong magnet in the washing station and washed several times followed by addition of isoluminol conjugated anti - human igg and again incubated for 9.5 minutes at 37c. the isoluminol conjugate is oxidized when sodium hydroxide solution and peroxide solutions (triggers) are added to the cuvette, and the flash of light produced from this reaction is measured as relative light units (rlus) by the bio - flash optical system. the rlus are proportional to the amount of isoluminol conjugate that is bound to the human igg, which is in turn proportional to the amount of anti - hmgcr antibodies bound to the antigen on the beads. a five - point calibration curve was used to convert optical density values into units. the cut - off was defined as the 99% percentile of level in a previous internal study based on disease controls. the titration of anti - hmgcr antibodies was performed using a luminex - based immunoassay, as described elsewhere. briefly, the recombinant human hmgcr catalytic domain was coupled to fluorescent bioplex cooh - microspheres (biorad, hercules, ca) with the bioplex amine coupling kit according to manufacturer 's protocol. a 10 l volume containing 1,250 beads was incubated with patient 's serum, in 96-well plates for 2 h. beads were collected by filtration and washed before adding biotinylated mouse anti - human igg ab. after 1 h incubation and washing, anti - hmgcr ab titers were calculated from the mean fluorescent intensity by comparison with a calibrator consisting in a human positive serum whose titer was arbitrarily set to 100 arbitrary units (a.u / ml). the threshold of positivity of this assay is 20 au / ml. all patients were also tested using assays for the detection of antibodies to extractable nuclear antigens (ena, bmd, and thermo fisher) and myositis related antibodies (scleroderma and myositis profile, d - tek, belgium). autoantibodies were detected by indirect immunofluorescence on hep-2000 cells (reference sa2014-ro, immunoconcepts, sacramento, ca, usa). sera were tested at 1/80 screening dilution in pbs buffer, using a fitc - coupled antibody against human igg (h + l). on these cells, the fluorescence pattern suggestive for anti - hmgcr antibodies is a finely granular cytoplasmic staining on a minority (3% or less) of cells with perinuclear reinforcement. autoantibodies to various peptides were studied using pepperchip technology (pepperprint gmbh, heidelberg, germany) [15, 16 ]. peptide arrays were blocked using blocking buffer (rockland blocking buffer mb-070 (60 min before the first assay). sera were diluted 1 : 1000 in incubation buffer (pbs, ph 7.4 with 0.05% tween 20 and 10% rockland blocking buffer) and incubated for 16 h at 4c and shaking at 500 rpm. arrays were then washed (2 1 min after each assay with washing buffer (pbs, ph 7.4 with 0.05% tween 20). secondary antibody (f(ab')2 goat anti - human igg (h + l) dylight680) diluted 1 : 5000 was added and incubated 30 min. identified epitopes were synthesized as soluble peptides, coated to elisa plates and tested with anti - hmgcr positive samples. by comparing the pepperprint sequence reactivity data to public domain structures of hmgcr the reactive sequences that were likely to be accessible to antibodies were determined to be gympipvgvagpl, 748gynahaanivtai, mattegclvastn, tdkkpaainwieg, clvastnrgcrai, and rgksvvceavipa. peptides were synthesized by biosynthesis (san diego, ca) as biotinylated constructs and tested via streptavidin elisa assay. the data was statistically evaluated using the analyse - it software (version 1.62 ; analyse - it software, ltd., leeds, uk). spearman 's correlation and cohen 's kappa agreement test were carried out to analyze the agreement between portions. as the first step, the inova hmgcr antigen was compared to the sigma antigen using western blot analysis to analyze the size, purity, and the reactivity pattern of both antigens. the western blot shows one anti - hmgcr positive sample for both inova and sigma antigens. using the sigma antigen, in contrast, using the inova antigen, only one distinct band is recognized and stained (~55 kda). next, the antigens were compared by elisa and the results obtained with the 40 samples were highly correlated (= 0.80, see figure 1). subsequently, anti - hmgcr antibodies were detected using albia, elisa, and cia and all three assays showed qualitative agreements of 100% (see figure 2). in addition, the levels of anti - hmgcr antibodies also showed significant correlation : elisa versus albia, = 0.84 (95% confidence interval, 0.720.91), albia versus cia, = 0.89 (95% ci, 0.800.94), and elisa versus cia, = 0.86 (95% ci, 0.750.92). briefly, their mean age was 54.4 years (range from 16 to 84 ; standard deviation 21.1 years) and 16/20 (80.0%) were females. in 18/20 (90.0%) of the patients, a diagnosis of necrotizing myopathy was established. the two remaining patients were not diagnosed at the last clinical follow - up but are highly suspected to suffer from myositis. the mean age at disease onset was 48.5 (sd 20.1 years, range 1283 years). nine out of 20 (45%) anti - hmgcr positive patients were on statin. all patients with anti - hmgcr antibodies were negative for all autoantibodies tested (ss - a, ss - b, sm, rnp, jo-1, scl-70, centromere, mi2, pm / scl, ku, pl-7, pl-12, and srp). testing various controls showed high specificity (99.3%). 3/518 apparently healthy individuals and 3/117 patients with sicca syndrome were positive (see figure 3) ; they all had low titers of anti - hmgcr antibodies. to investigate the staining pattern of anti - hmgcr antibodies a strongly reactive patient sample was used to stain hep-2 cells. on these cells, the fluorescence pattern suggestive for anti - hmgcr antibodies is a finely granular cytoplasmic staining on a minority (3% or less) of hep-2000 cells with perinuclear reinforcement (see figure 4). several potential epitopes were identified using the solid phase peptide arrays (see figure 5). a total of six sequences were selected based on surface exposure (gympipvgvagpl, gynahaanivtai, mattegclvastn, tdkkpaainwieg, clvastnrgcrai, and rgksvvceavipa), synthesized as soluble peptides and tested with positive sera and controls by elisa. reactivity found by solid phase peptide arrays could not be confirmed (data not shown). first described in 2010, anti - hmgcr antibodies represent a promising biomarker to aid in the diagnosis and treatment decision of idiopathic inflammatory necrotizing myopathies (iinm). the present study is the first to compare different methods for the detection of anti - hmgcr antibodies (albia, elisa, and cia), two of them manufactured in a precommercial setting and one based on albia. both, the elisa and the cia, use a 63 kda fragment of hmgcr which has previously been described as the epitope containing region of the antibodies. in addition, we studied the coexistence of anti - hmgcr antibodies with other msa and maa and found that in our patients anti - hmgcr antibodies were the only detectable autoantibody. our data on the association between statin use and anti - hmgcr antibodies confirms previous results. we also confirm that the majority of patients with anti - hmgcr antibodies have iinm. anti - hmgcr antibodies might become available as a single assay on a fully automated analyzer, as elisa and/or as part of multiparameter assays. a recent study showed that the majority of patients with and without statin exposure, including those with self - limited statin intolerance, do not develop anti - hmgcr antibodies. therefore, anti - hmgcr antibodies are highly specific for those with an iim. to further analyze the specificity especially against healthy and diseased controls, we performed a specificity study by elisa. using various disease controls (n = 824), very high specificity (99.3%) of the elisa test. therefore, anti - hmgcr antibodies detected by elisa, especially those moderate and high titers, are highly indicative of iinm. recently, an increasing prevalence of iinm was reported. in about half of the identified patients (45%) the majority of those patients were statin users (67%) compared to 18% in the group with detectable autoantibodies (i.e., jo-1, srp, pm / scl, and ro52). future studies are mandatory to investigate the pathophysiological mechanism of anti - hmgcr antibodies and the root cause for the increasing prevalence iinm. although we could not find any other autoantibody in our patients with anti - hmgcr reactivity, we can not rule out that the patients have autoantibodies that have not been tested in our study (i.e., anti - tif1 families, anti - mda5, anti - nxp2, and so forth). autoantibodies to intracellular antigens (referred to as antinuclear antibodies) are commonly tested using iif on hep-2 cells to aid in the diagnosis of systemic autoimmune diseases including myositis. consequently, we wanted to study if anti - hmgcr antibodies decorate certain structure on hep-2 cells. we found that patients with anti - hmgcr antibodies frequently stain cytoplasmic structures, however, not in all cells. in this context it is important to point out that the sensitivity for antibodies to cytoplasmic antigens (i.e., jo-1 or ribosomal p) is limited. in summary, further studies are needed to (1) confirm the observed staining pattern, to (2) analyze the pattern on slides from different manufacturers and to (3) assess the reliability (sensitivity) of iif hep-2 for the detection of anti - hmgcr antibodies. using solid phase peptide synthesis, we found several peptides reacting with anti - hmgcr antibodies contained in a serum of a patient with statin - associated necrotizing myopathies. in order to confirm the reactivity using a different method, we selected surface exposed epitope sequences for synthesis of soluble peptides. although we were unable to confirm the peptide reactivity using soluble hmgcr derived peptides, it can not be ruled out that anti - hmgcr antibodies bind linear epitopes. however, based on our data it is likely that conformational structures play a role in the formation of the major epitope. further studies are needed to analyze the nature of the epitope on hmgcr and to address the reason for the discrepant results between the two methods for the detection of antibodies to synthetic peptides. different technologies including phage or bacterial display, synthetic peptides, or recombinant proteins might prove useful. anti - hmgcr antibodies are strongly associated with iinm and to a lesser extent with statin use. testing for anti - hmgcr antibodies might prove useful in the diagnosis of iim and in the differentiation between self - limited and persistent statin associated myopathy which requires long - term immunosuppressive treatment. | diagnostic tests are needed to aid in the diagnosis of necrotizing myopathies associated with statin use. this study aimed to compare different technologies for the detection of anti - hmgcr antibodies and analyze the clinical phenotype and autoantibody profile of the patients. twenty samples from myositis patients positive for anti - hmgcr antibodies using a research addressable laser bead assay and 20 negative controls were tested for autoantibodies to hmgcr : quanta lite hmgcr elisa and quanta flash hmgcr cia. all patients were also tested for antibodies to extractable nuclear antigens and myositis related antibodies. to verify the specificity of the elisa, 824 controls were tested. all three assays showed qualitative agreements of 100% and levels of anti - hmgcr antibodies showed significant correlation : spearman 's rho > 0.8. the mean age of the anti - hmgcr antibody positive patients was 54.4 years, 16/20 were females, and 18/20 had necrotizing myopathy (two patients were not diagnosed). nine out of 20 anti - hmgcr positive patients were on statin. all patients with anti - hmgcr antibodies were negative for all other autoantibodies tested. testing various controls showed high specificity (99.3%). anti - hmgcr antibodies are not always associated with the use of statin and appear to be the exclusive autoantibody specificity in patients with statin associated myopathies. |
the prevalence of prostate cancer in korea quadrupled between 2002 and 2008, with the highest increased incidence rate in total forms of malignancy. the incidence of prostate cancer in korea increased up to 24.8 per 100,000 men in 2009 in comparison with 13 per 100,000 men in 2008. certain environmental elements have had an effect on the increased rate of prostate cancer, including the transition to western dietary habits among koreans and an aging population because of the rise in average life expectancy. in addition to environmental causes, the medical development of laboratory diagnoses and prostate - specific antigen screening campaigns by the korean urological association and other health organizations have helped to raise public awareness about the increase in prostate cancer in koreans. despite the rapid increase in prostate cancer incidence in korea, no published multicenter data on practical and clinical changes in korean patients with prostate cancer are available. in the united states, a database application project known as the cancer of the prostate strategic urologic research endeavor (capsure) was initiated in 1995 to establish a web - based database for longitudinal observations of prostate cancer patients in natural settings. the project began with 10 participating healthcare centers and increased to 26 centers in 1 year. currently, capsure is one of the most powerful prospective study groups for prostate cancer in the world consisting of approximately 14,000 registered prostate cancer patients. the most recent database study in asia, the japan study group for prostate cancer (j - cap), was developed in 2001. the j - cap database comprises 17,872 prostate cancer patients from prospective studies and was developed to improve patient care. against this background, the multicenter korean prostate cancer database (k - cap) was created in 2011 by combining several urologic healthcare institutions into a nationwide multicenter database for prospective studies about prostate cancer. the purpose of k - cap is to gather basic information about korean prostate cancer patients and analyze the clinical and oncological outcomes of prostate cancer to improve patient care. the purpose of this article was to declare the establishment of k - cap, to provide urologists with an overview of the k - cap methodology, and to present pilot test results from the first umbrella database comprising patient information from three participating institutions (gangnam severance hospital, seoul st. participating institutions include asan medical center, samsung medical center, seoul national university bundang hospital, seoul st. any institution that wants to participate in k - cap must first obtain approval from their ethics committees (or institutional review board). for every eligible institution, all patients with newly diagnosed prostate cancer will be registered in the k - cap web - based electronic server. all registered patient information will be updated periodically. after a diagnosis of prostate cancer, patients are invited by their urologists to join the study. all patients with biopsy - proven prostate cancer are offered enrollment in the study, regardless of disease stage, severity, or type of treatment. included in the database is all of the physician - gathered general information about a patient, including sociodemographic data. in addition, all of the patient 's clinical information at diagnosis is gathered, including diagnostic imaging, laboratory test results, pre - existing and postdiagnosis comorbidities, medical history, treatment types (e.g., active surveillance, androgen deprivation / hormonal medications, brachytherapy, cryotherapy, external beam radiation, hormone refractory and chemotherapy agents, and radical prostatectomy), neoadjuvant and adjuvant treatments, and other nontreatment - related medications. for patients who have undergone radical prostatectomy, detailed perioperative variables are measured and registered, including procedure types, intraoperative or postsurgical complications, surgical pathology results, and clinical and oncological survival outcomes. the patient files for the k - cap database include approximately 1,000 required clinical variables. the observational study of prostate cancer patients is the ultimate goal of the k - cap database. accordingly, the web - based electronic case report form (e - crf) was developed by using the startrial system, which is a commercially developed electronic data capture system. this e - crf system can be accessed over the internet on a 24-hour basis by coordinators and investigators at eight participating clinics (suppl. the system was developed at the catholic university of korea, south korea, and was previously tested with the help of staff at other participating sites. the database system was implemented with a microsoft sql server running on microsoft nt servers and was programmed with java. this was accomplished by directly partnering the electronic databases of the study 's participating institutions with the registered patient files. all legal requirements for privacy protection were respected in this procedure. according to the privacy rules of k - cap, cases for registered patients are to be updated once every 3 months in terms of test dates, changes in treatment, and progress data as a matter of course for maintaining the k - cap patient forms until death or patient withdrawal. follow - up with respect to morbidity and death from prostate cancer is assured by cooperation with national statistics. when patients die, the date, location, and cause of death are recorded in the database. after completion of the data control for registered patients, analytic reports are prepared for prospective studies and research to improve the care of prostate cancer patients. these data summaries evaluate clinical occurrences, patient quality of life, economic impact, and oncological outcomes and compare types of treatment by stage and practice among patients with prostate cancer in korea. the complex studies use well - sorted variables from the k - cap database, together with adjustment methods including case controls, standardization, regression trees, and multivariate regression approaches for optimal analysis. the entry form contains five categories : patient demographics, clinical parameters, pathological parameters, other treatment parameters (hormone therapy or radiotherapy), and parameters associated with follow - up. access given to an investigator allows extraction of only his or her patient 's data. the data format for data extraction includes csv format, ms excel (xls format), or ms access (accdb format). the system manager is able to select data by accessing the sql server system directly using the sql program (suppl. 2). to ensure the quality of the database, it is most important that accurate data be provided by the investigators. however, if a strange code is entered into the database, the database system automatically checks the input code and will not process the input of the strange data set. investigators are also able to compare their own raw data with data in the k - cap database at anytime, because each institution 's data can be extracted if the investigator has access rights. a pilot test was performed to determine whether to use the standard e - crf system based on the limited perioperative information of prostate cancer patients who underwent radical prostatectomy. between january 2006 and december 2010, 858 prostate cancer patients who underwent radical prostatectomy at three institutions (gangnam severance hospital, seoul st. mary 's hospital, or seoul national university bundang hospital) were registered in the e - crf system of k - cap. the database collected preoperative variables, including patient age, prostate - specific antigen level at diagnosis, preoperative gleason score, clinical stage, and the initial procedure chosen for radical prostatectomy. postoperative variables were also registered, including pathologic gleason score and stage. for each patient, the t stage was determined in accordance with tumor - node - metastasis (tnm) categories, as published in 2010 (american joint committee on cancer, 7th edition). all of the pathologic parameters that influence the determination of tnm staging were registered in detail. the first step was to gather the prostate cancer database files from each institution using the excel format and to send these files to an e - crf system manager controlling the data input / output of the web - based database. subsequently, the manager converted the coded excel files for use in the e - crf system. finally, we were able to obtain outcomes from the multicenter e - crf database according to several conditions that we initially established to fit the goals of k - cap. participating institutions include asan medical center, samsung medical center, seoul national university bundang hospital, seoul st. any institution that wants to participate in k - cap must first obtain approval from their ethics committees (or institutional review board). for every eligible institution, all patients with newly diagnosed prostate cancer will be registered in the k - cap web - based electronic server. participating institutions include asan medical center, samsung medical center, seoul national university bundang hospital, seoul st. eligible institutions include all urologic institutions in korea. any institution that wants to participate in k - cap for every eligible institution, all patients with newly diagnosed prostate cancer will be registered in the k - cap web - based electronic server. after a diagnosis of prostate cancer, patients are invited by their urologists to join the study. all patients with biopsy - proven prostate cancer are offered enrollment in the study, regardless of disease stage, severity, or type of treatment. included in the database is all of the physician - gathered general information about a patient, including sociodemographic data. in addition, all of the patient 's clinical information at diagnosis is gathered, including diagnostic imaging, laboratory test results, pre - existing and postdiagnosis comorbidities, medical history, treatment types (e.g., active surveillance, androgen deprivation / hormonal medications, brachytherapy, cryotherapy, external beam radiation, hormone refractory and chemotherapy agents, and radical prostatectomy), neoadjuvant and adjuvant treatments, and other nontreatment - related medications. for patients who have undergone radical prostatectomy, detailed perioperative variables are measured and registered, including procedure types, intraoperative or postsurgical complications, surgical pathology results, and clinical and oncological survival outcomes. the patient files for the k - cap database include approximately 1,000 required clinical variables. the observational study of prostate cancer patients is the ultimate goal of the k - cap database. accordingly, the web - based electronic case report form (e - crf) was developed by using the startrial system, which is a commercially developed electronic data capture system. this e - crf system can be accessed over the internet on a 24-hour basis by coordinators and investigators at eight participating clinics (suppl. the system was developed at the catholic university of korea, south korea, and was previously tested with the help of staff at other participating sites. the database system was implemented with a microsoft sql server running on microsoft nt servers and was programmed with java. this was accomplished by directly partnering the electronic databases of the study 's participating institutions with the registered patient files. all legal requirements for privacy protection were respected in this procedure. according to the privacy rules of k - cap, cases for registered patients are to be updated once every 3 months in terms of test dates, changes in treatment, and progress data as a matter of course for maintaining the k - cap patient forms until death or patient withdrawal. follow - up with respect to morbidity and death from prostate cancer is assured by cooperation with national statistics. when patients die, the date, location, and cause of death are recorded in the database. after completion of the data control for registered patients, analytic reports are prepared for prospective studies and research to improve the care of prostate cancer patients. these data summaries evaluate clinical occurrences, patient quality of life, economic impact, and oncological outcomes and compare types of treatment by stage and practice among patients with prostate cancer in korea. the complex studies use well - sorted variables from the k - cap database, together with adjustment methods including case controls, standardization, regression trees, and multivariate regression approaches for optimal analysis. the entry form contains five categories : patient demographics, clinical parameters, pathological parameters, other treatment parameters (hormone therapy or radiotherapy), and parameters associated with follow - up. access given to an investigator allows extraction of only his or her patient 's data. the data format for data extraction includes csv format, ms excel (xls format), or ms access (accdb format). the system manager is able to select data by accessing the sql server system directly using the sql program (suppl. 2). to ensure the quality of the database, it is most important that accurate data be provided by the investigators. however, if a strange code is entered into the database, the database system automatically checks the input code and will not process the input of the strange data set. investigators are also able to compare their own raw data with data in the k - cap database at anytime, because each institution 's data can be extracted if the investigator has access rights. after a diagnosis of prostate cancer, patients are invited by their urologists to join the study. all patients with biopsy - proven prostate cancer are offered enrollment in the study, regardless of disease stage, severity, or type of treatment. included in the database is all of the physician - gathered general information about a patient, including sociodemographic data. in addition, all of the patient 's clinical information at diagnosis is gathered, including diagnostic imaging, laboratory test results, pre - existing and postdiagnosis comorbidities, medical history, treatment types (e.g., active surveillance, androgen deprivation / hormonal medications, brachytherapy, cryotherapy, external beam radiation, hormone refractory and chemotherapy agents, and radical prostatectomy), neoadjuvant and adjuvant treatments, and other nontreatment - related medications. for patients who have undergone radical prostatectomy, detailed perioperative variables are measured and registered, including procedure types, intraoperative or postsurgical complications, surgical pathology results, and clinical and oncological survival outcomes. the patient files for the k - cap database include approximately 1,000 required clinical variables. the observational study of prostate cancer patients is the ultimate goal of the k - cap database. accordingly, the web - based electronic case report form (e - crf) was developed by using the startrial system, which is a commercially developed electronic data capture system. this e - crf system can be accessed over the internet on a 24-hour basis by coordinators and investigators at eight participating clinics (suppl. the system was developed at the catholic university of korea, south korea, and was previously tested with the help of staff at other participating sites. the database system was implemented with a microsoft sql server running on microsoft nt servers and was programmed with java. this was accomplished by directly partnering the electronic databases of the study 's participating institutions with the registered patient files. according to the privacy rules of k - cap, cases for registered patients are to be updated once every 3 months in terms of test dates, changes in treatment, and progress data as a matter of course for maintaining the k - cap patient forms until death or patient withdrawal. follow - up with respect to morbidity and death from prostate cancer is assured by cooperation with national statistics. when patients die, the date, location, and cause of death are recorded in the database. after completion of the data control for registered patients, analytic reports are prepared for prospective studies and research to improve the care of prostate cancer patients. these data summaries evaluate clinical occurrences, patient quality of life, economic impact, and oncological outcomes and compare types of treatment by stage and practice among patients with prostate cancer in korea. the complex studies use well - sorted variables from the k - cap database, together with adjustment methods including case controls, standardization, regression trees, and multivariate regression approaches for optimal analysis. the entry form contains five categories : patient demographics, clinical parameters, pathological parameters, other treatment parameters (hormone therapy or radiotherapy), and parameters associated with follow - up. access given to an investigator allows extraction of only his or her patient 's data. the data format for data extraction includes csv format, ms excel (xls format), or ms access (accdb format). the system manager is able to select data by accessing the sql server system directly using the sql program (suppl. to ensure the quality of the database, it is most important that accurate data be provided by the investigators. however, if a strange code is entered into the database, the database system automatically checks the input code and will not process the input of the strange data set. investigators are also able to compare their own raw data with data in the k - cap database at anytime, because each institution 's data can be extracted if the investigator has access rights. a pilot test was performed to determine whether to use the standard e - crf system based on the limited perioperative information of prostate cancer patients who underwent radical prostatectomy. between january 2006 and december 2010, 858 prostate cancer patients who underwent radical prostatectomy at three institutions (gangnam severance hospital, seoul st. mary 's hospital, or seoul national university bundang hospital) were registered in the e - crf system of k - cap. the database collected preoperative variables, including patient age, prostate - specific antigen level at diagnosis, preoperative gleason score, clinical stage, and the initial procedure chosen for radical prostatectomy. postoperative variables were also registered, including pathologic gleason score and stage. for each patient, the t stage was determined in accordance with tumor - node - metastasis (tnm) categories, as published in 2010 (american joint committee on cancer, 7th edition). all of the pathologic parameters that influence the determination of tnm staging were registered in detail. the first step was to gather the prostate cancer database files from each institution using the excel format and to send these files to an e - crf system manager controlling the data input / output of the web - based database. subsequently, the manager converted the coded excel files for use in the e - crf system. finally, we were able to obtain outcomes from the multicenter e - crf database according to several conditions that we initially established to fit the goals of k - cap the multicenter k - cap test database, which contains data for a total of 858 prostate cancer patients who underwent radical prostatectomy at three institutions, represents a single source for comprehensive information about the patients and the disease. a java program was developed to upload excel data into the e - crf database by matching each column name. if the uploaded excel file executes the java program, the data is automatically entered into the designated column. the first step is to compare the number of records between the uploaded excel file and the sql server database. the second step is to transform data from the sql server into excel format and then compare it with the original data set. we confirmed that the process of converting data to the e - crf system from excel files is easy and exact and that output data from the web - based database system are quickly retrievable according to many weighted limitations. complete treatment histories and patient information allow for comparison of different outcomes. as the number of patients enrolled in the database increases, the database will help to categorize patients and identify historical controls for various purposes. in existing research about prostate cancer, patients have been selected with indiscriminate consideration of treatment type or stage, regardless of the varying aims of specific studies. for example, the life expectancy of korean prostate cancer patients with multiple bone metastases is not yet known. in addition, we do not yet know the exact clinical course of korean prostate cancer patients after treatment. even though we reviewed the results of many studies and made conclusions about disease progression [7 - 12 ], the results were from single - institution cohort studies with small sample sizes. furthermore, despite the many studies of prostate cancer in koreans, most of our knowledge is still based on studies from a western database of prostate cancer. thus, we needed a prostate cancer database of our own, and we believe that k - cap is that database. however, under a system of longitudinal observation, the collection of visible results for analysis will require more time than is required for studies focusing on retrospective design. we expect that at least 5 years will be required to obtain tangible and qualitative results from the k - cap database. if the initial results are positive, however, lasting products will exist in the database on an ongoing basis. in the case of capsure, the first published results about trends in prostate cancer treatment (2003) were published 7 years after initial development of the capsure project. currently, a pubmed search with the term " capsure " delivers more than 100 relevant articles, including articles on the j - cap project in japan. achievement of this long - term plan requires verification of the web - based database system as the first step toward the establishment of k - cap. the pilot test with prostate cancer patient databases from three institutions showed the feasibility of a web - based system for k - cap. the important checkpoint is that the results of the pilot test are not intended to be used to make analytical conclusions about the specific subjects from the three institutions. the intent of the pilot test was to ensure that the k - cap database system was operating accurately before other participating institutions provided proprietary data. as this article goes to press, patient data from the remaining institutions in the korean healthcare system are being registered. we estimate that approximately 4,000 cases from participating institutions will be registered as the basic background data for the k - cap database (620 patients from asan medical center, j.h.h., c.s.k. ; 1,400 patients from samsung medical center, h.m.l. ; 340 patients from seoul national university bundang hospital, s.k.h. the patients with newly diagnosed prostate cancer from the participating institutions will be registered, including the required entry information for k - cap. according to the patients ' privacy agreement, basic information about the patients will be encrypted by using a program unique to the k - cap database and will remain unknown to our team and even to the database manager. thus, the privacy of the patients registered in k - cap is guaranteed. developing a uniform format for the k - cap database was one of the main concerns of the project staff at each of the participating institutions. for example, because of different policies at each institution, differences in preoperative prostate volume measurements (by transrectal ultrasonography) existed between the participating institutions. some institutions measured prostate volume preoperatively in all patients, but the procedure was performed at different clinical stages at some institutions. furthermore, each institution had different postoperative follow - up periods over which to conduct patient orientation surveys, such as the international index of erectile function questionnaire. in addition to policy differences between the institutions, internal problems concerning a uniform database format also existed. some pathologists reported the precise weights of the tumors in grams, whereas others reported approximate volumes using relative measurements (e.g., v1, v2, and v3). importantly, we previously identified several problems, such as those mentioned above, and have discussed solutions intended to achieve a uniform format for the database. we will continue to revise and develop the k - cap format in an effort to provide the flexibility needed to achieve the long - term goals of the database. another important consideration in the management of the k - cap database is how best to maintain continuous updates about patients. it is difficult to predict how many new prostate cancer patients from each participating institution will be enrolled in k - cap. likewise, it is difficult to update k - cap data periodically if the number of enrolled patients is too large, even before the k - cap settings are ready. however, it is well known that the design of a longitudinal observational study ensures that the quality of the data is more important than the quantity of the data. to solve this problem, we enrolled a limited number of prostate cancer patients representative of a particular institution rather than all of the prostate cancer patients at that institution. after determining the appropriate k - cap settings through trial and error, we intend to enroll all prostate cancer patients in the k - cap database and to include patients from any institution that wants to participate in k - cap. one last consideration is the importance of k - cap 's collaboration with capsure and j - cap. k - cap is intended to be used not only for domestic investigation but also for international participation and collaboration. even though k - cap is still in the preparatory stage, we have already received valuable advice and support from the working staff and chairmen of capsure and j - cap. collaboration will best be achieved if the format of the k - cap database is analogous to that of the other databases. the k - cap database was developed considering the original excel database of capsure and j - cap. the capsure database has many variables, for which each treatment of prostate cancer was equally taken into account. this probably reflects the different trends in prostate cancer treatment between the united states and japan. the ability to reference these different databases will help to enrich the k - cap database, so that our database can cover many fields. in this article, we announced the establishment of k - cap on behalf of all the participants in the k - cap project. the main purpose of k - cap is to establish and maintain a large database of randomized clinical trials and observational longitudinal studies of korean prostate cancer patients. several retrospective studies about prostate cancer in korea have been published in domestic and international journals. we believe that mutual cooperation is needed to establish the k - cap project as being equal to the capsure and j - cap projects in the united states and japan, respectively. although our beginnings have been humble, we are confident that our dedication and ongoing efforts will result in k - cap becoming a world - renowned longitudinal observation database, along with capsure and j - cap. this article announces the development and establishment of k - cap as the first database for comprehensive data collection about prostate cancer patients in korea for the purposes of research and improved patient care. this study tested the web - based system of the k - cap database to analyze coded excel files from three institutions. the system operated precisely, and the pilot test verified that the web - based database system is suitable for k - cap. the system processes will run successfully as long as sufficient and updated data is continuously provided to the system manager. as soon as possible, complete statistical results of registered prostate cancer patients will be reported for basic background data. | purposethe purpose of this article was to announce the establishment of the multicenter korean prostate cancer database (k - cap) and to provide urologists with details about k - cap 's methodology.materials and methodsthe initial participating k - cap institutions include five medical centers in korea. first, we registered prostate cancer patients who underwent radical prostatectomy as the basic background data. k - cap is poised to combine these initial observational longitudinal studies with those of other eligible institutions as the database grows. all current prostate cancer patients in korea are able to be registered into the web - based database system and thereby have a role in several observational studies. the structure of the database for k - cap was developed by matching it with the respective data from different studies. the operability of the k - cap database system was verified by using the existing databases from three participating institutions.resultsthe analysis of clinicopathologic characteristics of patients with the use of the web - based database was successfully conducted. we confirmed the accurate operation of the web - based database system without any difficulties.conclusionswe are announcing the establishment of k - cap the first database of comprehensive observational longitudinal studies about prostate cancer in korea. the database will be successfully maintained by sufficiently and continuously updating all patient data covering several treatments. complete statistical results for registered prostate cancer patients are forthcoming for the basic background data to establish the database. even though much trial and error are expected during the development process, we expect that k - cap will eventually become one of the most powerful longitudinal observation databases. |
posterior vitreous detachment (pvd) is defined as the separation of the posterior hyaloid from the internal limiting membrane. it is age - related and becomes noticeable after the sixth decade of life (up to 63% prevalence) and is related to synchysis senilis. vitreous separation can cause visual symptoms such as photopsia from vitreoretinal traction and floaters resulting from the presence of condensed vitreous collagen. about 30% of patients have floaters after development of pvd ; however, most patients tolerate their symptoms [1, 2, 4 ]. a minority of patients report that these floaters are troublesome, in particular young myopic patients and those whose work requires detailed visual tasks. pseudophakic patients frequently report floaters, which may be explained by the improved postoperative contrast sensitivity related to the intraocular monofocal lens, which increases the perception of floaters in the visual field [2, 4 ]. pvd incidence is also increased after cataract surgery by phacoemulsification with implantation of a posterior chamber intraocular lens (iol). multifocal iols were designed to reduce the need for spectacles by providing two or more points of focus. the apodized diffractive multifocal acrysof restor iol (alcon, fort worth, tx) was designed specifically to reduce glare and halos and provide increased dominant distance vision for patients with large pupils. this lens has a central 3.6 mm apodized optic area with 12 concentric diffractive zones on the anterior surface for gradual reduction of the diffractive increments from the center to the periphery. rayner m - flex multifocal iols (rayner, london, uk) are based on multizoned refractive aspheric optic technology, with either four or five annular zones (depending on the iol base power [7, 8 ]). the multifocal iol design with concentric rings of optical zones creates positive dysphotopsias, also called photic phenomena. researchers believe the brain adjusts to the altered visual input over time through neural adaptation. to experience the full visual benefits of multifocal iols, most patients require a neuroadaptation period of about 6 months [10, 11 ]. in the present study, the authors investigated the role of sutureless pars plana vitrectomy treatment in patients with symptomatic pvd in terms of visual acuity (va) and visual satisfaction (measured through a standardized questionnaire) in patients who previously underwent bilateral implantation of the restor + 3 (alcon, fort worth, tx) or the m - flex iols (rayner, london, uk). this prospective case series included patients who were not satisfied with their va and complained of halos, glare, and floaters for at least 6 months after having undergone bilateral phacoemulsification using a 2.2 mm microincision technique and implantation of restor + 3 (alcon, fort worth, tx) or m - flex iols (rayner, london, uk). patients underwent a complete preoperative ophthalmologic examination including evaluation of refractive status, measurement of far uncorrected va (ucva) and best - corrected va (bcva), slit - lamp examination, goldmann applanation tonometry, and indirect ophthalmoscopy. ultrasonography, automated visual field measurement using the humphrey 750i visual field analyzer (zeiss, germany), and optical coherence tomography (oct, spectralis oct, heidelberg, germany) were also performed, as well as a corneal aberrometry measurement (galilei dual scheimpflug analyzer, ziemer ophthalmology, switzerland). the national eye institute visual function questionnaire 25 (nei vfq-25) all eyes were submitted to yag - laser capsulotomy (alcon, usa) at distinct periods after cataract surgery. patients were informed about the possibility of being submitted to a sutureless ppv surgery to clear media opacities to obtain better focus of the multifocal iol on the retina. the study protocol was approved by the ethics committees of the federal university of so paulo. patients were evaluated on postoperative days 1 and 7 and at 1, 3, and 6 months postoperatively. patients were included in the study if they were previously submitted to cataract surgery with multifocal iol implantation and had been diagnosed with symptomatic pvd of at least 6 months of duration. symptomatic pvd was defined as pvd detected by indirect ophthalmoscopy and fundus biomicroscopy revealing vitreous debris (which could include a weiss ring but that was not mandatory) in a patient with pvd - related visual symptoms (floaters and/or photopsias). additionally, ultrasonography had to disclose pvd in the axial, temporal, nasal, inferior, and superior planes. the refractive error based on objective and subjective dynamic refraction could not exceed 0.25 of spherical and no cylindric refractive errors. patients also had to have aberrometry showing a root mean square (rms) of less than 1.2 in both eyes. in addition, macular / retinal diseases had to be ruled out by spectral - domain oct, fluorescein angiography, and automated visual fields (patients excluded if mean deviation values were not between + 1.00 and 7.00 decibels). exclusion criteria were diabetes mellitus, age less than 45 years, a previous stroke or neurosurgical procedure, glaucoma or uveitis, previous ocular surgery other than cataract and multifocal iol implantation, intraoperative complications during a previous phacoemulsification such as capsular tears, previous corneal diseases or scars, irregular corneal astigmatism, iris abnormalities, macular degeneration, neuroophthalmic disease, and postoperative complications such as macular edema and retinal detachment. four - port ppv using three 23-gauge valved trocars (dorc, amsterdam, netherlands) was performed. a tornambe (synergetics, missouri, usa) chandelier light pipe connected to a photon ii light source the surgical procedure was performed using the stellaris pc vitrectomy system (bausch & lomb, usa) and a standard 23-gauge vitrectomy probe and the binocular indirect ophthalmomicroscope (biom) (oculus, germany) for visualization of the vitreous cavity. a core vitrectomy was performed using 5,000 cuts / minute and an aspiration rate of 200 mmhg for approximately 5 minutes followed by central posterior capsulectomy. in all cases, a flush of 0,3 ml of triamcinolone acetonide 4 mg / ml (ophthalmos, brazil) was used to confirm the posterior hyaloid removal ; if still present, it was detached by direct aspiration with the vitrectomy probe with a vacuum rate set at 500 mmhg. scleral indentation was performed and aspiration was reset at 200 mmhg for complete removal of the vitreous base and identification of possible retinal breaks. in bilateral cases, the questionnaire of vision quality (nei vfq-25) was applied before and after ppv, in a 1-month interval. nei vfq-25 includes 25 questions that measure different components of visual function, with six additional optional items that enhance the reliability of the near and distance activity subscales. these were included in the minimally classic / occult trial of the anti - vegf antibody ranibizumab in the treatment of neovascular amd (age - related macular degeneration) study and anti - vegf antibody for the treatment of predominantly classic choroidal neovascularization in amd clinical trial. the scores ranged from 0 (worst vision) to 100 (best, perfect vision - related function). there are 12 subscales : one general health subscale and 11 vision subscales, including general vision, difficulties related to near and distance vision activities, difficulties related to driving, vision - specific dependency, social functioning, role difficulties, limitations in peripheral and color vision, ocular pain, and mental health issues related to vision. the overall composite score was calculated using the mean of the subscales, excluding the general health subscale. a portuguese version of the questionnaire was applied and reliability was assessed using cronbach 's alpha coefficient, intraclass correlation coefficient, and interrater reliability coefficient. to estimate the power of the test, we considered simple random sampling and normal distribution for the vfq-25 subscale scores. given these assumptions, the estimated power of the test was between 50 and 90%, depending on the subscale. the preoperative and postoperative nei vfq-25 scores and ucva using logarithm of the minimum angle of resolution (logmar) charts were compared using the nonparametric wilcoxon test. the analysis was performed using spss software v. 21.0 (ibm, new york, usa). sixteen eyes of eight patients were included in the analysis and all patients had improvement in ucva after vitrectomy. all studied eyes were submitted to previous yag - laser capsulotomy during the 6-month evaluation before the vitrectomy. all of the refraction values ranged from 0.25 to + 0.25 diopters with no astigmatism preoperatively and did not change after ppv ; hence, there were no differences between ucva and bcva values. table 2 shows the changes in the postoperative vfq-25 scores on its subscales and the comparison between the preoperative and postoperative scores of the vfq-25. va significantly improved from a median value of 0.17 preoperatively to 0.09 postoperatively (p = 0.017) (tables 1 and 2). all patients with symptomatic pvd confirmed by ultrasonography (figure 1) who underwent ppv reported not only improvement of va but also improvement regarding halos, glare, and floaters (each of these symptoms was reported preoperatively by all patients). the following postoperative nei vfq-25 subscales median scores significantly improved : general vision (from 60 preoperatively to 80 postoperatively, p = 0.023), near activities (from 75 to 92, p = 0.043), distance activities (from 67 to 100, p = 0.041), mental health (from 75 to 94, p = 0.011), role difficulties (from 88 to 100, p = 0.042), and driving (from 67 to 92, p = 0.016) (table 2 and figure 2). at 6-month follow - up, there were no complications reported ; specifically, no cases of rhegmatogenous retinal detachment and/or macular edema occurred in our series. vitreous floaters are common symptoms that are classically treated by observation only. floaters may even be considered physiologic and age - related. however, they can be inconvenient and decrease the va and visual quality of many patients. these symptoms may be exacerbated by multifocal iols, since both pvd and these lenses are known to be responsible for increasing light scattering in ocular media [15, 16 ]. we hypothesize that symptomatic pvd associated light scattering [1, 18, 19 ] enhances this phenomenon by diffracting light rays in many directions thus worsening va and quality of vision (figure 3(a)). when there are no media opacities (such as after ppv), both near and far light rays can reach the retina at the same point of focus (figure 3(b)). it has a well - known risk profile and, justifiably, there is reluctance to perform this surgery to treat floaters. however, technological advances in both instrumentation and techniques have made ppv a much safer procedure [20, 21 ]. small - gauge vitrectomy offers the advantages of minimal invasiveness, reduced postoperative inflammation and complications, and faster recovery. plenty of evidence suggests that quality of life is improved in successfully operated patients [2225 ]. the real issue remains, that is, the safety of this procedure [20, 26 ]. induction of pvd during surgery is a major risk factor for the development of postoperative rhegmatogenous retinal detachment. in the current small series, we tried to include only eyes with complete pvd to reduce the risk of retinal detachment. however, even with preoperative ultrasonographic examination, 12.5% of eyes in our series still required intraoperative induction of pvd. however, we should remain cautious given the small sample size and the relatively short follow - up. the authors think that the inclusion of only cases of bilateral disease was coincidental, or there is also the possibility that these patients have more intense symptoms related to pvd. the results of the current study demonstrate the benefits of ppv in specific eyes with a multifocal iol and symptomatic pvd. a statistically significant (p = 0.017) improvement in ucva (tables 1 and 2) was observed following ppv. the authors chose to report ucva instead of the most common bcva levels because usually patients with a multifocal iol are expected to have a satisfactory good visual acuity without the need to use glasses or contact lenses ; also, all patients presented no significant refractive error. all patients reported improvement regarding halos, glare, and floaters. to evaluate quality of vision, we compared the results of the vfq-25 before and after vitrectomy in eyes that underwent previous bilateral multifocal iol implantation (table 2). the results showed significantly (p < 0.05 for all comparisons) improved postoperative vfq-25 scores in all subscales tested (near and distance activities, mental health, role difficulty, and driving) (table 2). a comparison between the vfq-25 results in the pre- and postoperative periods at only 1-month interval after vitrectomy was performed in order to avoid the fact that binocular neurosensory adaptation could play a role in vision improvement ; additionally at least 6 months was used as inclusion criteria since the initial symptoms, after the cataract surgery, until the indication of vitreoretinal surgery. some limitations of the current study must be addressed : first, the small number of patients enrolled, the short follow - up period, and absence of control subjects, since this was a pilot investigation ; second, the lack of contrast sensitivity data, which was not included since the main objective was to observe the response of patients to surgery in terms of visual acuity and quality of life (as measured by the vfq-25) ; third, the inclusion of posterior capsulotomy / capsulectomy in all eyes (capsulotomy at the preoperative period using the yag - laser and capsulectomy during ppv). additionally, we hypothesized that residual capsular fragments or residual vitreous present at the posterior surface of the multifocal lens could also be related to these symptoms reported. finally, the explanation of lack of wavefront analysis is based on the argument that we believe the clinical exam, the absence of astigmatism, and also the rms value limit used (less than 1.2) are enough evidences to exclude that possible corneal problems, could be the cause of decrease in quality of vision. despite these, our preliminary results are exciting. to the best of our knowledge, this is the first study to address the importance of the posterior vitreous surgery in va and quality of vision in eyes implanted with bilateral multifocal iols. on the basis of these preliminary data, it is reasonable to conclude that the status of the vitreous is important in patients who may undergo multifocal iol implantation. preoperative evaluation of candidates for these intraocular lenses implantations should include an assessment of the vitreous. patients with symptomatic pvd who are not satisfied with their va and quality of vision may benefit from small - gauge ppv in specific situations reported. however, additional clinical trials with large series of patients performed by different surgeons are necessary to confirm these preliminary observations. | purpose. to determine the efficacy of 23-gauge pars plana vitrectomy (ppv) for symptomatic posterior vitreous detachment (pvd) on visual acuity (va) and quality after multifocal intraocular lenses (iols). methods. in this prospective case series, patients who developed symptomatic pvd and were not satisfied with visual quality due to floaters and halos after multifocal iol implantation underwent ppv. examinations included logmar uncorrected visual acuity (ucva), intraocular pressure, biomicroscopy, and indirect ophthalmoscopy at baseline and 1, 7, 30, and 180 days postoperatively. ultrasonography and aberrometry were performed. the visual functioning questionnaire 25 (vfq-25) was administered preoperatively and at 30 days postoperatively. both the postoperative ucva and questionnaire results were compared to preoperative findings using the wilcoxon test. results. sixteen eyes of 8 patients were included. va significantly improved from 0.17 to 0.09 postoperatively (p = 0.017). all patients reported improvement of halos, glare, and floaters. vfq-25 scores significantly improved in general vision (p = 0.023), near activities (p = 0.043), distance activities (p = 0.041), mental health (p = 0.011), role difficulties (p = 0.042), and driving (p = 0.016). conclusion. ppv may increase ucva and quality of vision in patients with bilateral multifocal iols and symptomatic pvd. larger studies are advised. |
as result of improved surgical techniques and newer immunosuppressive regimens contributing significantly to better graft survival, exocrine pancreas transplantation remains the standard treatment of choice for patients with diabetes mellitus complicated by end - stage renal disease. histological assessment continues to play an important role in the diagnosis of graft complications after pancreas transplantation, especially for evaluating allograft rejection where histopathology is still considered the gold standard. a review elaborates on the current types of pancreas transplants and focuses on the patterns of allograft injury that are encountered in posttransplantation pancreas biopsies along with the pertinent differential diagnoses. in addition to optimal histological assessment, as in any other organ transplant setting, clinical information including indication and duration of transplant as well as other serologic work - up must be taken into consideration during clinical decision making for optimal graft outcome. several lines of laboratory and clinical evidence suggest that in a carefully selected group of patients, long - term glycemic control and allograft function are similar to that observed for pancreas transplants performed for type 1 diabetes. the first successful pancreas transplantation in conjunction with a simultaneous kidney transplantation (spkt) was performed by kelly and lillehei from the university of minnesota in 1966. one of the alternative options to spkt for type i diabetics with renal failure is sequential transplant of a living donor kidney followed by a deceased donor pancreas transplant (pancreas after living donor kidney transplant, palk). adults (age 18 - 59 years) with type i diabetes who were waitlisted for kidney - pancreas and received a spkt or palk between 2000 and 2007 were now studied. of 11,966 patients who received a kidney transplant, 807 received a palk and 5580 received a spkt. median time to pancreas from kidney transplant was 336 (25 - 75 % : 185 - 602 days) days. average hospital stay for spkt recipients was 13 15 days, whereas for palk recipients was 6 4 days and 10 8 days for kidney and pancreas transplants, respectively. after controlling for confounding factors, patients receiving palk had better patient survival (hr 0.52 ; 95 % confidence interval 0.39 to 0.70) and kidney survival (hr 0.48 ; 95 % confidence interval 0.39 to 0.60) but worse pancreas survival (hr 1.37 ; 95 % confidence interval 1.16 to 1.62) compared with spkt. thus, among those who were waitlisted for a kidney - pancreas transplant, 53 percent received a kidney - pancreas transplant. of those who received a kidney - pancreas transplant, 87 percent patients underwent spkt and 13 percent underwent palk. it was found an inferior pancreas graft survival and longer total transplant hospitalization in palk. pancreas transplantation provides the best glycemic control option for diabetes mellitus but is associated with significant morbidities related to infectious disease. it was performed a retrospective study of a cohort of consecutive pancreas transplantation recipients in whom pancreas transplantation was performed from 1998 to 2006 (n=216) and followed up them until 2008. data regarding infections, rejection, infection chemoprophylaxis, graft failure, absolute lymphocyte counts, and mortalities were collected. simultaneous pancreas and kidney, pancreas transplantation alone, and pancreas after kidney (pak) transplantations were performed in 42, 67, and 107 patients, with a mean age at transplantation of 47, 41, and 44 years. of the simultaneous pancreas and kidney, pancreas transplantation alone, and pak transplant recipients, 55 percent, 37 percent, and 59 percent were men. overall, 63 percent developed a serious infection during the median follow - up of 6.4 years. mean (range) number of infectious episodes was 2.3 (1 - 12), with mostly bacterial infections both within (68 %) and after 1 year (78 %). incidence of bacterial and viral infections was greatest in the first 3 months after transplantation. bladder exocrine drainage was significantly associated with higher risk of infection (hazard ratio 2.5). infection within the first 3 months after transplantation was related to higher mortality after the first 3 months (hazard ratio 3.19). absolute lymphocyte counts was significantly associated with the risk of first infections and bacterial infections. therefore, the incidence of infections after pancreatic transplantation was 63 percent and mostly bacterial. | a pancreas transplant is a surgical procedure to place a healthy pancreas from a donor into a patient whose pancreas no longer functions properly. exocrine pancreas transplantation remains the standard treatment of choice for patients with diabetes mellitus complicated by end - stage renal disease. the use of pancreas transplantation for type 2 diabetes mellitus is an emerging concept. a pancreas transplant is often done in conjunction with a kidney transplant. even if pancreas transplantation provides the best glycemic control option for diabetes mellitus, it is associated with significant morbidities related to infectious disease. the present article provides with a review of pancreatic transplantation. |
a 71-year - old man visited us with a chief complaint of a 1-year history of a mass in the right scrotum. the patient had a 15-year - history of stroke and hypertension, for which he was receiving medical treatment. the patient also had an 8-year - history of hypertensive end - stage renal disease, for which he was receiving hemodialysis three times a week. on the physical examination at the time of admission, a hard, painless mass was palpated in the right scrotum that could not be differentiated from the testis. on hematology, tumor markers such as alpha - fetoprotein, beta - human chorionic gonadotropin, and lactate dehydrogenase were all found to be normal. scrotal ultrasonography showed a giant mass of 12 cm in size in the right scrotum. abdominal computed tomography scans showed a solid, contrast - enhanced mass in the right scrotum. we suspected that a tumor had developed in the scrotum and made an incision in the right scrotum with the patient under spinal anesthesia. we identified a tumor that was definitely separated from the right testicle and excised it. the removed tumor was 13106 cm in size and had an oval shape ; it was encapsulated and its cross - section was composed of white mucoid components. the sporadic presence of a solid part was confirmed, but there was no concurrent necrosis or bleeding inside. small and medium - sized blood vessels were shown under light microscopy. in the adjacent area, two months postoperatively, the patient is currently being followed up in an outpatient setting and has had no local recurrence. embryologically, various tissues such as the epithelium, mesothelium, and other tissues are derived from the mesoderm in the scrotum. for this reason, these tumors have different biological characteristics, for which a differential diagnosis between benign and malignant tumors and an accurate diagnosis can not be easily made. of the tumors occurring in the scrotum, angiomyofibroblastoma was first described by fletcher in 1992, which is a tumor that occurs rarely in the reproductive system of middle - aged women. it particularly occurs in the vulva, labium major, vagina, areas around the clitoris, and perineal region. despite a lower rate of local recurrence, it has histopathological characteristics that are similar to those of cellular angiofibroma and infiltrative angiomyxoma. histopathologically, there is a mixed presence of hypocellular or multi - cellular regions and the cellular morphology is diverse. besides, there is also a mixed presence of spindle - shaped cells, satellite cells, epithelioid cells, and locally polygonal cells. these cells are arranged around the blood vessels or epithelioid - shaped regions and are accompanied by the clear presence of myofibroblastic differentiation. eight cases have been reported in the vulva of female patients in korea. to date, however, no cases have been reported in male patients. compared with the angiomyofibroblastoma - like tumors that occur in women, those occurring in male patients are seen in elderly people and have immunohistochemical differences. histopathologically, the cellular matrix of an angiomyofibroblastoma - like tumor is more dense and is abundant with collagen. immunohistochemically, in more than half of the reported cases, tumor cells are positive for cd34 and are various for desmin. however, they are negative for s-100 protein. in the present case, because of the presence of histopathological characteristics that are similar to those of angiomyofibroblastomalike tumors, a differential diagnosis of infiltrative angiomyxoma can not be easily made. an accurate diagnosis is therefore mandatory. to date, only a small number of cases of angiomyofibroma and infiltrative angiomyxoma have been reported. it is assumed, however, that local surgical removal will be sufficient for a complete recovery. angiomyofibroblastoma - like tumors occurring in male patients have not been reported in korea until now, and their incidence is extremely low. despite the presence of benign findings on histopathology, in cases in which such tumors occur in the scrotum, both a surgical removal and a histopathologic examination are mandatory for a differential diagnosis. furthermore, in cases of infiltrative angiomyxoma, for which the histopathologic findings are similar, a meticulous monitoring of local recurrence is essential. an appropriate follow - up observation of the long - term clinical course after the surgery is also essential. | various tumors can occur in the scrotum. of them, angiomyofibroblastoma - like tumors are very rare mesenchymal tumors. angiomyofibroblastoma - like tumors can not be easily differentially diagnosed from other malignant tumors invading the male genital tract on the basis of clinical characteristics and imaging study. therefore, surgical removal and a histopathologic diagnosis must also be performed. |
the hemoglobinopathies are the most common inherited single - gene disorders worldwide, resulting from the heterozygote advantage against malaria (1). among the hemoglobin disorders, -thalassemias are found with high gene frequencies in african, asian and the mediterranean populations, whereas -thalassemias are particularly prevalent in asia and the mediterranean natives (2). the -thalassemias are a group of anemia which affect the rate of synthesis of normal -globin chains. -thalassemia occurs due to the mutations in the -globin gene cluster located on short arm of chromosome 11 (3). more than 500 mutations have been characterized in -globin gene region associated with the -thalassemia disease (omim 141900). however, it is revealed that each population has its own spectrum of mutations (4). the beta globin gene cluster region contains five expressed genes with the same transcriptional orientation and one pseudogene () between a and genes in the order of 5--g-a----3 (5 - 6). moreover, the cluster region contains several polymorphic markers throughout chromosome 11, which could be used as useful tools for linkage analysis and molecular diagnosis of beta thalassemia disease (7). -thalassemia is one of the most common public health problems in iran, having the frequency of about 10% in the iranian population (8). therefore, molecular diagnosis of the disease is highly requested in most molecular diagnosis centers in iran. the disease is diagnosed both by direct sequencing and indirectly by the use of polymorphic markers present in the beta globin gene cluster. however, to date there is no reliable information on the application of the markers in the iranian population. in this study, the aim was to investigate the presence or absence of the restriction sites are represented by (+) and (-) symbols in this table the population indices of five most commonly used polymorphic markers located in the beta globin gene cluster region including xmni, hindiiia, hindiiig, rsai and hinfi in the -thalassemia carrier individuals from central and south provinces of iran. blood samples were collected from 552 unrelated -thalassemia carriers referred to the isfahan medical genetics center, isfahan, iran. also, the ethical committee of university of isfahan has approved the study (approval number 790205). the selected individuals were from the provinces of isfahan (n=176), chaharmahal - o - bakhtyari (n=201), khuzestan (n=97) and hormozgan (n=78). genomic dna from each individual was obtained from peripheral leucocytes by salting out procedure (10). five rflp markers including xmni 5 to the gene, hindiii in the ivsii of and genes, rsai 5 and hinfi 3 to the -globin gene the statistical analysis of data was performed by use of the popgene32 software version 1.31 (available at http://www.ualberta.ca/~fyeh/-download.htm) and pypop program (13). the allele frequency, the expected and observed heterozygo - sity, shannon s information index (i), fixation indices and the ewens - watterson test were estimated. the d values between possible pairings of studied markers were obtained by means of the 2ld program (14). blood samples were collected from 552 unrelated -thalassemia carriers referred to the isfahan medical genetics center, isfahan, iran. also, the ethical committee of university of isfahan has approved the study (approval number 790205). the selected individuals were from the provinces of isfahan (n=176), chaharmahal - o - bakhtyari (n=201), khuzestan (n=97) and hormozgan (n=78). genomic dna from each individual was obtained from peripheral leucocytes by salting out procedure (10). five rflp markers including xmni 5 to the gene, hindiii in the ivsii of and genes, rsai 5 and hinfi 3 to the -globin gene the statistical analysis of data was performed by use of the popgene32 software version 1.31 (available at http://www.ualberta.ca/~fyeh/-download.htm) and pypop program (13). the allele frequency, the expected and observed heterozygo - sity, shannon s information index (i), fixation indices and the ewens - watterson test were estimated. the d values between possible pairings of studied markers were obtained by means of the 2ld program (14). blood samples were collected from 552 unrelated -thalassemia carriers referred to the isfahan medical genetics center, isfahan, iran. also, the ethical committee of university of isfahan has approved the study (approval number 790205). the selected individuals were from the provinces of isfahan (n=176), chaharmahal - o - bakhtyari (n=201), khuzestan (n=97) and hormozgan (n=78). genomic dna from each individual was obtained from peripheral leucocytes by salting out procedure (10). five rflp markers including xmni 5 to the gene, hindiii in the ivsii of and genes, rsai 5 and hinfi 3 to the -globin gene the statistical analysis of data was performed by use of the popgene32 software version 1.31 (available at http://www.ualberta.ca/~fyeh/-download.htm) and pypop program (13). the allele frequency, the expected and observed heterozygo - sity, shannon s information index (i), fixation indices and the ewens - watterson test were estimated. the d values between possible pairings of studied markers were obtained by means of the 2ld program (14). five polymorphic markers in the beta globin gene region including xmni, hindiiia, hindiiig, rsai and hinfi were genotyped in two central populations of iran, isfahan and chaharmahal - o - bakhtyari, as well as two south populations, khuzestan and hormozgan (figure 1). as shown in table 1, allele frequency of the markers did not differ significantly among the four iranian subpopulations. the expected and observed hetero - zygosity, shannon s information index (i), and fis value of the markers are shown in table 2. the overall observed heterozygosity ranged from 0.1706 (hindiiia marker) to 0.4484 (rsai marker). negative fis was observed for hinfi markers in khuzestan and rsai marker in hormozgan, indicating an excess of heterozygotes. all these polymorphic markers showed the shannon index < 1 in the studied populations, revealing low heterozygosity of studied markers in these populations of iran (table 2). a typical illustration of hinfi marker genotyping in four dna samples from the iranian population. the digestion products were resolved on 2% agarose gel and visualized following ethidum bromide staining. lane 1 : homozygous -/-, lanes 2 and 3 : homozygous + /+, lane 4 : heterozygous + /- m represents 100 bp dna ladder the frequency distribution of rflp markers located within -globin gene cluster region in four subpopulations of iran the observed and expected heterozygosity, shannon s information index (i) and inbreeding coefficient (fis) of five rflp markers located within -globin gene cluster region in four subpopulations of iran fixation indices fis, fst, fit were used to analyze genetic variation. as shown in table 3, the fst varied among studied markers from 0.0064 (hindiiia) to 0.0355 (xmni) with a mean of 0.0231. the fit values ranged from 0.0134 in rsai marker to 0.2553 in hindiiig polymorphic marker. however, mean fis (0.1025) and fit (0.1232) were both positive and greater than zero. the subscripts i, s and t refer to individual, population and total population, respectively. the mean number of migrants per generation among populations (nm) was reported maximum for the rflp marker hindiiia (38.9648) and minimum for xmni marker (6.7938). in ewens - watterson test of neutrality for these studied rflp markers, the observed homozygosity (f) lied inside the limit of 95% confidence region, and the normalized deviate of the homozygosity (fnd) was estimated negative for all these polymorphic markers (table 4). f - statistics for five rflp markers located within -globin gene cluster region in the iranian population the ewens - watterson homozygosity test of neutrality for five rflp markers located within -globin gene cluster region in the iranian subpopulations pairwise linkage disequilibrium values were also estimated using the 2ld programs (zhao 2004). the number of samples of hormozgan population was insufficient, and linkage disequilibrium values were only assessed for isfahan, chaharmahal - o - bakhtyari and khuzestan populations. d values ranged from 0.01516 (hindiiia - hinfi) to 0.81098 (xmni - hindiiig) in the studied iranian subpopulations. linkage disequilibrium values for possible pairings of markers located within -globin gene cluster region in four iranian subpopulations the usefulness of the polymorphic markers in linkage study and prenatal diagnosis as well as carrier detection of genetic diseases depends on the frequency and informative status of the markers in each population. as shown in table 1, the frequency of the minor alleles exceeds 0.3 for hindiiig and rsai markers. these data indicate that these two polymorphic markers, hindiiig and rsai, have a high degree of polymorphism in the iranian population. significant difference between the observed and expected heterozygosity was not observed for almost any of the rflp markers in the studied iranian population. the observed heterozygosity was lower than the expected heterozygosity for hindiiig and rsai in hormozgan and chaharmahal - o - bakhtyari populations and for xmni and hindiiig in the khuzestan population. the observed heterozygosity of the rflp markers was lower than 50% in all the studied populations, indicating that these markers are less informative in prenatal diagnosis and carrier detection of -thalassemia. however, haplotyping of the markers could be more useful than separately genotyping them in the iranian population. analysis of shannon index indicated that the highest value was observed for rsai polymorphism in all iranian subpopulations studied. these data revealed that rsai polymorphism was the most useful markers among the five rflp markers investigated. the hindiiia polymorphism in all the studied populations, and xmni and hinfi polymorphisms in hormozgan and chaharmahal - o - bakhtyari populations showed a very low shannon index. these results reveal that these markers have little application in carrier detection and prenatal diagnosis of -thalassemia disease in the iranian population. moreover, the studied rflp markers did not show significant deviations from hardy wein- berg equilibrium (hwe) except hindiiia and hindiiig polymorphisms in chaharmahal - o - bakhtyari population and hindiiig polymorphism in hormozgan population. once the subpopulations were considered as one population, the population only deviated from hwe at xmni, hindiiia and hindiiig polymorphic markers. in the calculation of hwe, it should be noted that the selected individuals were unrelated with no consanguineous relationship (their parents were unrelated). however, whether their parental marriage was random or not, is not clear. therefore, we can not definitely exclude any population inbreeding and claim that all marriages in that population were random. fst is the most suitable statistic for studying the overall level of genetic divergence (15). when nm is higher than 1, gene flow is able to offset the differentiation among populations caused by isolation and genetic drift (16). in the iranian population, f value lied inside the lower and upper limits of the 95% confidence region of expected f values for all these rflp markers (table 4), signifying that these polymorphic markers in -globin gene region were not under genetic hitchhiking and selection force operating on another locus could not change allele frequency and heterozygosity of these polymorphic markers in this population. as shown in table 4, the expected homozygosity () was higher than the observed homozygosity (f) value for all these polymorphic markers in the -globin gene cluster. these data could support the presence of balancing selection at the -globin gene cluster region in the iranian population. the results obtained from ld estimation indicated that d values for three pairings of the markers, xmni - hindiiig, hindiiia - hindiiig and rsai - hinfi, were higher than 0.5 in all three populations. these ld values could provide useful information for selecting suitable markers in molecular diagnosis of beta thalassemia disease. the d values close to zero for other pairings of markers showed that they were in linkage equilibrium. therefore, the two - marker haplotypes xmni - rsai, hindiiia - rsai, hindiiig - rsai, xmni - hinfi, hindiiig- hinfi and hindiiia- hinfi should be used with caution in diagnosis of disease. in summary, the iranian population showed heterozygosity deficiency for the rflp markers including xmni, hindiiia, hindiiig, rsai and hinfi, located within the -globin gene region. indeed, the haplotype phasing is more applicable than genotyping markers separately in the iranian population. although a large number of unrelated individuals from different provinces of iran were included in the present study, one should note that the data can not be applied to the whole iranian population. the data obtained from this study could provide additional insight into the genetic structure of -globin gene region in the iranian population. | objective(s):iran is considered as one of the high - prevalence areas for -thalassemia with a rate of about 10% carrier frequency. molecular diagnosis of the disease is performed both by direct sequencing and indirectly by the use of polymorphic markers present in the beta globin gene cluster. however, to date there is no reliable information on the application of the markers in the iranian population. here we report the results of an extended molecular analysis of five rflp markers, xmni, hindiiia, hindiiig, rsai and hinfi, located within the -globin gene cluster region in four subpopulations of iran.materials and methods : a total of 552 blood samples taken from the iranian subpopulations including isfahan, chaharmahal - o - bakhtiari, khuzestan and hormozgan were genotyped using pcr - rflp and sequencing. the allele frequency, the expected and observed heterozygosity, and shannon s information index (i) of these markers were calculated.results:distribution of the allele frequencies for xmni, hindiiia, hindiiig, rsai and hinfi polymorphic markers did not differ significantly among the subpopulations examined. overall observed heterozygosity ranged from 0.1706 for hindiiia to 0.4484 for rsai. the shannon index was < 1 for all the polymorphic markers in the populations studied. the data indicated that heterozygosity of these markers was low in the iranian population.conclusion:the results suggested that genotyping of these markers is not informative enough once used as single markers for prenatal diagnosis and carrier detection of -thalassemia in the iranian population. however, haplotyping of these markers may provide more useful data in linkage analysis and prenatal diagnosis as well as carrier detections for -thalassemia in iranians. |
epidemiological data indicate that mortality is higher than 2530% in patients with sepsis and 4050% in patients with septic shock. delayed diagnosis and inadequate antimicrobial therapy choosing optimized antimicrobial therapy according to the causative pathogen of sepsis can drive significant improvements in the prognosis of sepsis. therefore, identifying the causative pathogen is critical for the successful antimicrobial treatment of sepsis. however, bacteremia is confirmed via microbiological examinations in only about 30% of patients with sepsis. systemic inflammatory markers, such as c - reactive protein (crp) and erythrocyte sedimentation rate (esr), have poor sensitivity and specificity in diagnosing bacterial infections. hence, a biomarker to rapidly and accurately identify causative agents is warranted for use in the clinical setting. procalcitonin (pct), the protein precursor of calcitonin, is synthesized and released by c - cells in the thyroid gland. serum pct levels have been found to increase within 4 h, reach peak levels within 6 h and maintain a plateau through 24 h after healthy volunteers are injected with gram - negative bacterial endotoxin. pct appears to have higher sensitivity and specificity for predicting bacterial infection than other markers. whereas, pct levels are normal in patients without infection. hence, pct is a useful biomarker to predict systemic inflammatory response syndrome (sirs), bacteremia, and even sepsis. serum pct levels differ in patients with bacterial or fungal infections and are significantly elevated in patients with gram - negative bacteremia. pct levels have been shown to be able to distinguish gram - negative bacterial (gram - negative) sepsis from gram - positive bacterial (gram - positive) and fungal sepsis. here, we evaluated the diagnostic accuracy of different inflammatory markers to discriminate sepsis caused by different pathogens within a large patient population. this study was approved by the ethics committee of zhongnan hospital of wuhan university, china. written informed consent was provided by each participant. the researchers retrospectively analyzed medical records from january 2013 to september 2014 in zhongnan hospital of wuhan university, china, and all patients with sepsis were included. (1) confirmation of infection based on microbiological evidence (positive blood culture results) and (2) a diagnosis of sirs, which required the presence of at least two of the following clinical symptoms : fever (38c) or hypothermia (2.44 ng / ml for pct could distinguish gram - negative sepsis from gram - positive sepsis with a sensitivity, specificity, ppv, and npv of 77.1%, 68.4%, 74.0%, and 65.1%, respectively. hence, significantly higher pct levels can serve as a marker to diagnose gram - negative sepsis. charles. reported that an optimal pct cut - off value of 16 ng / ml resulted in 75.0% sensitivity and 82.2% specificity in distinguishing gram - negative from gram - positive sepsis. the mechanism underlying different pct levels in response to gram - negative and gram - positive bacteria remains confusing. differences in the membrane composition of gram - negative and gram - positive bacteria might partly explain this. the major membrane component of gram - negative bacteria is lipopolysaccharide (lps) (the major component of endotoxin) and that of gram - positive bacteria is peptidoglycan (pgn). lps and pgn, as pathogen - associated molecular patterns (pamps), are recognized by pattern recognition receptors (prrs) in the innate immune response. prrs include several families such as toll - like receptors (tlrs) and c - type lectin receptors (clrs). the ligation of pamps to tlrs initiates signaling pathways and induces the release of pro - inflammatory cytokines. lps is sensed as a ligand by tlr4, and pgn is sensed by tlr2. lps binding to tlr4 activates the classical myd88-dependent signaling pathway and induces the production of cytokines, such as tumor necrosis factor- (tnf-) and interleukin-6 (il-6). demonstrated that both lps and sepsis - related cytokines increased pct expression in human peripheral blood mononuclear cells (pbmcs). in pbmcs cultured with lps, tnf-, or il-6, pct mrna was detected at levels 4- to 230-fold, 2- to 90-fold, or 2- to 35-fold, respectively, higher than in control cultures. in addition, gram - positive pathogens induce relatively poor cytokine levels (tnf- and il-6) than gram - positive ones do, owing to tlr2-dependent simulation. these factors might mediate the expression of serum pct concentrations in response to gram - negative bacterial infections. e. coli and k. pneumoniae are the most common causative pathogens of gram - negative bacterial infections. approximately 54% of e. coli and 27% of k. pneumoniae were confirmed to possess extended - spectrum beta - lactamase (esbl) in zhongna hospital (unpublished data). esbl - producing e. coli and k. pneumoniae are resistant to beta - lactam antimicrobials. moreover, these pathogens might markedly prolong hospital stays and exacerbate the financial burden. in this study, pct levels in patients with e. coli (12.74 ng / ml [iqr : 1.7358.88 ]) or k. pneumoniae (15.46 ng / ml [iqr : 5.21144.48 ]) sepsis were significantly higher than in patients with other gram - negative, gram - positive or fungal sepsis. reported similar ranges of pct levels in e. coli and k. pneumoniae sepsis, namely 15.30 ng / ml (iqr : 2.7038.0 ng / ml) and 15.23 ng / ml (iqr : 5.4543.61 ng / ml), respectively. elson. demonstrated that for e. coli and k. pneumonia, tlr4/myeloid differentiation factor-2 (md-2) ligands were dominant. furthermore, serum pct levels could be used to distinguish e. coli or k. pneumoniae sepsis from other bacterial sepsis. for e. coli, 76.2% sensitivity and 91.5% npv were achieved with an optimal cutoff value of 2.32 ng / ml. for k. pneumoniae, 89.3% sensitivity and 98.3% npv were achieved with an optimal cut - off value of 2.81 ng / ml. hence, higher - than - average pct concentrations might indicate e. coli or k. pneumoniae infection. further studies are warranted to elucidate the mechanisms underlying the significant increase in pct concentrations in patients with e. coli or k. pneumoniae sepsis. according to several studies, low concentrations of pct in critically ill patients might suggest fungal infection. in this study, pct levels could be used to sensitively and specifically distinguish gram - negative sepsis from fungal sepsis. the diagnostic accuracy of pct for this differentiation was determined by roc analysis, revealing an optimal cut - off value of 3.11 ng / ml, resulting in 63.9% sensitivity and 93.3% specificity. in a similar study, leli. found that an optimal pct cut - off value of 1.6 ng / ml could be used to distinguish gram - negative infections from fungal infections with 77% sensitivity and 96% specificity. martini. also demonstrated that an optimal pct cut - off value of 2 ng / ml could be used to distinguish candida sepsis from bacterial sepsis with 92% sensitivity and 93% specificity. pct levels in patients with gram - positive (0.48 ng / ml [iqr : 0.15 - 2.16 ng / ml ]) and fungal (0.60 ng / ml [iqr : 0.142.06 ng / ml ]) sepsis differed, albeit not significantly (p = 0.602). although serum pct concentrations were moderately elevated in fungal and gram - positive sepsis, mechanisms of pct expression were different. tlrs were sensed by both gram - positive bacteria and fungi, but the major receptors recognizing fungi are clrs. the secretion of cytokines, such as il-1, il-10, and il-6, is induced by the activation of the clr / syk - card9 signaling pathway, resulting in moderately elevated pct levels after fungal infections. in addition, serum interferon - gamma (ifn-) levels are markedly elevated in patients with fungal infections, ifn- is a critical factor for defense against fungal infections. in vitro, pct secretion has been found to be inhibited by ifn-. thus, pct concentrations in fungal sepsis might be regulated by ifn- levels. furthermore, patients treated with broad spectrum antibiotics and invasive surgeries have a greater risk of developing fungal infections, and such patients with elevated pct levels should, therefore, be screen for fungal infections. crp can be used as a sensitive indicator of systemic inflammation during early stages of infection. here, crp concentrations were significantly elevated in all groups and could be used to distinguish between different causative pathogens. an optimal cut - off value of 59.25 mg / l for crp levels resulted in 74.4% sensitivity and 65.4% specificity, in discriminating gram - negative sepsis from gram - positive sepsis. an optimal cut - off value of 52.25 mg / l for crp levels resulted in 79.1% sensitivity and 83.3% specificity in discriminating gram - negative sepsis from fungal sepsis. although crp levels can be used to accurately distinguish causative pathogens, confounding factors limit the broader application of this marker. hence, crp levels were considered inferior to pct levels in distinguishing causative pathogens of sepsis. esr is also influenced by inflammation, serum concentrations of fibrinogen and immunoglobulin and erythrocytes characteristics. in this study, we did not observe any significant correlation between esr and the causative pathogen of sepsis. first, the majority of the patients were treated at different departments, and only some of the patients received a sequential organ failure assessment score. the diagnosis of severe sepsis and septic shock was defined by the guidelines of the german sepsis society. patients in each group were divided into independent subgroups according to the severity of sepsis (sepsis subgroup, severe sepsis subgroup, and septic shock subgroup). no differences in pct levels were observed in the severe sepsis subgroup, and pct levels could not be used to distinguish between patients with severe sepsis caused by different pathogens (p = 0.308, data not shown). we can not explain whether the severity of sepsis would influence the diagnostic accuracy of pct in discriminating between different causative pathogens. second, cons are common contaminants in blood cultures. because of technical limitations, distinguishing between cons contaminants and cons infections was difficult. seventy cases of cons bacteremia were identified in our study, and some of these cases might actually represent cons contamination. more rigid inclusion criteria should be adopted in future research to obviate the influence of cons contaminants. serum pct levels were significantly higher in patients with gram - negative sepsis than in patients with gram - positive or fungal sepsis with the exception of patients with severe sepsis. we provide evidence that pct is a sensitive biomarker that can be used to distinguish gram - negative sepsis from gram - positive and fungal sepsis. it should be noted that the importance of microbiological examinations remains unchanged. blood culture and antimicrobial susceptibility testing are required in clinical applications to positively identify causative microorganisms and assess their sensitivity to different antibiotics. sl contributed to the conception of the work, conducting the study, recruiting patients, processing data, and writing the manuscripthr contributed to the conception of the work, recruiting patients, processing data, and preparing the tables and figuresqg contributed to the conception of the work and collecting the results of blood - culture and serum inflammatory biomarkersyc contributed to the conception of the work, processing data, drawing roc curves, and preparing the figuresgz contributed to the conception of the work and recruitment of patientsjy contributed to the conception of the work and revising the draftall authors agree to all aspects of the study and approve the final version of the manuscript for publication. sl contributed to the conception of the work, conducting the study, recruiting patients, processing data, and writing the manuscript hr contributed to the conception of the work, recruiting patients, processing data, and preparing the tables and figures qg contributed to the conception of the work and collecting the results of blood - culture and serum inflammatory biomarkers yc contributed to the conception of the work, processing data, drawing roc curves, and preparing the figures gz contributed to the conception of the work and recruitment of patients jy contributed to the conception of the work and revising the draft all authors agree to all aspects of the study and approve the final version of the manuscript for publication. | background : serum procalcitonin (pct) levels differ in patients with bacterial or fungal infections and are significantly elevated in patients with gram - negative bacteremia. we evaluated the diagnostic accuracy of different inflammatory markers to discriminate sepsis caused by different pathogens.materials and methods : we included 328 episodes of bacteremia from 292 patients with sepsis and 31 patients with suspected sepsis in this study. medical records of patients who had bacteremia caused by gram - negative bacteria (gram - negative), gram - positive bacteria (gram - positive) or fungi were reviewed, and information about pct and other inflammatory markers was recorded. the diagnostic performance of inflammatory markers was calculated via receiver operating characteristic (roc) curves.results:serum pct levels in gram - negative, gram - positive, and fungal sepsis were 7.47 (interquartile range [iqr ] : 1.0941.26) ng / ml, 0.48 (iqr : 0.152.16) ng / ml, and 0.60 (iqr : 0.142.06) ng / ml, respectively (p < 0.001). roc analysis revealed an optimal cut - off value of 2.44 ng / ml for pct in discriminating gram - negative sepsis from gram - positive sepsis, which yielded a sensitivity of 68.4% and a specificity of 77.1%. an optimal cut - off value of 3.11 ng / ml for pct in discriminating gram - negative sepsis from fungal sepsis, led to a sensitivity of 63.9% and specificity of 93.3%. neither pct nor other inflammatory markers could be used to distinguish between gram - positive and fungal sepsis.conclusion:serum pct levels were significantly higher in patients with gram - negative sepsis than in those with gram - positive or fungal sepsis. pct is a potential sensitive biomarker for distinguishing gram - negative sepsis from gram - positive and fungal sepsis. |
the red reflex test is an effective screening test in the early diagnosis of various illnesses and disorders of eyes, such as cataracts, glaucoma, retinoblastoma, retinal disorders. the base of the red reflex test is that if the ophthalmoscope light directly placed on the optic axis of the dilated pupil, the pupil area would seem in a uniform light orange (close to red) color. the term red reflex, is the reflection of the fundus color (the color combination of vascular area and choroid pigments), which returns to the front via the transparent eye path (vitreous, lens, aqueous, and the cornea). any resentment placed within this path in the eye, partially or completely prevents the reflection, and would appear as a black mark or a shadow. darkening the examination room normally allows the pupil to be dilated enough so that the central fundus could be examined. dilating the pupil via drugs could greatly increase the sight, and enables a wider test in the sclera area. the royal college of pediatrics and the royal college of ophthalmologists in england have suggested that the eyesight examination, with the red light reflex, must be a part of general examination for all neonates after birth, and also at the 68 week of life. also, the american academy of pediatrics suggested that all neonates must be subjected to the eye s red reflex test by a pediatrician or a first aid doctor, over the first 2 months of life. there is no exact data in iran to show the method of application and analysis of this test, or on genetic and ethnic - racial variations. the goal of this study was to analyze the sensitivity and specificity of the red reflex test among neonates and analyzing of the achieved results by pediatrician for detecting the eyesight problems and finally comparing it to the results achieved by ophthalmologist, few days after birth. for conducting this prospective analytic study, all neonates born in the martyr mustafa khomeini teaching referral hospital in ilam, iran between july 2011 until march 2012, were tested, hours after their birth by previously trained pediatricians in a non - standard condition (at the delivery room, beside mother and without the eyes being dilated), to detect the presence or absence of the red reflex via a indirect ophthalmoscope with zero lens (riester ophthalmoscope, made in usa), and after three days, were transferred to the ophthalmology clinic of imam khomeini referral teaching hospital, in ilam, and were examined by an ophthalmologist in standard conditions (a dark room with the dilated eyes). eventually the findings reported by pediatric examination at the first few hours and in a non - standard condition were compared to the results obtained by ophthalmologic examination after few days and in a standard condition. also the association between red reflex findings and neonatal variables was analyzed via chi - square or t - test appropriately. the color gradient was intended to facilitate the registration and description of the characteristics of the reflex visualized in the test, making them the closest to the findings observed by the primary examiner and ophthalmologist, therefore in our study red reflex scorings were : bold red (10/10), orange to reddish (5/1010/10), orange (5/10), light yellow with fix dark spot (0/105/10) and milky white or asymmetry of reflexes (0/10). only the measured scores of 10/10 at the first screening examination by pediatricians have been considered as normal. data were collected by prepared forms and using spss 16 both conditions were compared for differences and sensitivity rates. this study was approved by the ethics committee of ilam university of medical sciences, under the number ec/92/h159 and informed consent was obtained from the parents of all neonates. totally 255 neonates including 141 (55.3%) boys and 114 (44.7%) girls were investigated of which 144 (56.5%) were born by cesarean section (cs). the mean age and standard deviation of participants was 28.2013.87 (5 to 79) hours (table 1). neonates with disturbed red reflex had a mean apgar score 8.851.38, whereas those with normal red reflex showed an apgar of 9.140.213. there was no statistical significant relationship between the red reflex results, and mean apgar score (p=0.05). demographic characteristics of participants in red reflex study in ilam city sd : standard deviation according to the results of this study, at the non - standard conditions, 34.1% of neonates had a 10/10 red reflex, which increased to 90.6% in the standard conditions, and also the disturbed red reflex amount at the non - standard condition was 65.9%, which dropped to 9.4% under standard conditions (table 2). disturbed red reflex under non - standard conditions was reported in 168 cases, of whom in the final examination under standard conditions only 51 were diagnosed with ocular lesions. also, of 87 cases reported as having normal red reflex, 9 neonates were identified to have ocular lesions. in examinations under standard conditions, 24 cases of abnormal red reflex were reported which was reduced to 11 cases at the final investigation and 231 cases of normal red reflex reported in the first observation reduced to 68 cases at the final examination (table 2). red reflex test results under non - standard condition by pediatrician and standard condition by ophthalmologist among participants in red reflex study in ilam city for the rate of ocular lesions under standard and non - standard conditions according to the red reflex test results see table 3. examination at the non - standard environment revealed that 72.9% of neonates born by cs, and 56.8% of those with vaginal delivery had a disturbed red reflex, with a statistically significant difference (p=0.007) ; however, these figures changed to 11.8% and 6.3% respectively during examination under standard condition a few days later with statistically non significant difference (p=0.1). distribution of red reflex test results under non - standard and standard conditions in participants in red reflex study in ilam city results indicated that, among neonates born by a prolonged delivery process, 100% of neonates examined at nonstandard condition and 19.2% of those at standard condition had a disturbed red reflex, but for those without a prolonged birth process, these figures accounted for 57.1% and 6.9% respectively. there was a significant relation between prolonged birth process and red reflex results in both standard and non - standard examinations (p<0.0001). among those with a history of difficult labor, 98.6% and 6.5% of neonates examined at non - standard and standard conditions respectively had a disturbed red reflex, indicating a significant relation between difficult labor and the red reflex disturbances in both examination methods (p<0.01). the results of sensitivity and specificity of red reflex examination under non - standard condition is shown in table 4. there was a significant relationship between method of delivery, duration of delivery, complicated delivery and intensity of red reflex sensitivity. a significant difference in results (identification of ophthalmic problems) was seen in neonates inspection in the first hours under substandard conditions compared to further inspections in standard conditions particularly from 3 day of life. a significant variety of red reflex could be present among children from different races or nations because of the various levels of ocular fundus pigmentation. this is a very sensitive test and the examiner must pay an absolute attention to prevent any false positive or false negative results, because there are no specific national instructions on this matter. the results of our study indicated that there was a significant difference between the examinations by pediatrician, and the results obtained by an ophthalmologist and there was no significant relation between age, weight or apgar score and red reflex under standard or non - standard conditions. a study by de aguiar and colleagues specified that 187 out of 190 neonates born in a general hospital had no changes or variations, while 3 cases were suspicious. this study also reported, 50 (26.3%)cases were red, 34 (17.9%) orange to reddish, 92 (48.8%) orange, 11 (5.8%) light yellow and 3 (1.6%) cases were milky white and indicated a significant statistical relation between the color gradient and neonatal variables, such as : weight (p=0.03), gestational age (p=0.02), and oxygen therapy (p=0.02). the results of current study in the standard conditions revealed that 11 out of 24 cases reported for disturbed red reflex had ocular lesions in the final examination and among 231 cases reported as normal red reflex 68 individuals had ocular lesions, in accordance with the study performed in brazil by rodrigues and colleagues. they clarified that during one year study ; only 16 out of 29 children reported with red reflex disorders were confirmed. there was no significant relationship between retinal hemorrhage and type of birth, prolonged birth process or dystocia during neonatal examination under non - standard conditions ; however, a significant relationship was revealed between the above mentioned factors during their examination under standard conditions. in our study, 31% of the neonates had ocular lesions, which included 22.7% retinal hemorrhage, 3.9% reduced vessels, 2.4% salt paper and 2% retinal discoloration, which indicated a higher prevalence of retinal hemorrhage among neonates at the early birth postpartum examinations. this was lower than the results achieved by li and colleagues with a prevalence of 21.5% for retinal hemorrhage among 3573 examined neonates. in a study inducted by sotomi and colleagues in 2007, it was shown that among 27 neonates detected with congenital and infantile cataracts 17 infants (63%) were diagnosed with bilateral disease, while the remainder were unilateral 10 (37%). most of the cases 17 (63%) were diagnosed following presentation with parental / career concerns about visual function (usually a squint). the remaining cases of congenital cataracts were diagnosed by general practitioners 8 (24%), pediatricians 4 (12%), ophthalmologists 3 (9%) or school medical officer (1, 3%). none of the neonates with congenital or infantile cataract were detected by newborn screening tests. eventov - friedman and colleagues during a study performed the red reflex test to examine the neonates and reported 5 neonates with cataract were detected within days 45 of their life and suggested that the red reflex examination must be placed as a part of neonatal examinations. in our study there was a significant difference between the examinations under non - standard conditions by pediatrician, and the results obtained under standard conditions by an ophthalmologist (p<0.0001). it may be related to this fact that pediatricians do not have enough experience or it may be due to ocular and tear film problems in the first day of life that gave rise to disturbance of red reflex and disappeared within few days especially in babies with difficult deliveries or low apgar scores. based on the incidence of corneal edema and its severe effects on the red reflex over the first hours of life, early application of the red reflex examination under standard condition (dark room and dilated pupil) is highly important. also by time passing and increasing age of neonates, the corneal edema is gradually reduced and better conditions are prepared for ophthalmologic examinations that can results in increasing test accuracy and higher sensitivity in detecting neonatal eye defects. applying a red reflex test in the first hours of life by a pediatrician in all neonates is important, and therefore it is recommended as an early diagnostic tool in detecting neonatal eye defects. this can reduce the delayed referrals to the ophthalmologist. in case of any abnormality in the red reflex test further examinations under standard conditions by ophthalmologist is necessary. performing this test with a standard method (dark room and dilated pupil), could lead to improving the diagnostic screening results compared to that under non - standard conditions. of course, potential threats of using the cycloplegic drugs in neonates and infants must be mentioned. it is suggested to examine all neonates with red reflex test before being discharged from hospital and 6 weeks after birth under standard conditions. the pediatricians should be trained and educated enough to lower this difference with specialists and increase the accuracy of red reflex assessment. m. mussavi : designed the first concept and prepared data and helped in the first draft of manuscript. k. asadollahi : analyzed and interpreted the data, participated in the manuscript preparation and critical revision. | objective : red reflex test is an effective screening tool in the early diagnosis of neonatal eye abnormalities. the aim of this study was to detect the sensitivity and specificity of red reflex assessment in neonates, performed by pediatricians (or other care providers) in comparison with ophthalmologists. also association between red reflex findings and neonatal variables is evaluated.methods:by a prospective study all neonates born from july 2011 until march 2012 in mustafa hospital, a general teaching hospital in ilam city, iran, were evaluated. neonates were firstly investigated by pediatrician in substandard conditions at the first day of birth and several days later by ophthalmologist in standard conditions.findings:totally 255 neonates including 141 boys and 114 girls were investigated, 144 of whom were born by cesarean section. there was a significant relationship between method of childbirth (72.9% disorders in cs vs 56.8% in vaginal delivery (p<0.007)), duration of delivery (disorders in prolonged : 100% and 11.8% vs no prolonged : 56.8% and 6.3% in standard and non standard conditions respectively (p<0.0001)), difficult delivery (98.6% disorders vs 6.5% in standard and non standard conditions respectively (p<0.01)) and increase or decrease of red reflex sensitivity test. a significant difference (identification of ophthalmic problems) was seen among neonates inspections in primary hours and substandard conditions compared to further inspections in standard conditions particularly from 3rd day of birth.conclusion:due to a considerable difference between the results of ophthalmic examination of neonates in different conditions, red reflex examination by pediatricians is suggested for all neonates to early identification of ophthalmic problems at the first step. it is also suggested a red reflex screening for all neonates before being discharged from hospital as well as 6 weeks later and in case of any problem to be referred to ophthalmologist. |
approximately 150,000 species of flies are described in the world and more than 24,000 species are described in the neotropics. the genus zaprionus (diptera, drosophilidae) consists of approximately 60 species of which about 10 are grouped in the subgenus anaprionus and 50 in the subgenus zaprionus. phylogenetic relationships within groups and subgroups of drosophilids as well as the phylogenetic position of zaprionus within the drosophilidae family are still uncertain. although most phylogenetic analyses associate the genus zaprionus to the subgenus drosophila, new comparative analyses are needed to test the robustness of this association. ultrastructural sperm analyses are important tools for study of the taxonomy and phylogeny of insects. characterized the primary evolutionary radiation that occurred in the drosophila tripunctata group based on the ultrastructure of the mitochondrial derivatives and the number of sperm per cysts. the authors highlighted the need for new ultrastructural studies of the gametes of these insects to provide additional clarification of their evolutionary relationships. ultrastructural analyses of sperm in zaprionus are restricted to the species zaprionus indianus and zaprionus sepsoides. the authors described important characteristics of these drosophilid gametes, such as the presence of granules in the peritoneal sheath, the presence of 2 mitochondrial derivatives of different sizes, the presence or absence of paracrystalline material in the derivatives, the arrangement of the axoneme, and the number of sperm per cyst. this study aimed to characterize the ultrastructure of sperm of 6 other species of zaprionus (z. africanus, z. camerounensis, z. davidi, z. gabonicus, z. megalorchis and z. tuberculatus) and the species drosophila willistoni (subgenus sophophora). scaptodrosophila latifasciaeformis (subgenus scaptodrosophila) was used as an outgroup (table 1). in addition, data from the literature for other species of zaprionus and drosophila were used to help understand the relationships between zaprionus and drosophila. all species showed globular granules in the cytoplasm of the coating layer of the testicular envelope, called the peritoneal sheath (fig. 1a, supplementary figs. 16), except for z. davidi (fig. 1b, supplementary fig. these pigmented granules are responsible for the color of the peritoneal coating sheath of the testes and for the formation of a physical barrier that can protect the testes and store nutrients. note peritoneal sheath (ps) filled with granules (gr) of different sizes and electron densities in z. africanus and their absence in z. davidi. scale : figure a : 11000 x ; figure b : 10000 x. tem micrographs of zaprionus testes. note peritoneal sheath (ps) filled with granules (gr) of different sizes and electron densities in z. africanus and their absence in z. davidi. scale : figure a : 11000 x ; figure b : 10000 x. the color of the peritoneal sheath of the testes is critically important for taxonomy. in the genus drosophila, the peritoneal sheaths of the zaprionus species analyzed in this study and those of d. willistoni and s. latifasciaeformis showed yellowing. yellowing has also been observed in the sheaths of z. indianus, z. sepsoides and z. spinipilus that were analyzed previously. however, z. vittiger was polymorphic for sheath color, which may be yellow or brownish purple. the sperm of pterygota (a primitive group of insecta) has 2 mitochondrial derivatives that flank the axial filament. in drosophilids, used the size of these mitochondrial derivatives as an evolutionary tool to understand the radiation of the genus drosophila. they observed that the 2 derivatives differed in size and that this size difference was greater in sophophora than in drosophila. in the present study, although measurements were not taken, the simple observation of prints showed that our results are consistent with those of mojica. : the difference in the size of these mitochondrial derivatives was greater in d. willistoni (which belongs to the sophophora subgenus) (fig. suggested that the relative size of the mitochondrial derivatives may have changed as drosophila species have evolved. figure 2.ultrastructure of transverse sections of the spermatozoal tail of z. gabonicus (a) showing the paracrystalline material (p) on both mitochondrial derivatives ; the axonemes in d. willistoni (b) and z. davidi (c) have the arrangement of 9 + 9 + 2 microtubules ; the cysts containing 64 spermatozoa in z. camerounensis (d) and z. tuberculatus (e). scale : figures a, b, c : 84000 x ; figures d, e : 10000 x. figure 3.ultrastructure of transverse section of the spermatozoal tail of s. latifasciaeformis (a), z. tuberculatus (b) and z. megalorchis (c) note the presence of the axoneme (ax) and 2 mitochondrial derivatives of different sizes : larger mitocondrial derivative (lm), smaller mitochondrial derivative (sm) ; the accumulation of paracrystalline material (p) is visible in s. latifasciaeformis and z megalorchis. scale : figures a, b, c : 84000 x. table 1.studied species and their geographical origin.subgenusgroupcomplexspeciesgeographic location of the collectionzaprionusvittigerindianuszaprionus africanuskibale (uganda)zaprionusvittigerindianuszaprionus gabonicusmakokou (gabo)zaprionusvittigerindianuszaprionus megalorchiscongozaprionusvittigervittigerzaprionus camerounensisamani (tanznea)zaprionusvittigerdavidizaprionus davidiso tom (so tom)zaprionustuberculatustuberculatuszaprionus tuberculatusithala (south africa)sophophora willistonidrosophila willistonimato / sp (brazil)scaptodrosophilascaptodrosophila latifasciaeformisso jos do rio preto / sp (brazil) table 2.ultrastructural parameters of sperm used for comparisons of species. mitochondrial derivativesparacrystalline materialaxoneme 64 spermspeciesgranulessame sizedifferent sizelargest derivativesmallest derivative9 + 9 + 2per cystzaprionus africanus++++++zaprionus camerounensis++++++zaprionus davidi+++zaprionus gabonicus++++++zaprionus megalorchis++++++zaprionus tuberculatus++++zaprionus indianus++++++zaprionus sepsoides++++++drosophila willistoni+++++drosophila melanogasterna++++drosophila simulansna++++drosophila cardinina+++drosophila dunnina+++drosophila hydeina++++drosophila subobscurana++scaptodrosophila latifasciaeformis++++++note.rego. 2000), noguchi and miller (2003), pasini. (1996), henning (1992), ramamurthy. (1980), hauschteck - junguen and maurer (1976), na : not analyzed. ultrastructure of transverse sections of the spermatozoal tail of z. gabonicus (a) showing the paracrystalline material (p) on both mitochondrial derivatives ; the axonemes in d. willistoni (b) and z. davidi (c) have the arrangement of 9 + 9 + 2 microtubules ; the cysts containing 64 spermatozoa in z. camerounensis (d) and z. tuberculatus (e). scale : figures a, b, c : 84000 x ; figures d, e : 10000 x. ultrastructure of transverse section of the spermatozoal tail of s. latifasciaeformis (a), z. tuberculatus (b) and z. megalorchis (c) note the presence of the axoneme (ax) and 2 mitochondrial derivatives of different sizes : larger mitocondrial derivative (lm), smaller mitochondrial derivative (sm) ; the accumulation of paracrystalline material (p) is visible in s. latifasciaeformis and z megalorchis. scale : figures a, b, c : 84000 x. studied species and their geographical origin. (2000), noguchi and miller (2003), pasini. (1996), henning (1992), ramamurthy. (1980), hauschteck - junguen and maurer (1976), na : not analyzed. except for z. davidi and z. tuberculatus (figs. 2c and 3b), the analyzed species have paracrystalline material on both mitochondrial derivatives (figs. this same characteristic has been observed in z. indianus, z. sepsoides and d. hydei. however, in most species of the genus drosophila (table 2), the paracrystalline material is present only in the larger mitochondrial derivative. the structure of the sperm axoneme is of great importance for phylogenetic studies in insects. most species of insects present the 9 + 9 + 2 arrangement, consisting of one pair of central microtubules and 9 double peripheral microtubules, surrounded by 9 additional accessory microtubules, although some species have a peculiar number, such as 9 + 9 + 3 in neuroptera, 9 + 9 + 1 in culicidae (diptera), 9 + 7 in tricoptera, and 9 + 0 in ephemeroptera. moreover, as the majority of species of the suborder brachyocera, all drosophilids species analyzed had an axoneme structure of the 9 + 9 + 2 configuration, which is the typical arrangement of 9 + 2 internal microtubules surrounded by 9 additional accessories microtubules (figs. spermiogenesis in all analyzed species occurs within cysts where the sperm are organized and exist at the same developmental stage (fig. this phenomenon is referred to as cystic spermatogenesis and is characterized by synchronized cell division within a given cyst. so far, studies have indicated that all insects have cystic spermatogenesis. in triatominae, the cysts develop independently ; that is, a cyst does not influence the developmental stage of neighboring cysts. in drosophilids, as described for plalycentropus (trichoptera : limnephilidae), we suggest that neighboring cysts are also synchronized for cystic spermatogenesis (fig. for all of the species analyzed, we observed the presence of 64 cells inside a cyst (fig., this number varies from 44 to 56 sperm per cyst ; in d. cardini, it varies from 36 to 40 sperm per cyst ; in d. melanogaster, this fixed number is 64 ; and in d. subobscura, this number can reach up to 128 sperm per cyst. in the cluster analysis, on the basis of the analyzed characteristics, 5 species of zaprionus as well as 6 species of drosophila were grouped, suggesting their relatedness within each genus (fig. although an increase in the number of characteristics and species is necessary to validate these results, they are already indicative that the ultrastructure of sperm is a promising tool for phylogenetic and taxonomic studies of insect groups. figure 4.cladogram obtained from cluster analysis using a presence - absence matrix for the characteristics analyzed in the present study and taken from literature. cladogram obtained from cluster analysis using a presence - absence matrix for the characteristics analyzed in the present study and taken from literature. the species and strains of zaprionus and other species used in this study and their geographic location are shown in table 1. the testes of 24 3-day - old adults of each species were processed according to the methods of cotta - pereira. with modifications. ultrathin sections of 70 nm, contrasted with uranyl acetate and lead citrate, were examined with a transmission electron microscope. a cluster analysis was conducted using the euclidean distance and joining method (statistica ; statsoft inc.) with the data from table 2 from which a presence - absence matrix of the characteristics was generated. jean david (center national de la recherche scientifique (cnrs) / gif - sur - yvete / france) for providing some of the lines of zaprionus, rosana silistino souza (academic support assistant ii) for technical assistance in sample preparation and luis fernando segala (assistant professor) for help with statistical analyses. this paper was supported by fundacao de amparo a pesquisa do estado de sao paulo (fapesp) (process number 2010/011939) and coordenadoria de aperfei, coamento do pessoal do ensino superior (capes). | abstractthe genus zaprionus consists of approximately 60 species of drosophilids that are native to the afrotropical region. the phylogenetic position of zaprionus within the drosophilidae family is still unresolved. in the present study, ultrastructural features of spermatozoa of 6 species of zaprionus as well as the species drosophila willistoni and scaptodrosophila latifasciaeformis were analyzed. the ultrastructure revealed that the species have the same flagellar ultrastructure. two mitochondrial derivatives, one larger than the other, close to the axoneme were present, primarily in d. willistoni (subgenus sophophora). except for z. davidi and z. tuberculatus, the analyzed species had paracrystalline material in both mitochondrial derivatives. moreover, the testes showed 64 spermatozoa per bundle in all of the species. in the cluster analysis, 6 zaprionus species were grouped closely, but there were some incongruent positions in the cladogram. the results indicated that sperm ultrastructure is an important tool for elucidating the phylogeny and taxonomy of insects. |
allergy nurses work with various tests aimed at diagnosing allergic diseases and other hypersensitivity disorders. common tests are skin prick test (spt), where different allergens are used, penicillin (pc) provocation tests, and tests with various respiratory irritants, such as methacholine (mch). many allergy nurses also perform allergen specific immunotherapy (asit) by injecting the allergen concentrates. in the clinical working situation with the patients, it is common to be exposed to drops of allergen extracts and to dried allergen on extract bottles and tissues used when performing spt and asit. efforts have been made to improve the working environment in some clinics, using fume or downdraft hoods for diagnostic tests, whereas such preventive and safety measures are missing in other clinics. there are no existing national guidelines for these specific tests concerning safety for the allergy nurses and local recommendations may vary. four main risk factors have been identified for developing occupational asthma (oa) : the causative factor of exposure to an agent at work, the predisposing factors of atopy and genetic predisposition, and the contributing factor of cigarette smoking. rhinoconjunctivitis is more likely to appear before the onset of ige associated oa, and ige sensitization and oa are most likely to develop in the first years of exposure. within the swedish association of allergy nurses (asta), discussions have been carried out concerning deficiencies in the working environment and there have been reports of members that have become sensitized or developed respiratory symptoms at work but there are few studies on this issue. previously, there was a case report concerning two nurses in sweden, who often conducted mch tests and who developed an increased bronchial hyperreactivity related to their work. in 1991, asta conducted a questionnaire survey among all members, studying the correlation between asthma and bronchial provocation tests. among the 259 (80%) respondents, 27% were working with mch and 15% with histamine tests. among those who performed the mch tests, 31% reported respiratory symptoms associated with the test situation and 4% (3 subjects) reported the development of asthma during the time they had been working with such tests. in cooperation with the department of occupational medicine in gothenburg, sweden, no evidence for a relationship between mch tests and development of new asthma was found, but it was concluded that individuals with bronchial hyperreactivity could experience respiratory problems associated with testing, especially if they did not use respiratory protection. there are a few international studies that indicate higher frequencies of asthma and bronchial hyperreactivity among respiratory therapists (rt) [5, 6 ]. an rt works to prevent, treat, and assist in the diagnosis of respiratory diseases and asthma is one of several assignments for the rt. the studies indicate that cleaning of instruments, such as bronchoscopes, in glutaraldehyde and administration of certain antiviral agents in aerosol form are the main risk factors [7, 8 ]. furthermore, a higher incidence of asthma among nurses has been reported in some studies [912 ]. the principal risks recognized are the handling of detergents and disinfectants, the use of latex gloves, and the administration of certain drugs in aerosol form. all these studies are only partially relevant for nurses working in sweden with asthma and allergy, since their main exposures are allergen extracts and respiratory irritants when performing, for example, spt, asit, and mch provocation tests and specific safety guidelines concerning work exposure to allergens, except for a recently published case report describing a compounding technician, with a known allergy to timothy grass, who experienced anaphylaxis after a needle stick from a stock bottle of timothy grass extract while preparing immunotherapy vials. the authors of this case report established that this raises a new question of occupational safety for allergy immunotherapy extract - compounding personnel safety not previously discussed in the allergy literature. they also considered a need for preemployment and regular screening for allergen sensitivity in these employees. in february 2010, asta organized a workshop on this subject and it was decided to perform a questionnaire survey among its members for the purpose of gathering information about these issues as a primary orientation to further discussions. the aims of the current study were toinvestigate the prevalence of allergies, asthma, and hypersensitivity symptoms among the members of asta;investigate the use of protective equipment and measures when working with allergen concentrates and respiratory irritants;gather data for further discussion concerning the working environment. investigate the prevalence of allergies, asthma, and hypersensitivity symptoms among the members of asta ; investigate the use of protective equipment and measures when working with allergen concentrates and respiratory irritants ; gather data for further discussion concerning the working environment. data were collected using a web - based questionnaire that contained 40 questions addressing personal characteristics, work situation, protection at work, and hypersensitivity symptoms. the questions on work situation and protection at work were constructed by the project group, while questions on hypersensitivity symptoms were taken from validated questionnaires, particularly those used in the european community respiratory health survey (ecrhs) ii. furthermore, one question concerned heredity (parents or siblings) for asthma and allergies. the questions used in the questionnaire to evaluate hypersensitivity symptoms are presented in table 1. similar symptoms were compiled in groups and these group definitions (table 1) are used in the presentation of the results. asta is an organization with members from most parts of sweden and represents a great majority of nurses working with asthma and allergy in sweden. webropol, a web - based survey tool provided by the county council of uppsala, sweden, was used to gather the data. an e - mail was sent out in february 2011 to all asta members containing information about the study and a link to the survey. a paper questionnaire was provided to a few members without an e - mail address. in both cases, after the data collection was completed, an excel file with all the data was compiled and the data were subsequently deleted from the webropol server. most of the data are descriptive in character, since the purpose of the study was to describe certain aspects of allergy nurses health and working environment. the alternative answers to the questions concerning frequencies of performing spt, pc, and mch provocation tests were classified into five groups : never, less than once a week, 15 times a week, 610 times a week, and more than 10 times a week. data were compiled into two groups : 5 times a week and > 5 times a week in order to identify nurses who had low or high exposure to these agents and to establish larger groups. subjects that had been working with specific tasks for different periods of time (02 years, 35 years, 610 years, and more than 10 years) were also compiled into two groups : nurses who had worked 5 years and > 5 years, for the same reasons as mentioned above. five years was chosen as suitable cut - off time to be able to include possible new onset asthma, allergies, and hypersensitivity symptoms that would appear after some years in the profession. differences in symptoms between groups were analyzed by test, and a significance level of 5%. there was no control group in this study but symptom prevalences among the allergy nurses were compared with data on women aged 3067 years old from the general population collected by the swedish part (stockholm, gothenburg, ume, and uppsala) of the ga2len survey. the questionnaire was sent to 585 nurses belonging to asta : 570 via e - mail and 15 via paper mail. after three reminders, 418 had answered, resulting in a participation rate of 71%. the average age of responders was 53 years (range 3067 years) and 98% were female. 71% reported that they had never smoked regularly, 28% were previously smokers, and 0.5% were current smokers. fifty - eight percent of the responders worked in primary care, 41% in hospitals, and 1% in both. thirty - four percent worked with children, 37% with adults, and 29% with both children and adults. forty - four percent worked with asthma and allergy approximately one day a week and 27% worked two to four days a week while 29% worked almost exclusively with asthma and allergy. a majority, 56%, had worked with asthma and allergy for more than ten years, and 75% for more than five years. among the requested tasks, spt was the most common task performed, followed by pc provocation tests and asit (figure 1). mch provocation test was the most common test among various hyperreactivity provocations, being performed by 11% (figure 1). asthma diagnosed by a doctor was reported by 17% of the responders to the questionnaire, while 18% had allergy to furry pets and 28% to pollens and 31% reported allergic rhinitis. thirty - four percent reported bronchial hyperreactivity to cigarette smoke, fumes, strong odors, and so forth. lower respiratory symptoms in the last 12 months, when not having a cold, were reported by in total 48%. symptoms of wheezing were reported by 21%, shortness of breath by 7%, and dry cough at night by 24%. itchy rash was reported by 15%, while 26% had nasal symptoms such as sneezing or a runny or a blocked nose and itchy eyes without having a cold or the flu. fifty - one percent acknowledged that they had a history of asthma, allergic diseases, or hypersensitivity symptoms in their family. thirty - three members (8%) reported that they had asthma, allergies, or hypersensitivity symptoms related to their working situation. of these, asthma or some kind of respiratory symptoms was reported by 28 subjects. twenty - one subjects reported allergic rhinitis and 25 nurses were bothered by nasal symptoms. two subjects had been out of work for more than ten days during the last six months due to colds or bronchial problems. of the 28 nurses who reported work - related asthma or some kind of respiratory symptoms, 19 developed these symptoms after entering the profession. two - thirds of the nurses, who had reported work - related symptoms, had a family history of asthma, allergic diseases, or hypersensitivity disorders. all asta members were also asked about when their hyperreactivity, respiratory, or asthma symptoms first appeared. fifty - seven percent of the responders reported that the symptoms occurred after some years in the profession, while 43% indicated that they had their symptoms before entering the profession. in the case of allergic rhinitis or nasal symptoms lower respiratory symptoms during the last 12 months were reported by 38% of those who performed spt more than five times a week compared with 28% for those who did fewer tests although not significant in test (figure 2). however, there was a tendency to reverse results regarding allergic rhinitis, where 29% of those who performed spt more than five times a week reported symptoms, compared with 34% for those who worked with spt less frequently, p = 0.3 in the test. there was a tendency that the nurses in asta who had worked with spt for more than five years, being 65% of those who performed spt, experienced more symptoms than those who had worked for a shorter period, although the differences were not statistically significant in the test (figure 3). pc provocations were performed by 40% of the respondents and 59% of these reported some kind of symptoms. there was no difference in the prevalence of lower respiratory symptoms in nurses who performed pc tests less than once a week compared with those who worked with this test one to five times a week. however, there was a tendency that those who had performed pc provocation tests for more than five years reported more lower respiratory symptoms, allergic rhinitis, nasal symptoms, and airborne allergy, although the differences were not statistically significant in the test (figure 4). mch provocation tests were performed by 44 (11%) of the asta members and 25 performed this task more than once a week. in the latter group, 10 nurses reported that they had asthma, hyperreactive airways, or lower respiratory symptoms and for seven subjects the symptoms had developed after they had started work as allergy nurses. nasal symptoms were reported by 14 of the nurses who performed this test more than once a week (25) and 8 reported allergic rhinitis. eighty percent of these nurses reported either airway or nasal symptoms, or both. a large majority, 38 of the 44 nurses who performed mch provocation tests, had worked with this test for more than 5 years. nasal and lower respiratory symptoms are common among nurses who have worked with mch provocation test for more than five years but due to few nurses in the groups, the differences are not statistically significant in the test (figure 5). the use of protective equipment during spt, asit, and pc provocation tests varied, as is shown in figure 6. a closed container is a container with a lid aimed to stop evaporation from used materials containing allergens and respiratory irritants, and a protective pad is a paper mat used to create a clean working area. approximately, a third of the responders did not take any protective measures when performing asit and pc provocation tests. protective equipment during mch provocation tests was primarily aimed at the presence of respiratory irritants. most of the responders, (73%) used a separate room or space with additional ventilation and the remainder mainly used the fume hood or downdraft hood. the results show that asthma and hypersensitivity symptoms are common among the members of asta, that most of the subjects have been in the profession for a long time, and that over half of the nurses have a family history of asthma, allergy, or hypersensitivity symptoms. in order to make a preliminary evaluation of the results concerning asthma allergies and hypersensitivity symptoms, a comparison has been done with the prevalence in the swedish general population [14, 16 ]. a general population is judged to be neutral and unexposed to these agents and working tasks. since almost all respondents in the present asta survey were women, a comparison with data from the swedish part of the ga2len survey for women, from 2008, the prevalence among asta 's members seems to be slightly higher for most symptoms (figure 7). about one - third of the responders to the questionnaire reported that they become hyperreactive in contact with cigarette smoke and so forth. another swedish survey on the general population showed a prevalence among women of the same magnitude, 31%. the high proportion of allergy nurses with a family history of asthma, allergy, or hypersensitivity symptoms suggests that there might be a biased selection of individuals entering the profession. if this is the case, it is very important that the working environment is adapted to their special needs. in addition to the general work environment act, the swedish work environment authority has also issued provisions on systematic work environment management 2001:01, workplace design, afs 2009:02, and the chemical working environment, afs 2011:19. the employer is responsible for establishing a working environment that does not expose workers to health risks and to investigate the working environment regularly to assess such risks. for example, hazardous chemicals that may occur at work should be identified and working conditions at such sites need to be planned to minimize the exposure. the working premises should have ventilation so that air quality in the breathing zone is satisfactory and air pollution generated in the operations should be effectively removed. however, the rules are very general. exposures, such as allergens and substances for respiratory provocations, which are relevant to allergy nurses, are not discussed explicitly. furthermore, the professionals in health care have established recommendations for performing these tasks, but there appear to be few recommendations in such methodology manuals and so forth concerning how these substances should be handled from an occupational perspective. the present survey found that the use of protective measures varied between different working tasks. the proportion of lack of use of any protection was the highest (39%) for those working with pc provocations, compared with 10% for those working with spt. there was also a higher proportion who reported symptoms among those who carried out the pc provocation tests (59%), compared with that for spt (52%). the highest risk for symptoms (80%) seemed to be caused by performing mch tests more regularly, even though protection measures, in many cases, had been used. the reason why nurses have not used protection measures might be linked to the fact that occupational safety for allergy nurses has not been discussed in a sufficient way, neither in the clinical settings nor in the scientific literature. health problems linked to the working situation can not have been a major concern for a majority of the asta members, since 56% had been in the profession for ten years or more. it may be difficult to establish a connection between work and perceived symptoms when the symptoms are the same as those perceived by the general population. the specific knowledge owned by allergy nurses may have had an impact on their assessment of their own hyperreactive symptoms. however, 8% considered that they had developed allergies or hypersensitivity disorders as a result of their work and 4.5% (19 nurses) reported that their respiratory problems had started after initiating work with allergens or provocations. the number of new cases of asthma among women in the general population has been estimated to be approximately 1.8/1000 person - years and the number of new cases of rhinitis to be approximately 7/1000 person - years [22, 23 ]. on the basis of these figures, 0.7 new cases of asthma and 2.8 new cases of rhinitis per year could be predicted in a group of 400 women. we do not know exactly for how long time the nurses had worked with asthma and allergy, but more than half of them had worked for more than 10 years. if we assume an average experience of 15 years, the number of new cases of asthma would be 11, while at 20 years of experience it would be 14 new cases. the corresponding figures for rhinitis would be 42 or 56 new cases. based on these conceptual calculations, the number of responders, who reported that they had new hypersensitivity symptoms which they attribute to their work, is not significantly higher than the incidence of new symptoms in the general population. nevertheless, the frequency of those reporting current symptoms was higher than in the general population. furthermore, we found an increase in symptoms among those having performed provocation tests for a longer period. the validated questions taken from echrs ii have been suitable for the purpose of this study, but the questions on work tasks that were constructed for this study could have been more specific. the classification concerning frequencies of work tasks was somewhat arbitrary and might have influenced the outcome of the results. however, when comparing symptoms in groups with different number of years in service, the use of different cut - points gave similar results (a tendency to increased symptoms with numbers of years in service). the participation rate was 71% which should be satisfactory but only 29% of the respondents worked exclusively with asthma and allergy and 44% worked just one day a week. consequently, a large proportion of the studied group had a regular but not a high exposure to allergens and respiratory irritants. healthy worker effect, since the study was only directed towards present members of asta, thus not giving any information from nurses who have left the profession and asta. however, the purpose of the study was to give some general descriptive information of an issue which has not been previously highlighted. among the members of asta, there is a tendency towards increased prevalence of lower respiratory symptoms, asthma, allergic rhinitis, nasal symptoms, and eczema and more than half of the nurses had a family history of these diseases and symptoms. with this knowledge, it is of great importance to be observant of the work environment and of hypersensitivity disorders in the nurses. the results of this study imply that further work should also be carried out to establish recommendations concerning protective measures for allergen management and other tasks. additional studies are needed to evaluate the validity of these results and to be able to answer more specific questions concerning causal relationship between the working conditions and the occurrence of respiratory and allergic symptoms. | background. allergy nurses are exposed to allergens and respiratory irritants, and there are no national guidelines addressing personnel safety when working with these agents. objective. to investigate the prevalence of allergies, asthma, and hypersensitivity symptoms among allergy nurses and the use of protective equipment and measures when working with allergen concentrates and respiratory irritants. methods. a questionnaire survey was performed among the members of the swedish association of allergy nurses. results. diagnosed asthma was reported by 17%, while 18% had allergy to pets, 28% had allergy to pollens, and 26% reported nasal symptoms. fifty - one percent reported a history of asthma, allergic diseases, or hypersensitivity symptoms in their family. exhaust ventilation was used by 24% during skin prick tests, 17% during allergen specific immunotherapy, and 33% when performing methacholine challenge tests. tightly closed containers for disposable waste were used by 58% during skin prick tests, by 60% during immunotherapy, and by 40% during pc provocation tests. conclusion. allergy nurses had a tendency to increased prevalence of lower respiratory symptoms, asthma, and allergic rhinitis and more than half of the nurses had a family history of asthma, allergic diseases, or hypersensitivity symptoms. additional studies are needed to evaluate the validity of these results. |
policy makers across the political spectrum, as well as many clinicians and physician professional associations, have proposed that better information on comparative clinical effectiveness should be a key element of any solution to the us health - care cost crisis. this superficial consensus hides intense disagreements over critical issues essential to any new public effort to promote more comparative effectiveness research (cer). this article reviews the background for these disputes, summarizes the different perspectives represented by policy makers and advocates, and offers a framework to aid both practicing and academic internists in understanding the key elements of the emerging debate. disagreements rage over whether value or cost effectiveness should be a consideration, and how specific patient perspectives should be reflected in the development and the use of such research. the question of how to pay for cer invokes controversies over the role of the market in producing such information and the private (e.g., insurers and employers) versus public responsibility for its production. the financing debate further highlights the high stakes of comparative effectiveness research, and the risks of stakeholder interests subverting any public process. accordingly there are a range of proposals for the federal government s role in prioritization, development, and dissemination of cer. the internal medicine community, with its long history of commitment to scientific medical practice and its leadership in evidence based medicine, should have a strong interest and play an active role in this debate. despite the seriousness of the nation s current economic woes, prominent health policy makers and obama administration officials see rising health - care costs as the key long - term fiscal challenge to the us.1,2 furthermore, many health service researchers and policy analysts assert that if medical practice were more evidence - based, substantial savings could be achieved without loss of access or quality of care.3,4 accordingly, better information on comparative clinical effectiveness is seen to be a critical element of the solution to the national health - care cost crisis,57 and the recently enacted american recovery and reinvestment act of 2009 committed $ 1.1 billion over 2 years to expand these efforts.8 since the marketplace does not produce sufficient quantities of high - quality clinical effectiveness research,6,9 a broad range of stake holders agree on the need for public intervention to provide more and better information on comparative clinical effectiveness.3,4,6,7,1012,13 underneath this broad consensus, however, debate rages on what kind of cer should be promoted, who should fund it, and how it should be conducted. the resolution of this debate has significant implications for both the quality and quantity of future evidence guiding clinical practice. this article will summarize key elements of the current debate, focusing on the policy issues of greatest importance to internists seeking better evidence to inform decisions at the bedside. policy makers have defined comparative effectiveness research (cer) as a rigorous evaluation of the impact of different options that are available for treating a given medical condition for a particular set of patients. such a study may compare similar treatments, such as competing drugs, or it may analyze very different approaches, such as surgery and drug therapy. the analysis may focus only on the relative medical benefits and risks of each option, or it may also weigh both the costs and the benefits of those options.9 the focus of cer is effectiveness under average conditions in diverse populations and clinical practice settings, in contrast to traditional clinical trials investigating intervention efficacy under ideal conditions. comparative effectiveness research will necessarily employ a broad range of research methodologies. these include not only randomized, controlled clinical trials, but also systematic reviews of existing research, practical clinical trials, medical registries, coverage with evidence development projects by payers, and observational studies using large data sets (such as electronic health records from hmo networks).7,9,1416 these raise a variety of methodological challenges as well as controversies regarding the standard of evidence needed to promote, or restrict, use of a clinical service.14,1620,2024 beyond these substantive methodological issues, there is considerable policy debate regarding other aspects of the potential scope for comparative effectiveness research. most advocates insist that cer should have an emphasis on developing information useful from the health - care consumer s point of view (as opposed to the perspective of the payer or the investigator).2527 thus, cer should be informative about the outcomes that patients most care about, such as ability to function and the quality (not just length) of life. advocates are also concerned that cer provides more information than simply the perspective of the average patient ; accordingly, many consumer advocacy groups urge that cer explicitly address the specific needs of various sub - populations (e.g., women, children, ethnic minorities).2528 perhaps no issue in the cer debate is more controversial than the role of assessment of the comparative value or cost of clinical services. this subject was explored in depth in a recent series of annals articles,2931 and anxieties of manufacturers and some patient advocacy groups have been highlighted in the recent editorializing over the comparative effectiveness research provisions in the american recovery and reinvestment act of 2009.3235 in brief, opponents of cer view the inclusion of cost or value within the context of comparative effectiveness research as equivalent to payer - oriented cost - effectiveness analysis (cea). such cea is assumed a precursor to nationwide coverage determinations and rationing of access to expensive but effective therapies. advocates for patients with rare conditions, and for disease groups with particularly expensive treatments, are of course particularly alert to these concerns, as are the developers of expensive innovations.36 this fear of centralized national coverage determinations has even complicated discussion of the nature of reports to be developed by publically supported cer. some advocates would prefer that any publically funded cer effort should provide only summaries of studies, rather than publish recommendations (such as the united states preventive services task force does) or clinical practice guidelines.11,25,28,37 clinical research is time - consuming and costly, thereby contributing to its underproduction by existing private interests.6,9 manufacturers are not required by the food and drug administration to demonstrate comparative effectiveness of new drugs or devices, and physicians routinely introduce new practices and procedures without strong evidence of relative clinical effectiveness. since robust cer studies could result in a loss in market share, manufacturers and providers are not motivated to invest in the full range of clinical effectiveness information of greatest benefit to patients and health professionals. furthermore, health - care purchasers like health plans and large employers can not justify substantial corporate investments in cer. to adequately inform the practice community, therefore, individual health - care purchasers can not gain a unique financial advantage from investments in cer.6,9 similarly, it is difficult to envision a consumers report type of business model using patient subscriptions or per - use charges to finance cer. with over 16% of gross domestic product (gdp) devoted to health care, the research on comparative clinical effectiveness involves high stakes, resulting in winnersproviders or manufacturers whose health care services are found to be superior as well as losers whose services are of lesser value, or even harmful. in a future where health - care decisions are strongly guided by comparative effectiveness research, studies will have enormous financial and other consequences for important health - care stakeholders. consider the implications of a negative study on a manufacturers key source of revenue (e.g., erythrocyte - stimulating agents for amgen) or on a clinical dominant service line (e.g., angioplasty for interventional cardiologists ; hip and knee surgery rehabilitation for inpatient rehabilitation facilities). not surprisingly, the typical annual congressional appropriations dedicated to cer have been miniscule ; support for the only explicit federal cer program, the effective health care program of the agency for healthcare research and quality (ahrq), was only $ 30 million for 2008 compared to over $ 29 billion for the national institutes of health (nih).9,11,16,25 despite the one - time infusion of cer funds to the national institutes of health (nih), ahrq, and the department of health and human services (hhs) under the 2009 recovery act, policy makers are skeptical regarding the feasibility of the annual congressional appropriation ever being a viable source for substantial, sustained investments in cer.38 the appropriations process, a yearly political exercise weighing the allocation of government funds to activities as diverse as space exploration, cancer cures, road construction, veterans care, and head start, is fraught with risks for political intrusion into the selection, production, or dissemination of cer. the political backlash directed through the appropriations process at the agency for health care policy and research (ahcpr) is a case study of these risks. established in 1989, ahcpr had statutory responsibilities for such cer - type activities as outcomes research and practice guideline development. it received substantially increased funding over its predecessor, the national center for health services research, to initiate major initiatives like patient outcomes research teams. these multidisciplinary centers focused on particular medical problems, reviewed and synthesized available research, analyzed practice variations and patient outcomes, and evaluated the effects of disseminating their findings. by 1995, port back pain research and related back pain management guidelines had questioned the value of back surgery in many clinical circumstances as well as the growing use of new surgical appliances like the pedicle screw. the hostility of back surgeons and device manufacturers to this work led to calls to eliminate the agency as an unwarranted intrusion into the physician - patient relationship. signaling this displeasure, in june of 1995 the house / senate budget committee conference report called for elimination of ahcpr. subsequent negotiations in the latter stages of the appropriations process prevented complete de - funding of ahcpr. nonetheless, as a result of this controversy, the agency suffered a severe budget reduction, with attendant loss of programs in outcomes research and clinical guidelines development, and, in 1999, a name change (to ahrq).39,40 this example is the best known of a variety of failed efforts in the us to undertake publically supported comparative assessments of health - care services. informed by these experiences, cer policy makers have sought to secure the advantages of public financing for cer without the complications of annual congressional appropriations. this requires the establishment of a dedicated trust fund, or some other mechanism of mandatory recurring funding, such as a tax on private health insurance, a new payroll tax, or a mandatory tap on the general federal revenue. while these approaches remove the public financing of comparative effectiveness research from the annual appropriations process, they introduce new challenges regarding how these funds should be managed.6,7,9,41 because of the high stakes nature of cer, as well as recent examples of pressure placed on congress and/or federal agencies regarding the interpretation and use of clinical evidence,39,40,42,43 policy makers have proposed a variety of governance options, weighing the risks of political intrusion into the selection, creation, and dissemination of comparative effectiveness information. the cer process will have a variety of steps, each of which will be of great interest to one or more stakeholders. these include the selection of priority topics for research (which drugs, procedures, and/or devices should be scrutinized first), the framing of study questions (e.g., study drug effects on blood pressure control or on quality - adjusted life - years), the choice of research method (e.g., commission a systematic review or a practical clinical trial), the phrasing of study conclusions (e.g., trend toward benefit for glucose control vs unlikely to have any meaningful impact on patient quality of life), and the dissemination of findings.6,7,9,16 special interests will be tempted to exert influence on all these processes, and various executive branch officials could provide a point of contact to steer the results in the direction preferred by a key stake holder. and of course congress can express pleasure or displeasure with study findings through various investigative or legislative actions. in considering options for governing the conduct of comparative effectiveness research, policy makers have identified a number of important principles, exemplified in the institute of medicine (iom) report knowing what works in health care.11 these include accountability, consistency, efficiency, feasibility, objectivity, responsiveness, scientific rigor, and transparency. while each of these principles is relevant to cer, they often represent competing, albeit important, priorities. for example, responsiveness might compete with transparency or efficiency (as defined by avoiding unnecessary duplication of resources), and scientific rigor almost always competes with feasibility and ability to respond quickly. a more complex challenge addressed in the iom report is that of coordination of efforts in order to ensure the authoritativeness of comparative effectiveness research studies. in the us, competing or conflicting clinical research findings may give providers broad discretion to make preference - sensitive or supply - sensitive medical decisions.44,45 accordingly these conflicting lines of clinical evidence may contribute substantially to wasteful variations in health - care expenditures and to inflationary increases in health - care spending. in light of these issues, policy experts have proposed several organizational options to manage this high - stakes scientific work.6,9,16,41,46 one proposed approach to governing publically funded cer is a federally - funded research and development center (ffrdc).46 ffrdcs are hybrid organizations47 that came to prominence at the close of world war ii through national defense and nuclear energy laboratories (e.g., oak ridge, los alamos). they provide executive branch departments with the option of establishing a government funded entity that is operated by a non - federal organization unfettered by civil service rules or other federal management laws. the theoretical advantages of an ffrdc for cer include close government oversight combined with some freedom from regulation. transparency and accountability may suffer, however, since ffrdcs are not subject to laws governing the establishment and management of advisory committees nor to federal employee regulations. furthermore, it is unclear how an ffrdc could achieve the iom s goal of coordinated activities across agencies, having neither authority over executive branch employees nor the ability to readily transfer funds to other government agencies with relevant cer capabilities [e.g., ahrq, nih, veterans administration (va), centers for disease control and prevention (cdc) ]. furthermore, since the ffrdc contract must be periodically renewed, executive branch officials will have ample opportunity to express displeasure and exert influence over cer processes. congress can also use its oversight authority to challenge findings as well as exert annual appropriations pressure on the federal agency parent of the ffrdc. thus, the integrity and clinical utility of the findings of an ffrdc for cer could be highly dependent on the agency, the ffrdc contractor, and the advisory board established to manage it. in the 110th congress, proposals in both the house48 and the senate49 have referred to an ffrdc model as a potential strategy to address the need for enhancing cer. another proposed approach to managing cer is a congressionally chartered non - profit organization,47,50 such as the national academy of sciences with its institute of medicine. a congressionally chartered cer institute could conduct timely and responsive projects on behalf of a governing board and could ensure consistency in developing and applying standards to cer. since such an institute would exist outside the executive branch, however, it would have no authority to coordinate with existing federal cer efforts or infrastructure, nor (at least under current law) to contract with federal agencies to conduct cer projects on its behalf. accordingly, the new institute likely would need to duplicate at least some, and perhaps much, of the functions and infrastructure of existing cer - related federal research agencies to achieve its mission, hardly the most efficient strategy. while achieving a high degree of political insulation congressionally chartered corporations are not subject to federal advisory commission rules or other federal regulations (unless these are explicitly addressed in enabling legislation), so the institute could exert considerable latitude in how it chose to define and implement key iom principles like accountability, objectivity, scientific rigor, and transparency. accordingly, the quality and clinical implications of its cer reports would be highly dependent on the governing board for the institute, the staff it employed, and the research contractors it selected. once such a cer institute were established, it would not be easy to correct deficiencies ; any change in the charter or in the funding would require a law to be passed by both the house and the senate and signed by the president. this barrier to interference gives the institute strong protection from the political process ; given the us history of past political interference in cer efforts and the increasingly high stakes involved in the production of evidence, this approach has its strong proponents. the s. 3408 : comparative effectiveness research act of 2008 introduced by senators baucus and conrad has such a structure, establishing a health care comparative effectiveness research institute with a 21-member board of governors.51 the most conventional approach to a public cer effort is that proposed in the iom report knowing what works in health care : congress should direct the secretary of the us department of health and human services to designate a single entity (the program) with authority, overarching responsibility, sustained resources, and adequate capacity to ensure production of credible, unbiased information about what is known and not known about clinical effectiveness. and the secretary of health and human services should appoint a clinical effectiveness advisory board to oversee the program.11 legislatively, this approach is similar to the house - passed version of comparative effectiveness research, section 904 of the children s health and medicare protection act.9 this approach may achieve a number of the iom s proposed cer principles. accountability is achieved through a federal agency (e.g., ahrq) managing the cer portfolio with the oversight of an advisory board. hhs could achieve efficiencies by using extant infrastructure at ahrq52,53 (e.g., evidence - based practice centers, centers for education and research on therapeutics, etc.) and at the nih (e.g., development and implementation of large clinical trials) to answer priority research questions. transparency would be enhanced through the federal advisory committee act (faca), while the management of the research process by federal agencies contracting with university researchers ensures public accountability. nonetheless, because of the constitution 's separation of powers, a non - political advisory board outside the president s control can merely advise, not direct the actions of a federal research agency ; executive branch leadership retains ultimate control over the agency s responsiveness to recommendations, timeliness of projects, adherence to standards of evidence, and coordination and consistency of cer reports. congress can manage this risk, however, through close oversight and legislative intervention if advisory board recommendations were ignored. for public cer efforts to be successful, there must be substantial and sustained federal investment. in an era of skyrocketing federal deficits, severe economic recession, and demands for increased public investments in traditional biomedical research, achieving such financing will be a challenge. even if the cer financing and governance problems are satisfactorily resolved, other controversial policy issues remain, including the range of stake holder perspectives and expertise to include in the governing board ; the processes to assure proper public input into cer projects and publications ; the development of findings and implications (i.e., recommendations, guidelines, etc.) ; and the role for value (e.g., cost, quality of life, and cost effectiveness) in evaluating and reporting comparative clinical effectiveness. of course, for any new public investment in cer to realize a return to the taxpayers, the new information must actually be put to use by payers, providers, and patients to increase the use of highly effective treatments and minimize the use of ineffective or inefficient ones.4,9 in considering solutions to other health policy dilemmas, leaders in the american college of physicians (acp) and elsewhere have recently looked across our borders to find useful models abroad.54 there are several vibrant and robust examples of policy and practice - relevant cer in other countries,11,55 but while these models are encouraging, they likely provide insufficient guidance on us implementation. the reasons involve not just differences in law and polity, but more substantively, the much larger role health care plays across many sectors of the us economy. continued health care expenditure growth and technology dissemination are built into the business plans of many prominent corporations and institutions. therefore, the stakes for cer may be much higher for more, and more prominent, advocates in the us than in many other industrialized countries, and the challenges to manage the process thereby more complex. internal medicine, with its long history of commitment to scientific medical practice and its leadership in evidence based medicine we can not countenance, nor can the nation afford, another federal effort in cer to be paralyzed by a twenty - first century version of ahcpr s back pain malady, nor can we allow an unaccountable and opaque cer institute to use private contractors to serve special interests ; such an outcome could also be harmful, risking misinformation to patients and clinicians while wasting money on redundant projects and bureaucracy. to solve our health - care problems, the us would be well served by reestablishing the appropriate function of federal health research agencies in public concerns. given the broad range of interests in cer, it is appropriate, even necessary, for an independent, multi - stakeholder process (with a prominent role for clinicians and consumers) to identify the clinical questions that the american people most need answering. and then the substantial infrastructure already developed through past investments in ahrq, nih, va, cdc, etc., must be put to effective use. these resources will be critical both in answering the clinical effectiveness questions confronted by patients and their physicians, as well as solving the implementation science and knowledge translation problems familiar to administrators and policymakers. | backgroundpolicy makers across the political spectrum, as well as many clinicians and physician professional associations, have proposed that better information on comparative clinical effectiveness should be a key element of any solution to the us health - care cost crisis. this superficial consensus hides intense disagreements over critical issues essential to any new public effort to promote more comparative effectiveness research (cer).methods and resultsthis article reviews the background for these disputes, summarizes the different perspectives represented by policy makers and advocates, and offers a framework to aid both practicing and academic internists in understanding the key elements of the emerging debate. regarding the fundamental question of what is cer, disagreements rage over whether value or cost effectiveness should be a consideration, and how specific patient perspectives should be reflected in the development and the use of such research. the question of how to pay for cer invokes controversies over the role of the market in producing such information and the private (e.g., insurers and employers) versus public responsibility for its production. the financing debate further highlights the high stakes of comparative effectiveness research, and the risks of stakeholder interests subverting any public process. accordingly there are a range of proposals for the federal government s role in prioritization, development, and dissemination of cer.conclusionthe internal medicine community, with its long history of commitment to scientific medical practice and its leadership in evidence based medicine, should have a strong interest and play an active role in this debate. |
yoga therapeutics is an increasingly appreciated discipline, particularly in india where it is overseen by the ministry of health and family welfare 's department of ayurveda, yoga and naturopathy, unani, siddha and homeopathy (ayush). not many studies have assessed the influence of integral yoga practices on psychological and health variables, establishing many possible benefits. this study apart from establishing the benefits of internal yoga investigates the relationships between the variables themselves. up till now, this correlation aspect has not been much investigated, since this requires simultaneous measuring of many variables for a large group, and then to correlate. it measured many variables on a group of volunteers large enough to identify significant correlations between variables. specifically it evaluated correlations between measures of sustained attention, emotional intelligence, general health and guna personality before and after a one - month yoga instructor 's course. sustained attention is the capacity to attend to a task for a required period of time. the ability may be related to steadiness of focus, which in turn is a sign of mental stability. sustained attention may thus depend on emotionality. a self - controlled study on 20 male volunteers measured immediate effects of three yoga - breathing techniques on performance of a letter cancellation task (lct). practice of alternate nostril yoga breathing (nadi suddhi pranayama) and right nostril yoga breathing (surya anuloma pranayama) improved task performance. the authors concluded that anxiety - reducing effects of pranayama might have contributed to better lct performance, since this requires selective attention. patil and telles measured the effects of two yoga - based relaxation techniques on six letter cancellation (slc) test performance, a task requiring selective attention, concentration, visual scanning abilities and repetitive motor response. khemka in a two separate control studies measured immediate effects of two relaxation techniques and immediate effect of kapalbhati and breath awareness on healthy volunteers on performance of slc task and digit letter substitution (dls) task and found significant increase in both task performances of attention in all four interventions of two studies. emotional intelligence has been variously defined as the ability to control one 's own and other 's feelings and emotions, to discriminate amongst them, and to use this information to guide one 's thinking and actions, the capacity for recognizing our own feelings and those of others, for motivating ourselves, and for managing emotions well in ourselves and in relationships. goleman claims that about 80% of a person 's success in life depends on emotional competencies as measured by eq. a controlled study of 170 subjects by kumari assessed the effects of a six week part - time self management of excessive tension (smet) program for managers on their emotional well - being. general health is not just absence of disease, but is well being on physical, mental and social levels. many studies have found improvements due to yoga practice. a small randomized control trial of sahaja yoga evaluated effects on depressive disorders. anxiety and depression scores reduced more in the yoga group. in a study of yoga 's effects on distress symptoms in survivors of the 2005 tsunami, self - rated fear, anxiety, sadness and disturbed sleep decreased significantly along with heart and breathing rates for indigenous people. a review of sudarshan kriya yoga for stress, anxiety, and depression concluded that the technique enhances well being, mood, attention, mental focus and stress tolerance, and seems beneficial as a low risk, low cost adjunct to treatment of many conditions, and for criminal rehabilitation. a study of hatha yoga 's effects in 107 healthy adults found improved physical and mental health and well - being. a controlled pilot study evaluating a comprehensive yogic breathing program observed reduced anxiety, depression and stress, and increased optimism in the experimental group. an rct comparing meditation stress - management and education programs as adjuncts to pharmacotherapy for anxiety disorder found reduced anxiety and depression. guna personality variables are three vedic personality patterns named sattva, rajas and tamas : sattva brings calmness, lightness, illumination, control, and the beginning of selflessness, all triggering constructive action ; rajas is a more compulsive tendency to action and selfishness, producing pain, and a restless mind ; tamas manifests as lethargy, drowsiness or sleepiness, blocks, and stagnation. according to vedic psychology, these gunas constantly control a person 's tendencies : everyone is helplessly driven to action by the gunas. an evaluation of yoga 's impact on the gunas, and on self - ideal disparity found significant correlations between self and ideal self for the yoga group, but not for controls. another study found that the hare krishna mantra increased sattva and decreased rajas and tamas. generally, yoga aims to improve guna quality, until sattva dominates all the time. correlations between attention and gunas : gunas are related to cognitive characteristics, perceptual acuity and field independence, intelligence, memory, and attention - concentration. negative correlations are reported between tamas and field independence, short - term memory, intelligence and attention - concentration, and positive correlations between sattva and general intelligence, short - term memory and attention - concentration. correlations between health and gunas : a study of rajas and tamas in psychological disturbance found the two predominant factors in the patient population, suggesting that elevated levels of rajas and tamas give rise to mental ill health. it is generally agreed that predominance of rajas and tamas leads to psychological disturbance. sattva guna, on the other hand, leads to positive mental health and optimal functioning. similarly, a study of 100 cancer patients found that patients high in rajas or tamas are more prone to cancer. on the other hand, an rct on gunas and health found sattva improved more in the yoga group than in controls. general health status improved in both groups, consistent with the finding that, while sattva correlates positively with health, rajas correlates negatively. based on previous studies on various variables and their correlations the slc and dls tests assess selective, focused and sustained attention, visual scanning and activation and inhibition of rapid responses. the tests identify major components of performance : detection, perception, recognition, processing and integration. n. k. chadha developed the adapted emotional intelligence (eq) test in 2003 as discussed by dalip singh. he compiled situations experienced by people in real life situations deemed neutral with regard to social desirability, and selected to avoid response bias. the general health questionnaire (ghq-28) contains 28 items in four 7-item sub - scales : a - somatic symptoms, b - anxiety / insomnia, c - social dysfunction, and d - severe depression. the guna - based personality test uses an inventory of 88 items, characteristic of sattvic, rajasic and tamasic personalities collected from the literature. no previous study has evaluated the effect of integral yoga in a residential set up on healthy volunteers and consistency of correlations between these variables before and after a yoga intervention and hence this study was undertaken. the slc and dls tests assess selective, focused and sustained attention, visual scanning and activation and inhibition of rapid responses. the tests identify major components of performance : detection, perception, recognition, processing and integration. n. k. chadha developed the adapted emotional intelligence (eq) test in 2003 as discussed by dalip singh. he compiled situations experienced by people in real life situations deemed neutral with regard to social desirability, and selected to avoid response bias. the general health questionnaire (ghq-28) contains 28 items in four 7-item sub - scales : a - somatic symptoms, b - anxiety / insomnia, c - social dysfunction, and d - severe depression. the guna - based personality test uses an inventory of 88 items, characteristic of sattvic, rajasic and tamasic personalities collected from the literature. no previous study has evaluated the effect of integral yoga in a residential set up on healthy volunteers and consistency of correlations between these variables before and after a yoga intervention and hence this study was undertaken. subjects comprised 108 healthy volunteers (male and female, aged 17 - 63 years, mean 31 10.75) attending one - month, residential, yoga instructor 's courses at swami vivekananda yoga anusandhana samsthana (svyasa), bangalore during august 2005 and february 2006. poor health as indicated by personal data, taking medication, or initial ghq value over 5. subjects were assessed in a single group before and after the yoga practice. an integrated approach to yoga therapy (iayt) module includes yogasanas, pranayama, meditation, kriyas, lectures, singing, and yogic games. lecture topics included the four paths of yoga, pancha - koshas, diet, and stress management.[3133 ] subjects took the five tests named below in a single sitting at the same time of day immediately before and after the one - month yoga training period. they were not allowed to talk after receiving test sheets, and instructed to remain in place until everyone had finished, so as not to disturb others. six letter cancellation (slc) test.digit letter substitution (dls) test.emotional intelligence (eq) test.general health questionnaire (ghq) test.guna personality (tamas, rajas, sattva) test six letter cancellation (slc) test. guna personality (tamas, rajas, sattva) test the slc test comprises a worksheet specifying six target letters to be cancelled from a working section of 22 rows by 14 columns randomly arranged letters of the alphabet. subjects are asked to cancel as many target letters as possible in 90 s. the dls test consists of a similar test worksheet, containing a key, pairing digits 1 - 9 with letters of the alphabet. subjects are asked to substitute as many target digits as possible in 90 s. a similar digit letter substitution task has been used on an indian population confirming its validity to study immediate effects. chadha 's eq test consists of 15 questions, based on 15 socially neutral situations, offering five possible answers for each carrying scores 0 - 20. interpretation is as follows : the test was standardized on broad populations in indian society ; hence it is used here. the ghq 28 questionnaire provides individual diagnostic profile information : four 7-item sub - scales are based on factor analysis, with factor structures consistent with the original studies. internal consistency, and reliability : cronbach 's alpha, 0.85, and validity, 0.76. better than usual, same as usual, worse than usual, and much worse than usual, scored : 0 - 0 - 1 - 1. pathak developed a triguna - based personality test, with items taken from guna depictions in sankhya karika. it consists of 88 items : 24 sattva, 34 rajas and 30 tamas, scored on a five point scale ranging from not at all to very much. low between scale correlations were reported. test reliability coefficients were reported : sattva r = 0.62, rajas r = 0.83 and tamas r = 0.70. poor health as indicated by personal data, taking medication, or initial ghq value over 5. an integrated approach to yoga therapy (iayt) module includes yogasanas, pranayama, meditation, kriyas, lectures, singing, and yogic games. lecture topics included the four paths of yoga, pancha - koshas, diet, and stress management.[3133 ] subjects took the five tests named below in a single sitting at the same time of day immediately before and after the one - month yoga training period. they were not allowed to talk after receiving test sheets, and instructed to remain in place until everyone had finished, so as not to disturb others. six letter cancellation (slc) test.digit letter substitution (dls) test.emotional intelligence (eq) test.general health questionnaire (ghq) test.guna personality (tamas, rajas, sattva) test six letter cancellation (slc) test. guna personality (tamas, rajas, sattva) test the slc test comprises a worksheet specifying six target letters to be cancelled from a working section of 22 rows by 14 columns randomly arranged letters of the alphabet. subjects are asked to cancel as many target letters as possible in 90 s. the dls test consists of a similar test worksheet, containing a key, pairing digits 1 - 9 with letters of the alphabet. subjects are asked to substitute as many target digits as possible in 90 s. a similar digit letter substitution task has been used on an indian population confirming its validity to study immediate effects. chadha 's eq test consists of 15 questions, based on 15 socially neutral situations, offering five possible answers for each carrying scores 0 - 20. interpretation is as follows : the test was standardized on broad populations in indian society ; hence it is used here. the ghq 28 questionnaire provides individual diagnostic profile information : four 7-item sub - scales are based on factor analysis, with factor structures consistent with the original studies. internal consistency, and reliability : cronbach 's alpha, 0.85, and validity, 0.76. better than usual, same as usual, worse than usual, and much worse than usual, scored : 0 - 0 - 1 - 1. pathak developed a triguna - based personality test, with items taken from guna depictions in sankhya karika. it consists of 88 items : 24 sattva, 34 rajas and 30 tamas, scored on a five point scale ranging from not at all to very much. low between scale correlations were reported. this found slc, eq, tamas, rajas and sattva pre - data to be normally distributed (p > 0.05), while those of dls, eq, and ghq were not normally distributed (p < 0.05). correlation coefficients and their significance are given in tables 2 to 4 where results for each class of data are presented. pre - post mean, sd, % change and p value of each variable n=108 correlations of emotional intelligence (eq) correlations of general health (ghq) and gunas correlations between gunas the effect of integral yoga practices shows significant improvement in all the variables but not much in sattva. improvement in sustained attention, emotional intelligence, general health and rajas changes were at p < 0.001 significant level while tamas at p < 0.01 significant level and sattva at p<0.447 insignificant level after integral yoga practices as shown in table 1. correlation coefficients range from -1.00 to + 1.00, extreme values indicating a precise algebraic relationship. similar measures are highly correlated for the two measures of sustained attention, slc and dls : positive (p < 0.001) both before (r = 0.616) and after (r = 0.55) the intervention. correlations between these and other variables were not correlated consistently before and after e.g. slc was significantly correlated only before the intervention with eq, tamas and sattva (p < 0.05), while dls was significantly correlated only after the intervention with eq (p < 0.05), a curious inconsistency. tables 24 list correlations among the other variables. table 4 gives internal correlations of gunas. sattva correlated negatively with tamas while rajas correlated consistently with tamas before and after yoga (see below, discussion section). finally, since pre and post intervention data are available, we present correlations between pre- and post - data for each variable in table 5. the results of the effect of the integral yoga practices on psychological and health variables showed significant improvement in all measures except sattva guna where improvement did not reach significant level. deshpande measured the same health variables (ghq) and guna variables in a randomized control trial in normal healthy volunteers in bangalore city and found significant improvement (p<0.001) on all the four domains in both yoga and exercise groups while increase in sattva in both the groups and decrease of rajas and tamas in yoga and physical exercise group after the intervention in a non residential set up with daily 1 h classes for eight weeks. ghq is scored according to negative health findings and so negative percentage value indicated tendencies to better health. both deshpande scores of ghq and gunas were almost in line with the result of this study. similarly sustained attention (slc and dls) scores of khemka controlled studies of integral yoga practices on healthy volunteers were also in lines with the result of this study. a controlled study of kumari who assessed emotional intelligence (eq) on corporate managers giving intervention of cyclic meditation found significant increase in eq as in this study of integral yoga practices. the results of all above studies on various psychological and health variables show beneficial effect of yoga practices in healthy volunteers. therefore, one may conclude that yoga imparts significant benefits to healthy people also. in correlation studies, pairs of variables are usually correlated if there is overlap between the various brain regions that each involves, even secondarily. sustained attention variables, though well correlated with each other, do not correlate significantly with most other variables. the reason is probably that there is little overlap between the principal brain regions utilized. sustained attention is primarily governed by cortical areas concerned with various stages of cognition. in healthy subjects, cortical areas concerned with variables like sustained attention is strongly dependent on tamas probably due to common influence of stress, anxiety or depression, on both task performance and personality. cognitive processing is mostly cortical, but emotions and imbalances are more connected to mid - brain centers, which also modulate cortical function. sattva on the other hand may be postulated to be a state where modulation of cortical processing is optimal. common use of brain regions, which tends to correlate variables with each other as seen between eq and guna personality, may be predicted from traditional guna analysis. detailed characteristics of guna personality types identify sattvic people as having higher eq, rajasic people as slightly negative, and tamasic people as low in eq. this sequence is reflected in pre - pre and post - post correlations between eq and sattva, rajas and tamas : positive for the first, a little negative for the second, and far more negative for the third. one way to understand yoga 's strong positive effect on eq is that it first increases alertness (exemplified by increases in sustained attention) ; next it erases negative influences on personality (exemplified by decreases in tamas) ; and finally this leads to increased sensitivity to others feelings and emotions (exemplified by increases in sattva). previous failure to observe the eq - guna correlations identified here may have been due to a more restricted sample : industry managers, with a more limited range of personalities. our observations of independence of sattva and rajas agree with previous results as do negative correlations between sattva and tamas. however, our study found high correlation between rajas and tamas, not seen previously. these correlations may be due to a restricted range of subjects (healthy), manifesting less tamas than a general population. kumari 's result suggests it is probably not due to inherent weakness in the questionnaire. when these correlations are compared with each other, many offer self - consistent results ; negative correlations between scores on the slc test and tamas, and between tamas and sattva are consistent with positive correlations observed between slc scores and sattva. remarkably, although the slc and dls tests are considered equivalent, they produced inconsistent correlations with the variables eq, tamas and sattva. it is not clear whether these are chance results or if they effectively distinguish between slc and dls tests. ghq is scored according to negative health findings, so high scores indicate susceptibility to disease, particularly psychic illness. thus, positive r - value correlations with ghq scores indicate a tendency to fall sick, while negative correlations indicate tendencies to better health. here the result that tamas correlate negatively with sattva confirms that the two oppose each other. post - post correlations between ghq and tamas increased, while correlations between ghq and sattva and rajas remained similar. the failure of tamasic personalities to improve in health increased the negative correlations between ghq and tamas. correlations observed between the gunas themselves do not necessarily indicate poor choice of variables, as normally holds for tests of individual differences in personality. rather, they tend to confirm that elevated levels of tamas (and to some extent rajas) prevent subjects from manifesting high levels of sattva. a single month of integral yoga practices imparts significant benefits to healthy volunteers in all psychological and health variables. it improves the personality of the healthy person by increasing sattva and decreasing rajas and tamas. cognitive processes involved in sustained attention tests did not correlate with health and personality tests. this may be because the major brain regions involved in cognitive abilities were sufficiently different from those of personality for this group of healthy subjects. enhancement in the functioning of one would not then seem to affect the other. on the other hand, it suggests that regular yoga practice benefits people for deeper reasons than immediate effects on the physical body. as yoga practice transforms people 's dominant guna making them more sattvic, their ability to spontaneously maintain high levels of health increases while not being affected by unhelpful emotions. thus, it is seen that integral yoga practices impart significant benefits and measuring correlations between different variables can yield significant information of potential importance that would not otherwise be available. | objective : certain psychological and health variables are commonly measured in india. this study evaluates the effects of integral yoga practices on these variables and also the consistency of correlations observed between them.materials and methods : the study was a pre - post intervention study. the variables were measured at the beginning and the end of a one - month yoga course. there was no control group.the study was carried out at swami vivekananda yoga anusandhana samsthana (s - vyasa) university, in its rural campus south of bangalore. based on health criteria, 108 subjects were selected out of 198 volunteers to form the experimental yoga group. ages ranged from 17 to 63 years. the yogasanas (postures), pranayama (breathing exercises), relaxation techniques, meditation, chanting and lectures were the components of yoga intervention. the variables measured were sustained attention, emotional intelligence eq, general health ghq, guna personality sattva, rajas and tamas.results:significant pre - post changes were found in all variables. significant correlations were found between the following pairs : the two sustained attention variables ; emotional intelligence and general health ; ghq and tamas ; sattva and tamas ; and rajas and tamas.conclusion:the study shows that there were significant changes in all variables (p < 0.001) except in sattva. it also confirms that eq and general health variables correlate significantly with each other and negatively with tamas. eq and tamas form positive and negative predictors of health respectively. sattva correlates positively with eq suggesting that a sattvic personality indicates better self - control. this suggests that, by improving guna personality, long - term yoga practice may stabilize eq. |
ethics : the animal care and use committee of the national institute of animal health (niah) approved all animal procedures prior to initiation of this study (authorization numbers : 12 - 027, 12 - 056, 12 - 081, 13 - 024, 13 - 054 and 14 - 009). all experimental infections were performed in cubicles whose sizes have approximately 14 m in a high - containment facility at the niah. experimental infections in non - vaccinated animals : full details of experimental infections (i), (ii) and (iii) have already been published [11, 14 ]. brief details of the experimental infections that provided serum samples for this study are as follows : (i) two 6-month - old holstein cattle housed in separate cubicles were inoculated with 1 ml of 10 50% tissue culture infectious dose (tcid50) of the fmdv o / jpn/2010 - 1/14c by an intradermal route. at 1 day post - infection (dpi), two additional 6-month - old holstein cattle were housed with the infected cattle. (ii) two 4-month - old japanese saanen goats were inoculated with 1 ml of 10 tcid50 of the o / jpn/2010 - 1/14c by an intradermal route. at 1 dpi, 2 additional 4-month - old japanese saanen goats were housed with the infected goats. (iii) thirteen 2-month - old pigs were inoculated with 1 ml of 10 or 10 tcid50 of the fmdv o / jpn/2010 - 1/14c by intranasal or intraoral route. experimental infections in vaccinated animals : (iv) seven 3-month - old holstein cattle were administered intramuscularly an inactivated fmdv vaccine (six 50% protection dose (pd50), serotype o, o manisa strain, aftpor, merial, lyon, france). at 3 or 30 days post - vaccination (dpv), the vaccinated cattle were inoculated with 1 ml of 10 tcid50 of the fmdv o / jpn/2010 - 1/14c by an intradermal route. (v) six 2-month - old pigs were administered intramuscularly the fmdv vaccine (6 pd50). at 3 or 30 dpv, the vaccinated pigs were inoculated with 1 ml of 10 tcid50 of the fmdv o / jpn/2010 - 1/14c by an intraoral route. collection of clinical samples from the animals : the clinical samples were collected routinely as shown in tables 1 table 1.detection of viral genes from clinical samples by rt - pcr assay and of antibodies by lpbe and the priocheck kit in infected cattle without vaccination, 2 table 2.detection of viral genes from clinical samples by rt - pcr assay and of antibodies by lpbe and the priocheck kit in infected goats without vaccination, 3table 3.detection of antibodies by lpbe and the priocheck kit in infected pigs by the intranasal and intraoral routes without vaccination, 4table 4.detection of viral genes from clinical samples by rt - pcr assay and of antibodies by lpbe and the priocheck kit in infected cattle with vaccination, 5table 5.detection of viral genes from clinical samples by rt - pcr assay and of antibodies by lpbe and the priocheck kit in infected pigs with vaccination. sera were collected from cervical veins using a vacuum blood collection tube (venoject ii, terumo corporation, tokyo, japan). saliva was collected from oral cavities using a roll - shaped synthetic saliva collector (salivette, sarstedt kk, tokyo, japan) and forceps. nasal swabs were collected from nasal cavities using a cotton swab (men - tip, jcb industry limited, tokyo, japan). rna extraction and rt - pcr : viral rnas were extracted from the clinical samples using the high pure viral rna kit (roche diagnostics, basel, switzerland). the fmdv - specific genes were detected by an rt - pcr assay using primers fm8 and fm9, which can amplify a portion of a 3d region of an fmdv genome. lpbe and nsp - elisa : an lpbe (institute for animal health, surrey, u.k.) was performed for detection of antibodies to structural proteins (sps) of fmdv according to manufacturer s instructions. the priocheck fmdv ns (thermo fisher scientific, waltham, ma, u.s.a.) was performed for detection of antibodies to the non - structural protein 3abc of fmdv according to the manufacturer s instructions. infected cattle without vaccination : in cattle 1 and 2, which were inoculated with the fmdv, clinical signs, such as vesicular development on the tongues, inside lips, snouts and feet, excess salivation, and lameness, were initially found between 1 and 6 dpi. viral genes were detected from the clinical samples between 1 and 9 dpi (table 1). the days when the antibodies were first detected in the priocheck kit were delayed for 2 days from those in the lpbe. in cattle 3 and 4, which were housed with the infected cattle, clinical signs, such as vesicular development on the tongues, inside lips, snouts and feet, and excess salivation, were initially found between 4 and 6 days post - contact (dpc). viral genes were detected from the clinical samples between 2 and 8 dpc (table 1). the days when the antibodies were first detected in the priocheck kit were delayed for 3 days from those in the lpbe. infected goats without vaccination : in goats 1 and 2, which were inoculated with the fmdv, clinical signs, such as vesicular development on the feet, were initially found between 3 and 4 dpi. viral genes were detected from the clinical samples between 1 and 8 dpi (table 2). in the priocheck kit, the antibodies were detected from 5 and 4 dpi, respectively. the day when the antibody was first detected in the priocheck kit was 1 day after that by the lpbe in goat 1 ; in goat 2, antibodies were detected by both tests on the same day. in goats 3 and 4, which were housed with the infected goats, clinical signs, such as vesicular development on the tongue, inside lip, snout and feet, and excess salivation, viral genes were detected from the clinical samples between 3 and 11 dpc (table 2). the antibodies were first detected 1 day later by the priocheck kit than by the lpbe. infected pigs by the intranasal route without vaccination : in pigs 124 to 126, which were inoculated with 10 tcid50 of the fmdv, clinical signs, such as vesicular development on the inside lips, snouts, teat and feet, excess salivation, lameness, depression and reduced appetite, were initially found between 3 and 9 dpi. the days when viral genes were detected in pigs 124 to 126 were as described previously. as previously reported, antibodies were detected from 8, 8 and 7 dpi in the lpbe, respectively (table 3). the days when the antibodies were first detected in the priocheck kit were delayed for 1, 1 and 6 days from those in the lpbe, respectively. in pigs 121 to 123, viral genes and lpbe antibodies were not detected during the experimental period as described previously. in the priocheck kit, antibodies were not also detected in this study. infected pigs by the intraoral route without vaccination : in pigs 131 to 133, which were inoculated with 10 tcid50 of the fmdv, clinical signs, such as vesicular development on the tongue, inside lips, snouts and feet, excess salivation, lameness and depression, were initially found between 7 and 10 dpi. the days when viral genes were detected in pigs 131 to 133 were as described previously. as previously reported, antibodies were detected from 13, 10 and 12 dpi in the lpbe, respectively (table 3). the days when the antibodies were first detected in the priocheck kit were delayed for 1, 1 and 2 days from those in the lpbe, respectively. in pigs 134 to 137, which were inoculated with 10 tcid50 of the fmdv, clinical signs, such as vesicular development on the snout and feet, excess salivation, lameness, depression and reduced appetite, were initially found between 1 and 6 dpi. the days when viral genes were detected in pigs 134 to 137 were as described previously. as previously reported, antibodies were detected from 6, 6, 7 and 7 dpi in the lpbe, respectively (table 3). the antibodies were detected from 7, 7, 9 and 13 dpi, respectively. the days when the antibodies were first detected in the priocheck kit were delayed for 1, 1, 2 and 6 days from those in the lpbe, respectively. infected cattle with vaccination : in cattle 0, 1 and 6, which were vaccinated at 30 days before virus infection (dbv), any clinical signs, such as vesicular development, excess salivation, lameness, depression and reduced appetite, were not found, except for development of lesions on sites of the virus infection. viral genes were detected from the clinical samples between 1 and 7 dpi ; however, they were not detected in cattle 1 during the experimental period (). antibodies were detected from 23 dbv in the lpbe. using the priocheck kit, the antibodies were detected from 6, 3 and 7 dpi, respectively. in cattle 3, 7, 2 and 9, which were vaccinated at 3 dbv, clinical signs, such as vesicular development on the tongues, inside lips, snouts and feet, viral genes were detected from the clinical samples between 1 and 7 dpi (table 4). antibodies were detected from 3, 3, 2 and 2 dpi in the lpbe, respectively. the antibodies were detected from 5, 5, 6 and 5 dpi, respectively. however, in cattle 2, no antibody was detected at 14 dpi in the priocheck kit. infected pigs with vaccination : in pigs 1 to 3, which were vaccinated at 30 dbv, any clinical signs, such as vesicular development, excess salivation, lameness, depression and reduced appetite, were not found. viral genes were detected from the clinical samples between 1 and 7 dpi (table 5). an antibody was first detected at 3 dbv. in pig 2, although an antibody was first detected at 20 dbv, there were several days when antibodies were not detected until 1 dbv. in pig 3, antibodies were detected at 10 and 17 dbv ; however, antibodies were not detected from 3 dbv to 4 dpi. in the priocheck kit, antibody responses of the pigs also varied. in pigs 1 and 2, antibodies were not detected during the experimental period in this study, except for 7 dpi in pig 2. an antibody was first detected at 18 dpi in pig 3. in pigs 5 and 6, which were vaccinated at 3 dbv, clinical signs, such as vesicular development on the tongues, snout and feet, however, any clinical signs, such as vesicular development, excess salivation, lameness, depression and reduced appetite, were not found in pig 4. viral genes were detected from the clinical samples between 1 and 18 dpi (table 5). antibodies were detected from 4, 11 and 6 dpi in the lpbe, respectively. in the priocheck, antibodies were detected from 11 and 7 dpi in pigs 5 and 6, respectively ; however, the antibodies were detected intermittently between 11 and 34 dpi in pig 4. the tests are in principle divided into 2 kinds of tests ; those detecting antibodies to sps and to nsps of fmdv, respectively. there are 7 serotypes in fmdv : a, o, c, sat1, sat2, sat3 and asia1. an appropriate antigen needs to be used for measuring antibodies to a seeking serotype. in contrast to tests for sps, tests for nsps are independent of serotypes. tests for nsps therefore may be more convenient in countries where several serotypes of fmdvs are prevalent. according to the terrestrial animal health code, tests for both sps and nsps can be used for serological surveillance in areas where animals are not vaccinated. in practice, tests for nsps have been frequently used for serological surveillance of cattle in south american countries [1, 2 ]. however, the results in this study showed that the first detection of antibodies by the priocheck kit was delayed compared to that by the lpbe. therefore, tests for sps should be used for surveillance, especially in an outbreak in fmd - free countries, because tests for sps may be able to detect fmdv infections earlier than tests for nsps. in our previous study, the sensitivities of three nsp - elisa kits were significantly low in infected animals without vaccination tested in the 2010 epidemic in japan. in contrast to our results, the sensitivities of several nsp - elisa kits were reported to be high in infected animals without vaccination [2, 3, 5, 16 ]. both of the results were completely inconsistent with each other. in our previous study, the antibody titers of serum samples in the lpbe were the focus of inquiry regarding the inconsistent results, because precise dates when the animals had been infected with the fmdv in the field were unknown. serum samples that showed relatively high antibody titers were speculated to have been collected from animals a longer time after virus infection. however, many serum samples that showed relatively high antibody titers in the lpbe showed negative results in the nsp - elisa kits. to explore the reasons for this inconsistency, we focused on the relationships between the results of the rt - pcr assay, lpbe and nsp - elisa kit in this study. as mentioned in tables 1 and 2, and the chapter of the results of infected pigs by the intranasal and intraoral routes without vaccination, basically, the viral genes by the rt - pcr assay, the antibodies by the lpbe and the antibodies by the priocheck kit were sequentially detected in infected animals without vaccination. detection of the viral genes by the rt - pcr assay therefore meant that the samples were collected early after the fmdv infection. in contrast, relatively high antibody titers did not usually mean that the samples were collected late after the animals were infected with fmdv, because high antibody titers were observed even at early dpi when the viral genes were detected simultaneously by the rt - pcr assay. from these results, early collection of samples after the animals were infected with an fmdv therefore may be the reason for the significantly low sensitivities of the nsp - elisa kits in the serum samples collected from the animals in the field in the 2010 epidemic in japan. antibody responses were influenced by viral doses for infection in the pigs (table 3). the antibodies in the lpbe were detected earlier in pigs 134 to 137 inoculated with 10 tcid50 of the isolate than in pigs 131 to 133 inoculated with 10 tcid50 of the isolate. in the antibody titers, the serum sample collected from pig 133 at 13 dpi showed the highest antibody titer, as 2048. the pig and pig 131 were assumed to be infected by horizontal transmission from pig 132. therefore, high viral dose infection from pig 132 may stimulate the highest antibody titer in pig 133. in previous studies [2, 3, 5, 16 ], sensitivities of several evaluated nsp - elisa kits were relatively high in infected animals without vaccination. on the other hand, sensitivities of the nsp - elisa kits were considerably low in infected animals with vaccination in the studies. as a reason for these differential sensitivities, it was speculated that viral replication is limited in infected animals with vaccination and that serological conversion to nsps occurs more slowly and in a lower proportion than in infected animals without vaccination. however, the precise reason why there are cases in which antibodies to nsps are not detected in infected animals with vaccination has not yet been confirmed. a report proposed that studies should be encouraged to define pathogenicity including antibody responses to nsps in infected animals with vaccination. in this study, antibodies were detected fully from serum samples collected from infected cattle with vaccination by the priocheck kit, although the first detection dates were later than those in the lpbe (table 4). the priocheck kit therefore may be applicable for serological surveillance in cattle after emergency vaccination would be practiced in an fmd outbreak. however, it should be noted that tests for nsps are generally considered reliable at a herd level [1, 16 ]. in addition, antibody responses to nsps in infected cattle with vaccination need to be further evaluated in different experimental conditions, such as viral infected doses and routes. on the other hand, the study of the infected pigs with vaccination showed the different results. the priocheck kit did not detect antibodies in pig 1 during the experimental period, and in pig 2, it detected antibodies at only 7 dpi, although both the pigs were confirmed to be infected with the fmdv by the results of the detection of the viral genes from the clinical samples (table 5). well - vaccinated animals may become subclinically infected, even if they are exposed to a sufficient virus challenge [8, 15 ]. similarly, in this study, pigs 1 to 4 did not show any clinical signs, even though viral infection occurred (table 5). the priocheck kit therefore may not be suitable to apply for serological surveillance in pigs after emergency vaccination would be practiced in an fmd outbreak, because infected pigs with vaccination may not be detected by either serological or clinical surveillance. if the priocheck kit would be applied as an assay for serological surveillance in pigs after emergency vaccination would be practiced in an fmd outbreak, statistical sufficient numbers of pigs should be examined by the kit. in addition, a high sensitive assay, such as an rt - pcr assay, should be applied simultaneously, because other pigs, which are in an acute stage of infection, may exist in the same premise. however, rapid sampling from the large numbers of pigs would be hard to perform during an outbreak. to reveal availability for serological surveillance in pigs, the priocheck kit needs to be further evaluated using infected pigs with vaccination before it is used for surveillance purposes. in addition, evaluation of these two elisa kits for antibody detection in infected goats with vaccination might be needed, although goats along with sheep are not major domestic animals in japan. in conclusion, the following was confirmed in this study : (i) the lpbe can detect antibodies in infected animals without vaccination earlier than the priocheck kit ; (ii) the significantly low sensitivities of the nsp - elisa kits in the previous study may have been due to the early collection dates of samples after animals were infected with fmdv ; and (iii) the priocheck kit may not be able to detect antibodies in infected pigs with vaccination. these results will be valuable for authorities in choosing adequate control measures for possible fmd outbreaks in the future. | an elisa kit for detection of antibodies to a nonstructural protein of foot - and - mouth disease (fmdv) was further evaluated using sequentially collected serum samples of experimentally infected animals, because the sensitivity of the kit used in a previous study was significantly low in field animals. the kit fully detected antibodies in infected animals without vaccination ; however, the first detections of antibodies by the kit were later than those by the liquid - phase blocking elisa that is used for serological surveillance in the aftermath of outbreaks in japan, for detection of antibodies to structural proteins of fmdv. additionally, although the kit effectively detected antibodies in infected cattle with vaccination, there were several infected pigs with vaccination for which the kit did not detect antibodies during the experimental period. taken together, the kit may not be suitable for serological surveillance after an fmd outbreak either with or without emergency vaccination in fmd - free countries. |
diffuse idiopathic skeletal hyperostosis (dish) is non - inflammatory systemic disease which leads to ossification of ligaments and entheses. especially ossification of longitudinal ligaments causes the ankylosing spine. according to utsinger, dish is diagnosed when radiographs present the ossification of at least four contiguous vertebral bodies. spinal fractures and following spinal cord injuries are easily caused by low - energy trauma in patients with dish because of immobilized spinal column [3, 4 ]. the pathogenesis is unknown and the prevalence of spinal dish varies depending on region and ethnic differences ; however, recently an age - related increase in prevalence is undisputed. several studies suggested that delayed neurological deterioration tends to occur in the fracture caused by hyperextension injury. here we report three cases of those who developed delayed leg paraplegia after hyperextension injuries of the thoracolumbar spine with dish. a 68-year - old male presented with back pain and no neurological deficits after the traffic accident. plain radiographs showed ankylosing spine at t1 to l2 level ; however, no fractures were identified at the first time visit to the hospital (fig. 1b), computed tomography (ct) scan revealed the hyperextension fracture at t12 (fig. magnetic resonance image (mri) also showed hyperextension fracture and epidural hematoma compressing spinal cord (fig. figure 1:plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at t12 ; mri t2-weighted image (d, e) showing the epidural hematoma compressing the dura matter. plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at t12 ; mri t2-weighted image (d, e) showing the epidural hematoma compressing the dura matter. a 77-year - old female presented with lower back pain and no neurological deficit after falling down. about 11 days after the injury, severe lower back pain and incomplete leg paraplegia of frankel grade b appeared. ct scan revealed ankylosing spine at t1 to l2 and the hyperextension fracture at l1 (fig. 2c). figure 2:plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at l1 and dislocation of it ; mri t2-weighted image (d, e) showing the severely compressed spinal cord at l1 level. table 1:summary of three casescasemechanism of injuryinjury levelcause of paraplegiaduration from injury to paraplegiafrankel grade alteration1high energyt12epidural hematoma<24 hc to d2low energyl1dislocation10 daysb to c3low energyl1dislocation14 daysa to b plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at l1 and dislocation of it ; mri t2-weighted image (d, e) showing the severely compressed spinal cord at l1 level. summary of three cases a 79-year - old female presented with lower back pain after falling down. about 14 days after the injury, she developed complete leg paraplegia of frankel grade a and she was admitted to our hospital. second plain radiographs showed no major change, but ct revealed ankylosing spine at t5 to l2 and the hyperextension fracture at l1 (fig. figure 3:plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at l1 and dislocation of it ; mri t2-weighted image (d, e) showing the severely compressed spinal cord at t12 level. plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at l1 and dislocation of it ; mri t2-weighted image (d, e) showing the severely compressed spinal cord at t12 level. a 68-year - old male presented with back pain and no neurological deficits after the traffic accident. plain radiographs showed ankylosing spine at t1 to l2 level ; however, no fractures were identified at the first time visit to the hospital (fig. 1b), computed tomography (ct) scan revealed the hyperextension fracture at t12 (fig. magnetic resonance image (mri) also showed hyperextension fracture and epidural hematoma compressing spinal cord (fig. figure 1:plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at t12 ; mri t2-weighted image (d, e) showing the epidural hematoma compressing the dura matter. plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at t12 ; mri t2-weighted image (d, e) showing the epidural hematoma compressing the dura matter. a 77-year - old female presented with lower back pain and no neurological deficit after falling down. about 11 days after the injury, severe lower back pain and incomplete leg paraplegia of frankel grade b appeared. ct scan revealed ankylosing spine at t1 to l2 and the hyperextension fracture at l1 (fig. 2c). figure 2:plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at l1 and dislocation of it ; mri t2-weighted image (d, e) showing the severely compressed spinal cord at l1 level. table 1:summary of three casescasemechanism of injuryinjury levelcause of paraplegiaduration from injury to paraplegiafrankel grade alteration1high energyt12epidural hematoma<24 hc to d2low energyl1dislocation10 daysb to c3low energyl1dislocation14 daysa to b plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at l1 and dislocation of it ; mri t2-weighted image (d, e) showing the severely compressed spinal cord at l1 level. about 14 days after the injury, she developed complete leg paraplegia of frankel grade a and she was admitted to our hospital. second plain radiographs showed no major change, but ct revealed ankylosing spine at t5 to l2 and the hyperextension fracture at l1 (fig. figure 3:plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at l1 and dislocation of it ; mri t2-weighted image (d, e) showing the severely compressed spinal cord at t12 level. plain radiographs on initial assessment (a) and on admission (b) ; sagittal ct image (c) showing the hyperextension fracture at l1 and dislocation of it ; mri t2-weighted image (d, e) showing the severely compressed spinal cord at t12 level. dish and ankylosing spondylitis are known as ankylosing spinal disorders, which lead to ankylosis of spinal column. the prevalence of dish is different between races ranging from 2.9% to 25% [6, 7 ], and its etiology is unclear. however, several studies showed association with advancing age, obesity and type 2 diabetes mellitus. dish patients are susceptible to spinal fracture with trivial trauma due to immobilization of vertebrae. the previous studies reported that most fractures are localized in cervical spine, uncommon at thoracolumbar junction [3, 4, 8 ]. hyperextension fracture is the most frequent type of fractures with dish patients (51.2%) and induces the anterior distraction of vertebral bodies upper and lower fused vertebral bodies make the stress concentration on fracture level, so spinal cord injury due to dislocation of fracture or epidural hematoma is easy to occur after an interval from first injury. therefore secondary deterioration of neurological status is observed [3, 4, 8 ]. however, it is difficult to diagnose the hyperextension fracture at the initial assessment only with plain radiograph. patients with unrecognized fractures commonly complain of only lower back pain until abrupt neurological deterioration occurs. once after the neurological deficits occur, surgical treatment does not have the great influence to the clinical outcome. we analyzed the mechanism of injury, fracture level, direct cause of leg paraplegia, the duration from onset to paralysis, surgical procedure, and neurological deficits before and after treatment. the mechanism of injury was high - energy trauma in case 1, and low - energy trauma in case 2 and case 3. only a few reports mentioned the epidural hematoma causing spinal cord injury. to the best of our knowledge, the cause of leg paraplegia was epidural hematoma within 24 h in case 1, and dislocation of fracture over 1 week in case 2 and case 3. a delay in diagnosis often occurs in hyperextension fractures with dish [3, 4 ] because it is difficult to diagnose the fractures in plain radiographs due to ankylosing spine. the absence of major trauma may also mislead the diagnosis, this leads to second neurological deterioration. all cases were treated conservatively at an initial treatment, because the hyperextension injuries in dish were not recognized only with plain photographs. we can find only a few reports that neurological deficits improved by more than two frankel grade after surgical treatment, again, the definitive treatment should be performed before the second neurological deterioration appears. we here reported three cases of the thoracolumbar hyperextension injuries in patients with dish, those who developed delayed leg paraplegia. the spinal fracture with dish is difficult to diagnose only with plain radiograph, therefore screening of spinal column with ct and/or mri should be recommended even if the fractures are not detected. | abstractdiffuse idiopathic skeletal hyperostosis (dish) is characterized by calcifications and ossification of ligaments and tendon insertions. the patients with dish are susceptible to spinal column injury with trivial trauma due to immobilization of vertebrae and this easily leads to spinal cord injury. however, only few reports on hyperextension injuries of the thoracolumbar spine with dish exist. here we report three cases of those who developed delayed leg paraplegia after hyperextension injuries of the thoracolumbar spine with dish. all cases complained only lower back pain without neurological deficits and plain radiographs showed no fractures at an initial assessment. the spinal fracture caused by hyperextension injury was diagnosed with computed tomography (ct) and magnetic resonance image (mri) after delayed leg paraplegia occurred. many of the fractures caused by hyperextension injuries are not detected only with plain radiographs in patients with dish. further investigations using ct and/or mri should be recommended. |
renal cell carcinoma (rcc) accounts for approximately 2 to 3% of cancer in adults of the general population. compared with other types of cancer, rcc is difficult to diagnose and shows different clinical patterns. in particular, rcc is associated with such a low survival rate in kidney transplantation patients that studies on the correlation between the transplantation and the cancer have recently been conducted. after diagnosis with end - stage renal disease (esrd), transplantation patients take immunosuppressive agents for preservation of the transplanted kidney, and the use of such agents since the 1970s has been reported to raise the occurrence of malignancy. immunosuppressant therapy was used in the past to prevent rejection, but this type of therapy has actually decreased the survival rate by increasing cancer occurrence. the incidence rate of cancer in patients using immunosuppressive agents is approximately 2 to 5 times higher than in healthy controls. according to a previous study, the incidence rate of cancer is 10 times higher in renal transplant recipients than in dialysis patients. acquired cystic kidney disease (ackd) is known to be frequently observed in dialysis patients with chronic renal failure and patients using immunosuppressive agents after transplantation. in particular, ackd needs careful follow - up as a pre - malignant lesion. ackd is found mainly in patients on dialysis, and its existence influences the occurrence of cancer. ackd was reported to be observed in 20% of patients on dialysis for 1 to 3 years and in > 90% of those on dialysis for 5 to 10 years. ackd increases the prevalence of rcc by around 20%. for transplant recipients among esrd patients undergoing dialysis, the incidence rate of rcc previous studies investigated only the occurrence of ackd and rcc in dialysis patients and the occurrence of rcc in the native kidney after kidney transplantation. there have been no reports comparing dialysis patients and transplant recipients among rcc patients. in the present study, we examined ackd development and the correlation between dialysis duration and the occurrence of rcc. two groups of patients were defined from among those who had been diagnosed and surgically treated for rcc : those who had undergone kidney transplantation (dialysis) and those who had undergone dialysis only. the purpose of this investigation was to determine whether there was a pathologic difference between the two groups. the study included 43 patients diagnosed with rcc during follow - up among patients undergoing dialysis owing to esrd and renal transplant recipients. the patients ' medical records were analyzed for age, age at radical nephrectomy, age at starting dialysis, duration of dialysis (months), duration of use of immunosuppressive drugs (months), gender, medical history, type of dialysis, dialysis after transplantation, routine examination, presentation of ackd, and symptoms for rcc evaluation, retrospectively. the patients were diagnosed with rcc and underwent radical nephrectomy or renal biopsy from may 1996 to december 2010. for the classification of rcc cell type, tumor stage was determined by the tumor size, metastasis to node, and metastasis classification and cellular grade was classified as g1 to g4. the chi - square test was used with nominal variables ; student 's t - test was used with continuous variables. statistical analyses were conducted by using ibm spss ver. 18.0 (ibm co., new york, ny, usa). this study compared dialysis patients and transplant recipients who had been diagnosed with rcc and had been surgically treated. as summarized in table 1, the mean age of the transplantation and dialysis groups was 5711 and 5211 years, respectively. comparison between the two groups did not show any significant differences in gender distribution (p=0.82), blood pressure (p=0.89), or the existence of diabetes (p=0.14) (table 1). in 42 cases among the total patients, rcc was diagnosed through radical nephrectomy, and in the other 1 case it was pathologically confirmed through renal biopsy. of the 43 patients, 21 underwent kidney transplantation after dialysis, and the other 22 were on dialysis. of the 21 patients who underwent kidney transplantation, 13 patients had histories of prior hemodialysis (hd) treatment. whereas six patients underwent only peritoneal dialysis (pd), two patients had undergone kidney transplantation without dialysis after being diagnosed with esrd. ten patients developed post - transplantation acute or chronic rejection, and six of these were on dialysis because of rejection of the transplanted kidney. the duration of dialysis following rejection was less than 1 month, and the transplanted kidney could be saved after dialysis or by using immunosuppressive agents. in addition, one patient developed membranoproliferative glomerulonephritis, which was dealt with by removal of the transplanted kidney and treatment with pd. the remaining patients were treated by immunosuppressive agents. among the 22 dialysis patients, seven men received hd, whereas 12 patients received pd. three patients switched during treatment from pd to hd (table 1). of the total of 7,009 dialysis patients in our hospital, 0.3% (22 patients) were surgically treated for rcc, and 82% of those (18 patients) had ackd. among the total of 2,757 renal transplant recipients in our hospital, 0.8% (21 patients) were diagnosed with rcc, and 5% (1 patient) of those had ackd (table 1). in the transplantation group, the subjects underwent transplantation after an average period of 54 months (range, 0 to 168 months) of renal replacement therapy, and the average period from transplantation to nephrectomy due to rcc was 119 months (range, 0 to 264 months). in the dialysis group, rcc was found after an average of 124 months (range, 2 to 228 months) of dialysis and was treated by nephrectomy. the transplantation subjects underwent mainly hd, and dialysis subjects in most cases received pd treatment. however, the difference in dialysis modality had no statistical significance with respect to the occurrence of rcc (p=0.14) (table 1). in the transplantation group, we took a routine examination by ultrasonogram or ct (22 patients). the dialysis group did not undergo a routine examination by ultrasonogram or ct except for one patient in whom another cancer was diagnosed (table 1). for symptoms leading to rcc evaluation, rcc was suspected coincidentally during follow - up by ct or ultrasonography in 8 patients (38%) of the transplantation group and in 9 (41%) of the dialysis group. when rcc characteristics were investigated after the surgery, there were no statistically significant differences in the cancers as to side of occurrence (p=0.2), cell type (p=0.85), fuhrman grade (p=0.91), t stage (p=0.27), n stage (p=0.36), or m stage (p=0.36) between the two groups (table 2). ackd was found more often in the dialysis group, and it affected the occurrence of rcc in the comparison with the transplantation group (p<0.01) (table 1, 3). since dunnill. first referred in 1977 to the correlation between rcc and ackd following esrd, some studies have confirmed a relationship between esrd and the occurrence of rcc. in 1992, chandhoke. found no correlation between the two. however, in 2006 moudouni. observed that the incidence of rcc in the native kidney was high in transplant recipients, and kojima. reported that the incidence rate was high in dialysis patients. in the latter study, it was shown that 44 (1.68%) of 2,624 esrd patients followed up during dialysis developed rcc an average of 11.2 years after dialysis, and 36 of these cases (81.8%) were associated with ackd. a positive correlation with the incidence rate of rcc was also found among transplant recipients. in 2007, ianhez. reported that 10 of 1,375 patients developed rcc of the native kidney an average of 8.4 years after renal replacement therapy, and all the rcc cases were associated with ackd. pope. showed in a study of 7 kidney transplantation patients and 13 dialysis patients in 1994 that the pathologic appearance was not significantly different between the two groups but the stage and mortality were higher in the kidney transplantation group. they suggested the use of immunosuppressive agents for the transplant recipients as a reason for the finding. in this study, analysis of all the pathological findings, including cell type (p=0.85), fuhrman grade (p=0.91), t stage (p=0.27), n stage (p=0.36), and m stage (p=0.36), showed no statistically significant differences between the transplantation and dialysis groups (table 2). one reason for this lack of significant difference could be the time at detection before the surgery. because the risk of cancer is high in transplant recipients, as shown in previous studies, our hospital routinely conducts ultrasonography. thus, we could detect abnormalities by ultrasonography earlier, and it is more usual to identify rcc in screening by ct after finding an abnormality by ultrasonography. in addition, most of the renal replacement patients with symptoms underwent ct for early detection of rcc. the correlation between dialysis and ackd should be mentioned. in 1990, matson and cohen investigated ackd development and its correlation with rcc among esrd patients. they demonstrated that ackd was closely correlated with the duration of renal replacement therapy but not with the type of dialysis that was performed. many studies on transplantation and ackd have also reported that ackd is observed more frequently, and the incidence rate of rcc is higher, in a kidney transplantation group than in a control group. moreover, heinz - peer. analyzed 96 patients with ackd detected by ultrasonography out of 385 renal transplant recipients in 1995 and found that the incidence of ackd was higher with longer duration times of dialysis before kidney transplantation in older subjects and in males. however, only a few studies have been performed to determine the effect of transplantation itself on ackd, and none have reported a clear effect on rcc. ishikawa., in their prospective study following up the native kidney of 61 transplant recipients by ct 1 to 2 years after transplantation, observed no renal cysts in 52.5% of the recipients, no change in the number of cysts in 14.8%, a decrease in the number of cysts in 14.8%, and an increase in the number in 17.9%. they explained that ackd was caused by epithelial hyperplasia following uremia and proposed that resolving the uremia in a successful transplantation could lead to the regression of ackd. in this study, the incidence of ackd was lower in transplant recipients than in dialysis patients. because the transplant recipients had undergone dialysis before, the effect of dialysis could not be excluded completely for this group. however, previous studies reported that ackd development increases cancer risk in dialysis patients and transplant recipients after kidney transplantation. in our study, one of the cases of ackd in a transplant recipient was found by routine examination (e.g., ultrasonogram or ct) (table 1). the incidence rate of ackd was lower in transplant recipients than in dialysis patients, and rcc not associated with ackd in the transplant recipients. although there have been some reports that transplantation may lead to the regression of ackd, it is difficult to say whether transplantation may reduce the risk of rcc, even though we found that there was a significantly longer time to the occurrence of rcc in the transplantation group than in the dialysis group. we considered that this was caused by the length of exposure to a uremic environment during dialysis before transplantation and the use of immunosuppressive agents after kidney transplantation. a comparison between renal transplant recipients and dialysis patients, all of whom had been surgically treated for rcc, except for one patient diagnosed by renal biopsy, found that the incidence rate of ackd was significantly higher in the dialysis group. ackd is known to be a potent risk factor for cancer among esrd patients, and this study has further shown that ackd is a cancer risk factor in dialysis patients. however, cancer without ackd was also observed in transplant recipients. considering that the recipients had also undergone dialysis before transplantation, prospective studies will be necessary to determine whether the use of immunosuppressive agents, other than ackd, may as also be a factor contributing to renal cancer risk. we propose that routine ultrasonograms or ct should be performed for follow - up in these high - risk patients. | purposein a group of surgery patients diagnosed with renal cell cancer, those who underwent dialysis were compared with those who received a kidney transplant.materials and methodsthe 43 subjects included in this study were patients who had been undergoing dialysis because of end - stage renal disease or had undergone kidney transplantation. the patients were diagnosed with renal cell carcinoma (rcc) during follow - up and underwent radical nephrectomy from may 1996 to december 2010. their medical records were retrospectively analyzed as part of the study.resultsin the transplantation group, the renal replacement therapy period averaged 54 months, and the period from transplantation to rcc averaged 119 months (range, 0 to 264 months). in the dialysis group, rcc was observed after an average of 124 months (range, 2 to 228 months) of dialysis, and nephrectomy was then conducted. acquired cystic kidney disease (ackd) was found more frequently in the dialysis group, and it had a statistically relevant effect on the occurrence of rcc by comparison with the transplantation group (p<0.01).conclusionsalthough the incidence rate of ackd was significantly higher in the dialysis group among patients undergoing surgery for rcc, cancer was found even without ackd development in some transplant recipients. considering that the transplant recipients also underwent dialysis, an informative prospective study will be necessary to determine whether other immunosuppressive agents besides ackd may function as a cancer risk factor. |
the subjects had been diagnosed as pd for the first time or diagnosed as pd by other clinicians but had never been treated before. the diagnosis of pd was established according to the criteria of the united kingdom parkinson s disease society brain bank.15 we excluded atypical or secondary parkinsonisms, and the diagnosis was confirmed during the follow up visits for more than 6 months. comorbid medical conditions that could affect blood pressure were also evaluated, which included hypertension, diabetes mellitus, and benign prostatic hypertrophy. severity and stage of parkisonism were rated using the unified parkinson s disease rating scale, the hoehn and yahr stage and the modified schwab and england activities of daily living. all patients were evaluated for the presence of oh, regardless of symptoms of orthostatic dizziness or syncope. to avoid the blood pressure lowering effect of dopaminergic drugs, all patients evaluated for oh before medication. after a 10 minute period of lying down, systolic and diastolic blood pressures were checked in the first, third, and fifth minutes after standing up. if the patient could not stand up by his or herself, a tilt table was used. oh was defined as a drop in systolic bp 20 mmhg or diastolic bp 10 mmhg at anytime following standing up. comprehensive assessment of autonomic dysfunction was also assessed using a systematized autonomic dysfunction questionnaire (adq).3 the adq covered gastrointestinal symptoms, urinary symptoms, sexual dysfunction, cardiovascular symptoms and thermoregulatory symptoms. the severity or frequency of each of the symptoms was expressed as scores (table 1). the subjects had been diagnosed as pd for the first time or diagnosed as pd by other clinicians but had never been treated before. the diagnosis of pd was established according to the criteria of the united kingdom parkinson s disease society brain bank.15 we excluded atypical or secondary parkinsonisms, and the diagnosis was confirmed during the follow up visits for more than 6 months. comorbid medical conditions that could affect blood pressure were also evaluated, which included hypertension, diabetes mellitus, and benign prostatic hypertrophy. severity and stage of parkisonism were rated using the unified parkinson s disease rating scale, the hoehn and yahr stage and the modified schwab and england activities of daily living. all patients were evaluated for the presence of oh, regardless of symptoms of orthostatic dizziness or syncope. to avoid the blood pressure lowering effect of dopaminergic drugs, all patients evaluated for oh before medication. after a 10 minute period of lying down, systolic and diastolic blood pressures were checked in the first, third, and fifth minutes after standing up. if the patient could not stand up by his or herself, a tilt table was used. oh was defined as a drop in systolic bp 20 mmhg or diastolic bp 10 mmhg at anytime following standing up. comprehensive assessment of autonomic dysfunction was also assessed using a systematized autonomic dysfunction questionnaire (adq).3 the adq covered gastrointestinal symptoms, urinary symptoms, sexual dysfunction, cardiovascular symptoms and thermoregulatory symptoms. the severity or frequency of each of the symptoms was expressed as scores (table 1). the subjects (17 men and 28 women, age of 63.8 10.1 years) showed mild disease [median hoehn and yahr stage 2.0 (13) ] with 1.3 1.1 years of disease duration. twenty - two patients had comorbid disease, which could affect blood pressure. hypertension (20/45 patients) was the most common, followed by diabetes (7/45) and benign prostatic hyperplasia (1/45). eighteen patients showed oh (oh group, 8 men, age 61.3 10.8 years) and twenty - seven patients (normotensive group, 9 men, age 65.5 9.4 years) did not. patients without oh were older and showed shorter duration of education, but those demographic characteristics were not significantly different between groups (table 2). parkinsonian symptoms and daily activities of the patients also showed no significant difference between groups (table 2). many dysautonomic symptoms were accompanied with oh in patients, and all the patients had more than two of those symptoms (table 3). although urinary incontinence was more prevalence in oh group, it did not reach statistical significance. total score in the adq was slightly increased in patients with oh compared to that of patients without oh (table 4). however, the differences did not reach statistical significance. in spite of the insignificant difference, the scores of all domains in the oh group were higher than those of the normotensive group. however, even in the questions of cardiovascular symptoms, orthostatic dizziness and history of syncope, there was no difference between groups. the subjects (17 men and 28 women, age of 63.8 10.1 years) showed mild disease [median hoehn and yahr stage 2.0 (13) ] with 1.3 1.1 years of disease duration. twenty - two patients had comorbid disease, which could affect blood pressure. hypertension (20/45 patients) was the most common, followed by diabetes (7/45) and benign prostatic hyperplasia (1/45). eighteen patients showed oh (oh group, 8 men, age 61.3 10.8 years) and twenty - seven patients (normotensive group, 9 men, age 65.5 9.4 years) did not. patients without oh were older and showed shorter duration of education, but those demographic characteristics were not significantly different between groups (table 2). parkinsonian symptoms and daily activities of the patients also showed no significant difference between groups (table 2). many dysautonomic symptoms were accompanied with oh in patients, and all the patients had more than two of those symptoms (table 3). although urinary incontinence was more prevalence in oh group, it did not reach statistical significance. total score in the adq was slightly increased in patients with oh compared to that of patients without oh (table 4). however, the differences did not reach statistical significance. in spite of the insignificant difference, the scores of all domains in the oh group were higher than those of the normotensive group. however, even in the questions of cardiovascular symptoms, orthostatic dizziness and history of syncope, there was no difference between groups. abnormal cardiovascular regulation, including orthostatic hypotension was frequently expressed in various stages of pd patients.16,17 the cardiovascular autonomic disturbances are following the gastrointestinal symptoms in frequency, but they have a higher impact on the quality of life in pd patients. lewy bodies, a pathological hallmark of parkinson s disease, have been found in autonomic regulatory regions of the brain or peripheral autonomic ganglia in pd patients.18 and, many studies have shown the evidence of cardiac sympathetic denervation in early pd patients by using cardiac sympathetic imaging.19,20 symptoms of autonomic dysfunction in pd patients were known to be related with older age, long disease duration, severe disease stage and anti - parkinsonian medication.7 however, allcock. showed that frequency of oh was independent of duration and severity of movement symptoms in the study of the frequency of oh in a community - based cohort of pd patients.13 the prevalence of oh in pd was variable according to criteria and methods. in 5 relatively large studies involving more than 80 patients each, the frequency of oh ranged from 30% to 58%.6 bonuccelli.21 showed the high prevalence of oh in de novo pd patients. in our study, prevalence of oh in the drug - nave pd patients was moderate (18/45 patients, 40%), and similarly shown in the patients without comorbid disease (9/23 patient, 39.1%). patients with oh were young and showed a short duration of disease, but those differences did not reach statistical significance, and the severity of parkinsonian symptoms was similar to those of patients without oh. the presence or severity of dysautonomic symptoms was not different between patients with or without oh, even in the cardiovascular domain. actually, the complaint of orthostasis was made by only six patients (13.3%), four of those patients showed oh. most patients with oh did not complain of orthostasis, and steven. showed that the majority of patients with profound orthostatic hypotension either did not show the typical symptoms (33%), or showed only atypical symptoms (24%).22 the unawareness of orthostatic hypotension stresses the importance of blood pressure monitoring irrelevant to complaints of patients. although we could not find statistical significance, patients with urinary incontinence were much higher in oh group (27.8% vs. 7.4%). this finding suggests that those patients can be diagnosed as multiple system atrophy (msa). we followed all the patients more than six months and confirmed the good response to dopaminergic treatment. because some patients with msa show good response to treatment in early period, we could not exclude the possibility of msa. further studies with longer period of observation and about the change of dysautonomic symptoms along the disease course are warranted. although pathological implications of autonomic dysfunctions in pd patients have not been established yet, clinical implications can be significant enough to impact the daily activities of pd patients. furthermore, most patients do not complain of noticeable symptoms, and the unawareness of patients warrants careful evaluation. appropriate management of oh can prevent further insult and can improve daily activities of pd patients. | background and purposeorthostatic hypotension (oh) is known to be present even in patients with early parkinson s disease (pd). to affirm the presence of oh and find correlation between oh and other dysautonomic symptoms in pd, this study has done in newly - diagnosed pd patients.methodsforty-five non - demented patients with no prior history of treatment for pd were recruited (17 men, 63.8 10.1 years of age). all the patients were evaluated for oh before starting medications. autonomic symptoms were evaluated with structured questionnaires. clinical characteristics of pd were evaluated (median hoehn and yahr stage 2.0 (13), 1.3 1.1 years of disease duration), and comorbid medical conditions that could affect blood pressure were also recorded.resultsoh was prevalent, and eighteen patients (40%) showed orthostatic hypotension, and twenty - seven (60%) did not (normotensive group). there was no significant difference in demographic and clinical characteristics between groups. the presence or severity of symptoms of autonomic dysfunction in the oh group also not differed from those of the normotensive group.conclusionsoh was prevalent even in the early stage of pd, and was not related to presence or severity of any other symptoms of autonomic dysfunction. our findings suggest that clinicians should pay attention to oh from the early stage of disease. |
nerve compression and traction may cause disorders of the intraneural microcirculation, lesions in the myelin sheath and the axon, as well as alterations in the supporting connective tissue. the entrapment of a peripheral nerve occurs as a result of its passage through an anatomical compartment that has become too tight, resulting in altered function within the nerve and dysfunction / damage of the nerve from the site of compression and beyond.18 median nerve entrapment in the carpal tunnel at the wrist is the most common example of this. these mechanisms are interacting and include the increased pressure in the tunnel, median nerve microcirculation injury, median nerve connective tissue compression, and synovial tissue hypertrophy. in addition to these mechanisms, the results of post - operative outcome of carpal tunnel release reported by ozkul,19 indicated that a less favorable outcome was found among diabetic when compared with non diabetic patients. these results suggested that cts in diabetic patients may also stem from internal factors deleterious to the nerve including hyperglycemia and deficiency of neurotrophic factors such as nerve growth factor.19 anatomically, there are 2 sites of median nerve compression : 1) at the proximal edge of the carpal tunnel, caused by wrist flexion and due to the change in thickness and rigidity between the antebrachial fascia and the proximal portion of the fr ; and 2) at the narrowest portion at the hook of hamate. normal pressure in the carpal tunnel has been recorded to range from 2 to 10 mm hg.20 dramatic changes of the fluid pressure in the carpal tunnel have been reported with wrist movement, with wrist extension increasing the pressure by 10-fold, and flexion increasing it by 8-fold.20 bauman utilized a wick catheter to demonstrate that the tunnel pressure was higher in idiopathic cts patients than in normal subjects. in neutral wrist posture, the average pressure that is registered in the patient s carpal canal is 32 mm hg. when the wrist is flexed, the pressure reaches a value of 94 mm hg, but is 110 mm hg when the wrist is extended. pathological changes occurring in the ligaments surrounding nerves including alterations in the amount and flexibility of connective tissue are thought to be the basis for increased pressure. experimental studies have suggested a dose - response relationship between median nerve dysfunction, and the duration, and amount of carpal tunnel compression.22 increased carpal tunnel pressure is thought to cause ischemic compression of the median nerve, and a number of experimental studies support the theory of ischemia due to externally applied compression and due to increased pressure in the carpal tunnel.23 seiler reported, by using laser doppler flowmetry, the restoration of normal pulsatile blood flow within the median nerve within one minute of transverse carpal ligament release. in idiopathic cts, the nocturnal increase in the tunnel pressure could result from several factors that include : redistribution of the upper limb fluids in supine position ; lack of muscle pump mechanism that contributes to the drainage of interstitial fluid in the carpal tunnel ; tendency to place the wrist in flexion thereby increasing intracanalicular pressure ; increased blood pressure in the second half of the night ; and fall of cortisol level.6 ischemic vascular injury and the breakdown in the blood - nerve barrier have also been identified as an essential component in cts. the blood - nerve - barrier is formed by the inner cells of the perineurium and the endothelial cells of endoneurial capillaries that accompany the median nerve through the carpal tunnel. these endoneurial microvessels are formed from nutrient branches that arise from the radial and ulnar arteries, proximal to the flexor retinaculum.25 an increase in pressure within the tunnel can cause a breakdown of vasculature within this barrier, causing an accumulation of proteins and inflammatory cells.25 this may induce a miniature closed compartment syndrome by increasing the permeability, contributing to increased endoneurial fluid pressure and development of an intra - fascicular edema.26 patients with vascular problems or prolonged exposure to static loading are particularly prone to a breakdown in the blood - nerve - barrier.27 mackinnon and coworkers have also described common progressive neurovascular changes occurring in a series of experimental studies looking at histological findings at common sites of entrapment.28 - 30 these included early perineurial and endoneurial microvessel thickening with basement membrane reduplication, renaut s body formation, perineurial and epineurial fibrosis, and patchy fibre loss associated with thinning of myelin, attributed to fibre demyelination and degeneration. one final etiological reason for the increased susceptibility of median nerve compression, particularly in diabetic patients, is changes in the microvascular structure of the nerve, exacerbated by biochemical disturbances, which could lead to a reduction in the endoneurial blood flow and oxygen tension.31 focal compression led to localized intraneural circulatory changes and increased permeability of endoneurial vessels as well as causing edema of the endoneurial space. the development of edema may lead to an increase in the diffusion distance for oxygen from the capillaries, which could lead to hypoxia.31 the resultant hypoxia could drive the upregulation of several angiogenic factors including hypoxia - inducible factor 1 (hif-1) and vascular endothelial growth factor (vegf). increased vegf levels have been demonstrated in neurones and schwann cells of experimental diabetic animal models.32 furthermore, endoneurial vessels in diabetic patients undergo characteristic microangiopathic changes such as hyaline thickening and increased deposition of a per - arnt - sim (pas)-positive substance within their walls. they also display endothelial hypertrophy, hyperplasia, basement membrane thickening, and pericyte loss. the thickened vessel wall, alongside the increased endoneurial vascular permeability and edema, would also increase the diffusion distance for oxygen to reach the nerve fibres and thereby induce more hypoxia.32 a schematic presentation for vascular mechanism of cts and median nerve injury is presented in figure 1. a schematic presentation for vascular mechanism of carpal tunnel syndrome and median nerve injury. these layers are the mesoneurium (not included in the peripheral nerve sheath), epineurium, perineurium, and endoneurium ; which is the most intimate layer. the extensibility of these layers is critical to nerve gliding (nerve gliding properties are due to the integrity of epineurium only), which is necessary to accommodate joint motion ; otherwise, nerves are stretched and become injured.33 it is estimated that in normal subjects, the median nerve at the wrist can move up to 9.6 mm between full flexion and extension of this joint but in the presence of stiff surrounding connective tissue, this is limited and exposes the nerve to shearing forces that could lead to injury.19 the longitudinal movement of the median nerve in the carpal tunnel was found to be 9.6 mm during flexion, and 0.7 - 1.4 cm in wrist extension. it can vary from 2.5 to 19.6 mm depending on the position of the shoulder, elbow, wrist, and fingers. the median nerve tension varies from 8% depending on the position of the shoulder, and 19% depending on the position of the fingers. in addition to the longitudinal movement, a transverse movement of the median nerve occurs with wrist position or during finger flexion against resistance. in compression and epineural adhesions, mobility is hindered, creating lesions due to repeated traction on the nerve during wrist movements. the complexity of the mechanisms underlying nerve compression and traction was described by lundborg and dahlin,34 who emphasized how a chain of events may set up a vicious cycle leading to nerve injury. a chronic increase in pressure in the nerve trunk produces a pressure gradient, which redistributes components of compressed tissue towards the side not compressed, with the subsequent stretching of the epineurial and vascular structures. the rapid development of edema, predominantly in the epineurium, leading to nerve swelling, would further restrict the movement of the nerve within the already narrow anatomical compartment. this scenario limits nerve gliding during movements of the extremities, further subjecting the nerve to more irritation, increased pressure on the nerve trunk and edema, initiating a vicious cycle. human studies of entrapment neuropathies during surgery or autopsies have found the nerves to be narrowed at the site of compression, whilst appearing enlarged at the proximal and distal segments.35 demyelination of the nerve develops in the compression site, and can then spread to the entire internodal segment, leaving the axons intact. a block of nervous transmission ensues (neuropraxia). if the compression persists, bloodflow to the endoneural capillary system may be interrupted, leading to alterations in the blood - nerve barrier, and development of endoneural edema. this starts a vicious cycle consisting of venous congestion, ischemia, and local metabolic alterations.27 axonal degeneration, macrophage attraction, and activation, release of inflammatory cytokines, nitric oxide, and development of chemical neuritis are all consequences of this viscous cycle if it continues for a substantial amount of time.36 the next stage includes axonal interruption, and distal wallerian degeneration. following surgical decompression, sensory recovery hypertrophy of the synovial tissue of the flexor tendons can also increase the pressure in the carpal tunnel and result in the development of cts.37 several histological and biochemical studies38,39 have reported tenosynovitis as a closely related risk factor to the development of idiopathic cts. this has been confirmed by the presence of increased expression of prostaglandin e2 and vegf in synovial biopsy tissue from patients with symptomatic cts.40 in response to this injury, there is an increase in fibroblast density, collagen fibre size, vascular proliferation, and type iii collagen in the synovial connective tissue.41 constrictive scar tissue would be formed around the median nerve,42 which in turn can result in tethering of the nerve. the inflammatory thickening of the synovial tissue increases the volume of tissue which in turn increases the fluid pressure within the carpal tunnel.43 the most profound thickening of synovial tissue has been reported to be at the entrance and exit regions of the canal where the tendons slide over a fulcrum of the flexor retinaculum.43 strain and micro - damage to the synovial tissue as well as the median nerve can occur due to the different degrees of excursion between the flexor tendons and the median nerve.39,44,45 these structural changes are aggravated by diabetes mellitus as non - enzymatic glycosylation of collagen is increased in diabetes, resulting in the alteration of packing, cross - linkage, and turnover of collagen. increased glycosylation adversely affects collagen degradation, resulting in the accumulation of less compliant connective tissue, and ultimately fibrosis.46 an increase in lysyl oxidase activity, an enzyme involved in collagen cross - link formation that adds to the fibrosis and stiffness, has also been suggested to play a role.47 this may lead to an increase in the intra - compartmental pressure and restrict the peripheral nerve gliding movement between tissues. also, double crush syndrome, was described by upton and mccomas,48 who initially proposed that focal compression of an axon often occurs at more than one level. they postulated that nonsymptomatic impairment of axoplasmic flow at more than one site along a nerve might summate to cause a symptomatic neuropathy. this was suggested by clinical observation that the majority of patients had a median neuropathy associated with evidence of cervicothoracic root lesions. other researchers have since reported series of patients supporting the frequent association of a proximal and distal nerve compression syndrome, including cts associated with cervical radiculopathy, brachial plexus compression, and diabetic neuropathy.49 mackinnon and dellon50 have also expanded the description of this syndrome to include a) multiple anatomic regions along a peripheral nerve, b) multiple anatomic structures across a peripheral nerve within an anatomic region, c) superimposed on a neuropathy, and d) combinations of the above.50 in conclusion, cts is a constellation of symptoms associated with the compression and traction of the median nerve in the carpal tunnel. however, the different pathophysiologic mechanisms presented in this review demonstrate that abnormally high carpal tunnel pressure and traction neuropathy is most likely to induce cts. compression and traction cause obstruction to venous outflow, edema formation, and ultimately, ischemia and nerve injury. further studies, particularly human studies, of these interacting pathophysiologic mechanisms are necessary to shed more light not only on pathogenesis but also on the prevention and control of this disabling disease. available from www.icmje.org the international committee of medical journal editors has recommended the following criteria for authorship ; these criteria are still appropriate for those journals that distinguish authors from other contributors. authorship credit should be based on 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data ; 2) intellectual content ; and 3) final approval of the version to be published. acquisition of funding, collection of data, or general supervision of the research group, alone, does not justify authorship. | carpal tunnel syndrome (cts) is the most common median nerve neuropathy, accounting for 90% of all neuropathies. carpal tunnel syndrome presents in 3.8% of the general population, with a higher prevalence among women. there are several risk factors associated with cts, including both medical and non medical factors. the pathophysiologic mechanisms involved in the median nerve compression and traction are thought to be complex, and as yet are not fully understood. the present review aimed to provide an overview of the pathophysiology of median nerve neuropathy in the carpal tunnel, and subsequent development of cts. |
acquired factor viii deficiency is caused by the development of autoantibodies directed against circulating coagulation factor viii, which inhibits its function [1, 2 ]. it may be associated with pregnancy, the postpartum period, autoimmune conditions, drugs, lymphoproliferative disorders, or malignancies. in approximately half of the diagnosed cases, we report a rare case of subdual hemorrhage in a man, who acquired factor viii inhibitors and developed coagulopathy after consuming toxin of the cobra, naja naja. a 49-year - old male was admitted with a 10-day history of subcutaneous hemorrhage of the right shoulder (fig. 1) and left thigh. the patient was being treated with losartan 50 mg / day for hypertension and metformin 500 mg twice a day for diabetes. he had eaten 10 g of dried gall bladder of a cobra, naja naja in thailand one month ago. laboratory test showed a prolonged activated partial thromboplastin time (aptt) exceeding 120 seconds (normal range, 30 - 47 seconds), not corrected with plasma mixing test. his prothrombin time was 10 seconds (normal range, 10 - 13 seconds), the level of fdp was 11.71 ug / ml (normal range, below 5 ug / ml), and d - dimer was 3.42 ug / ml (normal range, below 1.3 ug / ml). his hemoglobin was 7.0 g / dl and platelet count was 32310/l. the factor viii level was less than 1%, and his factor viii inhibitor antibody titer was 246 bethesda units / ml (normal range, 0 - 0.7 bethesda units / ml). lupus anticoagulant antibody, anti - nuclear antibody and anti - phospholipid antibody were negative. he was diagnosed with acquired factor viii deficiency. magnetic resonance imaging (mri) of the thigh showed hematomas in the left vastus medialis muscle (fig. the patient was treated with an intravenous infusion of factor viii 3,000 u (50 u / kg) every 8 hours. one month after treatment, the patient 's symptoms improved, but his factor viii inhibitor antibody titer was still high (147 bethesda units / ml). he had an aptt exceeding 120 seconds due to the presence of high titers of factor viii inhibitor (217 bethesda units / ml). brain ct scan revealed a mixed subacute subdural hematoma in the left fronto - temporo - occipital areas (fig. 3) and recombinant factor vii was infused at a dose of 360 kiu / day. despite treatment, the patient 's bleeding worsened. two weeks later, multifocal subarachnoid hemorrhages and intracranial hemorrhages in both cerebral hemispheres were revealed. it results from the development of inhibitors, that typically interfere with the coagulant properties of factor viii [1, 2, 7 ]. the clinical picture is characterized by spontaneous and often sudden onset of severe hemorrhage in a patient with no bleeding history. more than 80% of patients have hemorrhages into the skin, muscles or soft tissues, and mucous membranes. however, the spontaneous disappearance of inhibitors is unpredictable and may occur over many years. for a better prognosis, eradication of the inhibitor and maintenance of normal level of clotting factor viii are important. the therapeutic strategy is to stop the hemorrhage and reduce the autoantibody levels. bypassing products currently available for bleeding control are recombinant activated factor vii (rfviia) and plasma - derived activated prothrombin complex concentrate (apcc). in patients with a high inhibitor titer and severe hemorrhages, the extracorporeal removal of the autoantibody by therapeutic plasmapheresis can be used before factor concentrate treatment. there are two case reports in korea which were successfully treated with combination therapy that included therapeutic plasmapheresis. once hemostasis is achieved, autoantibody eradication is the second major goal of acquired hemophilia therapy. the inhibitor may also be eradicated with immunosuppressive agents, including corticosteroids and cytotoxic drugs, such as cyclophosphamide, azathioprine, 6-mercaptopurine, and vincristine. we report the first case of acquired factor viii deficiency in a patient, who suffered from coagulopathy after consuming the dried gallbladder of a cobra. coagulopathy is a significant cause of both morbidity and mortality in snakebite patients [13, 14 ]. a wide variety of venom components can act as procoagulants, activating the coagulation system in vivo, and causing the consumption of coagulation factors, resulting in clinical anticoagulation. fibrinogen clotting and fibrinolytic snake venom toxins exert a direct effect on the actual thrombus - forming protein, fibrinogen, but in varying ways. fibrinogen may be split into fibrin and then degradation products, or it may be only partially split, leaving an ineffective form of fibrinogen circulating, the end result being an increased bleeding tendency. snake venom inhibits platelet activity too, thus reducing their effectiveness in hemostasis [13, 14 ]. another study reported there is transient presence of an inhibitor to factors viii, ix and xi in cases of tiger snake envenomation. in our case, cobra toxin was probably the cause of development of factor viii inhibitor, since the acquired factor viii deficiency developed right after consuming the cobra toxin, and we did not find any other possible cause of acquired factor viii deficiency in this patient. perhaps cobra toxin entered the blood stream through damaged oral mucosa, and acted as factor viii inhibitor. in order to make a clear connection between cobra toxin and acquired factor viii deficiency, further evaluation about the effects of snake venom on individual coagulation factors is needed. | acquired factor viii deficiency is very rare, often fatal. it is associated with pregnancy, autoimmune diseases, malignancy, and drugs, although no underlying cause is found in 50%. a 49-year - old male was referred with right shoulder bruising. the coagulation test showed a prolonged activated partial thromboplastin time. the factor viii level was less than 1%, and the factor viii inhibitor antibody titer was 246 bethesda units / ml. the findings were compatible with acquired factor viii deficiency. he had consumed the dried gallbladder of a cobra, naja naja, for two weeks, it contained venom. after the initial treatment with factor viii, he did not take supplemental coagulation factor viii. the patient was readmitted with left forearm swelling. he lost consciousness suddenly and brain computed tomography (ct) revealed a subdural hematoma. despite administering recombinant factor vii, his bleeding was not controlled and he died. |
however, acetylcholinesterase inhibitors have several disadvantages, such as development of residual blockade and hemodynamic adverse effects. sugammadex, a -cyclodextrin derivative, is a new reversing agent that has been in use recently. sugammadex is preferred because of its advantages during extubation and recovery and low risk of residual blockade. it has been known that with sugammadex some changes in coagulation parameters occurred without documented clinical consequences. the aim of the present study is to evaluate the effects of sugammadex on postoperative nausea - vomiting, pain, coagulation parameters and amount of postoperative bleeding. this prospective study was approved by local ethics committee (2013/411) and informed consents were taken from all participants. fifty patients aged between 18 and 65 (mean age ; 34.58) scheduled for septoplasty operation were included in the study. all patients were in the asa i - ii (american society of anesthesiologists) class. patients taking antiaggregant / anticoagulant treatment, patients with history of bleeding disorder, patients with abnormal complete blood count and coagulation tests (prothrombin time (pt), activated partial thromboplastin time (aptt), international normalized ratio (inr)) were excluded from the study. patients were randomized preoperatively in to two groups : neostigmine (group n, n : 26) and sugammadex (group s, n : 24). randomization sequence was generated by using computer generated random numbers. initially according to the power analysis (for =0.05,desired power=95%) fifty - two envelopes prepared for probable sample loss (26 for neostigmine and 26 for sugammadex). two patients in group s were discarded (one patient did nt come to surgery and in one patient surgery was postponed because of recent upper respiratory tract infection). so study population included 26 patients in neostigmine group and 24 patients in the sugammadex group. when the patients arrived in the operating room, heart rate, noninvasive arterial pressure, pulse - oximeter, end - tidal carbon dioxide monitoring, and neuromuscular block monitoring (train - of - four (tof) watch sx monitor ; organon, dublin, ireland) were performed. standard anesthesia protocol was applied : after induction with propofol 22.5 mgkg, rocuronium 0.6 mgkg, fentanyl 0.5 gkg, patients were intubated when tof=0 was obtained. anesthesia was maintained with sevoflurane 2%, remifentanil 0.25 gkgmin infusion, 2l oxygen + 2l dry air with 0.8 mlkg tidal volume and freguency of 12/min to maintain end tidal volume co2 at 3035 mmhg. for routine analgesia paracetamol infusion 1 g/100 ml was applied when 15 minutes left to the end of the surgery in all patients. merocel (medtronic, xomed, fl, usa) intranasal packs were placed in both nasal passages. a separate nasal tip dressing was placed in the anterior part of the nose. at the end of surgery and when 2 responses were achieved on the tof stimulation, one of the study drugs was administered intravenously. neostigmine 0.05 mgkg + atropine 0.02 mgkg in group n or sugammadex 2 mgkg (bridion 200 mg/2 ml, n.v. organon kloosterstraat 6, holland) in group s. when tof 0.9 patients were extubated. after the extubation, mean arterial pressure (map ; mmhg), mean heart rate (mhr ; beats / min), peripheral oxygen saturation (spo2;%) and presence of nausea - vomiting and pain were documented in the postoperative recovery room. evaluation of pain was performed according to visual analog scale (vas), and nausea - vomiting according to likert scale. amount of bleeding was measured by evaluating the blood leak on the nasal tip dressing during 3 hours postoperatively at 30 min intervals during first hour then every hour during the next 2 hours. postoperatively nasal tip dressings were changed and amount of bleeding on the nasal tip dressing was measured by comparing the patients dressing with the previously 1, 2, 3, 4 and 5 milliliters blood soaked dressings. these measurements were done by the surgeon without knowing which drug was used and documented the result as milliliters for each time period. blood samples were taken 120 minutes after administration of sugammadex or neostigmine for pt (seconds) and aptt (seconds) measurements (half life of sugammadex is 120 minutes). the blood samples for routine preoperative analysis were taken into citrate including tubes for pt and aptt measurements. the blood samples were centrifuged at 2000g for 10 minutes at 4c and plasma samples were studied immediately. one - way anova with repeated measure was used for map, mhr, spo2 and postoperative bleeding in different time periods (during first hour, second hour and third hours) and fisher exact test was used for postoperative pain and nausea - vomiting. the blood samples for routine preoperative analysis were taken into citrate including tubes for pt and aptt measurements. the blood samples were centrifuged at 2000g for 10 minutes at 4c and plasma samples were studied immediately. one - way anova with repeated measure was used for map, mhr, spo2 and postoperative bleeding in different time periods (during first hour, second hour and third hours) and fisher exact test was used for postoperative pain and nausea - vomiting. there was no difference between patient characteristics such as age (p=0.719), gender (p>0.05), surgery duration (p=0.190) and asa classification (p>0.05) (table 1). the groups were similar regarding the postoperative pain (p=0.848), nausea - vomitting (p=0.512). spo2 (p=0.276), map (p=0.280) and mhr (p=0.697) values of two groups were not different (table 2). the preoperative and postoperative coagulation parameters (pt and aptt) were presented in table 3. the difference between preoperative and postoperative coagulation parameters (pt and aptt) were calculated for each group to evaluate the change in coagulation parameters. shapiro - wilk test was used to show normal distribution of the data. to evaluate the change in coagulation parameters no statistical difference was found between pt (p=0.953) and aptt (p=0.734) difference values of group n and group s (table 4). the amount of postoperative bleeding measured by nasal tip dressings in group s was significantly higher than group n in all measurement time periods (p=0.024, table 5). the average amount of total bleeding was 2.48 milliliters in neostigmine group and 4.13 milliliters in sugammadex group. the total amount of bleeding was also found significantly higher in group s than group n (p=0.033, table 6). septoplasty is a common operative procedure in otolaryngology that requires neuromuscular blockage and intubation when performed under general anesthesia. bleeding and respiratory complications residual neuromuscular blockade is one of the undesired effects of acetylcholinesterase inhibitors for the reversal of nondepolarizing neuromuscular blockade. it is preferred mainly because of its advantages over neostigmine during extubation and recovery period and should take place in the anestesia drawer. clinical trials on healthy volunteers has shown that sugammadex is a safe agent with rare and mild side effects. there are no reported data about interaction of sugammadex with laboratory tests except coagulation parameters (pt, aptt, inr,) and progesterone level. these reported interactions have been reported at blood levels achieved after administration of 16 mgkg sugammadex. however clinical significancy of these findings is unknown since number of clinical trials have been insufficent. according to the information supplemented by the european medicines agency, administration of 4 and 16 mgkg of sugammadex in healthy volunteers resulted in maximum and mean prolongations of the aptt by 17% and 22%, respectively and pt by 11% and 22%, respectively. and these mean aptt and pt prolongations were limited and of short duration (30 minutes). soon after the sugammadex administration, de kam. reported that after administration of sugammadex at doses 4 and 16 mgkg, a dose - dependent, limited, temporary, and clinically irrelevant prolongation in pt and aptt was observed. they stated that this effect may be related to decrease in factor xa activity but later they did not find any effect of sugammadex on xa activity in patients pretreated with heparin. in another study conducted by same authors on 26 healthy volunteers, aspirin and sugammadex raft. conducted a retrospective study performed in patients at high risk of postoperative bleeding (laparotomy for cancer surgery requiring suction drains) and they concluded sugammadex at doses of 2 and 4 mgkg was not associated with increased bleeding measured by amount of blood in suction drains and dressings. despite its limitations because of retrospective design, this study has been a remarkable study in this field. in 2014 in a study of patients undergoing joint surgery, compared the pt and aptt levels of patients given sugammadex, neostigmine with glycopyrrolate or atropine or placebo / spontaneous recovery and they found limited levels of increase and reported there was no other increase in incidence of bleeding. although routine preoperative assessment with coagulation tests (pt, aptt, platelet count) is recommended, it is not always possible to identify coagulation disorders and determine the postoperative bleeding risks. preoperative coagulation tests (platelet count, pt and aptt) were normal in our study population. sugammadex has an elimination half - life of 100150 minutes so blood samples were taken 120 minutes after administration of sugammadex for pt and aptt measurements in all patients. we did not investigate the postoperative platelet count since sugammadex has no reported effect on platelet count and aggregation. we found no significant change of pt and aptt values after administration of sugammadex or neostigmine. but amount of bleeding measured by nasal tip dressings was significantly higher in sugammadex group than neostigmine group without a change in pt and aptt values. anesthesiologist should consider the cons and pros of sugammadex use in patients with these risk factors. the transient increases in aptt and pt and inr reported in the previous studies were primarily the result of reversal with 16 mgkg sugammadex.. the increased amount of postoperative bleeding that we found in our study may be due to the fact that increased bleeding tendency can be observed without any change in standard coagulation tests. in conclusion, our study demonsrated that sugammadex increases postoperative bleeding, but without significantly affecting pt and aptt values. this study is one of the first to investigate the postoperative blood loss after sugammadex use. we think that future studies investigating the effects of sugammadex on haemostasis tests other than the routine coagulation tests can clarify the mechanisms leading to increased postoperative bleeding after sugammadex use. our results need to be supported with clinical studies that will be designed with low- and high - dose sugammadex in different surgery types. although life - threatening postoperative bleeding is uncommon after septoplasty, in operations like adenoidectomy and tonsillectomy, the safety of sugammadex as a reversing agent and the safe dose ranges need to be verified. | backroundsugammadex is a reversal agent with well known advantages but it s effects on haemostasis and bleeding have been a topic of interest. septoplasty is a common surgical procedure with postoperative respiratory complications and bleeding. the aim of this study is to investigate the effects of sugammadex on postoperative coagulation parameters and bleeding after septoplasty procedure.material/methodsin this randomized controlled study, fifty patients were grouped into two groups ; neostigmine (group n) vs. sugammadex (group s). for the evaluation of pt, aptt and inr, blood samples were taken for at the postoperative 120th minutes and alteration of these values with respect to preoperative values were documented. postoperative bleeding was measured by evaluating the amount of blood absorbed on the nasal tip dressing during 3 hours postoperatively.resultspostoperative bleeding amount was significantly higher in the group s compared to group n (p=0.013). no significant difference was observed between two groups according to coagulation parameters (pt ; p=0.953, aptt ; p=0.734, inr ; p=0.612).conclusionssugammadex was associated with higher amount of postoperative bleeding than neostigmine in septoplasty patients. in surgical procedures having high risk of bleeding the safety of sugammadex need to be verified. |
depressive symptomatology has been recognized as a feature of schizophrenia since bleuler first introduced this term in 1908. he described depressive symptoms as either directly triggered by the very process of the disorder in the acute stages (i.e. as essential symptoms of schizophrenia) or as secondary symptoms of this disorder. icd-10 classification recognizes two diagnostic categories that encompass the coexistence of schizophrenia and depression : post - schizophrenic depression (f20.4) and schizoaffective disorder, depressed type (f25.1). in 1994, dsm classification defined a post - psychotic depression, which is called a post - psychotic depressive disorder of schizophrenia in dsm - iv classification [2, 3 ]. this is, however, a limited approach since there are other depressive conditions associated with schizophrenia that have not been covered by these diagnostic classifications. depressive symptoms in patients with schizophrenia may be observed during each phase of the disorder : the prodromal phase, the acute episode, the post - psychotic phase and the chronic phase, over the long - term course. depressive symptoms are frequent in the prodromal period but they are most commonly associated with the acute phase of the disease. these symptoms most frequently occur before the beginning of the treatment and in as many as half of the patients with first psychotic episode who have not started medication [46 ]. the close relationship between depressive symptoms and acute episodes supports the hypothesis that these symptoms are an inherent characteristic of schizophrenia and suggests that depressive symptoms and typical schizophrenic symptoms may result from one and the same pathophysiological process [5, 710 ]. in the course of the chronic phase of the disease, the incidence of depressive symptoms is lower and ranges from 4 to 25% [1115 ]. post - psychotic depression, according to the current dsm definition, is occurrence of symptoms during the residual phase of depression i.e. it follows after the symptoms which fulfill criterion a for schizophrenia have dissolved. main clinical signs of post - psychotic depression are depressive affect and overall psychomotor retardation [3, 7, 16 ]. the majority of depressive symptoms that develop in various phases of schizophrenia fail to fulfill the criteria for major depressive episode of moderate or severe intensity. results from one study showed that in the subjects with schizophrenia the mild depression is predominant, being present in over a half of the subjects (52%) ; 46% of the subjects manifested moderate depression and 2% severe depression. this means that patients with schizophrenia more frequently develop depression of mild or moderate intensity. the majority of researchers have come to the same conclusion i.e. that mild and moderate depression are the commonest degrees of depression intensity in patients with schizophrenia, whereas severe depression is much less frequently reported in these patients [1, 1822 ]. data from literature show significant diversity in prevalence of depression in schizophrenic patients, ranging from 7 to 75%, depending on the definition for depression. in other words, depression may denote a description of mood, syndrome (cognitive, affective and neurovegetative symptoms) or a disorder (most commonly a major depressive disorder). the wide range of prevalence of depressive symptoms in schizophrenic patients is also due to the fact that the studies have been performed in different stages of the disease, that different methods have been applied, as well as the differences in the treatment conditions [21, 2327 ]. babinkostova and stefanovski concluded that the incidence of patients with depressive symptoms among the patients with schizophrenia was 54% i.e. out of 92 examined patients, 50 showed prominent depressive symptoms, assessed objectively using the 17-item hamilton rating scale for depression (score over 7). johnson showed that 70% from the sample of 30 subjects with schizophrenia had depressive episode in two years period and authors of another study that were assessing depression in 70 patients with first schizophrenic episode (followed in 5 year period), concluded that 75% of the patients had major depressive disorder at the beginning of the schizophrenic episode. depressive symptoms during the acute phase of the disorder are registered even before the start of the medication treatment and are present in about 50% of the unmedicated patients, but the percentage of the depressive symptoms in chronic schizophrenia (clinically stabile patients) is lower. in one study only 9% of the examined patients with schizophrenia, that were clinically stabile and socially accepted, had depressive symptoms. depression in schizophrenic patients has been shown to be associated with family history of depression, early loss of a parent, higher doses of depot antipsychotics, although no significant difference between sexes has been demonstrated [6, 16, 18, 30 ]. subotnik and coworkers have found that depression in schizophrenia correlates with a positive family history for depression indicating a genetic liability. conversely, other findings on a shared genetic liability between depression and schizophrenia are questioning the concept of genetic monocausality. many researchers report on a strong genetic predisposition and accentuate that patients with schizophrenia and depressive symptoms more frequently have first - line relatives with unipolar depression [7, 19, 32, 33 ]. one study demonstrated that in patients with schizophrenia and depressive symptoms positive history of depression is predominant (in 22%) and 20% of them have a family history of schizophrenia. depressive symptoms play an important role in schizophrenia as these contribute to a further worsening of already existing deficit state and further exacerbation of illness. it is important to define and clinically assess such symptoms accurately as there is now increasing evidence that they can be treated successfully [7, 18, 34, 35 ]. depression in schizophrenia is associated with poor overall outcome characterized by further deficits in psychosocial functioning, increased rate of relapse, more frequent and longer duration of hospitalizations, poor response to pharmacological treatments, significant work impairment, lower activity, dissatisfaction, less employment and poorer quality of life. cognitive impairment, poor social functioning, substance abuse, negative attributional style and suicide / suicide attempts have also been reported [13, 16, 26, 32, 36 ]. early diagnosis, adequate differential diagnosis and promptly initiated interventions have been shown to bring down the morbidity and mortality in schizophrenic patients with depressive symptoms, to reduce further deterioration of illness and to improve patients quality of life [7, 18, 32 ]. one of the most encouraging developments in schizophrenia management is the focused attention on aspects of the early detection of depressive symptoms and the importance of early interventions. structured psychiatric clinical interview is the first diagnostic procedure that should be conducted for early detection of depressive symptoms in patients with schizophrenia. common psychiatric rating scales for clinical evaluation of presence and prominence of depressive symptomatology in schizophrenia are calgary depression scale for schizophrenia (cdss) and hamilton depression rating scale (hdrs). the most appropriate dimensional assessment instrument today is calgary depression rating scale for schizophrenia, but hamilton depression rating scale, from which some modified items are also included in the cdss, is still widely used in clinical researches regarding this field. the calgary depression scale for schizophrenia is a clinician - rated depression scale specifically developed to assess the level of depression in schizophrenia [37, 38 ]. it has been extensively evaluated in both relapsed and remitted patients and appears sensitive to change. the cdss is a nine item scale, each item scored from 0 to 3. factor analysis showed that the cdss is unidimensional, has high internal consistency, and significant strong correlation with scores on the hdrs, beck and bprs depression scales. authors of one study compared the psychometric properties of the calgary depression rating scale and the hamilton depression rating scale for severity assessment of depression in schizophrenia. they investigated 119 inpatients with acute schizophrenia using the cdss, the hdrs and a global 4-point depression severity scale and they concluded that although both depression scales were correlated and highly effective in separating mild, moderate and severe depression, significant advantages emerged in favor of the cdss. thus the cdss should be used particularly for a sensitive detection of mild (for example subthreshold) depression at early stages or to assess schizophrenic patients with severe depression who require through follow - up assessment and specific interventions. also calgary depression scale for schizophrenia seems to have more efficiency and ability to distinguish between depression, negative and extrapyramidal symptoms. demonstrated the methodological superiority of the calgary depression rating scale in the assessment of depressive symptoms in schizophrenia over scales developed for nonschizophrenic populations such as the hamilton depression rating scale and the present state examination. the interview for the retrospective assessment of the onset of schizophrenia is a semi - structured interview which can help determine whether this is the case. the psychotic depression scale (pds) also can be useful for early detection of depression in schizophrenia. it has been shown that there is no overlap between negative or extrapyramidal and depressive symptoms assessed by the pds in schizophrenic patients. the psychotic depression scale is a 32 item scale and each item is rated from 0 to 7. some authors use the beck depression inventory (bdi), montgomery - asberg depression rating scale and depression subscale of the brief psychiatric rating scale (bprs) for evaluation of severity of depressive symptoms in schizophrenic patients [27, 4244 ]. kim. assessed the following four depression scales for their diagnostic validity as measures of depressive disorder in schizophrenia : the calgary depression scale for schizophrenia (cdss), the beck depression inventory (bdi), the hamilton rating scale for depression (ham - d), and the depression subscale of the panss (panss - d). their results suggested that the cdss may provide the best assessment for depression in patients with schizophrenia. hausmann and fleischhacer also recommend the calgary depression scale in schizophrenic patients with depressive symptoms and also hillside akathisia scale to help establish the right diagnosis. the positive and negative syndrome scale (panss), the simpson - angus rating scale (sars), barnes akathisia scale, abnormal involuntary movement scale (aims) and the rating scale for extrapyramidal symptoms (esrs) are used to differentiate depression from the negative and extrapyramidal symptom - related depressive phenomena in schizophrenia [1, 43, 45 ]. a major challenge in the clinical assessment of schizophrenia is the differentiation among depressive features, negative symptoms and neuroleptic side effects. when all of these possibilities have been excluded, there is evidence that depression is perhaps most often an integral part of the schizophrenic process itself [5, 7, 8 ]. it is essential to differentiate between depressive symptoms and previous mentioned conditions and to manage them accordingly, in order to reduce the risk of further morbidity and mortality [18, 29 ]. a key issue in the differential diagnosis of depressive symptoms in patients with schizophrenia is disentangling the contribution of negative symptoms. based on dsm - iv classification, negative symptoms of schizophrenia include affective flattening, alogia, avolition and anhedonia. there is thus a great overlap between symptoms of depression and negative symptoms of schizophrenia. diminished interest, pleasure, energy, or motivation along with psychomotor retardation and impaired ability to concentrate are relevant overlapping features. prominent subjectively low mood, suggesting depression, and prominent blunting of affect, suggesting negative symptoms, are the two features which are most helpful in differentiating the two syndromes. other symptoms that help to establish the diagnosis of depression include some of the main psychological features that occur in primary depressive illness, such as hopelessness, helplessness, worthlessness, guilt, anxiety and suicidal thinking. in schizophrenia, the biological features of the depressive syndrome, such as insomnia and retardation, are not always present and if they are present, they can be more difficult to disentangle from negative symptoms and can be an intrinsic part of the illness separate from any superimposed depressive syndrome [7, 16, 32, 49 ]. refers to patients in whom a full affective syndrome coincides with the florid psychotic syndrome but who also have substantial periods of psychosis in the absence of an affective syndrome [16, 32, 41 ]. dopamine blockade by a neuroleptic drug could theoretically lead to anhedonia and perhaps, depression. neuroleptic - induced akinesia is extrapyramidal side effect of neuroleptic treatment involving impaired ability to initiate and sustain motor behavior. patients with this form of akinesia may or may not have the classical parkinsonian feature of decreased accessory motor movements. they act as if their starter motor is broken, and they consequently appear to lack spontaneity. akinesia may even protect against suicidal behavior, but not against suicidal thinking [16, 32 ]. akathisia is another extrapyramidal side effect of neuroleptic treatment that can easily be confounded with depression. patients with akathisia behave as if their starter motor wo nt stop and often experience this state as substantially dysphoric. akathisia has been associated with both suicidal ideation and, perhaps as a consequence of a general tendency toward motor action, suicidal behavior [16, 32 ]. a number of medical / organic factors can present as depression in patients with schizophrenia. these include cardiovascular disorders, pulmonary infections, autoimmune diseases, anemia, cancer, metabolic, neurological and endocrine disorders. various pharmaceuticals used in medical treatment such as blockers, other antihypertensive agents, sedative hypnotics, antineoplastics, barbiturates, nonsteroidal anti - inflammatory drugs, sulfonamides, and indomethacin can cause depression as a side effect. depression can also accompany the discontinuation of other prescribed medications such as corticosteroids and psychostimulants. used or abused substances such as alcohol, cannabis, cocaine, or narcotics can contribute to depression either on the basis of acute use, chronic use, or discontinuation [16, 32 ]. reactions to disappointments, a sense of loss or powerlessness, or awareness of psychotic symptoms or psychological deficits can certainly present as or contribute to depression, especially when depression follows closely after a stressful event or exacerbation of schizophrenia. an acute reaction to disappointment or stress is suggested by the parallel history of a recent compatible event. it is characterized by hopelessness and helplessness, with a lack of confidence and feelings of incompetence. adjunctive psychotherapy, e.g. psychoeducational family therapy or cognitive behavioral therapy is the mainstay of helping patients to cope with demoralization syndromes [7, 16, 32 ]. in earlier times, the term postpsychotic depression was used to describe a dysphoric state that immediately followed a psychotic episode. dsm - iv now suggests that the term postpsychotic depression be used to describe depression that occurs at any time after a psychotic episode in schizophrenia, even after a prolonged interval [16, 32 ]. the appearance of a depression - like state as a prodrome of a new psychotic episode is a short - lived phenomenon, often lasting only a couple days to a couple weeks, before being superseded by more prominent and definitive psychotic symptoms [16, 32 ]. the therapeutic goal is significantly to reduce the excess morbidity and mortality associated with depressive symptoms and to improve individual patients quality of life. the first steps are to exclude cases of schizoaffective disorder and to treat them appropriately, to treat any medical conditions that are present and to consider the possibility of substance misuse as a contributing factor. any evidence that antipsychotic medication is producing akinesia should lead to a reduction in dosage and/or the introduction of anticholinergic medication. akathisia, with its concomitant feeling of dysphoria, should always be considered in patients describing subjective mood disturbance. the akathisia / dysphoria syndrome, if present, requires active management. other options include -adrenoceptor antagonists (e.g. propranolol), a benzodiazapine or a change in antipsychotic drug. if the above factors have been addressed and the clinician is sure that negative symptoms are not being mistaken for depressive symptoms, then the treatment options are largely dictated by the stage of the illness. during acute episodes, depressive symptoms should not be treated separately from other symptoms and are likely to resolve as the episode resolves. in the majority of cases increased antipsychotic medication, increased psychosocial support and, if necessary, hospitalization, will successfully treat depression as well as positive symptoms. moller hj in his comprehensive review reported that besides the potential risk of inducing depressive symptoms, the first generation antipsychotics seem to have a certain antidepressive effect, in the sense that depressive symptoms secondary to positive symptoms can be alleviated with the reduction of positive symptoms. the second generation antipsychotics seem to have no risk to induce depressive symptoms, appear to have better antidepressive effect and therefore represent a better option for the treatment of depressive symptoms in schizophrenic patients. there is accumulating evidence that the new atypical antipsychotics are more efficacious in treating the depression associated with an acute episode. olanzapine, for example, is superior to haloperidol in this regard [42, 52, 53 ]. other atypicals, such as risperidone, ziprasidone and zotepine, may also have a mood - elevating effect. the atypicals may prove to be useful for the depression that emerges during the chronic phase of the illness. clozapine has been shown to reduce hopelessness, depression and suicidality in patients with chronic schizophrenia [5459 ]. clinicians should consider switching patients to an atypical antipsychotic if they are not taking one already. in addition, dose optimization needs to be targeted towards depressive as well as positive and negative psychotic symptoms [16, 41, 60 ]. administration of antidepressants in combination with antipsychotics seems to be a meaningful option to treat depressive symptoms in schizophrenia. data collected show that treatment with antidepressants in addition to antipsychotic treatment is only of limited benefit [25, 61 ] there is a good case for the prescription of an antidepressant when the patient has persistent depressive symptoms and is not in a phase of acute illness. recent studies have shown that antidepressants are prescribed by clinicians to 30% of inpatients and 43% of outpatients of all ages with schizophrenia and depressive symptoms. ssris when compared with the triciclics appear to be the treatment of choice considering the side effects and efficacy [16, 18 ]. one needs to start with a low dose and then cautiously titrate upward to reduce depressive symptoms. if remission is not achieved after adequate treatment duration (812 weeks) or with an adequate dose (similar to that used for major depression without schizophrenia), switching to another agent or adding augmenting therapy is recommended. whitehead and coworkers in their systematic review of antidepressants for the treatment of depression in patients with schizophrenia concluded that the meta - analysis of the 5 suitable trials found that using antidepressants was beneficial and there was no evidence that antidepressant treatment led to a deterioration on psychotic symptoms in the trials. on the other hand, zisook. in their research paper compared the patients who were taking antidepressant medications to those who were not, on the 17-item hamilton depression scale total score and on the percentage of subjects classified as minimally, mildly and moderately to severely depressed and they found no difference between groups on any of these measures. also they stated that correlation between severity of depression and daily neuroleptic dose was low, negative and nonsignificant. siris reported that long - term maintenance treatment with antidepressants would be beneficial for patients who responded favorably to the initial treatment. he stated that besides the fact that some patients were resistant and even refractory to pharmacological treatment, it would be also probable that therapists found it difficult to administer high doses of antidepressants to these patients, because of the fear of recidivation of psychotic symptoms which lead to the use of subtherapeutical doses of antidepressants making it difficult to cute this syndrome and to a subsequent chronification of the condition. table 1 presents the main results from the conducted studies for antidepressant augmentation in schizophrenic patients with depressive symptoms. results from the several studies showed that citalopram augmentation of antipsychotic treatment in patients with schizophrenia and depressive symptoms improves depressive symptomatology [6567 ]. authors of several studies evaluated addition of venlafaxine to antipsychotic treatment in schizophrenic patients with depression and they concluded that the dual mechanism of this drug may contribute to a more complete antidepressant response than the single mechanism of selective serotonin reuptake inhibitor medication [68, 69].table 1antidepressant augmentation in schizophrenia with depressive symptomsstudyyearstudy designtype of antidepressantmain resultskasckow.2010randomized, placebo - controlledcitalopramimprovement in depressive symptoms, negative symptoms, social functioning and quality of life with citalopramzisook.2009double - blind, randomized, placebo - controlledcitalopramsignificantly more effective than placebo in improving depressive symptomsciobanu.2008randomized, prospectivevenlafaxin, fluvoxamine, mirtazapineimprovement in depressive symptoms with all antidepressants, quicker response with venlafaxinemazeh.2004open - label studyvenlafaxineimprovement in depressive symptomsaddington.2002randomized, double - blind, prospective, placebo - controlledsertralinelimited benefit of added sertralinekasckow.2001open - label studycitalopramsignificant improvement in hamd scoresmullholand.1997double - blind, placebo - controlledsertralineno significant benefit of added sertraline (limited benefit) antidepressant augmentation in schizophrenia with depressive symptoms psychosocial therapies in combination with pharmacotherapy are very important additional treatments needed for this patient population in order to alleviate residual symptoms and to improve social functioning and quality of life [16, 41 ]. cognitive therapy also has been shown to be effective, although its role in the treatment of depressive symptoms in particular has not been studied [7, 35, 41, 70 ]. several different concepts have been employed to explain the role of antipsychotics in schizophrenic patients with depression. it has been suggested that antipsychotics cause a so called pharmacogenic depression by a direct action on the dopaminergic system affecting the pleasure and reward pathways [32, 71, 72 ]. akinesia and other extrapyramidal adverse effects of antipsychotics, excluding tremor, accompanied by depressed mood or dysphoria may also mask depression. the so called akinetic depression accounts for 1015% of depressive symptoms and it includes a reduced level of activity as well as anhedonia, responding to anticholinergic medication. certain authors suggest that iatrogenic depression associated with the extrapyramidal adverse reactions of the antipsychotic treatment is less common nowadays than it used to be in the past [18, 73, 74 ]. although certain observations support the hypothesis of a pharmacogenic depression, suggesting that antipsychotics are responsible for several cases of depression in schizophrenia, the evidence against it carries more weight [75, 76 ]. depression may also occur in patients with schizophrenia who do not receive antipsychotics, and when antipsychotics are discontinued, the proportion of patients who need antidepressants is increased. several studies have demonstrated that there are no differences between depressed and non - depressed patients with schizophrenia in terms of the dose of the antipsychotic that they received [7, 24, 27 ]. there are several reasons to suspect that schizophrenia treated with atypical antipsychotics may prove to be a different condition than schizophrenia treated with conventional neuroleptics from the point of view of depression. since akinesia and akathisia figure prominently in the differential diagnosis of depression in schizophrenia, this issue could be responsible for a different expression of depression in schizophrenia. since atypical agents seem to rely much less exclusively on dopaminergic blockade for their therapeutic activity, they might circumvent the mechanism of neuroleptic - induced dysphoria that could contribute to the depression syndrome. atypical antipsychotics have frequently been reported to be superior to standard neuroleptics in the treatment of negative symptoms, which can sometimes appear similar to depression and it is possible that atypical antipsychotic agents have direct antidepressant activity on their own. combinations of any or all of these effects could potentially be responsible for more favorable depression profiles in patients treated with atypical antipsychotics versus conventional neuroleptics [16, 23, 28, 48 ]. the broad array of affinities for receptor sites attributable to the novel atypical antipsychotic agents (including a wide array of 5-hydroxytryptamine [5-ht ], dopamine [other than d2 ], and muscarinic sites as well as 1-noradrenergic and histamine-1 receptor sites) suggest a variety of potential mechanisms through which atypical antipsychotics might exercise antidepressant effects and that their antipsychotic and antidepressive effects are mostly based on different pharmacological mechanisms [16, 77 ]. the dopamine deficit in cortical prefrontal areas was a unifying hypothesis to explain both some symptoms of depression and negative symptoms of schizophrenia. studies in animal confirm this view and show that the association of an atypical antipsychotic drug and an ssri (olanzapine plus fluoxetine) increases synergistically the release of dopamine in prefrontal areas. harrow and coworkers observed that depression in schizophrenia correlated with first generation antipsychotic treatment and suggested that depression is associated with anhedonia and mesolimbic dopaminergic dysfunction, which may be potentiated by dopaminergic blockade induced by typical antipsychotics. although depressive symptoms are described in neuroleptic - naive schizophrenic patients having a first episode, patients receiving conventional antipsychotic drugs may have higher rates [41, 42 ]. by contrast second - generation antipsychotics may have some efficacy for depressive symptoms over improvement in positive and negative symptoms, perhaps due to their direct antidepressant effects [55, 77 ]. on the other hand, as reported by freudenreich. there was no difference in depressive symptoms between the different classes of antipsychotics used (typical, atypical antipsychotics and a clozapine group). also this study found that hamd scores did not vary between the group receiving antidepressant therapy (22% of the examinees) and the rest of the sample, nor did panss scores. authors of this study stated that treatment with second - generation antipsychotics did not confer benefit compared to first - generation antipsychotics with regard to depressive symptoms. similar to this, mauri and coworkers in their article refer to comparison of first and second generation antipsychotics did not found that atypical antipsychotics as a class are more effective on the depression dimension during the course of schizophrenia than typical ones. their results showed that none of the drugs seemed to have a real depressogenic effect. they did not confirm correlation between the severity of the depressive symptoms and the doses of the antipsychotics. they referred that all of the typical and atypical antipsychotics alone improved depressive symptoms, but the improvement was statistically significant in cases of fluphenazine decanoate, haloperidol, olanzapine, risperidone and l - sulpiride, which partially conflicts with some published data concerning the depressogenic effect of classical neuroleptics (pharmacogenic depression), especially fluphenazine decanoate and haloperidol. aguilar. in their article stated that second - generation antipsychotic agents appear to have a better potential for preventing suicide in schizophrenia. the strongest and perhaps unique evidence has been shown for clozapine, which seems to have a clinically relevant advantage over both first- and second - generation antipsychotics for reducing suicidality. suggestion that suicidality may be reduced in schizophrenic patients receiving clozapine is also confirmed by other authors [5559 ]. table 2 presents the main results from the studies that evaluated effects of the different types of antipsychotics on depression in schizophrenic patients. the strongest evidence for the benefit of atypical antipsychotics in the treatment of concurrent depressive symptoms in schizophrenic patients comes from trials of olanzapine and ziprasidone. first randomized double - blind study regarding efficacy and tolerability of atypical antipsychotics in the treatment of patients with schizophrenia and depressive symptoms, compared olanzapine with ziprasidone therapy in these patients. their results showed that for up to 8 weeks patients treated with olanzapine or ziprasidone had significant improvements on calgary depression scale for schizophrenia, but treatment group differences were not statistically significant. over 24 weeks, olanzapine treated patients showed significantly greater improvements in depressive symptoms and gaf scores. in a comparison of olanzapine and ziprasidone for the 6-week treatment of patients with acute schizophrenia or schizoaffective disorder, both antipsychotics were efficacious in improving positive, negative and depressive symptoms without significant treatment differences, suggesting that ziprasidone was as efficacious as olanzapine in improving the calgary depression scale for schizophrenia score. another study that compared olanzapine and ziprasidone in patients with schizophrenia demonstrated significantly greater improvement in olanzapine - treated patients on positive, negative and depressive symptoms at the 28-week study, suggesting that treatment response may differentiate over longer treatment durations. in a systematic review and meta - analysis, leucht. reported that olanzapine was found to have superior efficacy compared to aripiprazole, quetiapine, risperidone and ziprasidone. in addition, clozapine had superior efficacy compared to zotepine and risperidone when clozapine doses at 400 mg a day were used.table 2effects of antipsychotics on depression in schizophreniastudyyearstudy designtype of antipsychotic medicationmain resultskinon.2006randomized, double - blindolanzapine vs. ziprasidoneolanzapine showed significantly greater improvement in depressive symptomsbreier.2005randomized, controlled, double - blindolanzapine vs. ziprasidonesignificatntly greater improvement in olanzapine - treated patients on depressive, positive and negative symptomsmergui.2005case studyquetiapinequetiapine - associated depression in schizophrenic patientssimpson.2004randomized, controlled, double - blindziprasidone vs. olanzapineboth antipsychotics were efficacious in improving depressive, positive and negative symptomsemsly.2003randomized, controlledquetiapine vs. haloperidolquetiapine showed greater reduction in depressive scorestollefson.1999double - blind, randomizedolanzapine vs. risperidoneolanzapine was superior to risperidone in alleviating depressiontollefson.1998randomized, controlled, double - blindolanzapine vs. haloperidololanzapine was associated with a significantly higher improvement of depressive symptomsceskova.1993double - blind, randomizedrisperidone vs. haloperidolrisperidone was inferior to haloperidol in terms of bprs depression factor improvement effects of antipsychotics on depression in schizophrenia tollefson. in their double - blind, controlled clinical trial compared the use of olanzapine with that of haloperidol for up to 52 weeks of treatment in patients with schizophrenia, schizoaffective and schizophreniform disorder and they referred that the use of olanzapine was statistically significant superior to that of haloperidol in the early improvement of depressive signs and symptoms evaluated with the montgomery - asberg depression rating scale and there was no dose difference based on the depressive signs and symptoms. similar results presented other studies that evaluated the efficacy of olanzapine compared to risperidone and haloperidol in the treatment of schizophrenia - related depressive symptoms [52, 53 ]. most studies referred that olanzapine, risperidone or ziprasidone may have an antidepressant spectrum of activity in patients with schizophrenia [16, 43, 56, 7880 ]. one trial presented data suggesting that risperidone may be inferior to haloperidol in terms of bprs anxiety / depression factor improvement. it is noteworthy that quetiapine has been found to have an antidepressant effect. in one study patients suffering from schizophrenia derived greater reduction in depressive scores in comparison to haloperidol. in another study quetiapine was found to improve symptoms of depression, over the course of long term use. contrary to this the authors of one case report presented a quetiapine - associated depression in schizophrenic patients. it is the first description of depression associated with quetiapine treatment and their authors suggested that atypical antipsychotics may be a cause of depression, but further data based on controlled studies are required before one can draw definitive conclusions. in a recent review by furtado. the authors included randomized clinical trials of atypical antipsychotic drugs used for the treatment of patients with a diagnosis of both schizophrenia and depression. one trial found no significant differences between quetiapine and haloperidol for the outcome of less than 50% reduction in panss score. in a second trial, sulpiride was compared to chlorpromazine ; the group treated with sulpiride had more reductions in depressive symptoms based on scores of the comprehensive psychopathologic rating scale. in a third trial, jasovic used hamilton depression rating scale scores to compare clozapine to any other antipsychotic plus an antidepressant ; use of clozapine lead to greater reductions in hamilton symptoms. authors of this review article conducted a cross - sectional study which involved 92 patients with schizophrenic disorder, 50 of whom with prominent depressive symptoms (total score > 7 on the 17-item hamilton rating scale for depression) with the aim of evaluation of the effect of the different type of antipsychotic treatment administered on the depression intensity in schizophrenic patients. the group was created by randomization of inpatients and outpatients of both sex who were treated at the university clinic of psychiatry within a period of 6 months. for the purposes of the study, the test group was subsequently subdivided in several subgroups according to the type of used antipsychotic therapy (oral / depot ; typical / atypical ; olanzapine / risperidone / clozapine). 60% of the examinees were taking oral antipsychotics and 40% of them were administered depot antipsychotic treatment (long - acting atypical antipsychotic). according to the treatment with different classes of oral antipsychotics (typical / atypical), the results indicated that 20% of the oral antipsychotic group were taking typical antipsychotics (haloperidol), whereas 80% of these subjects received atypical oral antipsychotic medications. from the atypical oral antipsychotic subgroup 37.5% were taking olanzapine, the results obtained from this study suggested that subjects who received depot antipsychotics had less severe depression in comparison to the subjects who received oral antipsychotics, although this difference, when tested using the t - test for independent samples, was not statistically significant. contrary to this, some earlier studies in this direction showed a positive relationship between the intensity of depression and a higher dose of depot antipsychotics in patients with schizophrenia [6, 7, 18, 30 ]. further results of this study showed that subjects treated with depot antipsychotics had statistically significant lower depression intensity in comparison to the subjects treated with typical oral antipsychotics. figure 1 presents the difference in depression intensity between the subgroup with typical oral antipsychotics and the subgroup with atypical oral antipsychotics.fig. 1total score on hamd - typical vs. atypical oral antipsychotics total score on hamd - typical vs. atypical oral antipsychotics the tested difference in depression intensity between the subgroups treated with different types of oral atypical antipsychotics in this study showed that the subjects treated with clozapine had statistically significant lower depression intensity in comparison to the subjects treated with risperidone (fig. 2) and also they had lower depression intensity in comparison to the subjects treated with olanzapine, but statistically not significant. the patients treated with olanzapine had lower depression intensity in comparison to those treated with risperidone, but statistically not significant.fig. 2total score on hamd - risperidone / clozapine total score on hamd - risperidone / clozapine depressive symptoms are common in patients with schizophrenia and they can occur during any phase of the disorder. they are associated with a variety of undesirable outcomes and are a significant cause of higher rates of morbidity and mortality in these patients. early diagnosis, adequate differential diagnosis and promptly initiated interventions have been shown to bring down the morbidity and mortality in schizophrenic patients with depressive symptoms, to reduce further deterioration of illness and to improve patients quality of life. structured psychiatric clinical interview is the first diagnostic procedure that should be conducted for early detection of depressive symptoms in patients with schizophrenia. common psychiatric rating scales for clinical evaluation of presence and prominence of depressive symptomatology in schizophrenia are calgary depression scale for schizophrenia (cdss) and hamilton depression rating scale (hdrs). the most appropriate dimensional assessment instrument today is calgary depression rating scale for schizophrenia, but hamilton depression rating scale, from which some modified items are also included in the cdss, is still widely used in clinical researches regarding this field. the calgary depression scale for schizophrenia is a clinician - rated depression scale specifically developed to assess the level of depression in schizophrenia. the psychotic depression scale (pds) also can be useful for early detection of depression in schizophrenia. it has been shown that there is no overlap between negative or extrapyramidal and depressive symptoms assessed by the pds in schizophrenic patients. the positive and negative syndrome scale (panss), the simpson - angus rating scale (sars), barnes akathisia scale, abnormal involuntary movement scale (aims) and the rating scale for extrapyramidal symptoms (esrs) are used to differentiate depression from the negative and extrapyramidal symptom - related depressive phenomena in schizophrenia. an appropriate treatment approach to depression in schizophrenia begins with a consideration of the differential diagnostic possibilities. depressive symptoms should be adequately differentiated from negative symptoms, schizoaffective disorder, neuroleptic induced side effects, reactions to disappointments and a number of medical / organic factors. treatment of depressive symptoms in schizophrenia is accomplished through a combination of pharmacologic and psychosocial approach. atypical antipsychotics have advantages over typical in reducing depressive symptoms in the context of schizophrenia. they do not induce depressive symptoms, which is known side effect of traditional neuroleptics. their antidepressive potential is presumably related to their pharmacological mechanisms, which differ from those of traditional neuroleptics. the benefits of atypical antipsychotics might be of relevance also for reducing the risk of suicidality. from a pharmacologic perspective a lot of studies referred that clozapine, olanzapine, quetiapine and risperidone have an antidepressant spectrum of activity in patients with schizophrenia. most of the studies showed that antidepressant augmentation of antipsychotic treatment in patients with schizophrenia and depressive symptoms improves depressive symptomatology, especially ssri and snri augmentation. implementing psychosocial interventions in combination with pharmacotherapy is also an important part of patients treatment plan. we can conclude that in patients with schizophrenia early detection of depressive symptoms can be obtain conducting structured psychiatric clinical interview and using the appropriate assessment instruments regarding this topic in everyday psychiatric clinical practice. in schizophrenic patients with depressive symptoms preferable pharmacological treatment include use of atypical antipsychotics and antidepressants augmentation, particularly ssri and snri. pharmacological treatment in these patients should be combined with psychosocial interventions. early diagnosis and promptly initiated interventions have been shown to reduce further deterioration of illness and to improve patients quality of life. | depressive symptoms are common in schizophrenia and they can occur during any phase of the disorder. early diagnosis, adequate differential diagnosis and promptly initiated interventions have been shown to reduce further deterioration of illness and to improve patients quality of life. common psychiatric rating scales for early detection of depressive symptoms in schizophrenia are calgary depression scale for schizophrenia and hamilton depression rating scale, but the most appropriate assessment instrument today regarding this topic is calgary depression scale for schizophrenia. treatment of depression in schizophrenia consists of a combination of pharmacologic and psychosocial approach. atypical antipsychotics have advantages over typical in reducing depressive symptoms in the context of schizophrenia. most of the studies referred that clozapine, olanzapine, quetiapine and risperidone have an antidepressant spectrum of activity in patients with schizophrenia. antidepressant augmentation of antipsychotic treatment in schizophrenic patients with depressive symptoms improves depressive symptomatology, particularly ssri and snri augmentation. |
sample collection : 20 ml of fresh urine was collected from individual cattle in lopburi, chainat and nakhon nayok provinces in the central region of thailand from october 2012 to february 2014 for the propose of disease surveillance. after collection, the samples were kept in cool storage (4c) and transported to the national institute of animal health. all samples were subsequently examined with three methods including bacterial culture, real - time pcr and lamp assay. culture : one ml of urine samples was added to 9 ml of 1% bovine serum albumin diluent with 5 fluro the samples were cultured in emjh (johnson and harris modification of the ellinghausen and mccullough medium) semi solid media with 5 fluro uracil (becton - dickinson biosciences, detroit, mi, u.s.a.) at 30c for 1216 weeks. leptospira spp. were considered positive by the characteristic of thin helical structures with prominent hooked ends and motility under a dark field microscope. the suspected samples were further subcultured in the emjh liquid media and purified with 0.2 m pore dna extraction : dna in 10 ml of urine sample was extracted using dynabeads dna direct universal kit (invitrogen, oslo, norway) according to the manufacturer s instructions ; dna concentration was measured by qubit fluorometer (invitrogen). loop mediated isothermal amplification method (lamp) : the lamp was carried out using primer and condition previously described in. briefly, a total of 25 l reaction mixture contained 1.0 m outer primers f3 (5-gaaccctgaagcagcgac3) and b3 (5-gacttacatgtccgcctacg3), 1.6 m inner primers fip (5-cacatcgctgcttatttttccctgcgcgtgaacgatgaaggtct-3) and bip (5-cctgcctaaagcaccggctaacgcccaatgattccgaacaa-3), 0.8 m loop primer lb (5agccgcggtaatacgtatggtg3), 1x thermopol reaction buffer (new england biolabs, hitchin, u.k.), 4 mm mgso4 (new england biolabs), 1 m betaine (sigma - aldrich, st. louis, mo, u.s.a.), 0.4 mm dntps (new england biolabs), 0.5 mm mncl, 25 m calcein and 8 units bst dna polymerase (new england biolabs) and adjusted to a total volume of 25 l with sterile water (new england biolabs). the reaction mixture was incubated at 61c for 90120 min in a heating block and then was heated at 80c for 2 min to terminate the reaction. the presence of product was defined on the basis of green color detected with naked eyes. real - time pcr : real - time pcr was conducted according to by targeting the 241 bp region of lipl32 gene which encoded the major outer membrane of pathogenic leptospira spp. the light cycler nanoinstrument (roche diagnostic, mannheim, germany) was used for hydrolysis probe assay. the dna product was amplified using faststarttaq dna polymerase (roche diagnostic) with the primers lipl32 - 45f (5-aagcattaccgcttgtggtg-3), lipl32 - 286r (5-gaactcccatttcagcgatt-3) and probe189p ([fam ] 5-aaagccaggacaagcgccg-3[bhq1 ]. the reaction mixture consisted of 10 ng of extracted dna, 0.25 m each primers and probe and 1x faststart essential dna probe master and was adjusted to a total volume of 20 l with sterile water (roche diagnostic). the thermal condition of a holding stage was performed at 95c for 10 min and followed by 45 cycles of two step amplification from 95c to 55c for 30 sec. were calculated following ; accuracy of these tests was calculated according to the formulas proposed in. the formulas described in were used in positive and negative predictive values calculation. equations 17 were used in these analyses ; percent positivity ; pp=(tp+fp)/(tp + fp + tn + fn) (1) sensitivity ; se = tp/(tp + fn) (2) specificity ; sp = tn/(tn + fp) (3) gold standard prevalence ; gsp=(tp+fn)/(tp + fp + tn + fn) (4) positive predictive value ; ppv = (se gsp)/((se gsp) + ((1-sp) (1-gsp))) (5) negative predictive value ; npv = (sp (1-gsp))/(((1-se) gsp) + ((sp) (1-gsp))) (6) accuracy=(tp + tn)/(tp + fp + tn + fn) (7), where tp, fp, tn and fn refer to true positive, false positive, true negative and false negative, respectively. cohen s kappa statistics for agreement of all the tests were analyzed, and the strength of agreement was interpreted as proposed in. k=(po - pe)/(1- pe) (8), where k, po and pe represent cohen s kappa coefficient, relative observed agreement of the tests and hypothetical probability of agreement, respectively. all statistical analyses were carried out with program r version 3.2.2 (r development core team, vienna, austria). caret were used in the calculation of se, sp, ppv and npv, whereas cohen s kappa statistics were performed within the package fmsb. prevalence : a total of 533 urine samples were collected from 266 beef and 267 dairy cattle. it was found that 31, 45 and 58 animals were positive from leptospiral culture, lamp and real - time pcr, respectively. detection from these three assays was 5.8% (confidence interval (ci) 4.08.2), 8.4% (95% ci 6.211.1) and 10.9% (95% ci 8.413.8), respectively. accuracy : lamp and real - time pcr were evaluated using bacterial culture as a gold standard. the examination results comparing bacterial culture with two diagnostic assays are illustrated in table 1table 1. comparison of examination results from lamp and real - time pcr with culture method in the detection of leptospira spp. in urine samples collected from cattle in central region of thailandculture resultlampreal - time pcrno. lamp was more sensitive and more specific than real - time pcr as could be obviously seen that the sensitivity of lamp and real - time pcr was 96.8% (95% ci 81.599.8) and 74.2% (95% ci 55.187.5), while the specificity of lamp and real - time pcr was 97.0% (95% ci 94.998.2) and 93.0% (95% ci 90.395.0), respectively. 95.298.3) was significantly higher than that of real - time pcr (91.9% ; 89.394.1). both assays had higher negative predictive values (lamp : 99.8% ; 95% ci 98.7100.0 and real - time pcr : 98.3% ; 95% ci 96.699.2) than the positive ones (lamp : 66.7% ; 95% ci 50.979.6 and real - time pcr : 39.7% ; 95% ci 27.353.4). comparing these two assays, higher positive and negative predictive values agreement of the tests : to further assess the strength of agreement among all studied assays, cohen s kappa statistics were then analyzed. culture method and lamp substantially agreed with each other (77.4% ; 95% ci 66.588.3), whereas real - time pcr only moderately agreed with culture (47.7% ; 95% ci 32.762.7) and lamp (45.3% ; 95% ci 31.059.6). the present study evaluated the diagnostic accuracy of two rapid molecular methods including lamp and real - time pcr, in the detection of leptospira genetic materials in urine samples from cattle in central region of thailand. in this study, bacterial culture true prevalence. comparing the true prevalence with the prevalence derived from lamp and real - time pcr (table 1.), overestimated prevalence was found in both assays, especially from real - time pcr (10.9% ; 95% ci 8.413.8) ; it was almost two times higher than the true prevalence (5.8% ; 95% ci 4.08.2), whereas that of lamp fell in the middle at 8.4% (95% ci 6.211.1). our results corresponded with the previous study, suggesting that positivity in molecular techniques could be obtained with no positive culture results. regarding diagnostic accuracy of lamp and real - time pcr, lamp was more accurate method in the detection of leptospira spp. with higher sensitivity and specificity than real - time pcr. the lamp assay targeting 16s rdna gene used in the present study had a low detection limit at 10100 copies. therefore, its sensitivity was high as it could detect the pathogen even with low amount of genetic materials. in contrast, it was found that real - time pcr targeting lipl32 gene was a low sensitive method with the sensitivity of 43% (95% ci 3452%), and the detection limit of real - time pcr was found at 1001,000 copies per reaction under the author s conditions. the lower sensitivity of real - time pcr, compared to lamp, was attributed to the difference in lower detection limits of the two methods. however, the low sensitivity of real - time pcr in this study also resulted from the high number of false negative samples (25.8% ; 8/31) which were negative with real - time pcr but positive with culture. the plausible explanation was that a very low count in the original sample might stochastically present in the aliquot prepared for culture but not in that for real - time pcr. in terms of specificity, both lamp and real - time pcr were considered a highly specific assay which was consistent with other previous publications [4, 10, 24, 25 ]. the primers used in the present study were preliminarily tested with some intermediate species of leptospires and demonstrated some mismatched characteristics. however, the test did not guarantee that the primer set never amplifies the target gene from any other intermediate species. to prove this, the possible amplification of target gene from intermediate species by the lamp primer set may contribute to higher sensitivity of lamp than real - time pcr. apart from sensitivity and specificity, predictive values changed proportional to the prevalence changes. the predictive values gave a basic idea on how we can predict the results using a specific test with a specific population in a specific time. in this study, npv was high in both lamp (99.8% ; 95% ci 98.7100.0%) and real - time pcr (98.3% ; 95% ci 96.699.2%). in contrast, the ppv of these two tests was moderate to low (66.7% and 39.7% for lamp and real - time pcr, respectively). however, as the predictive values depended largely on prevalence, the values were not transferrable from one to another study with different spaces and times. based on the cohen s kappa statistics calculated in this study, real - time pcr this result was in agreement with the previous studies providing a notice on the possible disagreement of real - time pcr and culture [7, 25 ]. moreover, lamp was much more sensitive and slightly more specific than real - time pcr. therefore, the agreement of these two tests was only at moderate level. bacterial culture which was considered the gold standard in the present study was regarded as a technique with low sensitivity, laborious, time consuming and not practical in the early detection of leptospires. to confront with this problem, various molecular techniques have been employed to rapidly identify the pathogens including real - time pcr and lamp. however, real - time pcr was challenging in term of finance, technical and instrumental requirements and might not be applicable in developing countries where leptospirosis has been endemic [1, 26 ]. therefore, the less expensive method with less expertise requirement, such as lamp, has been developed and implemented for the early detection of leptospira pathogen. as demonstrated in our study, lamp was more effective than real - time pcr in the detection of pathogenic leptospira spp. in the urine of cattle with substantially less cost and more simplicity. accordingly, lamp was an excellent alternative for routine surveillance of leptospirosis in cattle. | leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic leptospira spp. many molecular techniques have consecutively been developed to detect such pathogen including loop mediated isothermal amplification method (lamp). the objectives of this study were to evaluate the diagnostic accuracy of lamp assay and real - time pcr using bacterial culture as the gold standard and to assess the agreement among these three tests using cohen s kappa statistics. in total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of thailand. sensitivity and specificity of lamp were 96.8% (95% ci 81.599.8) and 97.0% (95% ci 94.998.2), respectively. the accuracy of lamp (97.0%) was significantly higher than that of real - time pcr (91.9%) at 95% ci. with cohen s kappa statistics, culture method and lamp were substantially agreed with each other (77.4%), whereas real - time pcr only moderately agreed with culture (47.7%) and lamp (45.3%), respectively. consequently, lamp was more effective than real - time pcr in detecting leptospira spp. in the urine of cattle. besides, lamp had less cost and was simpler than real - time pcr. thus, lamp was an excellent alternative for routine surveillance of leptospirosis in cattle. |
mammals possess various muscle types that exhibit particular physiological properties to fulfill their diverse functions. for example, the heart muscle beats continuously throughout the life of the animal, slow skeletal muscle fibers support endurance exercises, and fast skeletal muscles empower peak forces but fatigue quickly. the major physiological and biophysical differences between muscle types are largely determined by differences in the expression patterns of structural proteins that build the contractile structures the myofibrils and sarcomeres. one prominent example is the transcriptional regulation of the various muscle myosin heavy chain genes in mammals, often used as the basis for muscle fiber - type classification 1. in addition to differential transcription, alternative splicing adds another level of regulation by creating a plethora of additional protein isoforms. in particular, alternative splicing of the large sarcomeric proteins, such as titin, contributes to physiological diversity. differential splicing between skeletal muscles and heart results in a short and stiff, heart - specific titin isoform that is implicated in the high passive stiffness of mammalian heart 2, 3. drosophila is a valuable model to study the mechanisms that instruct and execute muscle fiber - type diversity. the adult fly houses two different types of body muscles : fibrillar indirect flight muscles (ifms) and tubular body muscles. tubular muscles are similar to mammalian body muscle ; they contain laterally aligned sarcomeres and contract synchronously in response to motor neuron stimulation, which triggers calcium influx. by contrast, fibrillar ifms contain individual non - aligned myofibrils and use an asynchronous contraction mechanism. in addition to calcium influx, this mechanism requires physical stretch stimulation as a trigger. thus, ifms, similar to mammalian heart, display a high passive stiffness likely caused by a specific sarcomeric protein composition. together, these biophysical features of ifm myofibers achieve the very high contraction frequencies and large power output of ifms, enabling insect flight 4, 5, 6. we have shown previously that the zn - finger transcription factor spalt major (salm) is required and sufficient for fibrillar ifm fate choice during pupal development. loss of salm from ifms switches these muscles to a tubular fate, whereas gain of salm in tubular muscles converts them to the fibrillar fate 7. salm executes this switch by the regulation of targets on both the transcriptional and splicing level. however, as the initial study of the salm mutant ifms was performed by microarray analysis which provided limited coverage of the various gene isoforms 7, it remained unclear to what extent alternative splicing contributes to the muscle fiber - type switch. furthermore, it was unknown which rna - binding protein may instruct the ifm - specific splicing pattern. here, we provide a systematic analysis of the salm - regulated genes and gene isoforms in ifms by mrna - seq and identify a core set of more than 700 fibrillar - specific gene isoforms, many of which code for sarcomeric components. we show that the rna - binding protein arrest (aret, bruno) acts downstream of salm to regulate a large number of these genes by instructing their alternative splicing. these targets include stretchin (strn - mlck), sls / kettin, and wupa, which are incorporated into the growing sarcomeres during myofiber maturation. thus, aret ensures the proper isoform composition of the sarcomeric module during flight muscle development, enabling the construction of muscles fast and powerful enough to enable insect flight. wild - type ifms have a fibrillar morphology of their myofibrils, and their nuclei are spaced regularly between the myofibril bundles (fig1a). by contrast, leg or jump muscles display a tubular fiber morphology with their nuclei located in the center of the tube (fig1b, c). muscle - specific rnai - mediated knockdown of salm (salm - ir) or conditional deletion of salm with mef2-gal4 using a novel conditional salm allele that is flanked by 2 frt insertions (salm) results in a complete tubular conversion of the salm mutant ifms (fig1d and supplementary fig s1), which has been observed previously 7. to systematically identify the salm targets underlying the morphological and physiological differences between fibrillar ifms and tubular muscles, we dissected ifms, leg muscle, and jump muscle from wild - type adults, as well as salm and salm - ir ifms, and performed mrna - seq on biological duplicates. bioinformatic analysis using deseq2 to detect differential gene expression 8 identified 362 genes with a log2-fold change greater than 2 (log2fc > 2) whose expression are significantly enriched in wild - type ifms as compared to salm - ir ifms (fig2a). 133 of these genes are also significantly enriched in wild - type ifms as compared to leg and jump muscles (fig2a and supplementary table s1). thus, these 133 genes are fibrillar muscle specific, and their expression depends on salm function. a f wild - type fibrillar ifms (a) and tubular leg (b) and jump muscles (tdt) (c). knock down of salm leads to tubular conversion of the ifms (d), leg and jump muscles are unaffected (e, f). g t mrna - seq read counts showing expression of strn - mlck (g m) and kettin isoforms (n t) from wild - type ifms, leg muscle and jump muscle as well as salm - ir or salm conditional mutant ifms (g, n), and by genomic gfp - tagged isoform markers in strn - mlck (h m) and kettin (o t). note that the ifm - specific expression of strn - mlck - isor depends on salm (compare h and k), whereas the tubular muscle - specific kettin - isoa / d is gained in ifms upon loss of salm (compare o and r). insertion of the gfp tag is indicated by green arrows in g and n. hairpin sequence of strn - mlck - ir and the mimic insertion site are marked in g. note the fibrillar - specific exons marked with green boxes (g, n) and the tubular - specific exons in strn - mlck marked by a red box (g). a f wild - type fibrillar ifms (a) and tubular leg (b) and jump muscles (tdt) (c). knock down of salm leads to tubular conversion of the ifms (d), leg and jump muscles are unaffected (e, f). g t mrna - seq read counts showing expression of strn - mlck (g m) and kettin isoforms (n t) from wild - type ifms, leg muscle and jump muscle as well as salm - ir or salm conditional mutant ifms (g, n), and by genomic gfp - tagged isoform markers in strn - mlck (h m) and kettin (o t). note that the ifm - specific expression of strn - mlck - isor depends on salm (compare h and k), whereas the tubular muscle - specific kettin - isoa / d is gained in ifms upon loss of salm (compare o and r). insertion of the gfp tag is indicated by green arrows in g and n. hairpin sequence of strn - mlck - ir and the mimic insertion site are marked in g. note the fibrillar - specific exons marked with green boxes (g, n) and the tubular - specific exons in strn - mlck marked by a red box (g). scale bars are 5 m, and all images were cropped to the same size. a venn diagram comparing significantly differentially expressed (p - value 2). note that expression of 133 ifm - specific genes is salm dependent. b venn diagram comparing significantly differentially expressed (p - value 2. note that expression of most ifm - specific exons is salm dependent (n = 794). these exons combined with the genes in (a) define the core group of fibrillar - specific genes. c revigo treemap of go component analysis of the 703 core fibrillar genes versus all expressed genes showing enrichment for muscle, mitochondrial, and cytoskeletal terms. d tree of selected gorilla go component terms highlighting enrichment of muscle structural components. e hierarchical clustering of log2fc of all 319 exons from 53 sarcomeric genes that are significantly differentially expressed (p - value 2). b venn diagram comparing significantly differentially expressed (p - value 2. note that expression of most ifm - specific exons is salm dependent (n = 794). these exons combined with the genes in (a) define the core group of fibrillar - specific genes. c revigo treemap of go component analysis of the 703 core fibrillar genes versus all expressed genes showing enrichment for muscle, mitochondrial, and cytoskeletal terms. d tree of selected gorilla go component terms highlighting enrichment of muscle structural components. e hierarchical clustering of log2fc of all 319 exons from 53 sarcomeric genes that are significantly differentially expressed (p - value 2 that are significantly enriched in ifms as compared to leg or jump muscles and are dependent on salm (fig2b and supplementary table s1). together with the 133 genes regulated at the gene level, our analysis identified a total of 703 genes that are upregulated in a salm - dependent fashion in fibrillar versus tubular muscle. we define these 703 genes as core fibrillar muscle - specific genes or gene isoforms (supplementary table s1). interestingly, these 703 genes are highly enriched for cytoskeletal or mitochondrial components (fig2c, d). to investigate fibrillar versus tubular expression of the sarcomeric genes in more detail, we clustered the log2fc values of all exons from sarcomeric genes that are significantly differentially expressed (p - value 2 between aret - ir and salm - ir. b venn diagram comparing significantly differentially expressed (p - value 2 between aret - ir and salm - ir. note that expression of 78.6% (1119/1423) of salm - dependent exons is also aret dependent. c venn diagram comparing significantly differentially expressed (p - value 2 between ifm : aret - ir, ifm : salm - ir, ifm : leg, and ifm : jump muscle. only 24 fibrillar - specific genes are co - regulated by salm and aret. d venn diagram comparing significantly differentially expressed (p - value 2 between ifm : aret - ir, ifm : salm - ir, ifm : leg, and ifm : jump muscle. e correlation plot of the log2fc ifm : salm - ir versus ifm : aret - ir. all significantly differentially expressed exons (n = 5939, p - value 2 between wt ifm : aret - ir ifm at 30 h apf, 72 h apf and in 1-d adults. notably, 781 exons (491 uniquely) are regulated at the adult time point, while only 300 exons are regulated at each developmental time point. a venn diagram comparing significantly differentially expressed (p - value 2 between aret - ir and salm - ir. b venn diagram comparing significantly differentially expressed (p - value 2 between aret - ir and salm - ir. note that expression of 78.6% (1119/1423) of salm - dependent exons is also aret dependent. c venn diagram comparing significantly differentially expressed (p - value 2 between ifm : aret - ir, ifm : salm - ir, ifm : leg, and ifm : jump muscle. only 24 fibrillar - specific genes are co - regulated by salm and aret. d venn diagram comparing significantly differentially expressed (p - value 2 between ifm : aret - ir, ifm : salm - ir, ifm : leg, and ifm : jump muscle. e correlation plot of the log2fc ifm : salm - ir versus ifm : aret - ir. all significantly differentially expressed exons (n = 5939, p - value 2 between wt ifm : aret - ir ifm at 30 h apf, 72 h apf and in 1-d adults. notably, 781 exons (491 uniquely) are regulated at the adult time point, while only 300 exons are regulated at each developmental time point. many of the salm and aret co - regulated exons belong to sarcomeric genes, highlighting their key importance in building functionally different muscle types. tubular classes as observed for salm - regulated exons, highlighting the disruption of the normal fibrillar splicing program in aret - ir ifms (fig2e). the log2fc values are even more tightly correlated for sarcomeric protein exons (red dots in fig7e, pearson 's coefficient = 0.8365097 ; spearman 's coefficient = 0.8603421) than when comparing all exons significantly differentially expressed (p - value < 0.05) between aret - ir and salm - ir ifms (fig7e). we observe both loss and gain of exon expression in aret - ir ifms, although expression of more exons is lost, indicating that aret can both promote fibrillar exon inclusion and inhibit the use of tubular exons (fig7e). to support our bioinformatics data, we used our fosmid reporter lines and find that neither ifm - specific expression of act88f nor ifm- or leg muscle - specific splicing of lmpt depend on aret (supplementary fig s4a n). interestingly, both genes are already expressed at 30 h apf in ifms and hence already present during early phases of myofibril formation. by contrast, the ifm - specific strn - mlck - isor is entirely lost in aret - ir ifms (supplementary fig s5a e). strn - mlck - isor mrna is only expressed from 72 h onwards, correlating with myofibril maturation and the strong nuclear localization of aret protein (supplementary fig s5a and g). similarly, the tubular muscle - specific sls / kettin - isoa / d, which is present at low levels at 30 h apf in ifms but then entirely suppressed in ifms from 72 h apf onwards, is strongly gained in aret - ir ifms (supplementary fig s5f j). this demonstrates that aret is actively required to suppress splicing into the terminal exons of the short sls / kettin isoforms in developing ifms. we also identified wupa (troponin i, tni) as an aret target and generated a fosmid reporter line for the tubular muscle - specific isoform (supplementary fig s5k m). interestingly, aret is required for both splice suppression of a tubular muscle - specific exon and inclusion of a fibrillar muscle - specific exon from 30 h apf onwards (supplementary fig s5k). the wupa fosmid reporter confirms that the tubular wupa isoform is indeed gained in aret - ir ifms (supplementary fig s5n, o). in addition to confirming these complex changes in alternative splicing, we could also confirm an identified salm - dependent transcriptional change in aret - ir ifms. expression of the tubular muscle - specific mlp84b is strongly gained in aret - ir ifms (supplementary fig s4o s). since this gain only occurs after eclosion and not yet at 72 h apf, it is possibly promoted by an unknown transcription factor whose activity is regulated by aret, potentially via alternative splicing. statistically, we find that aret indeed regulates a large number of exons specifically in adult ifms (491), whereas only 129 exons are specifically regulated at 30 h apf during initiation of myofibrillogenesis and a smaller set of 52 exons are regulated at all analyzed developmental stages (fig7f). together, these data demonstrate that a large subset of the fibrillar muscle - specific salm targets are regulated by aret. this regulation happens mainly at the splicing level during later stages of flight muscle morphogenesis. mis - splicing of wupa (tni) is implicated in muscle fiber degeneration caused by muscle hyper - contraction 21, 22. interestingly, aret - ir ifms also display splicing defects in mhc and up (tnt), which are also implicated in muscle hyper - contraction and as a consequence can lead to muscle fiber loss 23 (supplementary fig s6). to test whether the aret - ir fiber degeneration phenotype is caused by uncontrolled myosin activity leading to muscle hyper - contraction, we crossed the ifm - specific mhc null allele, mhc, into the aret - ir background. at 90 h apf, the aret - ir ifm fiber morphology is comparable to wild - type ; however, aret - ir fibers are torn during the first days after eclosion (fig8a f). this fiber degeneration phenotype is entirely rescued by the additional removal of mhc from ifms, demonstrating that loss of aret causes uncontrolled myosin activity and ifm fiber hyper - contraction in adults (fig8g l). a l hemithoraces of 90 h apf pupae (a, d, g, j), 1-day adults (b, e, h, k) and 57 days aged adults (c, f, i, l). wild - type ifm fibers remain intact in aged adults (a c), whereas aret - ir fibers are successively ruptured and lost (d f, red arrow heads). this fiber loss is entirely suppressed upon removal of mhc function from aret - ir ifms using the mhc allele (g l). m r hemithoraces of ifm - specific strn - mlck knock down 90 h apf pupae (m), 1 day (n) and 57 days adults (o) and strn - mlck mimic 90 h apf (p), 1 day (q) and 57 days adults (r). note the progressive fiber degeneration upon aging (m r, red arrow heads). scale bars are 100 m. a l hemithoraces of 90 h apf pupae (a, d, g, j), 1-day adults (b, e, h, k) and 57 days aged adults (c, f, i, l). wild - type ifm fibers remain intact in aged adults (a c), whereas aret - ir fibers are successively ruptured and lost (d f, red arrow heads). this fiber loss is entirely suppressed upon removal of mhc function from aret - ir ifms using the mhc allele (g l). m r hemithoraces of ifm - specific strn - mlck knock down 90 h apf pupae (m), 1 day (n) and 57 days adults (o) and strn - mlck mimic 90 h apf (p), 1 day (q) and 57 days adults (r). note the progressive fiber degeneration upon aging (m r, red arrow heads). as the newly identified strn - mlck - isor protein is also strongly localized to the myosin filament (fig1h), we investigated its role in fiber contraction. we knocked down strn - mlck - isor by an isoform - specific hairpin with mef2-gal4 (see fig1 g) and found an ifm fiber degeneration phenotype after eclosion, remarkably similar to that of aret - ir (fig8m o). this strn - mlck - isor rnai phenotype was confirmed by a mimic insertion disrupting the ifm - specific isoform (fig8p r and see fig1 g). together, this suggests that strn - mlck - isor is a major aret target that regulates myosin activity and biophysical forces in adult ifms. functionally different muscle types are essential for normal life in higher animals. most insects require fast oscillating indirect flight muscles to enable flight. in drosophila and also in the beetle tribolium, salm or its tribolium homolog determines the fibrillar morphology of the ifms 7. our systematic mrna - seq data revealed that in order to achieve fibrillar muscle morphogenesis, salm controls the expression of a large core set of fibrillar genes (more than 700). these unique isoform combinations potentially determine the specific physiological and biophysical features of the different muscle types. as many of the salm targets, in particular the complex sarcomeric genes, are regulated at the level of alternative splicing, salm needs to instruct a fibrillar muscle - specific splicing program. our data suggest that this is largely achieved by ifm - specific expression of aret (supplementary fig s7). aret controls ifm - specific splicing of a very significant subset of sarcomeric genes, including the titin homolog strn - mlck, as well as repressing tubular - specific splicing in ifms, such as tubular - specific events of the titin homolog sls / kettin. interestingly, a number of these splicing events occur between 48 h and 90 h apf during which the myofibrils, initially housing thin and short sarcomeres, mature to myofibrils with long and thick sarcomeres, which can contract in a stretch - sensitive manner. as aret activity is essential for normal sarcomere growth and myofiber maturation, it is likely that incorporation of the aret targets such as strn - mlck or ifm - specific wupa (tni) instructs normal myofibril maturation (supplementary fig s7). aret lacks an obvious nuclear localization signal and until 60 h apf is found largely in the cytoplasm of the developing ifms. nevertheless, some aret is present within the nuclei, possibly in domains close to the nuclear membrane, where aret could regulate fibrillar - type splicing of targets like wupa. upon an unknown stimulus, most of the aret protein translocates to the nuclei by 72 h apf, and now most of the aret targets, including strn - mlck and sls / kettin, are spliced in fibrillar mode., it prevents muscle hyper - contraction and thus is essential for normal muscle fiber maintenance. it is well established that aret (bruno) can regulate mrna translation by binding to the 3utr of osk mrna to prevent its premature translation during transport of the rna from the nurse cells to the posterior pole of the oocyte in drosophila 24, 25. a similar function for aret in translational control of grk mrna in the oocyte has also been suggested 26. in both cases, however, it has been shown that upon a block of mrna export during oogenesis or upon overexpression of aret (bruno) protein, aret can be found in the nurse cell nuclei. this nuclear localization is more pronounced when the rna - binding motifs were mutated 15. this is consistent with our observations suggesting that aret can shuttle between cytoplasm and nucleus not only in oocytes, but also in flight muscles. the cytoplasmic function of aret in ifms, if any, remains to be determined. proteins containing rna recognition motif (rrm) domains are found frequently in the genome, with more than 250 examples in drosophila 15, 27. aret contains 3 rrms, 2 n - terminal and 1 more c - terminal, an organization shared with the elav family of proteins. drosophila elav is a well - established splicing factor that uses its rrms to regulate mrna splicing in neurons 28, 29. a role for aret in regulating splicing in drosophila was not known prior to this work. however, while this manuscript was in the final phase of preparation, a parallel study showed that aret regulates splicing of sls, wupa, and zasp52 in ifms. additionally, it can instruct the fibrillar splicing mode if expressed ectopically in tubular muscle or in s2 cells, suggesting that it regulates the splicing machinery directly 30. in vertebrates, alternative splicing is also a prominent feature of different muscle types 31. in particular in the heart, which shares some similarities with insect flight muscle, alternative splicing is very distinct to skeletal muscle and is one important mechanism to control the different physiological properties of both tissues. rbm20 regulates heart - specific splicing of titin by promoting exon skipping of the flexible pevk exons in titin 32. this is functionally important as human patients with a mutation in rbm20 suffer from hereditary cardiomyopathies 33. a similar role for muscle - type splicing in heart and skeletal muscle was recently identified for rbm24 34, highlighting the importance of muscle - type - specific splice regulation. while both rbm20 and rbm24 contain only a single rrm domain, the mammalian homologs of aret called celf 16 (cugbp, elav - like family) contain 3 rrms with a similar spacing as in aret. interestingly, they have been implicated in regulating alternative splicing in various tissues including splicing of troponin t in the heart 35. this indicates that the mechanism of aret - mediated alternative splicing is conserved to mammals, suggesting that insights gained in drosophila will also be applicable to vertebrate muscle biology. all antibodies and fly stocks are listed in figure legends and in the supplementary information. fosmids tagged with gfp were generated similarly as in previous studies 11, 12 and will be published in detail elsewhere. sarcomere length was quantified based on phalloidin staining in fiji (image j) and significance evaluated with unpaired student 's t - tests. for the mrna - seq analysis, ifms, jump muscles and entire legs marked with mef2-gal4, uas - gfp - gma were dissected at the indicated time points. briefly, total rna was isolated with tri - pure reagent (roche), mrna selected over oligo - dt beads (invitrogen), fragmented with peak length 300 bp, reverse - transcribed with the invitrogen superscript - iii kit and dutp labeled during second - strand synthesis. rna sequencing (rna - seq) was performed at the csf next - generation sequencing unit (http://csf.ac.at). reads were filtered and trimmed using the fastx toolkit and cutadapt and mapped to the ensembl bdgp5.25 genome assembly using tophat v2.0. reads were visualized on the ucsc server by normalizing to the largest library size (supplementary table s3). libraries were evaluated with featurecounts v1.4.2, and differential expression analysis was performed on the gene level with deseq2 and on the exon / isoform level with dexseq. additional data processing was handled in r. go analysis was performed with gorilla 38 and revigo 39. three supplementary datasets are provided listing : (1) all genes that are significantly differentially expressed in the deseq2 comparison of ifm, leg muscle, jump muscle, salm - ir ifm, and aret - ir ifm (supplementary raw data s1) ; (2) all exons that are significantly differentially expressed in the dexseq comparison of ifm, leg muscle, jump muscle, salm - ir ifm, and aret - ir ifm (supplementary raw data s2) ; (3) all exons that are significantly differentially expressed in the dexseq comparison of ifm to aret - ir ifm at 30 h apf, 72 h apf and 1-d adults (supplementary raw data s3). mrna - seq data are publicly available from ncbi 's gene expression omnibus (geo) under accession number gse63707. individual libraries are available from the sequence read archive (sra) under accession numbers gsm1555978gsm1555995. three supplementary datasets are provided listing : (1) all genes that are significantly differentially expressed in the deseq2 comparison of ifm, leg muscle, jump muscle, salm - ir ifm, and aret - ir ifm (supplementary raw data s1) ; (2) all exons that are significantly differentially expressed in the dexseq comparison of ifm, leg muscle, jump muscle, salm - ir ifm, and aret - ir ifm (supplementary raw data s2) ; (3) all exons that are significantly differentially expressed in the dexseq comparison of ifm to aret - ir ifm at 30 h apf, 72 h apf and 1-d adults (supplementary raw data s3). mrna - seq data are publicly available from ncbi 's gene expression omnibus (geo) under accession number gse63707. individual libraries are available from the sequence read archive (sra) under accession numbers gsm1555978gsm1555995. mls performed most of the experiments with important support by cb who also generated the salm allele. mls, ay, dg, as, and bhh performed the bioinformatic data analysis. cs contributed to aret and strn - mlck phenotypic analysis. if and ms generated the gfp fosmid clones. fs conceived and supervised the project ; mls and fs made the figures and wrote the manuscript. | in drosophila, fibrillar flight muscles (ifms) enable flight, while tubular muscles mediate other body movements. here, we use rna - sequencing and isoform - specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. we identify the rna - binding protein arrest (aret, bruno) as downstream of salm. aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of ifms. molecularly, aret regulates ifm - specific splicing of various salm - dependent sarcomeric targets, including stretchin and wupa (tni), and thus maintains muscle fiber integrity. as aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis. |
rheumatoid arthritis (ra) is characterized by synovial inflammation and hyperplasia, autoantibodies production (rheumatoid factor and anti - citrullinated protein antibodies [acpa ]), and cartilage and bone destruction. numerous lines of evidence support the potential contribution of fibroblast - like synoviocytes (flss) to the pathogenesis of chronic arthritis [1, 2 ]. in the joints, ra - flss display a constitutive proinflammatory phenotype that also persists in tissue culture in the absence of exogenous stimuli [35 ]. innocent mesenchymal cells to destructive aggressors, characterized by a number of unique and remarkable features, such as expression of adhesion molecules and mediators, contributing directly to local cartilage destruction and to the chronicity of synovial inflammation [6, 7 ]. resistance to apoptosis has also been associated with this specific phenotype of ra - flss, there is an imbalance between cell death and survival in ra - fls, and the microenvironment also contains, besides the proinflammatory cytokines, factors contributing to induce resistance to apoptosis. it has been suggested that a reduced rate of programmed cell death may provide an explanation for synovial hyperplasia and contribute to invasiveness of ra - flss. in the apoptosis resistance are involved the inhibitors of apoptosis proteins (iaps), a diverse family of proteins that have been implicated as regulators of apoptosis, mitosis, and inflammation [1113 ]. however, to date, the overwhelming majority of studies have focused on the ability of these proteins to modulate apoptosis, particularly in the context of tnf receptor engagement, and few studies have explored the role of the iaps in the production of proinflammatory mediators. because tnf is a major driver of inflammation in response to infection, as well as in the context of inflammatory diseases [14, 15 ], the role of iaps in shaping tnf - dependent inflammatory signalling is an important unresolved question. recent evidence indicates that ciap-1 and ciap-2 play particularly influential roles in repressing tnfr - induced cell death signals. these proteins are characterized by the presence of one to three baculoviral iap repeat (bir) domains responsible for the antiapoptotic activity of iaps because they bind to caspases 3, 7, and 9 and prevent assembly of caspases 8 and 10. smac, an endogenous proapoptotic protein, upon release from the mitochondria, binds and antagonizes several members of the iap family including xiap, ciap-1, and ciap-2. numerous synthetic smac mimetic compounds resemble the smac n - terminal avp1 sequence and bind to the bir3 domain on xiap and iaps. these compounds also promote iap ubiquitination and subsequent degradation [17, 18 ] and endogenous tnf alpha lowers maximum peak bone mass and inhibits osteoblastic smac activation through nf-b. in our previous work, we showed that the smac mimetic smac066 (figure 1 ; x = h) was able to induce apoptosis in ra - flss, proving its activity also in inflammatory diseases. in the present study we evaluated the proapoptotic effect of a more potent analogue, smac127 (figure 1 ; x = cf3). while smac66 and smac127 showed similar binding potency on bir3 domains from xiap and ciap-1 (figure 1) and were equally stable in biological media, the trifluoromethyl substituent in smac127 increased its lipophilicity and facilitated its permeation through cell membranes (indirectly measured by 30-fold increase in cytotoxicity for smac127 versus smac66 (figure 1)). finally, we demonstrated the anti - inflammatory activity of smac127 on ra - flss cultured in tissue medium alone or in the presence of ra synovial fluid. the synthesis of smac127 was carried out as described elsewhere [19, 20 ]. all samples of liquid and/or synovial membrane were taken from the materials considered waste during arthrocentesis or surgery, collected by a single team at ist division. gaetano pini orthopedic institute and university of milan approved whole study (approved on 27 march, 2012). was directly aspirated from the joints of ra patients ; the fluid was collected into heparinized tubes and spun at 1000 g for 10 min. we used pools composed of 10 ra patients ' fluid to reduce the variability in responses between the different fluids. sf pool was used at the final dilution of 1 : 8 in culture medium. synovial tissue was obtained from patients with ra (n = 22) during joint synovectomies. human synovial tissues were digested with collagenase in dulbecco 's modified eagle medium (dmem) (euroclone, pero, italy) for 2 hrs at 37c to isolate synoviocytes. dissociated cells were then centrifuged at 1000 g, suspended in dmem supplemented with 10% fetalclone 1 serum (fcs) (thermo scientific, usa), 2 mm l - glutamine, 100 units / ml of penicillin, and 100 g / ml streptomycin (euroclone, pero, italy), and plated. after overnight culture, nonadherent cells were removed, and adherent cells were cultivated in dmem supplemented with 10% fcs. the cultures were kept at 37c in 5% co2 and the medium was replaced every 3 days. the purity of the cells was tested by flow - cytometric analysis using phycoerythrin - conjugated anti - cd14 (pharmingen, san diego, ca, usa), fluorescein isothiocyanate phycoerythrin - conjugated anti - cd3, anti - cd19, and anti - thy-1 (cd90) monoclonal antibodies (r&d systems, minneapolis, mn). a facs calibur flow cytometer (488ex/620em) (becton dickinson, san jose, ca, usa) was used for the analysis. at passage 3, the cells were morphologically homogeneous and exhibited the appearance of fls, with typical bipolar configuration under inverse microscopy. most cells (> 98%) expressed the surface marker for fibroblasts (thy-1) and were negative for the expression of cd3, cd19, and cd14. we assessed apoptosis in ra - flss cultured in tissue medium alone or in sf (1 : 8 dilution in culture medium) for 5 days, in the presence / absence of smac127 (15 m) for 24 hrs. apoptotic cells were detected with the annexin v - fitc apoptosis detection kit (abcam, cambridge, uk), according to the manufacturer 's instructions. fitc - conjugated annexin v emission was collected in the flh-1 channel, and propidium iodide (pi, for detecting necrotic or dead cells) emission was collected in the flh-3 channel. the percentage of apoptosis was calculated, considering cells in both early (annexin pi) and late apoptosis (annexin pi). ra - flss were grown in culture medium or in sf (1 : 8 dilution in culture medium) for 5 days. smac127 (15 m) was added to the cultures 48 hrs before sacrificing the cells for iaps detections, while for caspase detection smac127 was added to the culture in medium alone 6 hrs before the sacrifice and to the culture in medium supplemented with sf 18 hrs before the sacrifice. staurosporine (20 m) (sigma - aldrich, st. cells were lysed in lysis buffer and protein concentration was measured by the bca method (thermo scientific, usa) according to the manufacturer 's instructions. the cell lysates were separated by sds - page on 412% tris - hcl precast gels for iaps detection, or by 10% tris - hcl precast gels for caspase 3 detection (life technologies, carlsbad, ca), and transferred onto nitrocellulose membranes (life technologies, carlsbad, ca). the membranes were blocked for 3 hrs with 5% nonfat dry milk (lab scientific) in pbs 0.1% tween-20 (sigma - aldrich, st. louis, mo) and probed with primary antibody overnight at 4c. the primary antibodies used were ciap1 (1 : 600) (r&d systems, minneapolis), ciap2 (1 : 600) (bd pharmingen, ma, usa) and xiap (1 : 400) (cell signaling technology, europe), and anticaspase 3 mouse monoclonal antibody (2 g / ml) (enzo life sciences, usa), whereas -actin (1 : 4000) (sigma - aldrich, st. secondary antibodies were conjugated to horseradish peroxidase (thermo scientific, usa) and the gels developed using western lightning plus ecl (perkinelmer, oh, usa). densitometry was performed using imagej software (national institutes of health, bethesda, usa). ra - flss were cultured in culture medium alone or in the presence of sf (1 : 8 dilution in culture medium) for 5 days and treated with smac127 24 hrs before the sacrifice ; then the cells were fixed and permeabilized. to avoid nonspecific binding, the samples were saturated with li - cor odyssey blocking buffer for 2 hrs with moderate shaking. the plate was washed four times with washing solution (pbs added with 0.1% tween-20). ra - flss were incubated with 50 l of several primary mouse monoclonal antibodies (anti - human il-15 (15 g / ml), il-6 (15 g / ml), and il-10 (15 g / ml) (peprotech, rocky hill, nj, usa)) (anti - mmp1(1 : 40), rankl (1 : 70), and opg (1 : 15) (novus, italy)). after 2.5 hrs, the plate was washed 5 times with pbs + 0.1% tween-20 and incubated for 1 hr with goat anti - mouse irdye 800cw labeled secondary antibodies (1 : 800 dilution ; li - cor) and with celltag 700cw (1 : 500 ; li - cor) for cell number normalization. after 5 washes with pbs + 0.1% tween-20, the plate was scanned simultaneously at 700 nm and 800 nm using the odyssey infrared imaging system (li - cor bioscience). the fluorescently labelled irdye 800cw secondary antibody was used for detection of a specific cytokine in the 800 nm channel, and two fluorescent cell stains are used in combination in the 700 nm channel to normalize for well - to - well variations in cell number. celltag 700 stain is a near - infrared, fluorescent, nonspecific cell stain that provides accurate normalization to cell number. differences with a confidence level of > 95% were considered statistically significant (p < 0.05). in our previous paper we showed that smac066 induced apoptosis in ra - flss ; now here we studied smac127. smac66 and smac127 showed similar binding potency on bir3 domains from xiap and ciap-1 (figure 1) and were equally stable in biological media, but the trifluoromethyl substituent in smac127 increased its lipophilicity and facilitated its permeation through cell membranes indirectly measured by 30-fold increase in cytotoxicity for smac127 versus smac66, which is endowed with greater lipophilicity and intracellular uptake. since casnici. emphasized the importance of using sf from ra patients in in vitro studies involving ra cells, in order to reproduce faithfully the physiopathological environment of ra joints, we tested smac127 on ra - flss cultured in sf as well as in tissue medium alone. our new smac mimetic compound induced a significant inhibition of cell growth, although lower when cells were cultured in sf (figure 2). then, we assessed apoptosis in ra - flss cultured in tissue medium alone or in sf, in the presence / absence of smac127. after 5 days of culture, the addition of smac127 before the annexin v test (24 hrs) induced apoptosis and the percentage of apoptotic cells observed in the cultures with sf was lower than those with medium alone but remained significant. taking into consideration that in the presence of sf a lower percentage of apoptotic cells was initially present than when medium alone was used, probably due to the presence in sf of antiapoptotic factors, the induction of apoptosis by smac127 was similar in both culture conditions (figure 3). since iaps are expressed at high levels in ra - flss and smac127 should downregulate these proteins and promote the induction of cell death, we analysed the effect of smac127 on these proteins. as shown in figure 4, smac127 downregulated ciap1, ciap2, and xiap expression in ra - flss cultured in tissue medium alone or in the presence of sf. as reported in figure 5, smac127 could also promote both the proteolytic activation of procaspase 3 and the enzymatic activity of mature caspase 3. as a positive control in ra joints, an imbalance between pro- and anti - inflammatory cytokine activities favours the induction of autoimmunity, of chronic inflammation, and thereby of joint damage ; therefore, we studied the effects of smac127 on the modulation of the cytokines expression. in particular, we analysed the production of inflammatory cytokines il-15 and il-6 and that of the anti - inflammatory cytokine il-10. reported in figure 6, smac127 induced a significant inhibition on the secretion of il-15 and il-6 while the production of anti - inflammatory il-10 dramatically increased, both in cells cultured in tissue medium alone and in presence of sf (figure 6). immune cells and flss are the main source of receptor activator of nuclear factor kappa - b ligand (rankl) in pathological conditions such as arthritic ra joints. the cartilage destruction in ra patients is partly mediated by metalloproteinases secreted by activated synoviocytes and chondrocytes. for this reason we also explored the influence of smac127 on the ra - flss production of metalloprotease 1 (mmp-1), rankl, and osteoprotegerin (opg), involved in bone resorption characteristic of ra pathology (figure 6). the maximum production of mmp-1 occurred when the fibroblasts were cultured for 5 days in sf, and this production was significantly antagonized by smac127 (figure 6). iaps are overexpressed in ra - flss and contribute to their expansion, inflammation, and disease progression and small molecules that disrupt the binding of iaps with their functional partners, such as caspases, should restore the cancer cell 's apoptotic response to proapoptotic stimuli [24, 25 ]. ra - flss are resistant to apoptosis and this resistance and the increased proliferation of flss might contribute to the pathogenesis of ra [8, 9 ]. in a previous paper, we demonstrated, for the first time, that flss are sensitive to smac mimetics ; here we evaluated the effects of more lipophilic, cell - permeable smac127. indeed, the trifluoromethyl substituent in smac127 increased its lipophilicity and facilitated its permeation through cell membranes. ra studies performed in the presence of sf in the culture medium recreate the physiopathological microenvironment of ra, because the composition of sf in ra pathology is very complex and strongly influences the microenvironment of joints. thus, it was important to evaluate how smac127 acted on ra - flss cultured in the presence of sf in the culture medium. to this purpose, we used a pool of sf from ra patients to minimize the variability in responses among the different individual sf samples. in this study we showed that smac127 induced a significant inhibition of flss growth and induced apoptosis in ra - flss cultured in tissue medium or in sf (figures 2 and 3). the percentage of apoptosis in cells cultured in sf was lower than in cells cultured in tissue medium alone, probably because of the presence in the sf of antiapoptotic factors. in our previous study we showed that resistance to apoptosis in ra - flss depends on upregulation of iaps. indeed, figure 4 shows that our molecule significantly inhibits the expression of iap1, iap2, and xiap in ra - flss cultured in presence of both tissue medium and medium added with sf. we propose that administration of smac127 could lead to rapid induction and proteasomal degradation of cellular iaps and to the inhibition of canonical nf-b pathway, leading to upregulation of other survival proteins including bcl-2 and bcl - xl. [26, 27 ]. smac127 enhanced the autoubiquitination of ciaps, leading to their rapid destruction and thus allowing the activation of caspases inhibited by iaps, when ra - flss were cultured either in tissue medium (figure 5(a)) or in sf (figure 5(b)). probably, smac127 disrupts iap interaction with caspases or decreases intracellular levels of iaps with distinct approaches, such as autoubiquitination - induced degradation, damaging the translation of iap proteins, or inhibiting the transcription of iaps. when ciap1/2 are eliminated by iap antagonists, a brief activation of the classical pathway nf-b occurs, presumably due to transient scaffolding from autoubiquitination, resulting in a short surge of tnf alpha expression, although tnf - independent apoptosis has been reported [24, 28, 29 ]. the overgrowth of flss results in the formation of typical ra pannus, which erodes surrounding bone, and in the production of proinflammatory cytokines, released by infiltrating mononuclear cells and lining synoviocytes, thus promoting and sustaining inflammation by both autocrine and paracrine pathways [29, 30 ], and the persistent imbalance between pro- and anti - inflammatory mechanisms leads to chronic inflammation and subsequent joint destruction. iaps influence the production of multiple inflammatory mediators, which are not only inhibitors of apoptosis, and here we demonstrated that smac127 was able to inhibit il-15 production in all tissue culture conditions. il-15 is a potent proinflammatory cytokine, constitutively expressed on the surface of ra - flss, that plays a role in the pathogenesis of ra. the blockade of il-15 secretion by smac127 is an important goal because it breaks the proinflammatory loop and decreases the resistance to apoptosis of flss. the levels of il-6 were very high in ra - flss cultured in the presence of sf, which reproduce the microenvironment within the inflamed joints, and this is consistent with the hypothesis of the presence of several prosurvival and proliferative factors, secreted by cells located within the joint [32, 33 ]. interestingly, we found that smac127 also inhibited il-6 production when ra - flss were cultured both in tissue medium and in the presence of sf. the intense synovial cell hyperplasia and proliferation are associated with a compensatory anti - inflammatory response characterized by the production of soluble tnf receptors, transforming growth factor (tgf-), il-1 receptor antagonist (il-1ra), and il-10. particularly, levels of il-10 are elevated in serum and sf of patients with ra and biologically significant quantities of functionally active il-10 are released in the suspensions of rheumatoid synovial cell cultures [3436 ]. our results demonstrate that smac127 significantly increased the production of il-10 (figure 6). this result is interesting because this cytokine has a dual anti - inflammatory activity, as, on the one hand, it decreases the secretion of proinflammatory cytokines il-15 and il-6 and on the other hand it stimulates its own secretion in ra - flss, particularly when cultured in sf. here we did not investigate whether the stimulation of il-10 secretion by smac127 was responsible of the downregulation of il-15 and il-6 or if such downregulation is a direct effect of smac127. the synergistic interactions among the different cytokines in the inflamed joint enhance the production of mmps and subsequent joint destruction. mmps determine the timing, the amplitude, and the combination of molecular signals that are released within a tissue, and crucially influence the availability of cell death or cell protective cues in the microenvironment, thereby affecting the outcome of an inflammatory challenge. since the regulation of mmps becomes aberrant in immune cells in many human inflammatory and autoimmune diseases [37, 38 ], we also evaluated the effect of smac127 on mmp-1 production. we observed that it reduced mmp-1 levels (figure 6) and, interestingly, this inhibition was maintained even in ra - flss exposed to sf, where mmp1 levels were very high. other authors have shown that iap antagonists may induce high turnover osteoporosis characterized by enhanced osteoclast and osteoblast activities in mice and may increase the risk of tumour growth and metastasis in the bone by stabilizing nf-b inducing kinase (nik) and activating the alternative nf-b pathway in osteoclasts. therefore, we sought to find whether the rankl / opg pathway was altered by smac127, both in cells cultured in tissue medium and in the presence of sf. as expected, rankl is upregulated in cells cultured in the presence of sf, but we found no statistically significant difference in rankl secretion after the addition of smac127., smac127 demonstrated to be effective in inducing apoptosis in ra - flss which are characterized by resistance to apoptosis and are responsible for the erosive pannus typical of ra. we also demonstrated that smac127 has a beneficial regulatory effect on the expression of proinflammatory cytokines and downregulates the production of mmps. we did not confirm previous observations regarding a possible side effect on bone due to upregulation of rankl and downregulation of opg. overall, these observations suggest a potential use of smac mimetic compounds in general, and smac127 in particular, for the control of inflammation and disease progression in ra. | rheumatoid arthritis (ra) is characterized by synovial inflammation and hyperplasia. fibroblast - like synoviocytes (flss) are apoptosis - resistant and contribute to the pathogenesis of ra by producing cytokines and proteolytic enzymes, which degrade the extracellular matrix. we evaluated the proapoptotic and anti - inflammatory activity of the small molecule smac127 on ra - flss cultured in synovial fluid (sf), in order to reproduce the physiopathological environmental characteristic of ra joints. in this context, smac127 induces apoptosis by inhibiting apoptosis proteins (iaps). this inhibition activates caspase 3 and restores the apoptotic pathway. in addition, smac127 induces a significant inhibition of the secretion of il-15 and il-6, stimulation of pannus formation, and damage of bone and cartilage in ra. also the secretion of the anti - inflammatory cytokine il-10 is dramatically increased in the presence of smac127. the cartilage destruction in ra patients is partly mediated by metalloproteinases ; here we show that the mmp-1 production by fibroblasts cultured in sf is significantly antagonized by smac127. conversely, this molecule has no significant effects on rankl and opg production. our observations demonstrate that smac127 has beneficial regulatory effects on inflammatory state of ra - flss and suggest a potential use of smac127 for the control of inflammation and disease progression in ra. |
airway management in the obese patient can be challenging, as airway difficulties are often complicated by obesity. the morbidly obese patients may require several attempts at intubation and have a greater likelihood of use of accessory airway devices. application of positive end expiratory pressure (peep) at induction of anaesthesia has been shown to prolong non - hypoxemic apnea time by preventing atelectasis in obese and non obese patients.[26 ] conventionally, facemasks with oropharyngeal airway (fm) have been used to ventilate patients following induction, prior to securing the tracheal tube. this is associated with continuous insufflation of gases into the stomach, which can further compromise the lung volumes and also increase propensity for regurgitation and aspiration. the proseal laryngeal mask airway (plma), by allowing an airtight seal, application of peep and drain tube for gastric decompression, can prove to be a useful tool in the airway management of morbidly obese patients.[79 ] although plma has been proven to be efficacious as a temporary ventilatory device in morbidly obese patients, its benefits have not been compared with facemask with oropharyngeal airway, more so in the context of arterial oxygenation, which has a direct influence on the non - hypoxemic apnea time. we hypothesized that when plma is used as a conduit prior to laryngoscope - guided intubation, there is improvement in the arterial oxygenation. we conducted this randomized prospective study with the primary objective of comparing the effect of ventilation with peep, through plma with fm, on arterial oxygenation in morbidly obese patients undergoing laparoscopic surgery under general anaesthesia. with the approval of the institutional ethics committee, and written informed consent from patients, a total of 40 patients with body mass index (bmi) > 35 kg / m, of either sex and age more than 18 years, scheduled for elective procedure under general anaesthesia were recruited for the study. this investigation was registered with the clinical trials registry - india (ctri) and had the following registration number : patients were randomly allocated (computer aided) to receive proseal laryngeal mask airway (plma) as airway device for group a and facemask with oropharyngeal airway (fm) of appropriate size for group b. patients with restricted mouth opening 25 ml in the morbidly obese. we included patients with no history suggestive of gerd. none of the patients were found to have gastric fluid volume greater than 20 ml. the mean volume of the gastric fluid aspirated did not differ between the two groups. some workers have suggested that the obese and non - obese patients are comparable in terms of resistance gradient between stomach and gastroesophageal junction, and gastric emptying. also, the use of cricoid pressure to prevent passive regurgitation has been challenged. patients with history suggestive of gastroesophageal reflux were not included in this study and cricoid pressure was not applied in any patient.[2630 ] the ph of the devices, immediately prior to laryngoscopy, did not differ. safe intubation of these patients requires careful attention to be paid to optimize pre - oxygenation and to prevent de - oxygenation under a controlled fashion. moreover, airway in the morbidly obese should be handled by anesthesiologists who have the expertise and who are aware of the limitations associated with it for the safe conduct of oxygenation. whether the plma can be used for conducting longer procedures application of peep using proseal laryngeal mask airway as a conduit prior to laryngoscopy is effective in increasing the oxygen reserves and can be suggested as beneficial when managing the airway in the morbidly obese. | background : the primary objective of this study was to compare the effect of ventilation using the proseal laryngeal mask airway (plma) with facemask and oropharyngeal airway (fm), prior to laryngoscopy, on arterial oxygenation in morbidly obese patients undergoing bariatric surgery.methods:forty morbidly obese patients were randomly recruited to either plma or fm. after pre - oxygenation (fio2 1.0) in the ramp position with continuous positive airway pressure of 10 cm h2o for 5 min, anaesthesia was induced. following loss of jaw thrust oropharyngeal airway, the fm and plma were inserted. on achieving paralysis, volume control ventilation with peep (5 cm h2o) was initiated. the difficulty in mask ventilation (dmv) in fm, number of attempts at plma and laryngoscopy were graded (cormack and lehane) in all patients. time from onset of laryngoscopy to endotracheal tube confirmation was recorded. hypoxia was defined as mild (spo2 95%), moderate (spo2 90%) and severe (spo2 85%).results : significant rise in po2 was observed within both groups (p=0.001), and this was significantly higher in the plma (p=0.0001) when compared between the groups. spo2 90% (p=0.018) was seen in 19/20 (95%) patients in plma and 13/20 (65%) in fm at confirmation of tracheal tube. a strong association was found between dmv and cormack lehane in the fm group and with number of attempts in the plma group. no adverse events were observed.conclusion : proseal laryngeal mask airway as conduit prior to laryngoscopy in morbidly obese patients seems effective in increasing oxygen reserves, and can be suggested as a routine airway management technique when managing the airway in the morbidly obese. |
fusion of haploid cells to form a diploid zygote is the defining event of sexual reproduction in eukaryotes (lillie, 1913). in organisms from every eukaryotic taxon, the plasma membranes of gametes of opposite sex or mating type come into intimate contact and then fuse to form the zygote (bianchi. very little is known about the molecular mechanisms of the membrane fusion reaction between gametes, and a bona fide fusion protein has not been formally identified. the best candidate to date is the ancient gamete plasma membrane protein hap2, whose presence in green algae, higher plants, unicellular protozoa, cnidarians, hemichordates, and arthropods (cole., 2014, ebchuqin., 2014, johnson., 2004, kawai - toyooka., 2014, liu., 2008, mori., 2006, steele and dana, 2009) indicates it was likely present in the last eukaryotic common ancestor (leca) (wong and johnson, 2010). hap2 was first identified in a screen for male sterility in the flowering plant arabidopsis thaliana (johnson., 2004) and later under the name gcs1 (mori., 2006)as a sperm - specific protein shown to be required at an unidentified step in sperm - egg fusion (mori., 2006, a screen for genes essential for gamete fusion in the green alga chlamydomonas independently uncovered hap2, showing that it is expressed only in minus gametes and is exclusively present on an apically localized membrane protuberance termed the minus mating structure (liu., 2008) (see figure 1a for a diagram of chlamydomonas fertilization). studies in chlamydomonas and plasmodium (the pathogen causing malaria in humans) revealed that hap2 mutant gametes in both organisms were fully capable of robust adhesion to gametes of the opposite mating type or sex, but merger of the lipid bilayers was abrogated (liu., 2008). in both organisms, adhesion relies on proteins that are species - limited : fus1 in chlamydomonas plus gametes and its unidentified receptor in minus gametes (misamore., 2003), and p48/45 in plasmodium berghei gametes (van dijk., 2001). based on these findings, which have since been confirmed in arabidopsis thaliana (mori., 2014) and the ciliated protozoan tetrahymena thermophila (cole., 2014), a model emerged positing that hap2, a single - pass transmembrane protein, functions after species - limited adhesion in the membrane fusion process between gametes (liu., 2008). furthermore, in all of these organisms, hap2 is required in only one of the two gametes, raising the possibility that it may function similarly to fusion proteins of enveloped viruses (wong and johnson, 2010, harrison, 2015). to understand the function of hap2 at the molecular level we carried out concerted bioinformatic, functional, and x - ray structural analyses of hap2 from chlamydomonas reinhardtii. initial bioinformatic analyses identified weak similarity to class ii fusion proteins, revealing a segment within a cysteine - rich portion of hap2 that could potentially correspond to the fusion loop. we demonstrate by mutational analysis and fusion - blocking antibodies targeting this segment that it has elements that are essential for hap2 function. finally, we show that the recombinant hap2 ectodomain is monomeric, but inserts into liposomes by concomitantly forming trimers, the x - ray structure of which revealed a class ii fusion protein fold in the typical trimeric post - fusion hap2 has 16 conserved cysteine residues with a signature distribution in the ectodomain (figure 1b). early alignments of hap2 family members identified a characteristic 50 aa domain (residues 352399 in chlamydomonas hap2) with several conserved residues that was designated the hap2/gcs1 pfam domain (pf10699) (see http://pfam.xfam.org/family/pf10699) (finn., 2016). a previous mutagenesis analysis in chlamydomonas failed to identify functional properties in the pf10699 domain, as the mutant proteins tested either were not transported to the mating structure, or were nearly indistinguishable from wild - type in their ability to support fusion with plus gametes (liu., 2015). database searches for additional conserved regions using the hhpred protein homology detection server (sding., 2005) indicated that a cysteine - rich region in the n - terminal half of the ectodomain exhibited weak similarity to class ii fusion proteins. in particular, hhpred identified a polypeptide segment in c. reinhardtii hap2 (170204, sqvwddtfgsskertranldcdfwsdpldiligrk) that fell in the fusion loop region of the flavivirus envelope protein e in the resultant amino - acid - sequence alignment (figure s1). analysis of hap2 orthologs showed that the sequence in this region is highly variable, with a number of deletions and insertions and is framed at each side by relatively conserved segments : amino acids (aa) 159167 upstream (including conserved cysteines 57) and aa 208219 downstream (including conserved cysteine 9) (figures 1b and 1c). only amino acids r185 and c190 (in bold in the sequence above) within the identified segment are conserved, suggesting that they may play a role in hap2 function. to investigate the functional importance of this segment we transformed chlamydomonas wild - type (wt) or mutant hap2 transgenes carrying an influenza virus hemagglutinin tag (ha) into a fusion - defective, hap2 mutant strain (liu., 2008) and assessed hap2-ha expression and trafficking to the mating structure as well as fusion of the transformed hap2 minus gametes with wt plus gametes. hap2-ha was detected in hap2 minus gametes transformed with wt hap2-ha as the expected doublet in sds - page / immunoblotting (figure 1d), the upper form of which was present on the cell surface as assessed by its sensitivity to protease treatment of live gametes (liu., 2008). all mutant proteins were expressed at levels similar to wild - type hap2-ha (figure 1d), trafficked to the cell surface as assessed by their sensitivity to trypsin treatment of live gametes (examples shown in figure 1e), and localized at the mating structure (example shown in figure 1f). thus, any defects in gamete fusion could be ascribed directly to the functional properties of the mutant hap2 proteins. hap2 with a deletion of residues 184tra186 (hap2-tra - ha), which includes the conserved r185 was non - functional and failed to rescue fusion in the hap2 mutant when mixed with wild - type plus gametes (figure 1d). a mutant hap2 with a lysine sustituted for the conserved r185 (the hap2-r185k - ha mutant) was fully functional, whereas the hap2-r185a - ha or hap2-r185q - ha mutants failed to rescue fusion (figure 1d). a reverse - order hap2-ra185 - 86 mutant (hap2-r185a - a186r - ha) also was non - functional, indicating that a positively charged residue at position 185 is essential for the fusion activity. finally, hap2 minus gametes expressing hap2-f192a - w193a - ha were impaired in fusion, although fusion was not abolished by these mutations, indicating that these nearby aromatic residues also play a role in hap2 fusion function (figure 1d). thus, the segment hap2170 - 204, bounded by a pair of conserved cysteines, contains residues that are dispensable for protein expression, folding and localization, but are essential for the membrane fusion activity. in an independent approach to examine the function of the hhpred - identified region, we generated a rabbit antibody against a synthetic peptide, hap2168 - 190, spanning the functionally important r185 residue. the affinity - purified antibody (-hap2168 - 190) immunoprecipitated epitope - tagged hap2-ha from lysates of hap2-ha minus gametes (figure 2a), confirming its reactivity with hap2. to test whether -hap2168 - 190 interfered with gamete fusion, we incubated minus gametes with undiluted antibody, mixed them with plus gametes, and determined the percentage of gametes that had fused to form zygotes pre - incubation of minus gametes with -hap2168 - 190 had no effect on motility or adhesion but inhibited gamete fusion by over 75%, whereas pre - incubation with a control igg had no effect on fusion (figures 2b and 2c). antibody dilution resulted in a loss of fusion - blocking activity, suggesting a low concentration of hap2-specific antibodies in the polyclonal mixture, probably due to a low immunogenicity of the synthetic peptide. pre - incubation of plus gametes with the antibody did not affect their ability to fuse ; and the fusion - blocking activity of -hap2168 - 190 was neutralized by pre - incubation with the hap2168 - 190 peptide, but not with a control peptide (figure 2c), further documenting the specificity of the antibody. finally, immunolocalization experiments using an anti - ha antibody showed that hap2-ha on -hap2168 - 190-treated minus gametes remained at the mating structure (figure 2d), indicating that -hap2168 - 190 did not alter hap2 localization, but directly interfered with its function. these functional studies with chlamydomonas mutant gametes and the anti - peptide antibody indicated that in its native conformation on live gametes, the 168190 segment of hap2 is accessible at the protein surface and its integrity and availability are essential for fusion of gametes. we used a drosophila expression system to produce a soluble form of c. reinhardtii hap2 aa 23592 (comprising almost the entire ectodomain, figure 1b) and purified it to homogeneity (see star methods ; figure s2). analysis by size - exclusion chromatography (sec) and multi - angle static laser light - scattering (malls ; figure s2) showed that the protein behaved as a monomer in solution (fraction labeled hap2e), but had a tendency to oligomerize with time (especially under high ionic strength conditions) to elute at a volume corresponding roughly to hexamers (hap2eh fraction in figure s2). the purified protein from the hap2e monomeric fraction efficiently neutralized the fusion - inhibition potential of the -hap2168 - 190 antibody (figure 2c), indicating that at least part of the 168190 segment is exposed in hap2e and accessible to the antibody. to detect membrane insertion, we incubated the recombinant protein with liposomes of a standard lipid composition (see star methods) and monitored binding by co - flotation on a sucrose gradient (figure 3e) followed by immunoblot detection of lipid - inserted hap2e using a monoclonal antibody raised against hap2e (mab k3 ; see figure s2 ; star methods). we observed efficient co - flotation of the monomeric hap2e fraction (figure 3e), but not of the multimeric hap2eh fraction (not shown). electron microscopy analysis showed that hap2e decorated the liposome surface as projecting rods about 12 nm long (figures 3b3d), which are similar to those formed by viral class ii fusion proteins in their trimeric, post - fusion form, such as the alphavirus e1 protein (gibbons., 2003). the size and shape of membrane - bound hap2e suggested that it had also oligomerized upon membrane insertion. of note, 3-fold symmetry was apparent in some top views of unbound proteins present in the background (figure 3d, arrowheads). we confirmed that hap2e had indeed trimerized upon membrane insertion by detergent - solubilizing it from the liposomes and analyzing it by native page (figure 3f) and by sec (figure s3). these results indicated that hap2e behaves similarly to the alphaviruses and flaviviruses class ii proteins, with membrane insertion concomitant with trimerization of a monomeric pre - fusion form (klimjack. 2002), except that hap2e did not require an acidic environment for lipid binding and trimerization. this difference is in line with the ability of hap2 to induce gamete fusion in the extracellular environment, whereas alpha- and flaviviruses require the acidic environment of an endosome for fusion. because the co - flotation experiments demanded relatively high amounts of purified recombinant protein for detection, it was impractical to assess flotation inhibition by the undiluted polyclonal -hap2168 - 190 antibody. but we tested the effect of the mutations described in figure 1 by recombinantly expressing the mutant hap2 ectodomains. these experiments showed that the mutations that impaired gamete fusion also affected the co - flotation capacity of the mutant hap2e (figure 3 g). the r185k hap2e mutant co - floated with liposomes as efficiently as wild - type, whereas those in which the charge at position 185 was removed the r185a and r185q mutants co - floated poorly (figure 3 g). co - flotation with the liposomes was also essentially abrogated with the hap2e-tra and hap2e - f192a - w193a mutants. the combination of the in vivo (figures 1 and 2) and in vitro (figure 3) analyses indicates that hap2 has the capacity to directly interact with membranes and that altering the conserved residues of the hhpred - identified segment affects this interaction. details of the crystallization and structure determination are described in the star methods, and the crystallographic statistics are listed in table s1. we determined the x - ray structure by the single isomorphous replacement with anomalous scattering (siras) method using a ptcl4 derivative. the experimental electron density map allowed the tracing of 442 amino acids out of 569 in the hap2e expression construct (aa 23592 ; figure 1b) from amino acids 24 to 581 with internal breaks at several disordered loops (listed in the star methods). the atomic model of c. reinhardtii hap2 revealed a trimer with unambiguous structural homology to class ii fusion proteins, featuring the three characteristic sheet - rich domains, termed i, ii, and iii, arranged in a hairpin conformation typical of class ii fusion proteins in their post - fusion form (bressanelli., 2004, modis., 2004, willensky., 2016) the overall shape of the hap2e trimer is totally compatible with the projections from the liposomes observed in the electron micrographs, with the predicted membrane interacting region at the tapered end of the rods (figures 3c and 3d). the monomeric hap2e (figure s2) used to grow the crystals obviously underwent the same conformational change to form trimers observed during insertion into liposomes (figure 3). a similar oligomeric rearrangement during crystallization was reported previously for the dengue virus e protein under acidic ph conditions (klein., 2013, nayak., 2009). in the case of hap2, acidification is not required ; the trigger for the rearrangement to the post - fusion form is not known. the comparison with class ii fusion protein trimers of known structure defines the membrane - facing side (top in figure 4) and the membrane distal end (bottom side) of the hap2 trimer, in agreement with the shape observed in the electron micrographs (figures 3b3d). the dali server (holm and park, 2000) yielded z scores ranging from 9 to 16 for up to 343 c atoms of class ii fusion proteins of viral and cellular origin (table 1), in the same range as those obtained in previous comparisons among known class ii fusion proteins (prez - vargas., 2014). domain i in hap2 is a sandwich of about 200 aa with ten strands in the two apposed sheets : the a0b0i0h0g0 sheet is buried and the j0c0d0e0f0 sheet is exposed in the trimer, and we refer to them as inner and outer sheets (figures 5a and 5d). a specific feature of the hap2 domain i is that the a0b0 -hairpin is long and projects out of the inner sheet to augment the outer sheet of the adjacent subunit, where strand a0 runs parallel to j0 (figure s5). these additional inter - subunit main chain -interactions are likely to confer extra stability to the post - fusion trimer. at the membrane - distal, bottom face of the trimer, the outer sheet projects very long loops, which are disordered in the crystal (dashed tubes in figures 4, 5, s4, and s5). the c0d0 loop projection displays six additional cysteine residues (presumably forming three disulfide bonds) in an insertion that is present essentially only in algal hap2 (liu., 2008) (figure 5d) ; the e0f0 loop projection is also an insertion with four potential n - linked glycosylation sites. domain iii is made of about 130 aa and has an immunoglobulin - like fold, with seven strands in two sheets (labeled abe and gfcc ; figures 5a, 5d, and s4). although the sheets are longer than in other class ii fusion proteins (figure 4), giving domain iii a more elongated bean shape, it is in a similar location at the side of the trimer (figure 4) as in other class ii fusion proteins of known structure. core, which is composed of domains i and ii arranged parallel to each other and interacting along their length about the 3-fold molecular axis. the latter central trimer interaction is postulated to exist during formation of an extended trimeric intermediate 2010), in which the fusion loops are inserted in the target membrane (plus gamete here), while the c - terminal tm segment is anchored in the minus gamete mating structure, at the opposite end. the final collapse into the post - fusion hairpin conformation of each protomer in the trimer brings domain iii to the sides of this core, projecting the downstream stem and tm regions toward the fusion loop (liao and kielian, 2005), in a fusogenic rearrangement of the protein analogous to an umbrella folding inside out. in this final, post - fusion location, domain iii buries an area of about 2,100 of its surface, divided roughly equally in contacts with the same and with the adjacent subunit (intra- and inter - subunit contacts). the observed contacts therefore can only form after the assembly of the central trimer core, in line with the proposed clamping role of domain iii, and resulting in irreversible trimerisation, as proposed for other class ii fusion proteins (liao and kielian, 2005, prez - vargas., 2014). domain ii is the largest domain (roughly 250 aa in total) and is made by two distinct segments emanating from domain i : the d0e0 and h0i0 strand connections of the outer and inner sheets of domain i, respectively (figure s4). as in all class ii proteins, the domain i proximal region of domain ii has a central sheet (aefg ; figures 5 and s4) flanked by additional short helices. the distal tip of domain ii contains sheet bdc, with the strands running parallel to the molecular 3-fold axis at the distal end of the d0e0 segment. the connection between strands c and d at the tip of domain ii (the cd loop) was shown to be the fusion loop in the viral proteins (reviewed in kielian and rey). the bdc sheet normally packs against the ij -hairpin (the distal end of the h0i0 segment), which in hap2 maps to the conserved hap2/gcs1 pfam domain pf10699) (figure 5a). although in hap2 strands i and j are absent, we still refer to this region as the ij loop (figures 5 and s4). the cd loop in hap2 is 40 aa long and has an intervening short -helix in the middle (0), in contrast to the standard class ii fusion proteins from the arthropod - borne viruses such as flaviviruses and alphaviruses (1015 residues long). in this respect, hap2 resembles the rubella virus class ii fusion protein e1, which has 50 aa in between stands c and d, with a couple of short intervening helices as well as an additional strand which results in two separate fusion loops (dubois., 2013). the presence of the 0 helix in hap2 also results in two loops (loops 1 and 2 in figures 5b and 5c), which project outward. although disordered in the crystal, these two loops are in position to project non - polar residues into the target membrane. the hhpred alignment was indeed quite close and pointed correctly to the cd loop (figure s1). the hap2168 - 190 peptide used for immunization spans loop 1 all the way to the end of helix 0, and the fact that the resulting polyclonal antibody blocks fusion is in line with this region being exposed at this end of the molecule. the hap2 crystal structure is therefore compatible with the mutagenesis data in this region, and with the effect of the -hap2168 - 190 antibody. moreover, a peptide derived from the corresponding region of tetrahymena thermophila hap2 was found to display properties typical of a fusion loop (see related paper in current biology, pinello., 2017), in line with our findings with c. reinhardtii. the structure shows that the functionally important residue r185 is at the n terminus of the 0 helix and its side chain points away from the two exposed loops and toward the core of the hap2 trimer. r185 makes a salt bridge and bidentate hydrogen bonds with the side chain of the strictly conserved e126 in strand b (figures 5b and 5c). furthermore, the r185 side chain is at the core of a network of interactions stabilizing the main - chain conformation of the ij loop. this network involves main chain atoms of f376, g382 and r385, together with the strictly conserved q379 side chain, which are part of the pf10699 signature segment that allowed the identification of hap2 in widely disparate organisms. the structure now shows that the conserved pattern of disulfide bonds of this signature element is required for the ij loop to adopt a convoluted fold acting as a framework underpinning the cd loop by interaction with the conserved r185 so that the two fusion loops project out at the membrane - interacting region. the latter region, in contrast, is variable and has multiple deletions and insertions in the various orthologs, some insertions being quite long (figure 1c). it is possible that the differences in membrane - interacting regions of hap2s across the broad spectrum of eukaryotic organisms reflect evolutionary adaptations required for fusion with different target gametes. we note that the fusion loop in viral class ii proteins is in general the most conserved segment of the protein within orthologs from a given virus genus, most likely because the same residues are also required for inter - subunit interactions in their pre - fusion form on infectious particles. but this comparison is not necessarily informative, since the analyzed hap2 proteins span eukaryotic taxa that are much more distantly related than are viruses within a given genus. the pre - fusion conformation of hap2 remains unknown, however, and it is possible that the non - polar residues of the two fusion loops are maintained unexposed until the time of fusion. the conserved interaction of the r185 side chain with that of the e126 residue in the tip domain may allow exposure of the two fusion loops only after a conformational change. in this context, the fusion - inhibiting -hap2168 - 190 antibody would bind only after a conformational change that exposes the fusion loop, but this remains to be explored. the identical topological arrangement of secondary, tertiary, and quaternary structure elements of hap2 with the viral class ii proteins indeed, the probability of convergence to the observed complex fold from independent origins, to result in two proteins displaying exactly the same topological arrangement throughout the entire ectodomain (as shown in figure s4) is extremely low and can be considered negligible. nature is parsimonious, and once a protein required for a complex function such as membrane fusion becomes available, the corresponding gene is used over and over again, most likely transferred via horizontal gene exchanges. this concept is supported by the observation that only three structural classes of viral fusion proteins have been observed so far, in spite of the enormous variety of known viruses. as one example, the membrane fusion proteins of the herpesviruses, rhabdoviruses and baculoviruses, which are otherwise totally unrelated viruses, were shown to be homologous by structural studies (heldwein., 2006, kadlec., 2008, roche., 2006). these fusion proteins most certainly derived from a distant common ancestor (class iii proteins) (backovic and jardetzky, 2011), whose genes must have been acquired via horizontal exchanges. within eukaryotic organisms, only a few other cell - cell fusion proteins have been positively identified. although the myoblast myomaker, a seven - pass transmembrane protein that governs fusion of myoblasts to form myotubes (millay., 2013, millay., 2016), has no obvious relation to viruses, the proteins involved in the two other cell - cell fusion events that have been characterized at the molecular level are virus related. cytotrophoblast fusion in mammals during placenta formation (blaise., 2003, holm and park, 2000) and epidermal cell - cell fusion in nematodes to form syncytia (mohler., 2002) both use fusion proteins also found in viruses. in the first case, the class i fusion protein involved is clearly derived from an endogenous retroviral element (denner, 2016). it is also possible that a similar process involving retrotranscription may have taken place in the case of the class ii caenorhabditis elegans fusion protein eff-1 (prez - vargas., 2014), as retroviruses of nematodes have been found to have an envelope protein related to that of the phleboviruses (frame., 2001,, 2000), which have class ii fusion proteins (dessau and modis, 2013). these observations suggest that retro - transcription, followed by integration into the genome, may have been an important pathway for gene exchanges between viruses and eukaryotic cells. hap2 is present in organisms responsible for several of the globe s most devastating human diseases, including plasmodium, trypanosoma, and toxoplasma. many arthropods that are vectors of human diseases or are agricultural pests, such as insects and ticks, also possess hap2. a strategy that used recombinant plasmodium hap2 fragments to induce transmission - blocking immunity in mice has been reported previously (blagborough and sinden, 2009, miura., 2013), but the expression systems were not sufficient for viable clinical developments. our results suggest that the use of a peptide spanning the hap2 fusion loops as immunogen might be sufficient to induce transmission - blocking immunity, similar to the antibodies we obtained here against chlamydomonas hap2. in conclusion, our data now open the way to a full mechanistic characterization of gamete fusion induced by hap2 and raise new questions, including the identification of the trigger for the hap2 fusogenic conformational change, the structure of the pre - fusion form(s) of hap2, and its organization on the mating structure of the minus gamete membrane. evolution through hundreds of millions of years may have led the different taxa to develop alternative solutions, and the metastable pre - fusion form of hap2 may be organized differently in the multiple organisms in which it is present, as shown for viruses of different families. in contrast, the post - fusion conformation described here appears as a universal feature of class ii fusion proteins. reagent or resourcesourceidentifierantibodiespolyclonal rabbit -hap2168 - 190 peptide abthis papern / amonoclonal mouse -hap2e ab k3this papern / achemicals, peptides, and recombinant proteinshap2168 - 190 (sssqvwddtfgsskertranldc)yenzym antibodiesn / ahap2control (ctqpprppwpprpppappps)yenzym antibodiesn / acommon lab reagentsn / an / adeposited datac. melanogaster : cell line s2 : s2thermo - fishercat # r690 - 07experimental models : organisms / strainschlamydomonas reinhardtii : strain 21 gr : mt+chlamydomonas culture collectioncc-1690chlamydomonas reinhardtii : strain 40d4 : hap2 mating type minus mutantchlamydomonas culture collectioncc-4552chlamydomonas reinhardtii : strain hap2-ha : hap2-ha plasmid - rescued 40d4 strainchlamydomonas culture collectioncc-5295recombinant dnapmt / bip / twinstrepkrey. - 592twinstrepthis papern / asoftware and algorithmsxdskabsch, 1988http://xds.mpimf - heidelberg.mpg.de / pointlessevans, 2006http://www.ccp4.ac.uk / ccp4collaborative computational project, 1994http://www.ccp4.ac.uk / autosharpvonrhein., 2010https://www.globalphasing.commolprobitychen., 2010http://molprobity.biochem.duke.edu / hhpredsding., 2005https://toolkit.tuebingen.mpg.de / hhpreddaliholm and park, 2000http://ekhidna.biocenter.helsinki.fi / dali_server / start requests for resources and reagents should be directed to flix a. rey ([email protected]). wild - type chlamydomonas strains 21 gr (mating type plus ; mt+ ; cc-1690), 40d4 (hap2 mating type minus mutant (liu., 2015)) and hap2-ha (hap2-ha plasmid - rescued hap2 strain (liu., 2015)) were used and are available from the chlamydomonas culture collection. vegetative growth of cells and induction of gametogenesis in gamete medium (m - n) were as described before (liu,. gametes were activated with dibutyryl - camp (db - camp) by incubation with 15 mm db - camp and 0.15 mm papaverine for 0.5 hr in n - free medium (ning., 2013). gamete fusion was assessed by determining the number of cells that had formed zygotes after being mixed with wild - type plus gametes for 30 min and was expressed as percent fusion using the following equation : (2 number of zygotes)/[(2 number of zygotes)+(number of unfused gametes) ] 100 (liu., 2015). a polyclonal antibody against hap2 peptide sssqvwddtfgsskertranldc (aa 168 - 190) was made in rabbits by yenzym antibodies (san francisco, ca ; -hap2168 - 190), under oversight by their institutional review board. the antibody was purified on a peptide - conjugated affinity column prepared by the company. to assay for antibody inhibition of gamete fusion, gametes that had been activated with dibutyryl - camp for 30 min (misamore., 2003) were washed once with gamete medium and the activated gametes (2 10 cell / ml) were incubated with 110 ug / ml peptide antibody (pre - dialyzed in gamete medium) for 2 hr followed by mixing with gametes of the opposite mating type gametes for the times indicated in the figure legends. the number of zygotes (detected as cells with 4 rather than 2 cilia) was determined by phase - contrast microscopy. at least 100 cells were counted each time with at least two counts per sample in at least two separate experiments. a rabbit igg (sigma) was used as a control antibody. to test the capacity of peptides or hap2e to neutralize the fusion - inhibiting properties of -hap2168 - 190, the antibody (220 ug / ml) that had been dialyzed in gamete medium was incubated overnight with control peptide ctqpprppwpprpppappps (200 ug / ml) (this peptide is encoded by cre03 g176961 whose transcripts are specific to minus gametes and upregulated during gamete activations (ning., 2013), hap2168 - 190 peptide (200 ug / ml), or hap2e protein (200 ug / ml) overnight and then added to activated gametes as above to determine percent gamete fusion. immunofluorescent staining of gametes was performed as described previously (belzile., 2013). the gametes were fixed in ice cold methanol ; the primary antibody was rat anti - ha (roche) and the secondary was alexa fluor 488 goat anti - rat (invitrogen). images were captured in dic or fitc channels as described previously (liu., 2015). for hap2-ha localization after pre - incubation in -hap2168 - 190 (see below), db - camp - activated gametes were incubated with the antibody for 2 hr, washed three times with gamete medium, and stained with ha antibody as above. for immunoprecipitation, gametes were disrupted by incubation at 4c for 30 min in ripa buffer (20 mm tris, 150 mm naci, 1% np-40, 0.5% deoxycholate, 0.1% sds) containing a proteinase inhibitor cocktail (roche). the samples were centrifuged at 12000 rpm for 30 min, the supernatants were subjected to immunoprecipitation with -hap2168 - 190 and protein a agarose beads using methods described previously (liu., 2010) and the immunoprecipitated proteins were subjected to sds - page and immunoblotting (liu., 2015). for trypsin treatment 5x10 live gametes / ml were incubated in 0.05% freshly prepared trypsin for 20 min at room temperature, diluted 10-fold with n - free medium, centrifuged, and resuspended in fresh n - free medium containing 0.01% chicken egg white trypsin inhibitor. for immunoblotting, the remaining cells were washed twice more with n - free media containing 0.01% chicken egg white trypsin inhibitor before analysis by sds - page and immunoblotting (misamore., 2003). tra184 - 6, r185a, r185k, r185q, ra185 - 6ar and fw192 - 3aa mutant forms of chlamydomonas hap2 (gi:288563868) were performed using standard pcr methods. the pcr fragments were inserted into the hap2-ha plasmid at bglii / nrui sites using in - fusion dry - down pcr cloning kit (clontech). plasmids were transformed into the 40d4 hap2 mutant using electroporation and selected on paromomycin plates (liu., 2015). colonies were selected based on pcr identification of the transgene and confirmation of hap2-ha expression by immunoblotting (liu., 2008). codon - optimized synthetic cdna corresponding to a soluble c - terminally truncated version of the hap2 ectodomain (hap2e) comprising residues 23 - 592 from chlamydomonas reinhardtii was cloned into a modified drosophila s2 expression vector described previously and transfection was performed as reported earlier (krey., 2010). for large - scale production, cells were induced with 4 m cdcl2 at a density of approximately 7x10 cells / ml for 8 days, pelleted, and the soluble ectodomain was purified by affinity chromatography from the supernatant using a streptactin superflow column followed by size exclusion chromatography using a superdex200 column in 10 mm bicine ph9.3. purified hap2e ectodomain was subjected to sec using a superdex 200 column (ge healthcare) equilibrated with the indicated buffers. online malls detection was performed with a dawn - heleos ii detector (wyatt technology, santa barbara, ca, usa) using a laser emitting at 690 nm. online differential refractive index measurement was performed with an optilab t - rex detector (wyatt technology). data were analyzed, and weight - averaged molecular masses (mw) and mass distributions (polydispersity) for each sample were calculated using the astra software (wyatt technology). balb / c mice were immunized subcutaneously with 10 g of recombinant hap2e in complete freund s adjuvant and boosted five times with the same antigen dose in incomplete freund s adjuvant. mice splenocytes were fused to p3u1 myeloma cells and growing hybridomas were selected in an elisa test on plates coated with 1 g / ml hap2e. monoclonal antibody k3 was purified using protein g hitrap columns (ge healthcare) according to the manufacturer s instructions followed by sec in pbs using a sdx200 column. dope (1,2-dioleoyl - sn - glycero-3-phosphoethanolamine), dopc (1,2-dioleoyl- sn - glycero-3-phosphocholine), cholesterol and sphingomyelin were purchased from avanti polar lipids. liposomes were freshly prepared by the freeze thaw and extrusion method using molar ratios of 1/1/3/1 of dope / dopc / cholesterol / sphingomyelin. 0.7 m purified hap2e were mixed with 8 mm liposomes and incubated 1h at 25c in 100 l pbs. samples were then adjusted to a final concentration of 20% sucrose, overlayed with a 5%60% sucrose gradient (in pbs) and centrifuged overnight at 4c at 152.000 x g. fractions from the top, middle and bottom of the gradient were analyzed by immunoblotting using specific anti - hap2 monoclonal antibodies and the bands quantified using the genetools syngene software. the percentage of hap2e in either fraction was calculated as the ratio between hap2e in individual fractions and total hap2e (sum of hap2e in top and bottom fractions). purified hap2e (c. reinhardtii) mixed with liposomes was spotted on glow discharged carbon grids (cf300, ems, usa), negatively stained with 2% phosphotungstic acid (pta) ph 7.4, analyzed with a tecnai g2 bio - twin electron microscope (fei, usa) and imaged with an eagle camera (fei, usa). for cryo - electron microscopy liposomes alone or liposomes mixed with purified hap2e were applied on a glow discharged lacey carbon grid (agar scientific, uk). samples were plunge - frozen in liquid ethane using an automated system (leica emgp, austria) and visualized on a tecnai f20 electron microscope operating at a voltage of 200 kv. image frames were recorded in low - dose mode on a falcon ii direct electron detector (fei, usa). purified recombinant hap2e in pbs and hap2e from the top fraction of the sucrose gradient solubilized with 4% chaps were independently purified by size - exclusion chromatography using a superdex 200 increase column. elution fractions corresponding to the respective proteins were further analyzed on a 4%16% native gradient gel using the nativepage novex bis - tris gel system (invitrogen) followed by silver staining. crystals of hap2e were obtained using in situ proteolysis as described before (dong., 2007). briefly, subtilisin dissolved in 10 mm tris ph8, 30 mm nacl at 10mg / ml was added to protein solution (12 - 14 mg / ml in 10 mm bicine, ph 9.3) on ice immediately prior to crystallization trials in a 1/100w : w ratio. crystals of hap2e were grown at 293k using the hanging - drop vapor - diffusion method in drops containing 1 l protein / protease solution mixed with 1 l reservoir solution containing 100 mm hepes ph7.5, 2% 2-propanol, 100 mm sodium acetate, and 12%14%w / v peg 8000. diffraction quality rod - like crystals appeared after 1 week and were flash - frozen in mother liquor containing 30% (v / v) mpd. data collection was carried out at the swiss light source (px i), the esrf (id30a-3), and the synchrotron soleil (proxima1). data were processed, scaled and reduced with xds (kabsch, 1988), pointless (evans, 2006) and programs from the ccp4 suite (collaborative computational project, 1994). a single - wavelength anomalous dispersion (sad) dataset was collected from a single crystal of hap2e from c. reinhardtii soaked for 6 hr in 2 mm k2ptcl4 solution in cryo buffer. data were collected at the liii edge of platinum (1.072) on a single crystal using low - dose (0.5 mgy per 360), high - redundancy (5 360) fine -sliced collection strategy using five crystal orientations by means of a high - precision multi - axis prigo goniometer (weinert., 2014). an initial set of experimental phases was obtained by the single isomorphous replacement method using autosharp (vonrhein., 2007) with the platinum derivative and a highly isomorphous native dataset. starting phases were improved by consecutive cycles of manual building and combination with phases derived from molecular replacement using the partial model as search model in phaser (mr - sad) (mccoy., 2007). after building an initial poly - alanine model accounting for 50% of the c atoms these phases were further refined using the anomalous signal of a highly redundant sulfur - sad dataset collected at a wavelength of 2.06641 on crystals of the native protein following a similar collection strategy as mentioned above (weinert., 2014). model building was performed using coot (emsley., 2010), and refinement was done using autobuster (bricogne., 2010) with repeated validation using molprobity (chen., 2010). the final model includes amino acids 24 to 581 (see linear diagram in figure 1b), with internal breaks at loops 69 - 97, 152 - 156, 167 - 182, 194 - 205, 238 - 283 and 330 - 345, corresponding to disordered loops that are marked with a gray background in the c. rheinhardtii sequence in figure 5d (top sequence) and as dashed tubes in the ribbon diagrams (figures 4 and 5a5c). clear electron density was observed for one n - linked and one o - linked glycan chain (attached to n497 and t577 in domain iii). data are presented as mean sd unless otherwise indicated in figure legends and experimental repeats are indicated in figure legends. the accession number for the atomic coordinates and structure factors reported in this paper is pdb : 5mf1. designed the mutant hap2 proteins and selected the peptide used to produce the fusion - blocking antibody. w.j.s., y.l., and w.l. performed the gamete fusion assays with hap2 mutants and antibodies and interpreted them with w.j.s. collected data and determined the hap2 x - ray structure and g.b. helped devise a collection strategy for derivative x - ray data. | summarysexual reproduction is almost universal in eukaryotic life and involves the fusion of male and female haploid gametes into a diploid cell. the sperm - restricted single - pass transmembrane protein hap2-gcs1 has been postulated to function in membrane merger. its presence in the major eukaryotic taxa animals, plants, and protists (including important human pathogens like plasmodium)suggests that many eukaryotic organisms share a common gamete fusion mechanism. here, we report combined bioinformatic, biochemical, mutational, and x - ray crystallographic studies on the unicellular alga chlamydomonas reinhardtii hap2 that reveal homology to class ii viral membrane fusion proteins. we further show that targeting the segment corresponding to the fusion loop by mutagenesis or by antibodies blocks gamete fusion. these results demonstrate that hap2 is the gamete fusogen and suggest a mechanism of action akin to viral fusion, indicating a way to block plasmodium transmission and highlighting the impact of virus - cell genetic exchanges on the evolution of eukaryotic life. |
autoimmune pancreatitis is a fibro - inflammatory form of chronic pancreatitis that accounts for 5%-6% of chronic pancreatitis. type i involves both genders but is more prevalent among males and associated with hyperimmunoglobulin g4, anti - nuclear antibody, anti - smooth muscle antibody, anti - lactoferrin antibody, and anti - carbonic anhydrase antibody. extra - pancreatic manifestations and association with other autoimmune diseases is also common in type i autoimmune pacreatitis. increased igg4 to levels above 135 mg / dl in type i differentiates autoimmune pancreatitis from other pancreatic disorders although high igg4 levels have been reported in pancreatic carcinoma and chronic pancreatitis. however in detecting autoimmune pancreatitis, pancreatic enlargement and narrowing of the pancreatic duct histopathological evaluations show lymphocytic and plasmacytic infiltration around the pancreatic ducts accompanied with lymphoid follicles and an increase in the thickness of the intralobular septate and fibrosis as well as obliterans phlebitis. although this condition can result in various extra - pancreatic lesions similar to primary sclerosant cholangitis it can be differentiated by detection of igg4 plasma cell infiltration around the pancreatic ducts and appropriate response to corticosteroid therapy. an endoscopy may reveal focal infiltration of plasma cells in the stomach, duodenum, and colon. autoimmune pancreatitis may be accompanied by other autoimmune diseases such as ulcerative colitis or autoimmune hepatitis. ct scan images may show various conditions including local or diffused enlargement of the pancreas, a pancreatic mass or a normal pancreas. autoimmune pancreatitis should be differentiated from pancreatic carcinoma by the absence of diffuse pancreatic duct stenosis in cases of pancreatic carcinoma. cholangiopancreatography demonstrates the presence of local or diffused pancreatic duct stenosis that may be associated with biliary duct stenosis. the diagnostic criteria for autoimmune pancreatitis are a combination of radiologic, laboratory, and histopathologic findings. short course corticosteroid treatment should be considered for patients who refer with typical autoimmune pancreatitis presentations even if serological and histopathological findings are absent. the presence of radiological and laboratory findings in accordance with autoimmune pancreatitis is sufficient to administer corticosteroid therapy. due to extra - pancreatic manifestations and association with other autoimmune diseases, autoimmune pancreatitis this case report introduces a patient with autoimmune pancreatitis who manifested with extra - pancreatic presentations similar to primary sclerosant cholangitis. it is important to consider autoimmune pancreatitis as a possible diagnosis for patients with jaundice and abdominal pain who do not have diagnostic results of routine laboratory tests and are unresponsive to conventional treatments. a 41-year - old female referred with a history of increasing abdominal pain and jaundice since 2 weeks prior. the patient did not have any history of alcohol consumption, drug abuse, previous liver and biliary or hematologic diseases, transfusion, high risk sexual behavior, and recent travel to high risk regions for infectious diseases. the patient had diffuse abdominal pain that increased with eating and the sclera was icteric. there was no evidence of fever, diarrhea, constipation, ascites, hematemesis, hematochezia, lymphadenopathy, hepatosplenomegaly, or weight loss. routine laboratory analysis revealed increased liver function test results, especially alkaline phosphatase (alp), total and direct bilirubin, and erythrocyte sedimentation rate (esr). abbreviations : wbc : white blood cells ; rbc : red blood cells ; hct : hematocrit ; fbs : fasting blood sugar ; ldl : low density lipoprotein ; hdl : high density lipoprotein ; tg : triglycerides ; cr : creatinine ; bun : blood urea nitrogen ; ast : aspartate aminotransferase ; alt : alanine aminotransferase ; alp : alkaline phosphatase ; pt : prothrombin time ; ptt : partial thromboplastin time ; inr : international normalized ratio for prothrombin time ; total bil. : total bilirubin ; direct bil. : direct bilirubin ; na : sodium ; k : potassium ; ca : calcium ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; hbsag : hepatitis b surface antigen ; anti - hcv : antibody hepatitis c virus ; anti - hav : antibody hepatitis a virus ; ana : antinuclear antibody ; anti - lkm : antibodies to liver - kidney microsomes ; igg4 : immunoglobulin g4 ; ama : anti - mitochondrial antibody ; u / a : urine analysis abdominal ultrasound evaluations reported a normal - size liver with increased tissue echo and mild dilatation of the intra- and extra - hepatobiliary ducts. mri imaging of the abdomen showed mild dilatation of the intra- and extra - hepatobiliary ducts(figure 1). by taking into consideration the observed biliary duct dialatation and absence of any intra - hepatic conditions, we requested laboratory markers for viral hepatitis, hemachromatosis, wilson s disease, celiac, and primary biliary cirrhosis - all had negative results. irregular dilatations and stenosis of the intra- and extra - hepatobiliary ducts and pancreatic duct results from mrcp, the presence of ana and igg4 were evaluated, both of which were both positive(figure 2). thus according to the association of clinical presentations that included abdominal pain and obstructive jaundice with the presence of specific autoimmune markers the patient underwent corticosteroid therapy, 30 mg daily, with a diagnosis of autoimmune pancreatitis accompanied by autoimmune sclerosant cholangitis. at the one month follow up visit post - treatment, the control laboratory tests and mrcp revealed normal liver function results along with normal igg4 and ana, and normal intra- and extra - hepatobiliary ducts. axial images from mri of the abdomen showed irregular narrowing and dilatation of the intrahepatic bile ducts (arrow). magnetic resonance cholangiopancreatography (mrcp) imaging study showed mild narrowing and dilatation of the intrahepatic bile ducts, mild dialatation of the cbd, and irregularity of the pancreatic duct (arrows). autoimmune pancreatitis can be diagnosed according to the following criteria : 1) narrowing of the pancreatic duct with segmental or diffused irregularity and localized or diffused pancreatic enlargement observed in imaging modalities ; 2) increase in serum igg4 levels and the presence of autoantibodies such as antinuclear antibody and rheumatoid factor (rf) ; and 3) intralobular fibrosis, plasma cell and lymphocytic infiltration around the pancreatic ducts and local lymph node enlargement. autoimmune pancreatitis is defined as the presence of criterion 1 with either criterion 2 or 3. however, the most recent criteria for diagnosis of autoimmune pancreatitis has changed as follows:3 imaging : diffused pancreas enlargement, segmental or diffused irregular narrowing of the main pancreatic duct.. a definite diagnosis is defined by either criteria a+c, a+b, a+b+c, or a+b+c+d, whereas a probable diagnosis is the presence of criteria a+d and suspicion of disease is only criteria a. recent mayo clinic studies have introduced additional criteria for the histopathological diagnosis of autoimmune pancreatitis ; predictive images in ct scan or pancreatography and increased igg4 level, and response to corticosteroid therapy. they have reported diffused enlargement of the pancreas in 27% of patients diagnosed with autoimmune pancreatitis. increased igg4 was present in 71% of patients with mean values of 134 mg / dl. autoimmune pancreatitis is more prevalent among males and commonly presents with mild abdominal pain without typical pancreatitis pain that sometimes is accompanied by obstructive jaundice. increased pancreatic and hepatobiliary enzymes may also be present. histopathological evaluations show intralobular fibrosis, lymphocytic and igg4 plasma cell infiltration, and obliterans phlebitis observed in biopsies and autopsies. none of these findings are present in chronic pancreatitis in alcoholics and those with sjgren s syndrome. specific inflammatory changes in patients with autoimmune pancreatitis strongly suggest a close relation to mutational fibrosclerosis. extrapancreatic manifestations of these patients include sclerosant cholangitis, retroperitoneal fibrosis, interstitial nephritis, chronic thyroiditis, interstitial pneumonia, in addition to mediastinal and peritoneal lymphadenopathy. autoimmune pancreatitis associated sclerosant cholangitis can be differentiated from primary sclerosant cholangitis by the presence of igg4 plasma cell infiltration around the pancreatic duct and a response to corticosteroid therapy. the current patient s pancreas imaging evaluations were normal however her igg4 level increased to 276 mg / dl. the presence of ana in conjunction with the above results and appropriate response to corticosteroid therapy fulfilled the diagnostic criteria for autoimmune pancreatitis. routine laboratory evaluations in this patient revealed a significant increase in alp and direct bilirubin ; the patient also had obstructive jaundice. sonography and mri showed dilatation of ducts secondary to stenosis and obstruction confirmed by mrcp as secondary dilatation and stenosis in the pancreas and biliary ducts which was similar to sclerosant cholangitis. a control mrcp at one month after corticosteroid therapy showed no evidence of dilatations and stenosis. this report introduced a case of autoimmune pancreatitis with pancreatic duct stenosis, increased igg4, positive antinuclear antibody, and appropriate response to corticosteroid therapy as well as its association with sclerosant cholangitis, which resolved after therapy. | autoimmune pancreatitis is a fibro - inflammatory form of chronic pancreatitis. it is diagnosed by the combination of imaging studies such as a ct scan and pancreatography, laboratory analyses that include igg4 and/or autoantibodies, histopathological evaluations and positive response to corticosteroid therapy. we report the case of a 41-year - old female with a history of jaundice and increasing abdominal pain for two weeks prior to her clinic visit. laboratory results were significant for an increase in alkaline phosphatase (alp) and erythrocyte sedimentation rate (esr). magnetic resonance cholangiopancreatography (mrcp) confirmed areas of stenosis and dilatation in the pancreatic duct and in the intra- and extra - hepatic bile ducts similar to primary sclerosantcholangitis. laboratory analyses showed increased levels of igg4 with thepresence of antinuclear antibodies. |
cancer is one of the major causes of childhood death in the developed and developing countries. it accounts for 4% of death of children under 5 years of age and 13% of children 5 - 15 years of age in the iranian population ; contributing to 15% of total loss of life in the under 15- year age group. however, despite the greatly improved clinical outcomes, children with cancer and their parents continue to experience significant distress throughout the course of the diagnosis and illness. a number of previous investigations demonstrated that parents of children with cancer experience elevated levels of distress [2 - 4 ]. findings vary based on measurement, but the most commonly identified types of distress are depression, anxiety, posttraumatic stress symptoms, and subjective symptoms of stress. it is estimated that moderate and severe symptoms of distress range from 15% to 51% [5 - 7 ], especially in mothers [8, 9 ] who report more emotional problems than fathers [10, 11 ]. to date, there have been a few studies that have used parents as facilitators during their children s medical procedures. in fact, some studies [12 - 14 ] have designed stress management programs specifically for parents of children undergoing medical procedures. intervention programs generally incorporate procedural information and coping skills training (i.e., muscle relaxation, deep breathing, visual imagery, and positive self - statements). mbct includes mindfulness practices designed to cultivate nonjudgmental observation and acceptance of bodily sensations, cognitions, and emotions. participants learn to engage in sustained observation of these phenomena, with an attitude of interest and curiosity, and to accept them as they are, without trying to change or escape them. mbct also includes elements of cognitive therapy that are consistent with nonjudgmental acceptance of the experience. a decentered view of thoughts is emphasized, in which participants are encouraged to view their thoughts as transient mental events rather than as aspects of themselves or as necessarily accurate reflections of reality or truth. it is claimed that this interventional method has just recently been invented by cognitive therapists. however, historically the method had been considered by islamic philosophers few centuries ago i.e : imam fakhr razi who suggested the theory of imagination without judgment. the present study sought to evaluate the effectiveness of mbct on reduction of depression and anxiety symptoms in mothers of children with cancer. efficacy studies examine the effects of treatment in randomized controlled trials, involving recruited patients, using a highly structured treatment manual for a narrow problem focus. effectiveness studies examine the effects of treatment conducted in non research based clinical settings and purposive sampling. the aim of such research is to maximize the external validity or generalization of results to various settings. inclusion criteria were (a) possessing 19 score and above in beck depression inventory - ii (bdi - ii) ; (b) 15 score in beck anxiety inventory (bai) ; (c) meeting criteria for anxiety and depression determined by the modified version of the structured clinical interview for dsm iv ; (d) medically stable. patients with comorbid current major depression, substance abuse and/or dependence and psychosis were excluded from the study because of low concentration and orientation. one child was a girl and three were boys (ages 5 - 12). one child was diagnosed by neuroblastoma, and three by acute lymphoblastic leukaemia (all). this paediatric treatment center had a social worker but s no treatment, systematic approach or recommendations were offered at this center regarding how mothers ' anxiety and depression should be handled during their children 's treatment procedures. (table 1) the method used in this study was the single - case experimental design. in analyzing the data in the single case study, the dependent variable for the possible changes resulting from the independent variable the first criterion is to draw the graphs of subjects ' functions at the baseline and the intervention phase, and then compare them ; and the second criterion is to consider the slopes in each of the two - step graph - line during the intervention. thus, any trends or slopes in each stage are examined. in this study, we subtracted the pretest scores from the post - test scores and then divided the attained number by the pretest score. at the baseline state including 3 times measurement following 5 days of treatment, and 10 days after the end of the treatment and 10 days after the end of the treatment for follow up, subjects completed self - report measures of anxiety, and depressive symptomatology. to monitor the changes, the measurements were also performed at end of second, forth, sixth and eighth sessions. intervention program mindfulness training teaches a way of being rather than of doing, allowing participants to step back from automatic behaviours and habitual thought patterns. mbct is a psychological therapy which uses features of cognitive therapy with mindfulness techniques of buddhism. mbct consists of accepting thoughts and feelings without judgment rather than trying to push them out of consciousness, with aim of correcting cognitive distortions. mbct was found by zindel segal, mark williams and john teasdale, who based mbct on a program developed by jon kabat - zinn called mindfulness - based stress reduction (mbsr), [21 - 23 ] which was adapted to use for patients with major depressive disorder. the aim of mbct is not directly provide relaxation or happiness, but is rather a " freedom from the tendency to get drawn into automatic reactions to thoughts, feelings, and events ". mbct programs usually consist of an eight week course with two - hour classes in each session with weekly assignments to be done after the sessions. the aim of the program is to enhance awareness so clients are able to respond to things instead of react to them. mindfulness practice helps us to see the patterns of the mind more clearly ; and to learn to recognize when our mood is beginning to go down. mindfulness teaches us a way in which we can get in touch with the experience of being alive. low mood can bring back memories and thoughts from the past, and make us worry about the future. mindfulness helps to halt the escalation of these negative thoughts and teaches us to focus on the present moment, rather than reliving the past or pre - living the future. when we start to feel low, we tend to react as if our emotions were a problem to be solved : we start trying to use our critical thinking strategies. when these do not work, we re - double our efforts to use them. we end up over - thinking, brooding, ruminating, and living in our heads. mindfulness helps us to enter an alternative mode of mind that includes thinking but is not just thinking. it teaches us to shift mental gears, from the mode of mind dominated by critical thinking (likely to provoke and accelerate downward mood spirals) to another mode of mind in which we experience the world directly, non - conceptually, and non - judgmentally. it helps develop our willingness to experience emotions, our capacity to be open to even painful emotions. it gives us the courage to let distressing moods, thoughts and sensations to come and go, without battling with them. increasing ability to rest within the present moment requires training in concentration and sustained attention. patients are trained to ground themselves in the moment as a starting point, for example, by paying attention to the movement of the breath or to body sensations. the body and the breath are constantly present, and the mind can return to them whenever awareness is lost. participants in the classes discover that they can not be fully aware of body sensations or the breath from moment to moment, if their minds wander off to another place or time. the body scan practice is designed to increase patients ability repeatedly to engage, sustain, and then disengage attention. participants move a focused spotlight of attention from one part of the body to another, as if they could breathe in to each location and explore sensations in depth just as they are before letting go and moving on. participants are encouraged to approach whatever sensations arise with an attitude of kindness, open curiosity, without judging them. when the mind wanders, they are invited simply to notice where it has gone and gently to shift attention back to the body. by staying with the body scan, mothers had an opportunity to notice how their experience changed from moment to moment and to practice a different way of responding to intense, uncomfortable sensations. developing nonjudgmental awareness of thoughts, body sensations, and physical stimuli (sights, sounds) facilitates adoption of this same nonjudgmental attitude when responding to negative thoughts, for example, about the self. participants develop an ability to see thoughts as mental events that pass through the mind, rather than as facts or central parts of their identity. for example, one exercise involves imagining thoughts that arise during sitting meditation as passing images on a cinema screen or as leaves floating past on a river. when mothers used this technique, they were surprised to discover that sustained but decenterd attention to the thoughts caused them to lose their ability to provoke an emotional reaction. the subjects found that many thoughts disappeared altogether as they watched them come and go ; and this made them to be keen to experience their thoughts and emotions by this technique in other situations. therapeutic package in this study, the interventions included in our manual were provided in eight sessions. in the first session, goals and techniques included building a rapport with the client, obtaining information from the client, providing psychoeducation on mindfulness, cbt, depression, and anxiety, identifying automatic thoughts and leading the client through a guided mindfulness meditation. in the second session, goals and techniques included helping the client recognize that most of her thoughts are not facts, teaching the client to use the thought record, educating client about cognitive distortion. goals and techniques in the third session included educating diaphragmatic breathing and sleep hygiene ; next, teaching the client a brief body scan exercise to reduce muscle tension. in the fourth session, goals and techniques included introducing mindful daily activity, teaching mindful eating and mindful labelling on thoughts, feelings and behaviours. in the fifth session, goals and techniques include to enhancing recognition of personal consequences of chronic worry, having the client perform a cost - benefit analysis of her chronic worry, scheduling worry time and mindful worry - free zones. goals and techniques in the sixth session included generating a hierarchy of worries, imaginary exposure with acceptance, incorporating in vivo exposure through increased participation in planned events. in the seventh session, goals and techniques included helping the client learn to identify and respond to early signs of relapse, collaborating with the client to generate responses to her early warning signs, helping the client practice developing a worry action plan. in the eighth session, goals and techniques included reviewing the insights and techniques found most useful by the client, identifying obstacles to practice mindfulness, providing a checklist of the techniques included in the program. beck anxiety inventory (bai) the bai (24) is a 21-item scale developed to address the need of an instrument that would reliably discriminate anxiety from depression while displaying convergent validity. the items are summed to obtain a total score which range from 0 to 63. numerous studies have reported alpha internal consistency and the results are consistent across a wide range of respondents. for example, alpha of 0.90 was found for undergraduates and alpha of 0.94 was found for outpatients with anxiety disorder. considering concurrent validity, the correlation between the beck anxiety index and the hamilton rating scale for anxiety is 0.47 and correlation with brief symptom inventory (anxiety scale) is 0.69. discriminate validity of scale was obtained in a study of psychiatric outpatients through their anxiety (94%) [24 - 26 ]. beck depression inventory - ii (bdi - ii) the beck depression inventory - second edition (bdi ii) is a 21-item scale and one of the most widely used self - report measures of depression. beck cited alphas of 0.93 for college students and 0.92 for outpatients, in another study steer. reported an alpha of 0.92 for the bdi - ii. beck reviewed 11 studies that showed the bdi is capable of discriminating between groups that differ in level of depression. a further 35 concurrent validation studies compared the bdi with other ratings of depression. fourteen studies reported correlations between the bdi and clinical ratings ; coefficients for psychiatric patients ranged from 0.55 to 0.96, with a mean of 0.72. the correlation between the earlier version of bdi and (bdi ii) was 0/93 and kappa agreement was 0.70. diagram 1 shows that the participant 's depression and anxiety level is 23 in bdi - ii and 34 in bai approximately at the baseline statement. these scores are severe and moderate rates of depression in bdi - ii and anxiety in bai. she obtained a score of 13 in bdi - ii and 15 in bai at end of session 2 ; and this reduction was continued except for session 6.between fifth and seventh sessions, her child was in chronic paediatric medical condition. her scores in posttest measurement was 10 in bdi - ii and 8 in bai that indicate reduction in symptoms of depression and anxiety. (figure 1) diagram 2 shows that participant 's depression and anxiety levels were 19 - 29 in bdi - ii and 26 - 29 in bai approximately at the baseline statement. these scores are in severe and moderate range of depression in bdi - ii and anxiety in bai. she obtained a score of 16 in bdi - ii and 18 in bai at end of session 2. her scores in post - test measurement were 6 in bdi - ii and 15 in bai that indicate reduction in symptoms of depression and anxiety. her improvement quotient was % 74 for depression and % 46 for anxiety.(figure 2) diagram 3 shows that participant 's depression and anxiety levels are 24 - 28 in bdi - ii and 31 - 38 in bai approximately at the baseline statement. these scores are in severe and moderate rate of depression in bdi - ii and anxiety in bai. she got the score of 22 in bdi - ii and 15 in bai at the end of session 2. her scores in post - test measurement were 14 in bdi - ii and 8 in bai that indicate reduction in symptoms of depression and anxiety. her improvement quotient was % 46/83 for depression and % 77/14 for anxiety.(figure 3) diagram 4 shows that participant 's depression and anxiety levels are 21 - 26 in bdi - ii and 30 - 37 in bai approximately at the baseline statement. these scores are severe and moderate range of depression in bdi - ii and anxiety in bai. she got the score of 12 in bdi - ii and 22 in bai at the end of session 2. her scores in post - test measurement were 7 in bdi - ii and 14 in bai that indicate reduction in symptoms of depression and anxiety. her improvement quotient was % 86/95 for depression and % 59/22 for anxiety.(figure 4) all participants showed scores of 21 - 28 at the baseline statement that indicated severe and moderate depression (19 - 29) in bdi - ii. post - test of three participants (a, b and c) is in low or no depression remain (less than 10) and one participant (d) remains in low or moderate depression (10 to 18 score).(figure 5) all participant possessed scores of 26 - 38 at the baseline statement that indicated severe anxiety (19 - 29) in bai. visual observation of diagrams demonstrated a decline of scores. post - test of all participants showed low or moderate anxiety (8 - 18 score).(figure 6) there has been limited research regarding psychosocial interventions about mothers of children with cancer because their problems to be sophisticated. however, studies demonstrated that the symptoms of depression and anxiety have been reduced using mindfulness - based cognitive therapy in these mothers. the current study, which is consistent with ingram, hayes, and scott theory (2000), explains the result in four areas to evaluate the performance of cognitive therapy. universality of change (what is the percentage of the improvement ?) differences between base line and post - test scores demonstrated that examinees show positive improvement on both beck depression and anxiety scales. the percentages of recovery on depression were : participant a : 78.87% ; participant b : 74% ; and participant c : 46.83%. percentages of recovery on anxiety were : participant a (76.92%) ; participant b : (46%), participant c : (77.14%) ; and participant d : (59.22%). generality of change (what are the changes in relation to critical situations and jobs ?) according to harding (1996), madan - swain and brown (1991), the studies showed that a child 's cancer effects parents in spending their energy on the patient diverted all his attention to care for the sick child and find himself detached from the other family members. at the end of the final sessions, the participant found himself having a better relationship with his spouse and child. however, it should be noted that the child seeks more attention from the mother. (2001) found that after using mindfulness - based cognitive therapy on patients with depression or dysthymia, recovery can be observed on their over general autobiographical memory. over general autobiographical memory is a capacity to recall categories of events in a coherent and continuous way when the subjects are asked to provide specific instances from their life (17). participant b had more difficult problems with her husband about how to care for their sick child. she was suffering as a result of arguments with her husband at the presence of their child and this led to a big fight between them. at the end, she showed a significant improvement in her over general autobiographical memory and marital satisfaction. she was suffering from lack of concentration and chronic fatigue in her office during work hours ; she was late for work every morning and used up all her breaks and vacations that resulted in a critical job situation. at the end of the therapeutic sessions, she has shown an improvement on her anxiety symptoms by (77.8 %) ; however, her anxiety improvement was poor compared to other subjects (47.7%), but still significant. participant d also showed (86.95%) improvement in her depression rate ; however, less recovery was observed on her anxiety level (59.22%). while she was undergoing therapeutic sessions for her own anxiety symptoms, she was facing her son chronic medical condition. therefore, the result shows a strong correlation between her anxiety scores and her son relapse condition. this indicated the real extent of her condition. at the end of her anxiety therapy session, mohammad valimirza indicated that those children whose parents dealt with their condition more effectively, experience less situational anxiety. comparing the participants scores at base line, post - test, and follow up in both depression and anxiety showed a great deal of improvement on the scales, and led to a full recovery. stability (treatment achievements) follow up result (30 days after the last therapy session and 5 days after post - test) indicated that depression of participants a and d both maintained in the score of less than 10 which indicates the state of no depression or least depression. participant b with the score of 10, and participant c with the score of 16 were categorized in a range of 10 - 18 with a diagnosis of mild to moderate depression ; and it can be stated that mbct provided them with a relative stability. anxiety of participants a, b, and c with the score of 11, 12, and 5 respectively indicated the effectiveness of this kind of treatment for anxiety. participant d, with 1 score above the cut of point (15), represented a poorer result in regards to the therapy achievements. in summary, the current study demonstrated that mindfulness - based cognitive therapy has a significant effect on both depression and anxiety on our sample. the anxiety and depression both changed in different ways. according to psychological interviews which the therapist carried out with mothers, this treatment approach significantly improved the relationship of these mothers with themselves, and their family, and also improved their social functions. the results have coherency with studies which emphasize the effectiveness of mbct for treatment of depression, anxiety and stress and to improve psychosocial adjustment of people [21- 23 ]. because psychological conditions of mothers can affect their children wellbeing, it is suggested that future research concentrate on direct effectiveness of mbct on the mothers and on indirect improvement of medical condition of children with cancer. as mentioned in the article, the root of mindfulness can be found in theories of some islamist logic experts such as imam fakhr razi. therefore, it is suggested that the whole procedure of mbct should be developed based on iranian- islamic culture. in summary, the current study demonstrated that mindfulness - based cognitive therapy has a significant effect on both depression and anxiety on our sample. the anxiety and depression both changed in different ways. according to psychological interviews which the therapist carried out with mothers, this treatment approach significantly improved the relationship of these mothers with themselves, and their family, and also improved their social functions. the results have coherency with studies which emphasize the effectiveness of mbct for treatment of depression, anxiety and stress and to improve psychosocial adjustment of people [21- 23 ]. because psychological conditions of mothers can affect their children wellbeing, it is suggested that future research concentrate on direct effectiveness of mbct on the mothers and on indirect improvement of medical condition of children with cancer. as mentioned in the article, the root of mindfulness can be found in theories of some islamist logic experts such as imam fakhr razi. therefore, it is suggested that the whole procedure of mbct should be developed based on iranian- islamic culture. | backgroundfindings demonstrated that parents of children with cancer experience elevated levels of distress, depression, anxiety, posttraumatic stress symptoms, and subjective symptoms of stress. in this study, we determined effectiveness of mindfulness - based cognitive therapy (mbct) on reduction of depression and anxiety symptoms in mothers of children with cancer. methodsfour mothers whose children had cancer were diagnosed to have depression and anxiety symptoms, using beck anxiety inventory, beck depression inventory ii, and structured clinical interview for dsm - iv. they were selected through purposeful sampling to participate in this experimental single - case study. after the baseline was determined, subjects attended an eight - session program of mindfulness - based cognitive therapy.resultsthe comparison of baseline and post - test showed that depression and anxiety symptoms decreased through mbct. improvement quotient for depression and anxiety of each subject was good. conclusiondata showed that mbct reduced depression and anxiety symptoms in mothers of children with cancer. |
chorea is the ceaseless occurrence of rapid, jerky, dyskinetic, involuntary flicking movement of hands, face, and other parts of body. huntington 's disease (hd) and drugs account for more than 50% of such patients. basic pathology involved in chorea and other hyperkinetic movements is damage to various parts of basal ganglia that ultimately leads to disturbances in neurotransmission. in chorea, there is degeneration of gamma aminobutyric acid (gaba) secreting neurons in the caudate nucleus and putamen of basal ganglia. the normal inhibition by gaba neurons is lost in globus pallidus, and substantia nigra resulting in over activity of excitatory transmitters causing the distortional movements. phenytoin sodium is an anti - seizure drug widely used for generalized tonic clonic seizures. few cases have been reported to cause hyperkinetic movements by various anti - seizure drugs. here, we report a case of phenytoin induced chorea in a child possibly due to interaction between anti - seizure drugs. a 3-year - old female patient weighing 10.5 kg was admitted in pediatric ward of sir takhtsinhji general hospital, bhavnagar, gujarat, with complaint of low grade fever for three days and convulsions with frothing, involuntary micturition, and altered sensorium for one hour. patient was treated with injection lorazepam (0.04 mg / kg) followed by injection phenytoin (5 mg / kg) intravenously to control the seizures. all investigations like total leukocyte count, differential leukocyte count, liver function tests, renal function tests, random blood sugar, and cerebrospinal fluid (csf) examination were normal except hemoglobin (9.0 g / dl ; reference value : 12.0 to 18.0 g / dl), positive c - reactive protein (crp), and ionized calcium (1.01 mmol / l ; reference value : 1.16 to 1.32 injection phenytoin (5 mg / kg / day) and injection phenobarbital (5 mg / kg / day) intravenously were given for five days and tablet clobazam (0.75 mg / kg / day) was given for three days to control seizures. injection acyclovir (40 mg / kg / day) and injection methyl prednisolone (2 mg / kg / day) were started as empirical therapy on 5 day. from 6 day, syrup phenytoin (30 mg/5 ml), 4 ml, 12 hourly, and syrup phenobarbital (20 mg/5 ml), 6 ml, 12 hourly, were started after stopping injectable anti - seizure drugs. on the 8 day, patient developed involuntary, continuous, uncontrolled, jerky movements of head and upper limbs, which was diagnosed as chorea. there was no family history of hd and diagnosis of viral encephalitis was not confirmed so it was suspected as drug induced chorea with phenytoin being an offending agent. patient was having an attack of convulsions two days after stopping phenytoin ; hence, sodium valproate (15 mg / kg / day) was added to treatment. naranjo 's scale showed that the relationship between phenytoin and chorea was probable. according to modified schumock and thornton 's criteria, this reaction was probably preventable and modified hartwig and siegel 's scale showed that the reaction was moderately severe (level 3). chorea and hyperkinetic movements are the rare side effects of phenytoin. in this case, recovery on de - challenging the phenytoin therapy and normal laboratory investigations are in favor of diagnosis of the phenytoin induced chorea. normal ct - scan report after emergence of reaction has ruled out any structural damage to basal ganglia. involuntary movements caused by phenytoin are mainly related to its high plasma concentration (40 - 50 prolonged phenytoin therapy or its high plasma concentration is related with increased dopaminergic and serotonergic activity in basal ganglia that may be considered as a cause of chorea. phenytoin toxicity depends on the route of administration, duration, exposure, drug interaction, and dosage. drugs that alter the functions of these enzymes can place the patient at risk of toxicity. addition of clobazam to the patients who had been taking maximum tolerable dose of phenytoin is reported to result in its intoxication. though, both are enzyme inducers, they may increase plasma concentration of each other by competitive inhibition of their metabolism. acyclovir decreases plasma concentration of phenytoin due to interference in absorption. in our case, acyclovir was started parentrally on 5 day and is less likely to affect the plasma level of phenytoin. use of phenytoin along with clobazam and phenobarbital may have resulted in increased plasma concentration of phenytoin due to an interaction between them which may have caused the reaction. combined use of phenytoin and lamotrigine has been reported to cause the hyperkinetic movement disorder. a possible interaction between phenytoin, phenobarbital and clobazam may have resulted in chorea in this case. it is important that therapeutic drug monitoring should be done and drug interactions should be kept in mind especially when two or more anti - seizure drugs are prescribed. early detection and withdrawal of suspected drug in such cases can help to prevent further harm to the patient. | a 3-year - old female patient developed chorea possibly due to an interaction between phenytoin, phenobarbital and clobazam used for generalized tonic clonic seizures. phenytoin withdrawal resulted in recovery within 24 hours. post reaction computerized tomography (ct)-scan of brain was normal. combined use of anti - seizure drugs and interactions between them may be responsible for the reaction. therapeutic drug monitoring is important while prescribing two or more anti - seizure drugs. |
cytosine methylation at cpg sites in the genomic dna is a well - characterized epigenetic modification involved in gene regulation and transposon silencing in mammals (1,2). upon replication of methylated dna, cytosines in the newly synthesized strand are initially unmethylated (resulting in a hemimethylated duplex), then become methylated by a maintenance - type dna methylase, dnmt1. in addition, the de novo dna methylases, dnmt3a and dnmt3b, also have a role in the maintenance methylation for some genomic regions (35). due to this template - dependent copying mechanism, in contrast, the level of dna methylation changes dynamically during germ - cell development. first, primordial germ cells (pgcs) undergo genome - wide erasure of methylation and then prospermatogonia, a direct derivative of pgcs in the male gonad, undergo extensive de novo methylation (6,7) (figure 1a). the prospermatogonia are mitotically arrested, premeiotic cells expressing high levels of the de novo dna methylase dnmt3a and its related protein dnmt3l (8,9). therefore, de novo dna methylation and its maintenance through cell division can be separately observed in these germ cells. oocytes are also non - dividing cells that undergo extensive de novo methylation (6,7), but the methylation marks in most genomic regions are not maintained through cell divisions after fertilization (10). mitotically arrested prospermatogonia undergo de novo methylation of cpg sites in the fetal testis, and mitosis resumes after birth. non - cpg methylation at three representative b1 loci detected by bisulfite sequencing in p0 prospermatogonia (see supplementary figure s1 for other loci). circles and squares represent cytosines in cpg and non - cpg (cph where h is a, t or c) sites, respectively. (c) alignment of the sequences of the 33 b1 loci to visualize the common sites of methylation. cytosines methylated over 65% at cpg sites are highlighted in black, and those methylated over 5% at non - cpg sites are highlighted in gray. positions of a- and b - box in the pol iii promoter and the binding sites for the ahr and slug transcription factors are indicated above. (d) methylation levels in different sequence contexts. the data for dinucleotides and trinucleotides are shown. (e) individual cpg and non - cpg sites are categorized according to their methylation levels, and counted. mitotically arrested prospermatogonia undergo de novo methylation of cpg sites in the fetal testis, and mitosis resumes after birth. non - cpg methylation at three representative b1 loci detected by bisulfite sequencing in p0 prospermatogonia (see supplementary figure s1 for other loci). circles and squares represent cytosines in cpg and non - cpg (cph where h is a, t or c) sites, respectively. (c) alignment of the sequences of the 33 b1 loci to visualize the common sites of methylation. cytosines methylated over 65% at cpg sites are highlighted in black, and those methylated over 5% at non - cpg sites are highlighted in gray. positions of a- and b - box in the pol iii promoter and the binding sites for the ahr and slug transcription factors are indicated above. (d) methylation levels in different sequence contexts. the data for dinucleotides and trinucleotides are shown. (e) individual cpg and non - cpg sites are categorized according to their methylation levels, and counted. short interspersed elements (sines) are a class of retrotransposons and are transcribed by rna plolymerase iii (pol iii). biochemical studies have shown that cpg methylation of the pol iii promoter inhibits transcription of trnas and human alu sine (1113). we previously showed that the bulk of cpg sites in b1 retrotransposons, a family of sines scattered over the mouse genome, become highly methylated in prospermatogonia and stay methylated throughout the later stages of spermatogenesis (14). we also showed that a mutation in dnmt3a or dnmt3l reduces the methylation levels in a locus - specific manner and that the level of b1 methylation is well correlated with the level of b1 expression (14). during the course of that study, we noticed that methylation at non - cpg sites is abundantly present in prospermatogonia. this is interesting because non - cpg methylation has been so far found in oocytes, embryonic stem cells (escs), induced pluripotent stem cells (ipscs) and the brain (3,1521). although non - cpg methylation dramatically decreases upon differentiation of escs and thus could have some relevance to pluripotency (3,19), the biological function of non - cpg methylation remains unknown. in this report, we characterize the non - cpg methylation that we found at b1 retrotransposons in prospermatogonia in detail. we show accumulation of the non - cpg methylation in the non - dividing prospermatogonia and its gradual loss after the resumption of cell division. we also provide evidence suggesting the absence of a template - dependent copying mechanism for non - cpg methylation. epcam - positive germ cells were collected from testes of c57bl/6 j mice at p0, p2, p5 and p7 as described previously (14). to collect fetal germ cells at e16.5, green fluorescent protein (genomic dna preparation, bisulfite conversion and pcr were done as described previously (14). typically, 100200 ng of genomic dna was treated with bisulfite, and an aliquot equivalent to 510 ng of genomic dna was used for pcr with extaq hs (takara bio, ohtsu, japan). to monitor the efficiency of c - to - t conversion, phage dna that had no methylated cytosine (promega, madison, wi) was included in the bisulfite reaction. all sequenced clones were used for the analysis. to validate the purity of germ - cell preparations, methylation of the imprinted lit1 differentially methylated region (dmr) was also analysed using the same bisulfite - treated dna. among at least 40 sequenced clones, none showed methylation in the lit1 dmr, suggesting a germ - cell purity of > 95%, given that only the maternal lit1 dmr allele is methylated in somatic cells and no allele is methylated in male germ cells. the sequences of the primers for the dmrs, cpg islands and control phage dna are shown in supplementary table s1. genomic dna (100 ng) was digested with taqi (new england biolab, ipswich, ma) and ligated with a synthetic oligonucleotide (5'-cgtgaactgaggtcggaagacctcagttca-3 ') that forms a hairpin structure. the ligation product was denatured at 98c for 10 min in 0.3 m naoh, treated with 9.17 m bisulfite (22) and used for pcr as described (14). the hairpin - bisulfite pcr primers for the three b1 loci are listed in supplementary table s1. epcam - positive germ cells were collected from testes of c57bl/6 j mice at p0, p2, p5 and p7 as described previously (14). to collect fetal germ cells at e16.5, green fluorescent protein (genomic dna preparation, bisulfite conversion and pcr were done as described previously (14). typically, 100200 ng of genomic dna was treated with bisulfite, and an aliquot equivalent to 510 ng of genomic dna was used for pcr with extaq hs (takara bio, ohtsu, japan). to monitor the efficiency of c - to - t conversion, phage dna that had no methylated cytosine (promega, madison, wi) was included in the bisulfite reaction. all sequenced clones were used for the analysis. to validate the purity of germ - cell preparations, methylation of the imprinted lit1 differentially methylated region (dmr) was also analysed using the same bisulfite - treated dna. among at least 40 sequenced clones, none showed methylation in the lit1 dmr, suggesting a germ - cell purity of > 95%, given that only the maternal lit1 dmr allele is methylated in somatic cells and no allele is methylated in male germ cells. the sequences of the primers for the dmrs, cpg islands and control phage dna are shown in supplementary table s1. genomic dna (100 ng) was digested with taqi (new england biolab, ipswich, ma) and ligated with a synthetic oligonucleotide (5'-cgtgaactgaggtcggaagacctcagttca-3 ') that forms a hairpin structure. the ligation product was denatured at 98c for 10 min in 0.3 m naoh, treated with 9.17 m bisulfite (22) and used for pcr as described (14). the hairpin - bisulfite pcr primers for the three b1 loci are listed in supplementary table s1. we collected prospermatogonia at postnatal day 0 (p0) and studied dna methylation states of 33 b1 loci by bisulfite sequencing. the bisulfite sequencing method is unable to distinguish between 5-methylcytosine (5mc) and 5-hydroxylmethylcytosine (5hmc), but hereinafter we regard bisulfite - resistant cytosines as 5mc, given that 5hmc is present much less than 5mc in cells / tissues examined so far, and almost exclusively at cpg sites in escs (23). as shown in figure 1b, c and supplementary figure s1, significant methylation was found at non - cpg sites, in addition to cpg sites, both within and around the b1 sequences. this was not due to imcomplete bisulfite conversion, because the failure rate was only 0.20.5%, as determined using the control unmethylated dna (phage dna) mixed to the samples. on average, the methylation level at non - cpg sites (total of 2700) in p0 prospermatogonia was 4.8%, which was higher than the levels in escs (2.6%), ipscs (3.2%), oocytes (3.43.8%) and the brain (1.52.2%) (16,20,21). of all methylated cytosines, 77% were at cpg sites and 23% were at non - cpg sites. when the methylation level was determined for each dinucleotide sequence, it was 94%, 9.8%, 1.3% and 0.4% at cpg, cpa, cpt and cpc sites, respectively (figure 1d). about half of cpa sites showed methylation levels of higher than 5%, whereas the vast majority of cpt and cpc sites showed no methylation (figure 1e). thus, cpa is the most preferred non - cpg sites. among the cpa sites, the cpapc (15%) and cpapg sites (12%) were more frequently methylated than the cpapa and cpapt sites (figure 1d). interestingly, despite that the 33 b1 loci that we analysed were from various genomic locations, some non - cpg sites at equivalent positions in the b1 sequences were more preferentially methylated (figure 1c). a subfamily of b1 sequences, designated as b1-x35s, has been shown to bind the ahr and slug transcription factors, and the binding was involved in the formation of a chromatin boundary (24,25). however, the preferentially methylated non - cpg sites did not overlap with these binding sites or the rna polymerase iii promoter (a - box and b - box) (figure 1c). to address whether the non - cpg methylation is specific to b1 sequences or not, we examined the methylation status of three paternally methylated dmrs (the h19, dlk1/meg3 and rasgrf1 dmrs) in p0 prospermatogonia. non - cpg methylation was detected in the dmrs with the levels comparable to those in b1 loci (supplementary figure s2a). we also examined seven cpg islands that are methylated in spermatozoa (26). whereas three of them were not methylated even at cpg sites (data not shown), the other four had methylation at non - cpg sites in p0 prospermatogonia (supplementary figure s2b). it is known that de novo cpg methylation of the dmrs and retrotransposons occurs in prospermatogonia between embryonic day 14.5 (e14.5) and p0 (27). the presence of non - cpg methylation in p0 prospermatogonia suggested their co - occurence with cpg methylation in fetal prospermatogonia. we selected eight b1 loci that showed high levels of non - cpg methylation in p0 prospermtogonia (b1_007, b1_014, b1_016, b1_018, b1_021, b1_042, b1_045 and b1_055) (supplementary figure s1) and examined their methylation status in prospermatogonia at embryonic day 16.5 (e16.5) (figure 2a and c). the average cpa methylation level at e16.5 was six times less than that at p0, indicating that these non - cpg sites become de novo methylated in prospermatogonia during late gestation. we previously showed that mutation in dnmt3a or dnmt3l affects cpg methylation at b1 loci (14). we therefore analysed p0 prospermatogonia from dnmt3l knockout mice (28) for the eight loci (figure 2b). the cpa methylation level was significantly reduced (> 5-fold, p cpt > cpc) in prospermatogonia is well correlated with the target preference of the recombinant dnmt3a protein (34,35). indeed, in human and mouse escs and mouse oocytes, a depletion of the de novo methylases resulted in greatly reduced non - cpg methylation (3,17,18,20, k. shirane. the numbers of sites with symmetric or asymmetric methylation are shown for cag (and ctg) and cg sites. the numbers of sites with symmetric or asymmetric methylation are shown for cag (and ctg) and cg sites. next, we studied the fate of non - cpg methylation after birth, during the transition from the prospermatogonia to spermatogonia. proliferating spermatogonia at p7 showed very low levels of methylation at all three non - cpg dinucleotides (0.20.4%) (figure 1d), which were comparable to the error rate of bisulfite conversion (0.20.5%). we then studied the time course of the loss of non - cpg methylation between p0 and p7. whereas the cpg methylation levels in the 33 b1 loci stayed around 95% at all time points, the non - cpg methylation levels declined gradually (figure 4a and supplementary figure s1). for example, the average cpa methylation levels were 9.8, 7.4, 1.5 and 0.4% at p0, p2, p5 and p7, respectively (figure 4b). these results suggest that the de novo methylation activity is not high in postnatal germ cells and that the lack of a copying mechanism for non - cpg methylation results in a gradual loss of this methylation after every dna replication. the dnmt3a and dnmt3l expression levels are high in prospermatogonia but become low after birth, and the dnmt3b expression level is relatively low in prospermatogonia (8,9). figure 4.loss of non - cpg methylation after the resumption of mitosis in postnatal development. (a) gradual loss of methylation at a representative b1 locus in the transition period from prospermatogonia to spermatogonia (p0, p2, p5 and p7). cpg sites are shown in circles and non - cpg sites are shown in squares, with open and filled (black or gray) symbols representing unmethylated and methylated cytosines, respectively. (b) methylation levels of the 33 b1 loci at cpg sites and cpa sites at p0, p2, p5 and p7. the boxes indicate 75 and 25 percentiles, with the thick line inside being the median. (a) gradual loss of methylation at a representative b1 locus in the transition period from prospermatogonia to spermatogonia (p0, p2, p5 and p7). cpg sites are shown in circles and non - cpg sites are shown in squares, with open and filled (black or gray) symbols representing unmethylated and methylated cytosines, respectively. (b) methylation levels of the 33 b1 loci at cpg sites and cpa sites at p0, p2, p5 and p7. the boxes indicate 75 and 25 percentiles, with the thick line inside being the median. the dashed line indicates the average. since a high level of non - cpg methylation was first detected in pluripotent cells such as escs and ipscs, a correlation of non - cpg methylation with pluripotency has been discussed (19). however, the detection of non - cpg methylation in oocytes, brain cells and prospermatogonia suggests that the above may not be the case. rather, the feature common to these cell types seems relatively high expression levels of the de novo methylase(s) (6,7,36,37). indeed, in human escs and ipscs, the level of non - cpg methylation is better correlated with the expression levels of the de novo methylases than those of pluripotency factors (20). in summary, we showed that non - cpg methylation accumulates within and around b1 sine sequences, as well as the paternally methylated dmrs and some cpg islands, in the mitotically arrested prospermatogonia during fetal development. the observed non - cpg methylation level was relatively high compared with other cells, but this does not necessarily mean that prospermatogonia have high levels of non - cpg methylation throughout the genome, as non - cpg methylation is relatively high in the alu sine sequences in human escs (20). our hairpin - bisulfite sequencing revealed that non - cpg sites including symmetrical cphpg sites are hemimethylated. therefore, it appears that no template - dependent copying mechanism exsists for non - cpg methylation in prospermatogonia. perhaps, the presence and the level of non - cpg methylation are determined by the balance between the de novo methylation activity and the rate of cell proliferation. in the case of b1 retrotransposons, the non - cpg methylation may not have a role in regulation of transcription or boundary function as shown above, but further studies are needed to know whether non - cpg methylation in other genomic regions has a function. supplementary data are available at nar online : supplementary table 1 and supplementary figures 1 and 2. grants - in - aid for scientific research from the ministry of education, culture, sports, science and technology of japan [22770009, 21200037 to k.i. and 20062010 to h.s. ] ; career center for women researchers hand - in - hand program at kyushu university (research assistants dispatching program to t.i.). funding for open access charge : grants - in - aid for scientific research from the ministry of education, culture, sports, science and technology of japan [20062010 to h.s. ]. | dna methylation is a well - characterized epigenetic modification involved in gene regulation and transposon silencing in mammals. it mainly occurs on cytosines at cpg sites but methylation at non - cpg sites is frequently observed in embryonic stem cells, induced pluriotent stem cells, oocytes and the brain. the biological significance of non - cpg methylation is unknown. here, we show that non - cpg methylation is also present in male germ cells, within and around b1 retrotransposon sequences interspersed in the mouse genome. it accumulates in mitotically arrested fetal prospermatogonia and reaches the highest level by birth in a dnmt3l - dependent manner. the preferential site of non - cpg methylation is cpa, especially cpapg and cpapc. although cpapg (and cptpg) sites contain cytosines at symmetrical positions, hairpin - bisulfite sequencing reveals that they are hemimethylated, suggesting the absence of a template - dependent copying mechanism. indeed, the level of non - cpg methylation decreases after the resumption of mitosis in the neonatal period, whereas that of cpg methylation does not. the cells eventually lose non - cpg methylation by the time they become spermatogonia. our results show that non - cpg methylation accumulates in non - replicating, arrested cells but is not maintained in mitotically dividing cells during male germ - cell development. |
anticipated pressures on fossil fuel resources in the coming decades mean that raw materials for the chemical industry must be found that can reduce our reliance on fossil - fuel - based feedstocks. hence, the development of new production processes for chemical syntheses from renewable resources is a challenge that should be a priority for all major economies. research into / application of carbon dioxide utilization (cdu) technologies has recently gained increased interest. cdu offers an attractive alternative to the well - established carbon dioxide storage (ccs) processes, however, scale of production means that cdu is unlikely to significantly reduce anthropogenic carbon dioxide emissions, but waste carbon dioxide under cdu protocols can be converted into useful chemicals or fuels. carbon dioxide is abundant, cheap and non - toxic when compared with other c1 building blocks, such as phosgene, and has been used in the manufacture of salicylic acid, urea and cyclic carbonates for 50100 years. however, because of the relative inertness of carbon dioxide these processes are significantly energy demanding with reactions taking place at high temperatures and pressures. therefore, if cdu technologies are to become viable processes reliance on very little energy input is required, hence processes at mild temperatures (99:1 selectivity for the monomer (table 1, entry 2). of the supporting electrolyte was required for good yield to be achieved (50 %, table 1, entry 7) and an increase in yield to 60 % could be achieved through a slight increase in the applied current (60 to 90 ma, table 1, entry 9). a series of experiments to look at the effect of temperature on the reaction were also carried out and finally we were able to obtain tmc 1 in 96 % yield, although the reaction was not quite 100 % selective for the monomer (table 1, entry 18). kleij and co - workers recently reported the synthesis of five - membered cyclic carbonates in excellent yield with either an iron- or aluminium - based catalyst at 102 atm co2. they also reported high yields and selectivity of oxetane to tmc 1 with the same systems but observed that addition of steric bulk to the oxetane substrate at the 3,3-position (two methyl groups, 4) dramatically decreased the yield and selectivity of the co2 incorporation reaction (typically 928 % yield, table 2 entries 1 and 2). darensbourg has also reported that 3,3-substituted oxetanes are slow substrates for co2 insertion at 35 atm but has not yet disclosed the yields when employing his vanadium - catalysed conditions (table 2, entry 3). when employing (salen)cr chloride complexes, darensbourg has reported a product distribution of 88 % in favour of copolymer 6, when exploring the equilibrium distribution of a range of oxetanes. a comparison of processes for 5,5-dimethyl-1,3-dioxan-2-one 5 synthesis[a ] conditions : [fe(tphoa)]2 (0.5 mol-%), bu4ni (5 mol-%), mek, co2 (102 atm), 85 c. conditions : al triphenolate catalyst (0.5 mol-%), bu4ni (2.5 mol-%), co2 (102 atm), 70 c. vo(acac)2 (5 mol-%), bunbr (5 mol-%), 60 c, co2 (35 atm). (salen)cr chloride complex, bu4nn3 (2 equiv.), co2 (35 atm), toluene, 110 c. conditions : cu cathode, mg anode, co2 (1 atm, balloon), bu4ni (1.0 equiv.), mecn, single compartment cell, 90 ma, 50 c. under our conditions the formation of 5,5-dimethyl-1,3-dioxan-2-one 5 proceeded relatively smoothly with an unoptimised 70 % yield being obtained at 50 c when employing tetrabutylammonium iodide and 60 ma (table 2, entry 4). a proposed mechanism is described in scheme 3 in which magnesium iodide is prepared in situ, following the reports of north and co - workers tributylamine is released under the reaction conditions[4 g ] and also activates co2 towards eventual incorporation to tmc, with magnesium iodide regeneration. we have developed, to the best of our knowledge, the first selective procedure for the formation of the tmc monomer 1 at atmospheric pressure carbon dioxide from oxetane. this process is one of only a handful of approaches that allow the conversion of oxetane to the cyclic carbonate rather than the thermodynamically more stable polymer 2. the equipment required to perform this co2 incorporation reaction is cheap and should be readily available in any undergraduate teaching facility, that is, copper wire, magnesium ribbon and a power supply. the protocol described affords the highest reported yield for the 3,3-dimethyl - substituted oxetane and we are currently exploring the co2 insertion into these challenging substituted oxetanes, for which there are currently very few commercially available. representative procedure for the formation of cyclic carbonates : trimethylcarbonate (tmc) (2) : tetrabutylammonium iodide (0.74 g, 2.0 mmol) was dissolved in acetonitrile (145 ml), the resulting solution was flushed with co2 for 1 h at room temperature and trimethylene oxide (0.12 g, 2.0 mmol) added as a solution in acetonitrile (5 ml). the reactor was heated to 75 c and potential was then applied to the system (constant current : 90 ma) for 6 h in a single compartment cell [mg anode and copper(0) cathode ]. on completion the reaction mixture was washed with aqueous hcl (0.1 m, 50 ml) followed by extraction with et2o (3 35 ml). the combined organic extracts were then dried with mgso4 and evaporated under reduced pressure to afford an amber oil, which was suspended in etoac (100 ml). after 1 h the precipitated bu4ni (70 %) was removed by filtration and the solvent evaporated under reduced pressure to afford an amber oil. this crude material was purified by column chromatography on silica gel eluting with ethyl acetate / light petroleum to give a colourless solid (0.196 g, 96 %). h nmr (400 mhz, cdcl3, me4si) : = 2.122.18 (m, 2 h), 4.454.48 (m, 4 h) ppm. c nmr (100 mhz, cdcl3, me4si) : = 21.8, 68.0, 148.5 ppm. ir (ch2cl2) : = 1252 (c o), 1740 (c = o) cm. as a service to our authors and readers, this journal provides supporting information supplied by the authors. such materials are peer reviewed and may be re - organized for online delivery, but are not copy - edited or typeset. technical support issues arising from supporting information (other than missing files) should be addressed to the authors | carbon dioxide utilisation (cdu) is currently gaining increased interest due to the abundance of co2 and its possible application as a c1 building block. we herein report the first example of atmospheric pressure carbon dioxide incorporation into oxetane to selectively form trimethylene carbonate (tmc), which is a significant challenge as tmc is thermodynamically less favoured than its corresponding co - polymer. |
cleft lip, cleft palate (clp), or both are the most frequently occurring congenital facial deformities, with an incidence rate of 0.65% in newborns, influenced by ethnic and geographic variations.[1 - 2 ] the nasopharyngeal region is impacted by complex interactions of bony compartments, muscular functionality, and soft tissues. this region also influences aesthetic facial harmony and provides the anatomical basis of speech and hearing. the size of pharyngeal space size is determined principally by relative growth and size of the soft tissues surrounding the dental and facial skeleton. comprehensive orthodontic diagnoses and treatment plans and orthognathic surgery treatment planning require an understanding of all functional variables, including the upper airway. volumetric imaging might be useful for planning surgical intervention in cleft patients to improve the patency of the upper airway.[6 - 8 ] clefts of the lip and palate frequently produce nasal deformities that tend to reduce the size of the nasal airway. approximately 70% of the cleft patients have nasal airway impairment and about 80% have mouth - breathing to some extent. craniofacial anomalies, including mandibular or maxillary retrognathism, short mandibular body, and backward and downward rotation of the mandible, may lead to reduction of the pharyngeal airway passage. the oral, nasal, and pharyngeal structures that are affected by cleft lip and palate during breathing are often compromised for speech. the nasal airway plays a significant role in controlling speech pressures when velopharyngeal function is impaired. the findings of the pilot study show that more variability exists in airway volume than in airway area. this observation strengthens the viewpoint of montgomery that reported single plane cephalometric radiographs do not reflect the airway volume in a reliable manner. cone beam computed tomography (cbct) is a new method which uses the reciprocal rotation of a two - dimensional receptor and a cone - shaped x - ray beam to gain volume data.the development of cbct and computer simulation in treatment planning provide the opportunity for a more precisely evaluating of cases compared with the conventional radiographs with less radiation, shorter acquisition scan times, easier imaging than a medical ct scan at a lower cost.[14 - 16 ] recent publications have demonstrated the ability of cbct to accurately image the airway to provide minimum cross section and total airway volume in obstructive sleep apnea patients and controls. previous studies have shown that 3d imaging using cbct is a simple and effective method to accurately analyze the airway.[18 - 20 ] currently, there are a few 3d studies to measure airway volume in individuals with clp and those studies used different methods.[21 - 23 ] cheung and oberoi suggested that there was no significant difference in pharyngeal airway volume and smallest cross sectional area between cleft and normal patients, while the airway length was significantly longer in clp children when compared to non - clp group. celikoglu demonstrated that patients affected by clp had decreased volumetric scores of nasopharyngeal, oropharyngeal, and total airways ; however, the oropharyngeal airway volume difference between the unilateral clp and the control groups was statistically significant. however, the study of xu. showed an enlarged nasopharynx in isolated clp in the sagittal plane and increased nasopharyngeal airway volume mainly around the palatal plane because of the increase in the sagittal diameter of the pharyngeal airway at this plane. the goal of this study was to find 3d differences of the pharyngeal airway using cbct in individuals with non - syndromic clp versus normal individuals. regarding the differences in the musculoskeletal and anthropometric proportions of individuals in different ethnics and concerning the current controversies over this subject, we planned to reconstruct another study to investigate this issue over a sample of iranian population. the research committee of the medical ethics group of shiraz medical science university approved this study (reference code ec - p-9365 - 6385). thirty cleft patients (19 men, 11 women ; 17 - 45 years) were selected from the department of orthodontics ; dental university (shiraz, iran) who had cbct scans of the head for other reasons (such as evaluating impacted teeth, missing teeth, or to assess the bone level). the control group included 30 individuals from the same center with class i angle occlusion who were matched for gender and age variables with the experimental group. the control group had already cbct scans from for orthodontic treatment purpose or assessment of impacted third molars or evaluating sinusitis. the exclusion criteria were diagnosis of a syndrome, craniofacial anomalies, previous orthognathic surgery or tonsillectomy or adenoidectomy, and detectable pathology along the upper airway through inspection of the images. all the cbct images have been made with similar unit and technique, by newtom vgi cone beam ct machine (qr srl company ; verona, italy) with the aid of a guide light. since the volume of the pharyngeal airway is influenced by head posture, all subjects were seated in the upright position and maximum intercuspation and the heads of patients were then centered and fixed in the cbct system. the midline laser beam of the cbct system was adjusted to the midsagittal plane of the skull. the horizontal laser beam was parallel to the frankfort plane (fhp) of the skull. the maxillofacial regions were scanned by using tube voltage of 110 kvp, tube current of 6.35 mas, and scan time of 18s. all images had a full field of view [15 12 ] that allowed visualization of the cranial base. this method was previously used in normal individuals by grauer. to assess the relationship of the pharyngeal airway volume with facial morphology and study to analyze the airway volume. to reconstruct the 3d models of the airways for the subjects, the anonymous cbct data in dicom format were transferred to itk - snap 2.4.0 (kitware, new york, usa) pc software for semiautomatic segmentation that is used for medical image processing. the images were oriented as a line 6 down from sellanasion as the horizontal axis. the interactive steps of the segmentation are selection of an initial thre - shold (which consist of the anterior border, a vertical plane through posterior nasal spine perpendicular to the sagittal plane ; posterior border, the posterior wall of the pharynx ; lateral border, the lateral walls of the pharynx ; lower border, a horizontal plane from the most caudal medial projection of the third cervical vertebra) (figure 1) and then placement of references points (figure 2). following the edge detection, the growth of the reference points in the single intensity value, fill up the airway structure (figure 3). it uses the contrast differences between the airway and surroundings structures on the grey scale images. after segmentation, the airway was subdivided into superior and inferior compartments by a plane from the posterior nasal spine to the lower border of the first cervical vertebra (figure 4). subsequent to the segmentation, a 3d graphical model of the volumetric object was generated by the software and the volume in mm3 of the segmented 3d model was obtained. landmark identification and segmentation were measured by the same investigator and measurement was repeated two times with one month interval by the same investigator to scrutinize intra - observer error. superior and inferior border of pharynx placement of references points growth of the reference points to fill up the airway structure segmentation to superior and inferior compartments the databases were collected. pearson s correlation coefficient was used to assess the relationship between the superior airway volume and inferior airway volume. thirty patients with unrepaired clp (19 men, 11 women) and 30 normal controls (20 male and 10 female) were enrolled in this study. the average volume of the pharyngeal airway of normal adult was 23.4 cm (sd, 8.7 cm), with mean volumes of 9.7 cm (sd, 2.3 cm) for the superior component and 13.7 cm (sd, 7.2 cm) for the inferior component. the average volume of the pharyngeal airway of cleft group was 18.6 cm (sd, 9.9 cm), with mean volumes of 6.8 cm (sd, 3.5 cm) for the superior component and 11.3 cm (sd, 5.8 cm) for the inferior component. the total and superior airway volume of cleft group were significantly lower than non - cleft groups (p= 0.008, p=0.00, respectively) but the inferior airway volumes were not significantly different between the cleft and non - cleft groups (p= 0.2). data of the measured and adjusted volumes are summarized in table 1and figure 5. the airway volumes (mm3) in cleft patients and normal individuals in 3d cbct and p - value according to mann - whitney test graph depicting airway volume (mm3) in clp (cleft palate) vs. non - clp sup pharynx vol = superior pharyngeal airway volume inf pharynx vol= inferior pharyngeal airway volume there was a significant and positive correlation between superior airway volume and inferior airway volume in clp patients (r=+0.786, p < 0.001) and control group (r=+0.575, p= 0.001). figure 6 shows the scatter diagram as well as regression line for the relationship between superior airway volume in clp patients (figure 6a) and control (figure 6b). in assessing the intra - observer errors, correlations between the first and second measurements ranged from 0.965 to 0.989 (p < 0.005). a : scatter diagram of the relationship between superior airway volume and inferior airway volume in clp (cleft palate) group, b : scatter diagram of the relationship between superior airway volume and inferior airway volume in control group regarding the differences in the musculoskeletal and anthropometric proportions of individuals in different ethnics and concerning the current controversies over this subject, our study investigated this issue over a sample of iranian population. in the study of grauer., using the same method as the current study, the measurement of the average volume of the pharyngeal airway with the same method was 20.3 cm in normal individuals. close to their results, in our study the mean airway volume was 23.4 cm for the normal group. the 3d study of cheung and oberoi demonstrated the mean airway volume of 18.1 cm for the cleft group similar to our result (18.6 cm), however, they reported a 15.1 cm for the non - cleft group which significantly differ from the results yielded by the current study (23.4 cm). moreover, they stated that there was no significant difference in pharyngeal airway volume between cleft and normal patients. the differences might be attributed to their different method or different referenced points or fewer sample size of the two compared studies. the results of diwakar.s study showed that the volume of the nasopharyngeal airway was significantly reduced in the clp group when compared with the non - cleft group which is in agreement with the results yielded by current study. that showed the patients affected by unilateral cleft lip and palate had decreased volumes of oropharyngeal and total airways compared with the well - matched control group without unilateral cleft lip and palate. the 2d study of imamura. showed that there was no significant difference between the adenoidal tissue of adults with clp and control groups. moreover, the upper airway of adults in the clp group was significantly smaller than that in the control group. they concluded that the size of adenoid tissue may not affect airway size of cleft patients. xu y. demonstrated that volumetric analysis of the total volume, volume above the palatal plane, and volume between the palatal plane and c2 plane were larger in patients with isolated clp, but there was no significant difference below the c2 plane. in their 3d imaging study, the most posterior points of the palatal process was selected to define the posterior nasal spine (pns), resulting in a pns that was anatomically more anterior to the pterygomaxillary palatinum which may influence the result. yoshihara. who used cbct for the evaluation of airways in 15 unilateral clp girl patients reported that the mandible and the oropharyngeal airway were larger in the adolescent controls than in the juvenile controls without cleft lip and palate, but there were no significant differences between the adolescent and juvenile patients with cleft lip and palate. the small size and retrognathic position of the mandible in the cleft group compared to the control group might be expected to narrow and reduce the volume of the pharyngeal airway. the evaluation of the nasopharyngeal airways of unilateral cleft lip and palate patients who was previously reported by aras. using 3d and 2d methods in which significant difference was detected between the cleft and non - cleft patients in nasal volume. this study also concluded that the upper and middle pharyngeal airway areas were found to display significant larger areas in the non - cleft group on cephalometric images. hence it seems that the 2d data can be insufficient and therefore might be deceiving. consequently, they showed that even though the radiation dose of 3d imaging systems was greater ; they have benefited from the advantage of superior diagnostic outputs. there are several methods to calculate the 3d airway volume which may affect the accuracy of the pharyngeal analysis. our segmentation method has been described, validated, and tested for accuracy, and according to study of weissheimer., itk - snap showed less than 2% errors in volumes compared with the gold standard. it is claimed to be more accurate than the conventional manual tracing method and other 3d analysis methods. shi. also demonstrated that automatic segmentation of the airway imaged using cbctwas feasible and could be employed to assess airway cross - section and volume similar to measurements yielded by manual segmentation. this selection was in accordance with the study enrolled by grauer. which reported that in skeletal class ii patients, inferior compartment airway volume was smaller than class i and class iii patients, but there were no significant differences in airway volume among the long, normal and short face - height groups. imamura. suggested that adenoidal tissues in the clp have similar size compared with normal adults so we didnot consider adenoid size as an affective factor on airway volume. reiser. found no correlations between the size of the initial cleft in infancy and size of the nasal airway in adulthood. so in adult patients who were born with uclp, the size of the cleft in infancy does not seem to affect the size and function of the nasal airway in adulthood. our analysis included only patients who underwent cbct for orthodontic planning, and no cbcts were performed solely for the purpose of studying airway volume. 3d imaging using cbct is a reliable diagnostic imaging tool to provide accurate data regarding airway size to allow clinicians to assess and screen the airway structures in clp patients. through this 3d analysis the measurement of the pharyngeal airway volume showed that the nasal and total airway is restricted in individuals with clp but the inferior airway is not compromised in these individuals. | statement of the problem : individuals with cleft lip and cleft palate mostly have airway problems. introduction of cone beam computed tomography (cbct) and imaging software has provided the opportunity for a more precisely evaluating 3d volume of the airway. purpose : the purpose of this study was to analyze and compare 3d the pharyngeal airway volumes of cleft palate patients with normal individuals using cbct. materials and method:30 complete cleft palate patients were selected from the department of orthodontics ; dental university (shiraz, iran) who had cbct scans of the head. the control group included 30 individuals with class i angle occlusion who were matched for age and gender with the experimental group. itk - snap 2.4.0 pc software was used to build 3d models of the airways for the subjects and measuring airway volumes. the statistical analyses were performed using spss software (version 19). mann - whitney test was adopted with p < 0.05 as statistical significance. results : the average volume of the pharyngeal airway of cleft group was 18.6 cm3, with mean volumes of 6.8 cm3 for the superior component and 11.3 cm3 for the inferior component. the total and superior airway volume of cleft group were significantly lower than non - cleft groups (p= 0.008, p= 0.00, respectively) but the inferior airway volumes were not significantly different between the cleft and non - cleft groups. there was a significant and positive correlation between superior airway volume and inferior airway volume in cleft palate patients (r=+0.786, p < 0.001) and control group (r=+0.575, p= 0.001). conclusion:3d analysis showed that the nasal and total airway was restricted in individuals with cleft palate but the inferior airway was not compromised in these individuals. this would be a crucial data to be considered for surgeons during surgical planning. |
the publication of the human genome sequence was a landmark in biological research (lander., 2001 ; venter., 2001 ; www.ornl.gov/sci/techresources/human_genome/home.shtml). it provides the basis for understanding the role of all genes in normal physiology and disease pathology, particularly when coupled to the sequencing of the genomes of cancer cells (stratton., 2009) as well as infectious organisms (berriman., 2009) and other species (www.ensembl.org/info/about/species.html). chemical tools are playing an important role, alongside molecular biology and genetic techniques, in functional annotation of the human genome (http://nihroadmap.nih.gov/molecularlibraries/index.asp ; austin., 2004) and in expanding the druggable genome (hopkins and groom, 2002 ; overington., 2006). an ambitious and inspiring early grand challenge that was set for the emerging fields of chemical genetics and chemical biology was to identify small molecule probes for the products of all human genes (schreiber, 1998). the discovery and exploitation of chemical probes has evolved from the more traditional ad hoc pharmacological approach where advantage was taken of individual natural products and drugs to explore cellular processes to the current higher throughput approaches that are now in widespread use in both academia and industry (austin., 2004 ; frearson and collie, 2009) and that facilitate interrogation of chemical and biological space in a more systematic way (schreiber, 2003 ; dobson, 2004). although terms such as molecular pharmacology, chemical genetics, and chemical biology can be useful to indicate nuances of approach, the overall unifying principle is the use of small molecules to understand the function of genes and proteins and their role in physiology and pathology. there are major synergies between more basic chemical biology research and drug discovery (anonymous, 2009). from a drug discovery perspective, chemical probes are key players in validating new molecular targets for therapeutic exploitation and in providing proof of concept for potential druggability of a molecular target, pathway, or process by small molecules. they can help to minimize the technical and biological risk for a biological target or pathway of interest. chemical tools can also serve as pathfinder molecules in drug discovery projects, informing the design and evaluation of biological assay cascades and the identification of useful biomarkers. importantly, chemical probes are highly complementary to the use of rna interference (rnai), in particular in being able to inhibit a specific function of the target protein rather than removing the whole protein, thus avoiding multiple function or scaffold effect issues ; in giving an immediate inhibition rather than a delayed knockdown ; and in providing greater control over the extent and kinetics of inhibition (weiss., 2007). both chemical probes and the use of rnai can have off - target as well as on - target effects. use of these approaches in parallel, as well as making mutated alleles of target proteins (bishop., 2000), can give us much greater confidence in functional annotation and target validation. over a long and distinguished history, the use of small molecule chemical tools has led to advances in biological understanding and therapy in such diverse areas as the cytoskeleton (colchicine, palitaxel), mitosis (monastrol), immunophilins and immunosuppression (fk506, cyclosporin), mtor (rapamycin), histone deacetylases (trapoxin, vorinostat), protein kinases (phorbol esters, staurosporine, tyrphostins, and many others), ppar (thiazolidinediones, 2-chloro-5-nitrobenzanilide gw9662), and very recently, stem cell reprogramming (emre., 2007). from the examples cited above and others, it seems that the use of chemical probes has been extremely effective and is having a growing impact as a result of powerful new technologies. so why do we need rules or guidelines introduced into what seems to be a productive process ? few would dispute that to be valuable in chemical biology and drug discovery research, chemical tools must satisfy at least some basic criteria, such as permeability (getting to the site of action in the cell), potency (inhibiting the target at reasonable concentrations) and selectivity (not being unacceptably promiscuous). if a biologist uses a lousy probe, then the interpretation of biological results will likely be flawed. recent publications have suggested objective guidelines for what makes a useful chemical probe for application in biological research (oprea., 2007 ; cohen, 2009 ; edwards., 2009 ; frye, 2010 ; kodadek, 2010). the emergence of guidance for probes is analogous to guidelines (sometimes referred to as rules, although they are more rules of thumb) that have proved to be of real practical value for assessing the suitability of fragment or high - throughput screening (hts) pharmaceutical leads for progression to drug candidates, and also for judging the candidates themselves (lipinski., 2001 ; rees., 2004 ; davis., the potential need for guidelines for probes has been stimulated recently by the increase in public screening efforts and in particular, the assessment of the output of the large national institute of health molecular library and imaging initiative (nih mli) (http://nihroadmap.nih.gov/molecularlibraries/index.asp ; austin. crowdsourcing review (oprea., 2009) of the burgeoning output of the pilot phase of the nih mli that ran from 2004 to 2008 at an estimated cost of us $ 385 million, a significant proportion (25%) of the 64 chemical probes generated were considered to have a high dubiosity rating, with low confidence in their value as probes, whereas 25% and 50% were viewed as inspiring medium- and high - confidence, respectively. a recent conference presentation (bologna, 2010) identified concerns with a high proportion of the nih mli hits and noted the very low citation rate for most nih mli probes proposed to date. criteria for the nomination of a chemical probe were made more stringent during the pilot phase. especially given the increasing involvement in the production and use of probes of scientists who may have less experience in this area than pharmaceutical industry professionals, the development of guidance for assessing potential probes does seem appropriate (see kaiser). on the other hand, nobody and not just the more anarchic or rebellious fringe wants a chemical biology thought police that dictates overly prescriptive rules that stifle innovation (hoffmann and bishop, 2010). rules are unlikely to work. moreover, as we will discuss later, probes evolve with time and need to be given a chance to be improved, especially in new research areas. a balance needs to be struck between allowing freedom for creativity and establishing sensible guidelines that eliminate at least the worst offenders among flawed probes and that encourage good practice in the community. against the above background, we discuss the recently emerging views concerning the desirable properties of chemical probes. after reviewing proposed guidelines and offering our own recommendations, we will illustrate these with several case histories of biological targets that have benefited particularly well from the use of progressively enhanced chemical probes. the selection of these cases is made from the cancer therapeutic area because, with outcomes from the human genome sequence now being reviewed 10 years on from the initial announcement, cancer is seen clearly to be the area that has benefited most in terms of the discovery and implementation of personalized, genome - based medicines (collins, 2010 ; golub, 2010). chemical probes have contributed considerably to the progress made with targeted cancer therapies and many of the drugs or analogs thereof have, in turn, served as small molecule tools for use in the lab. note, however, that although the illustrative examples are taken from oncology, the views expressed on chemical probes should be of generic relevance across basic, translational and drug discovery research. we advocate a fit - for - purpose approach to the properties of chemical probes, recommending fitness factors for probe evaluation. when combined with rigorous ongoing characterization and transparent reporting of the advantages and limitations of chemical probes together with open availability of reagents and data, a flexible, evidence - based strategy that is geared to the current understanding of the particular biological area should encourage innovative chemical biology research while minimizing the generation of poor quality and misleading biological data. progressive characterization and iterative refinement of chemical tools by the international scientific community can then follow in parallel with increased comprehension of the particular biological area, to the benefit of basic and translational research and drug discovery. in this section, we discuss recent proposals for the preferred properties of tool compounds and recommend what we term we build on previous guidelines that have been put forward for determining the use of chemical probes and the confidence in results derived from them (e.g., see cohen, 2009 ; frye, 2010 ; kodadek, 2010 and references in the legend to figure 1). cohen and colleagues have particularly focused on choosing high quality protein kinase inhibitors for interrogating targets in cells, where selectivity of the agents is paramount (cohen, 2009 ; davies. bain., 2007). recognizing the challenge of specificity given the more than 500 protein kinases in the human genome, in his recommendations, entitled guidelines, cohen (2009) describes essential and desirable criteria for kinase probes. frye (2010) was careful to draw a distinction between rules, which he argued were unrealistic in view of the numbers of parameters involved, in contrast to the more practical use of a small number of relatively simple principles or guidelines that could be of great value. these principles covered the biological and physicochemical properties needed in chemical probes that are intended for use in establishing the broader biological consequences of modulating a molecular target. there is an overlap with established practice to weed out promiscuous compounds identified from hts assays (baell and holloway, 2010 ; inglese., 2007), especially in avoiding unspecific chemical reactivity (rishton, 2003) and aggregation due to poor aqueous solubility (mcgovern., 2003 avoiding these undesirable properties will reduce the risk of a chemical probe exhibiting nonspecific effects. the importance of emphasizing these issues, which are well known in the pharmaceutical industry, is illustrated by the fact that significant problems with these properties were identified in the recent assessment of nih mli probes (oprea. specific guidelines for probes from the nih mli program are available (lazo., 2007 ; oprea. it is interesting that in the recent crowdsourcing analysis of nih mli probes there was a fascinating divergence of views on individual putative probes among the various expert panel members (see supplementary material in oprea., 2009). thus there will be an important element of judgment based on experience involved in the assessment of probes, even with guidelines available. the various existing guidelines mentioned above are interpreted and augmented with our own suggestions for fitness factors in figure 1. we define these fitness factors as the key properties of chemical probes that should be evaluated in relation to their intended use to give confidence that they are fit - for - purpose. the performance of a probe with respect to the separate fitness factors taken together provides an assessment of the suitability of a given probe and of potential uncertainties in interpreting biological data obtained with it. figure 1 highlights the four main categories into which the fitness factors are classified. the four main categories are : (1) chemical properties, (2) biological potency, (3) biological selectivity, and (4) context of use. we also raise questions of experimental use that are important to consider in assessing the quality of chemical probes. whereas the fitness factors are expressed for convenience as a checklist and commonly discussed threshold values are presented in table 1, we do not imply that a rigid adherence to fixed values is always appropriate. rather, they are criteria that may be used to facilitate judgments of the robustness of data generated with particular probes in particular settings, and are intended to help estimate the risks that may be carried forward in building on data generated from the use of new chemical probes where, of necessity, incomplete characterization is available. the need for well - characterized chemical identity and purity of chemical probes is clear cut. it is also important that reliable and reproducible procedures for the synthesis of the compounds are available. the chemical stability of the compounds in relevant media should be evaluated, particularly with regard to sensitivity to acid or base. covalently reactive functionality may be less problematic for chemical probes than drugs (kodadek, 2010), especially if linked to a specific target mechanism, but caution is needed because nonspecific chemical reactivity may promote stress - related phenotypes in the cell. thus a lack of chemical functional groups and physicochemical properties known to elicit oxidative stress, redox chemistry, membrane destabilization, or irreversible protein binding is very desirable (price. aqueous solubility and membrane permeability are of great importance in providing usable chemical matter for cell-, tissue-, or whole organism - based research, and assays for these properties are now readily accessible (kerns and di, 2008). for leads and drugs, adequate solubility and permeability are required to achieve intestinal absorption and oral bioavailability but they are also essential for probes of intracellular targets to be useful in vitro. the achievement of pharmaceutical lead - like pharmacokinetic properties may not be absolutely essential in a chemical probe, but there are considerable advantages in probes that can be progressed with confidence to an in vivo whole animal setting. similarly, the avoidance of adverse off - target toxicological effects is important in a tool for use in animal models ; physicochemical properties as well as toxicophore groups are important for this, with an increased likelihood of toxic events being observed for less polar, more lipophilic compounds across a broad range of chemical space (hughes., 2008). thus the stringency of the fitness factors for in vivo probes is higher than for biochemical or cellular probes. the value of defining the physicochemical behavior of compounds in drug discovery is well established. the widely used lipinski guidelines (known as the rule of five) are aimed to increase the likelihood that small molecules will have physicochemical properties compatible with the biological environment, especially for oral bioavailability (lipinski., 2001). on the other hand, such probabilistic guidelines have many exceptions and must be applied in context, and similar diligence should be applied when assessing chemical probes according to physicochemical parameters. evidence - based guidelines for the desirable properties of pharmaceutical lead - like molecules are summarized in table 1 and compared with those for typical drug - like molecules (collins and workman, 2006) and those emerging for chemical probes. the discrimination between lead - like (or fragment - like) and drug - like chemical space in terms of calculated molecular properties (size, polarity, conformational rigidity, efficiency of biological interaction) has been useful in drug discovery where a fragment or hts lead is anticipated to grow in size and complexity as it is engineered to a clinical candidate. a satisfactory probe may arise as a primary hts hit and not require addition of further functionality to enhance properties, although iterative improvement to refine the probes is increasingly expected (edwards. analyses of the footprint of existing probes in chemical property space has shown interesting concordance with that of high quality pharmaceutical leads (oprea. and there is recognition that an iterative process of refinement of initial hts hits is required to generate high quality chemical probes just as for the progression of a pharmaceutical lead to a drug. although the experimental performance of a molecule is the prime consideration, calculated property values may be useful in guiding the design of new screening libraries for both pharmaceutical leads and chemical probes to increase the probability of finding fit - for - purpose compounds. however, the necessarily retrospective nature of such analyses biases the definition of chemical space toward what is well known. because chemical probes are intended to be used at the leading edge of biological research, it will be important to revise definitions of appropriate chemical space to encompass as yet relatively unexplored classes of target interactions, e.g., protein protein interaction inhibitors (dobson, 2004 ; sperandio., 2010). a useful consensus has emerged in favor of 10100-fold over related targets ; table 1) may be more stringent than those for pharmaceutical leads because no further engineering of the former molecules is envisaged, and therapeutic polypharmacology is more likely to be acceptable (and sometimes may even be essential, as in multi - targeted protein kinase inhibitors) in a drug (kodadek, 2010). large scale in vitro selectivity profiling is often recommended, particularly for kinase inhibitors (where several tens or hundreds of kinases may be tested) but also for modulators of other protein superfamilies (fabian., 2005). we suggest that selectivity testing against at least 50 carefully chosen kinases is appropriate for assessing kinase inhibitor probes. although this profiling can yield valuable information, it is important not to generate a false sense of security. for example, biochemical kinase inhibition profiles require interpretation to predict the likely cellular consequences (smyth and collins, 2009) and inhibitors may interact with other target families that may not be looked at (bantscheff., 2007). nevertheless, at some point on their journey chemical tool compounds ideally need to be well characterized in terms of broad ligand pharmacology, including effects on g protein coupled receptors, nuclear receptors, ion channels, kinases, phosphatases, proteases, and ubiquitin ligases (entzeroth., 2000). such broad profiling can rule out promiscuous pharmacology or activity on particular anti - targets the cost of selectivity screening in focused or broader pharmacology panels may be restrictive, particularly for academic groups and small companies. useful pharmacological data on approved drugs can often be found in the disclosures that accompany regulatory approval (www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm). nonbiased global gene expression profiling can provide a valuable means of assessing on- and off - target effects in cells, with connectivity maps linking the profiles to targets (clarke. selectivity screening can be supplemented by using control compounds in biological experiments, especially inactive or weakly active analogs from the same chemical scaffold, sometimes conveniently referred to as a chemotype, together with active analogs of a different chemotype. examining concentration - dependency and structure - activity relationships (sar) from these analogs compensates for the deficiencies of any one compound and thus triangulates the target - dependent phenotype, especially when the sar for molecular biomarker changes that are associated with target modulation also correlate with cellular outcome (cohen, 2009 ; frye, 2010 ; kodadek, 2010). this can be seen as an application of the pharmacological audit trailused to build confidence during drug development at the biochemical and cellular level (workman, 2003 ; yap., 2010). these days, chemical probes frequently arise from large scale library screening that will often produce clusters of chemically related compounds of varying activity, so access to suitable analogs is not necessarily restrictive for academic groups (inglese., 2007). interrogating the burgeoning databases that annotate chemical structure with biological information, 2006 ; huryn and cosford, 2007 ; petri seiler., 2008 ; keiser., 2009 ; http://www.ebi.ac.uk/chembl). it will be important to apply the fitness factors under discussion here in evaluating historical data retrieved by such chemoinformatics methods, and to assess the reliability of the associations uncovered depending on the suitability and application of the probes concerned., we emphasize that the appropriateness of a probe 's use can not necessarily be extrapolated from one biological system to another. the intended use will govern the suitability of a probe as well as the compound 's intrinsic properties (table 1). what is desirable is a suite of chemical probes and techniques, appropriate to the target and scientific question under investigation, i.e., fit - for - purpose, that allows a consensus to be built about the role of a given target in a particular mechanism from multiple approaches. thus complimentary genetic methods, such as specific rnai or mechanism - based mutants of the target protein are recommended to reinforce the data from chemical probes while remaining aware of the differences between the techniques (weiss. an example of the dependence on context in oncology is the assessment of the activity of targeted probes or drugs in large panels of cancer cell lines with defined mutational, gene expression and other molecular characteristics (see http://www.sanger.ac.uk/genetics/cgp/cosmic/ for information on cancer gene mutations). markedly differential effects on cell proliferation or apoptosis are seen depending on a given compound 's selectivity profile combined with the cell 's genetic / molecular profile (sharma., 2010). indeed, there is considerable power in interrogating many different cell types with a range of well - validated probes. the chemical probes are used to help understand the biology of the cell lines and the effects in the cell lines help to credential the probes (http://www.sanger.ac.uk/genetics/cgp/translation). that repetition of biological profiling in multiple relevant biological systems and in different laboratories is a key part of chemical probe validation is to be emphasized. an important fitness factor for context is to consider the question : is this the best probe to use for this purpose ? there may be several probes available targeted to a particular pathway or molecular target, and the properties of certain compounds may be better matched to some biological systems than others and more capable of yielding a robust test of the biological hypothesis under investigation. for example, minimizing the risk of inhibition of particular confounding anti - targets in the biological system by avoiding a chemotype with those activities. in addition to the uses of biomarkers described earlier, considerable value can be added to the probing of probes by using biochemical and molecular biomarkers to characterize the target cells of interest and then to evaluate the effects of the probes for on - target and off - target pathways in those cells (e.g., banerji., 2005 ; guillard., 2009 ; raynaud., biomarkers provide an important step in the pharmacological audit trail referred to earlier, helping to establish that the chosen chemical probe is fit for purpose in a specific context. ideally, quantitative biomarker methods should be used so that the degree of target modulation can be determined and results interpreted accordingly. the interpretation of the effects of a probe on a cellular phenotype (e.g., proliferation, apoptosis, differentiation, migration, or other specific functions) is much more powerful if validated biomarkers are used to demonstrate that the probe is actually modulating the intended target and cognate biochemical pathway (e.g., solit., 2006). in addition, of course, the probe compounds can be used to validate the proposed biomarkers. the origin, identity, and properties of a compound proposed as a chemical probe should be disclosed fully, and it should be available in appropriate amounts for follow - up studies. the different availabilities of chemical probes may in practice be the dominant factors in determining which compound is used, and availability is therefore an important fitness factor. widespread availability of a probe and its associated data throughout the global scientific community is highly desirable. provided that structures and synthetic methods are fully disclosed in scientific publications and patents, it should generally be possible to make the required compound, or have it made. there is an increasing tendency for probe compounds and drugs to be sold by commercial suppliers. commercial supply can reduce the otherwise onerous demands on academic labs and companies to provide compounds from finite resources. some pharmaceutical companies have taken the initiative of making probes available via commercial vendors (e.g., http://investor.sigmaaldrich.com/releasedetail.cfm?releaseid=451709). there may, however, be a need for new thinking to further improve the availability of probes throughout the research community, taking into account legal considerations around research use and supply of proprietary compounds. for a given target, we strongly recommend the use of probes from more than one chemical scaffold and also inclusion of negative control analogs. despite their importance, companion inactive or other analogs are rarely available commercially. in the light of the selectivity fitness factors discussed above it could be desirable to consider chemical probes as reagent sets consisting of a pair of active and inactive compounds from the same chemotype, ideally together with an active compound from another chemical scaffold, in the same way that gene silencing studies using small interfering rna reagents is typically conducted with multiple effective rna oligomers together with appropriate negative control sequences. several previous publications rightly stress the need for transparency in reporting all relevant data on proposed new probes as well as the desirability of unrestricted or availability (oprea., 2007 ; edwards. transparency on reporting findings is important so that data from all probes, whether the compound is freely available or proprietary, can be assessed with confidence and equal rigor. much of this work will be carried out using conventional support in academia and industry. making probes widely available will mean that the international scientific community can put them through an enormous range of biological assays, thus building up a profile of information in an open access fashion that is similar to other fields such as software development and genome sequencing. maintaining quality and having databases and tools to access this information is increasingly important (e.g., http://pubchem.ncbi.nlm.nih.gov/ ; www.ebi.ac.uk/chembl/). the nih mli is one approach funded publically that seeks to build on success with large scale genome sequencing projects (austin, 2003 ; oprea., 2009). (2009) discuss united states funding mechanisms that might be used to support probing the probes. private partnerships have been initiated as a new funding model with the aim of developing open access chemical probes against pioneer targets such as chromatin modifying enzymes (edwards., 2009). we emphasize that the fitness factors in figure 1 and the threshold values in table 1 represent guidelines rather than proscriptive rules. particularly when a molecular target or biological area is new, useful information can still be gained with a chemical probe that is not yet ideal (see examples later), and indeed may have obvious limitations with respect to fitness factors. unfit ones will be discarded whereas fit - for - purpose tools will be taken up and used. improved probes will then emerge into the light and the chemical tools will evolve alongside the biological understanding. it is clearly important that when new probes do emerge they are compared with the current best in class and that the added value is clear (oprea., 2009). potentially important probes in a new biological area must not be damned too quickly because they have a few rough edges. on the other hand, considering the fitness factors can help decide when a probe is fit - for - purpose, should encourage good practice and should avoid the worst examples that continue to contaminate the literature. examples of the sort of experience that has been gained with probing probes and evolving tools will be found in the oncology case histories discussed in the following sections. as an illustration, figure 2 shows the limitations in fitness factors for selected early chemical probe inhibitors of the three classes of important targets that will be considered in detail, namely protein kinases, the pi3k family of lipid kinases, and the hsp90 molecular chaperone. in each case, the early probes were flawed but nevertheless proved useful, and the fitness factor profile was enhanced considerably during subsequent probe evolution, such that the liabilities were effectively removed. the close links between chemical biology and drug discovery are amply illustrated by the coevolution of protein kinase inhibitor tool compounds and pharmaceutical leads. selectivity has been a key fitness factor consideration in protein kinase research, as will be highlighted. the natural product staurosporine (tamaoki., 1986) (for the chemical structure of this and other selected protein kinase inhibitors, see figure 3) has been a widely used probe of signal transduction biology in cells but has significant liabilities in its fitness factors (figure 2). the compound and its analogs are now seen as unacceptably promiscuous (bain., 2007 ; frye, 2010). very high binding to human plasma proteins was seen in clinical trials of the staurosporine analog ucn-01 (fuse., 2005). hypotheses based on the activities of the indolocarbazoles have nevertheless been productively investigated, and over time compounds with better utility have been developed (nakano and omura, 2009). the distinctive effect of the compounds on tumor cell cycle progression and apoptosis was originally interpreted as inhibition of protein kinase c (pkc), but it became apparent that a much wider range of protein kinases, or nonkinase targets, were inhibited (akinaga., 2000 ; prudhomme, 2003). substantial medicinal chemistry efforts were made to tease apart the activities of the indolocarbazole early leads (roffey., 2009). for example, the bisindolylmaleimide ruboxistaurin (ly333351) was generated with somewhat improved selectivity for pkc (jirousek. more recently, sotrastaurin (aeb071) has been reported as a potent and highly selective inhibitor of pkc isoforms (wagner., 2009), as illustrated in the kinome profiles in figure 4. interestingly, the anilinopyrimidine scaffold that led ultimately to the bcl - abl inhibitor imatinib, the first approved kinase - targeting anticancer drug, was discovered initially in a project to find new pkc inhibitors (capdeville., 2002). it is also important to note that for structural biology studies involving wide comparisons across the kinase superfamily, the promiscuity of staurosporine has actually been exploited as a useful property (tanramluk., 2009). in addition, probably because of its promiscuous effects, staurosporine is frequently used as a control compound in studies of apoptosis. elucidation of the multifactorial effects of the staurosporine analog ucn-01 on tumor cells led to an understanding of the role of the cell cycle regulator chk1 (senderowicz, 2000). ucn-01 and more selective inhibitors of checkpoint kinases, such as isogranulatimide (jiang., 2004), abrogate the s and g2 checkpoints induced by dna - damaging agents and thus potentiate their cytotoxicity. this exemplifies the use of multiple compounds to build confidence in the association of an effect to a particular target even where specificities are not high (collins and garrett, 2005). selective inhibitors of chk1, or dual inhibitors of chk1 and chk2, have now been developed and clinical trials are ongoing with agents including azd7762, pfz00477736, and sch900776 (bucher and britten, 2008 ; dai and grant, 2010), whereas highly selective inhibitors of chk1 have been identified as chemical probes with good performance across the fitness factors, e.g., sar-020106 (walton., 2010). thus it is clear that with all the limitations of staurosporine, it has provided the inspiration for a new generation of robustly fit - for - purpose probes with excellent fitness factor profiles for in vitro and in vivo use, as well as drugs in the clinic. an early breakthrough in the development of chemical probes for kinases came with the rational design of the tyrphostin inhibitors of receptor tyrosine kinases (rtk) (gazit., these compounds have poor chemistry fitness factors due to the presence of functional groups widely considered as undesirable, in particular polyphenolic residues associated with nonspecific protein binding and redox metabolism, as well as chemically reactive benzylidenemalononitriles. some tyrphostins were later shown to uncouple the mitochondrial electron transport chain leading to atp depletion and generalized inhibition of intracellular kinase signaling (soltoff, 2004). nevertheless, the tyrphostins were important pathfinder compounds that provided impetus for research into kinase inhibition, showing the ability to achieve selectivity at the atp site, and leading to more refined chemical probes, as in the pioneering example of the epidermal growth factor (egfr) rtk enzyme (yaish. following on, the anilinoquinazolines were an early, more drug - like scaffold providing stable and selective rtk inhibitors with much improved fitness factor profiles (fry., 1994). this chemical class has since provided a plethora of excellent in vitro and in vivo probes and clinical candidates, including the approved drugs gefitinib, erlotinib, and lapatinib that show cellular and clinical activity in the context of genetic mutation or amplification of the egfr and erbb2 rtks (barker., 2001 ; pollack., 1999 ; xia., a number of chemical probes for protein kinases evolved from structures inspired by the purine cofactor atp (vesely., 1994). the development of purine - derived inhibitors is an early example of the successful generation of novel probes through the application of combinatorial library synthesis combined with structural biology (gray., 1998). several purines, including seliciclib (r - roscovitine ; meijer., 1997) that is now in clinical trial for cancer (whittaker., 2004 ; benson., 2007), are appropriate probes for pan - cyclin dependent kinase (cdk) inhibition (bain., 2007). the identification and refinement of other cdk inhibitor chemotypes has provided isoform - selective compounds, such as the pyridopyrimidinone cdk4/6-selective inhibitor pd 0332991 or the aminothiazole cdk2/7/9 inhibitor sns-032 (toogood., 2005 ; misra., importantly, the molecularly targeted drug discovery efforts again progress hand - in - hand with the evolution of better chemical probes. to find highly selective chemical probes for kinases, there may be advantages in specifically targeting distinct inactive conformations of the atp - binding site (liu and gray, 2006) or true allosteric modulatory sites, as demonstrated with selective akt inhibitors (cherrin., 2010). the atp noncompetitive inhibitor of mek1/2 (mkk1/2), u0126, was identified from a cell - based screen for inhibitors of ap-1 mediated transcription (duncia., 1998 ; the compound prevents activation of mek1/2, leading to high potency in cells for inhibition of the raf - mek - erk cascade. the 1,4-diamino-2,3-dicyanobutadiene moiety is an interconvertible mixture of steroisomers in solution, where activity resides uniquely in one isomer. although unstable structures may be generally undesirable in chemical probes, the chemical behavior was reported and understood at an early stage, and u0126 has proved a useful chemical tool (davies., 2000 ; hts for other chemotypes of mek ligand identified the chromenone pd 098059 as an alternative noncompetitive inhibitor with improved chemical fitness factors (dudley., 1995), and mek inhibitor development has led to enhanced, highly selective chemical probes and clinical candidates, including pd 184352, pd 0325901, and azd6244 (arry142886) (bain. structural biology studies with an analog of pd 184352 (pd 318088) showed the inhibitors to bind in an allosteric pocket adjacent to the atp - binding site of mek1/2, locking the enzymes in the unphosphorylated, inactive form (ohren., 2004). the inhibitor pd 0325901 has been used as a cellular mechanistic probe to reveal biology context - dependent, selective therapeutic effects in that tumor cells driven by mutant braf have a unique vulnerability to mek inhibition (solit., 2006)an effect also recapitulated recently both in cells and in the clinic with selective atp - competitive braf inhibitors, whereas enhanced tumor progression is seen in cancers with nras mutations (heidorn., 2010). (2007) recommend the use of pd 184352 or pd 0325901, with confirmation by the structurally unrelated u0126, as probes to inhibit mek in cells. an example of the development of protein kinase inhibitor chemical probes highlights the complementarity between chemical and genetic approaches. the pyrazolopyrimidines pp1 and pp2 were identified as inhibitors of src family kinases (hanke., 1996). structural studies showed the importance of a small gatekeeper residue and accessibility of the interior hydrophobic pocket of the kinase in determining the selectivity of the pyrazolopyrimidines (liu., 1999). exploiting this paradigm, mutant protein kinases where a large gatekeeper residue is replaced by alanine can be rendered sensitive to the pyrazolopyrimidines nm - pp1 and na - pp1 that bear large substituents not generally tolerated by wild - type kinases (bishop and shokat, 1999). transfection of cells with the drug - sensitized mutants thus allows highly selective, rapid, and reversible pharmacological inhibition of the target enzyme to be probed. however, nm - pp1 and na - pp1 do inhibit some wild - type kinases and this should be taken into account in interpreting results from gatekeeper mutant proteins (bain., 2007). in addition to inhibitor - sensitized mutants, drug - resistant mutant kinase alleles have also proved to be very useful (brown., 1995 ; are recommended for additional security as regards selectivity, alongside the use of two distinct chemotypes and inactive controls when interrogating kinase probe selectivity in cells (cohen, 2009). for example, the cellular expression of braf alleles with mutant gatekeeper residues rendering them resistant to inhibition by small molecules has clearly shown that the multi - rtk inhibitor sorafenib does not exert its cellular antiproliferative effects through inhibition of braf, in contrast to the more recently developed inhibitor plx4720 (whittaker., 2010). protein kinase inhibitors provide arguably the best examples of context - dependent effects, with agents (such as imatinib in bcr - abl positive or kit mutant cancer cells, or the egfr and erbb2 inhibitors in cells with mutated and amplified targets) exhibiting selective antiproliferative and apoptotic outcomes in cells that have become addicted through, for example kinase mutation, amplification or translocation (weinstein, 2002 ; collins and workman, 2006). phosphatidylinositide 3-kinases (pi3ks) are key components of signal transduction pathways controlling a wide range of biological phenotypes and are deregulated in several diseases including immune inflammation and cancer. these lipid kinases phosphorylate the 3-hydroxy position of the inositol ring of phosphoinositides (pi) generating pi second messengers downstream of rtks and g protein coupled receptors. in parallel with biochemical and genetic studies (cantley, 2002), our current understanding of the biological roles of pi3ks has benefited enormously from the use of chemical probes over the last 20 years (workman., starting from probes that are now known to have significant limitations but which have proved useful in thousands of studies, these have evolved into more sophisticated chemical tools with much more attractive fitness factor profiles and also into the first pi3k drugs entering the clinic for cancer treatment (workman., 2010). the fungal furanosteroid metabolite wortmannin (see figure 5 for the chemical structure of this and other selected pi3k inhibitors) was identified in 1987 as a potent inhibitor of the respiratory burst in neutrophils and monocytes (baggiolini., 1987) and subsequently found to inhibit pi3k by covalent binding to a specific atp site lysine (wymann., 1996). ly294002, a synthetic chromone related to the bioflavonoid and broad spectrum protein kinase inhibitor quercetin, was discovered in 1994 by targeted screening of analogs using a biochemical assay and was found to inhibit pi3k competitively at the atp site (vlahos., 1994). wortmannin is potent but unstable and was shown by profiling to inhibit smmlck, plk1, pi4k, and mtor (bain., 2007). like wortmannin, it is only a weak pi3k inhibitor with a ki of 1.6 m, and activity in cells at 1050 m, and is also active against torc1, ck2, plk1, pim1, pim3, hipk2, and gsk3, as well nonkinase off - targets, at concentrations similar to those that inhibit pi3k (davies., 2000 ; despite these limitations, wortmannin and ly294002 were used to generate invaluable understanding of physiological and pathological processes and to validate pi3k as a druggable target. over the last few years, combinations of screening, medicinal chemistry, and structure - based design approaches have generated an exciting series of enhanced probes and drug candidates with markedly improved properties in respect of all of the fitness factors (shuttleworth., 2009). a compound that is now recommended (bain., 2007) as a chemical tool for class i pi3k / mtor is the pyridofuropyrimidine pi-103 that has single digit nanomolar target potency, shows a high degree of selectivity against at least 70 kinases, and exhibits activity against animal models of cancer (hayakawa. optimization of pi-103 to reduce metabolic clearance and improve its pharmacokinetic properties resulted in the thienopyrimidine gdc-0941 that is now in phase i clinical trials for cancer (folkes. 2009). as with pi-103, detailed and quantitative molecular biomarker studies confirmed inhibition of the pi3k pathway in cells. gdc-0941 has the overall profile of a potent class i pi3k inhibitor (acting at low nanomolar concentrations on all the class 1a isoforms p110, p110, and p110 and the single class 1b isoform p110) with very high selectivity versus class ii and iii pi3k superfamily members. in contrast to pi-103, gdc-0941 also has low activity on the class iv pi3k superfamily protein kinases, including mtor. broader profiling showed that gdc-0941 is highly selective for pi3k with respect to a panel of 228 protein kinases. as would be required of a clinical candidate, gdc-0941 has negligible effects on cyp1a and cyp3a4 with no significant blockade of the herg channel by patch clamp assay. a number of pi3k inhibitors are now in the clinic (yap., 2008 ; shuttleworth., 2009), including the imidazoquinoline nvp - bez235 that evolved by target hopping from a pdk1 inhibitor lead (liu., i lipid kinases in the pi3k superfamily has been an important goal for probes and drugs. in a landmark chemical biology study, large numbers of pi3k inhibitors from journal articles and patents were synthesized and profiled against multiple enzymes (knight., 2006), revealing intriguing cryptic homologies across pi3k targets and chemotypes. clear selectivity trends were uncovered that were not predicted from the amino acid sequences. in terms of the use of these agents as chemical probes, the matrix of pi3k inhibitors was used to confirm the key role of p110 in insulin signaling, consistent with the mutant p110 mouse knock - in studies published around the same time (foukas., 2006)an example of chemical probe and genetic studies validating contemporaneously the same cutting edge biological result. of note, structural biology and modeling approaches have played a leading role in the design of pi3k inhibitors and in the interpretation of their potency and selectivity fitness factor properties (workman., 2010). (1999) for the apo and atp - bound forms of p110 and for early probes like wortmannin and ly294002 bound to this isoform (walker., 2000). the x - ray structure of gdc-0941 explained its improved potency (folkes., 2008). very recently, structures of inhibitors complexed with the p110 subunit have been determined, e.g., the quinazoline ic87114, which facilitated the design of new propeller - shaped compounds selective for the more conformationally flexible atp site of this isoform (berndt., 2010). agents selective for p110 and p110 have potential in diseases such as immune inflammation and cancer, as well as being powerful chemical probes, including the p110 clinical drug cal-101 derived from ic87114 (shuttleworth., 2009). the very recently solved crystal structure of the class iii pi3k vps34, which is involved in autophagy, membrane trafficking and cell signaling, shows why it is so difficult to inhibit (miller., 2010). a very weak vps34 inhibitor, 3-methyladenine, has been used at 10 mm as a new cocrystal structures of vps34 with inhibitors have pointed the way to novel compounds with greater potency and selectivity over class i pi3ks, highlighting the potential for probes with much better fitness factors for blocking vps34 and autophagy in cells. the testing in parallel of more than one probe chemotype, e.g., wortmannin plus ly294002 or pi-103 for class i lipid kinases, has been quite common in pi3k research. the application of companion inactive derivatives has been much less common, although early cellular work with ly294002 used analogs that were substituted in the essential morpholine ring to reduce hinge - binding (vlahos., 1994). in terms of cellular context, cancer cells with mutations in the pik3ca gene encoding p110, or loss of the counteracting phosphatase pten, may be more sensitive to pan - class i selective pi3k inhibitors, whereas mutations in kras seem to confer resistance (workman., 2010). as with protein kinases, the pi3k family is another excellent example of a target group for which the fitness factors of the probe compounds were initially less than we would now desire, but which were nevertheless sufficient to move the field forward markedly, leading through iterative improvements to the highly potent and selective probes that are available commercially and drugs that are now in the clinic. hsp90 is a molecular chaperone that helps to control the stabilization and degradation of its client proteins, as well regulating their activated states (workman., 2007). it has emerged as an exciting oncology target because inhibiting hsp90 causes depletion of multiple oncogenic clients, e.g., mutant kinases, leading to blockade of many key cancer - causing pathways and the antagonism of the hallmark pathological traits of malignancy. cancer selectivity is achieved by exploiting oncogene addiction as well as the stressed state of tumor cells (workman., 2007). hsp90 is an outstanding example of a contemporary pioneer drug target for which chemical probes played a leading role in the elucidation of the physiological and pathological functions of the protein, as well in establishing its druggability and reducing biological risk as perceived by industry. in particular the natural products geldanamycin and radicicol (figure 5) were found in landmark studies in the 1990s to bind hsp90, specifically at its unusually shaped ghkl class of atp - binding site, and thereby to inhibit the essential atpase - driven chaperone cycle, resulting in client protein degradation (whitesell., 1994 ; roe., 1999). these valuable early natural product hsp90 probes turned out to be quite potent and selective for hsp90 with respect to the isolated biochemical target and also in cells. however, they nevertheless have other significant fitness factor limitations, particularly the metabolically labile quinone in geladanamycin (figure 2) and the reactive epoxide group in radicicol, as well as other metabolism / stability issues, efflux pump and cytochrome p450 liabilities, and general toxicity problems. in parallel with the 17-allylamino analog of geldanamycin (17-aag, tanespimicin) being developed for clinical evaluation, efforts were initiated to identify synthetic small molecule inhibitors that lacked the obvious undesirable features of the pathfinder natural products (see figure 5 for the chemical structures of hsp90 inhibitors discussed in text). the purine pu3, designed by structure - based modeling, led the way ; although showing only 1520 m potency on hsp90, it depleted specific chaperone clients in cancer cells, confirming an on - target mechanism (chiosis., 2001). optimization produced more potent purines, such as the in vivo active pu24fcl (vilenchik., 2004) and the nanomolar potent, orally active clinical candidate biib021/cnf-2024 (kasibhatla., 2007 ; zhang., the pyrazole / isoxazole resorcinol class of synthetic small molecule inhibitors was identified by biochemical screening. the original hts hit cct018159 was atp - competitive with a kd of 0.5 m and, like pu3, caused client depletion in cancer cells ; an indication of selectivity was shown by the relative lack of activity on the related ghkl class protein topoisomerase ii and 20 protein kinases (cheung., 2005 ; x - ray crystallography confirmed a resorcinol - anchored binding mode identical to the natural product radicicol. structure - based multiparameter optimization yielded the clinical candidate nvp - auy922, now in phase ii trials (brough. this had a kd of 2 nm, showed mechanism - based inhibition of cancer cell proliferation at 9 nm and exhibited potent antitumor activity in animal models. a high level of selectivity for nvp - auy922 was shown for the hsp90 and isoforms, as compared to the closely related hsp90 family chaperones grp94 and trap-1, the ghkl superfamily member topoisomerase ii and the structurally distinct molecular chaperone hsp72, as well as 13 representative kinases, 14 additional enzymes, and 67 receptors (eccles., 2008). a range of additional hsp90 inhibitory chemotypes have emerged recently (biamonte., 2010), including clinical candidates like the pyrazolobenzamide snx-5422 (huang., 2009a) and the imidazopyridine cudc-305/debio 0932 (bao., 2009). the mutually enabling advances in basic, translational and drug discovery research on hsp90 have been facilitated especially by the combined use of chemical probes and mechanism - based molecular biomarkers. early work identified the depletion of clients and activation of heat shock proteins as a direct result of hsp90 inhibition and these were also used to show target modulation by 17-aag in cancer patients (banerji., 2005). further on - target versus off - target effects were revealed by comparing the unbiased global expression profiles for tumor cells treated with 17-aag, an inactive analog and radicicol as an alternative active chemotype (maloney., 2007). interestingly, the upregulated gene products observed in that profiling study included members of the hsp70 family, hsp72 and hsc70, which have chaperone and antiapoptotic properties. this has led to these proteins being validated recently as cancer targets using rnai together with several chemical probes of varying quality that collectively give some reassurance of potential druggability (powers., 2008 ; in the supplemental information available online, we highlight briefly a number of chemical probes (see figure s1) for a range of additional oncology targets of different structural and functional types. these are selected as being of current interest to both basic and drug discovery research. protein interaction targets ; chromatin - modifying enzymes, particularly histone deacetylases (see figure s2), phosphatases ; poly(adp)ribose polymerase (parp) ; and tankyrase. some of them provide excellent additional examples of context - dependent biological effects of chemical probes and drugs, exploiting oncogene addiction and synthetic lethality in certain cancer cells in the context of particular mutation profiles. the field of chemical biology has matured in approach but is still growing fast in scale and in a way that makes the emergence of guidelines for chemical probes almost inevitable. given that analogous guidelines have been useful with fragments, leads and drugs in pharmaceutical discovery research, it seems likely that comparable guidance for probes will also be very valuable. taking deliberately extreme positions on this, the upside is that the following of sensible guidelines will result in probes that do what it says they should do on the tin and hence will prove to be useful research reagents ; whereas the downside is that slavish adherence to a rule book could stifle innovation, particularly in the early stages of work on a new target or pathway for which probes are lacking. we recommend a common sense, fit - for - purpose approach and provide easy to use guidance on fitness factors for small molecule probes (figure 1). these fitness factors encompass four important areas chemical properties, biological potency, biological selectivity, and context of use that between them define the appropriateness of using a given chemical probe for exploratory biology. it is clearly not absolutely essential for all the requirements to be met at the level proposed by us and others. the case histories described here show that valuable progress can be made with initial probes that may well be suboptimal. at the same time, striving toward highly potent and selective cell permeable probes that are free of unnecessary chemical or biological baggage is highly desirable. proponents of new probes and those who use them should be aware that suboptimal probes carry with them significant risk of off - target biological effects that may be general or context - dependent. the field should support transparency and availability of chemical probes so that the profiling and evidence - based refinement of these can proceed in an open source fashion by scientists around the world. at the same time, it is recognized that there is often a strong overlap between probes, leads and drugs and that some of this work will have to operate within the constraints that inevitably accompany pharmaceutical research if patients with serious diseases are to benefit from important new drugs. the reality is that bad probes will bite the dust whereas better probes will evolve, thrive and prosper until they too are replaced by fitter and more powerful progeny in a true darwinian process. improving the speed and efficiency of probe evolution will bring great benefits to basic, translational and drug discovery research as we move forward into the second decade of exploiting the human genome sequence. | chemical probes for interrogating biological processes are of considerable current interest. cell permeable small molecule tools have a major role in facilitating the functional annotation of the human genome, understanding both physiological and pathological processes, and validating new molecular targets. to be valuable, chemical tools must satisfy necessary criteria and recent publications have suggested objective guidelines for what makes a useful chemical probe. although recognizing that such guidelines may be valuable, we caution against overly restrictive rules that may stifle innovation in favor of a fit - for - purpose approach. reviewing the literature and providing examples from the cancer field, we recommend a series of fitness factors to be considered when assessing chemical probes. we hope this will encourage innovative chemical biology research while minimizing the generation of poor quality and misleading biological data, thus increasing understanding of the particular biological area, to the benefit of basic research and drug discovery. |
decision - making strategies for optimal treatment of recurrent ovarian cancer (roc) are complex as new cytotoxic drugs and an increasing number of biological agents become available. this presents challenges in defining the optimal timing and sequencing of the drugs or treatment regimen. a controversial issue of roc treatment concerns the lack of an os advantage observed with a number of investigational compounds and regimens, and they can be often associated with increased toxicity and no improvements in patients ' qol. therefore, current treatment options for patients with roc are frequently guided by safety considerations and convenience. a sub - analysis of the ova-301 study showed that for platinum - sensitive patients (pfi > 6 months), median progression - free survival was 9.2 months with the trabectedin + pld combination vs 7.5 months with pld monotherapy (hazard ratio, 0.73 ; 95% ci, 0.56 to 0.95 ; p=0.0170). in fact, the median overall survival in the patient subgroup with a platinum - free interval in excess of 12 months (considered to have very ps disease) was 36.5 months with the combination, which was in the range of that obtained with platinum combinations. although therapeutic regimens that combine platinum - based therapy with other cytotoxic agents are the current standard of care for advanced ovarian cancer patients, the cumulative toxicities of cisplatin and carboplatin can present barriers for the long - term use of these agents [2, 3 ]. as previously discussed, there are women with relapsing disease (particularly the pps subgroup) who would benefit from a delay in platinum retreatment to possibly enhance their response to platinum in a future application, or who are not ideal candidates for platinum - based therapy due to the toxicity profile of platinum or due to platinum - induced toxicity. there appear to be three main limitations to using carboplatin combinations, including allergy to carboplatin (hypersensitivity), renal toxicity, and ototoxicity. hypersensitivity reactions (hsrs) to carboplatin are a particular concern, and have been reported in approximately 15 - 20% of women. symptoms of carboplatin hsrs are highly variable and can be mistakenly attributed to other agents, particularly when they are used in combination. they include itching, rash, chest tightness, emesis, blood pressure changes and facial swelling. the onset of the carboplatin - associated hsr is also highly variable, occurring either as soon as the infusion starts or after it is completed. when expectations for a positive outcome with platinum are good, then desensitisation protocols may be useful to continue platinum - based therapy, and success rates in excess of 50% have been reported. however, for example a 12-step desensitization protocol that administers the total dosage of carboplatin at increasing doses and faster rates over an extended period of time, requires adequate support in an intensive care unit or allergy department. all with the attendant burden on staffing and resources, and of course patient preference [1, 2 ]. other clinically significant sequelae such as neurotoxicity, severe cumulative myelosuppression, renal toxicity and ototoxicity are commonly caused by platinum - based chemotherapy. indeed neurotoxicity, which influences patient 's quality of life, is a dose - limiting adverse effect for all platinum compounds, and there is a high probability of persistent neurotoxicity including residual neuropathy (~20% of oc patients) associated with carboplatin plus paclitaxel treatment. carboplatin causes dose - limiting and cumulative myelosuppression, characterised by frequent and severe thrombocytopenia, granulocytopenia and anaemia. likewise, cisplatin is associated with several cumulative and irreversible toxicities, including dose - dependent renal tubule toxicity and neurotoxicity. cumulative doxorubicin and paclitaxel exposure must also be monitored to minimize the risk of patient morbidity due to cardiotoxicity and neuropathy. gemcitabine has many overlapping toxicities with other agents, and care must be taken with combination regimens to avoid synergy of these adverse effects. toxicity issues should be carefully taken into account before considering platinum re - treatment, as the platinum - associated cumulative and irreversible toxicities may jeopardise its long - term intervention on subsequent relapses. consideration of platinum - induced cumulative toxicity takes on greater significance as the number of salvage regimens increase, as it can be given only to those patients for whom the toxicities would be acceptable. this underscores the need for alternative therapeutic options, including an efficacious non - platinum regimen with an acceptable toxicity profile. other factors that influence treatment decisions in this heavily - treated patient population have to be taken into account. for example, efficacy (clinical or symptom benefit), safety (specifically limitations with carboplatin due to hypersensitivity or residual toxicities) and qol need to be considered. this could include health - related qol, patient - reported outcomes regarding symptoms, and time without symptoms or toxicity. for example, although temporary in nature, alopecia is a visible reminder of cancer treatment and is extremely upsetting for many women. a recent pan - european survey into the qol of 1743 patients with ovarian cancer by oskay - ozcelik. reported that alopecia was the single most troublesome side effect, with 42% patients reporting being bothered by alopecia. furthermore, there is now an increasing body of evidence which shows that extending the platinum - free interval in relapsing patients with partially platinum sensitive ovarian cancer, also improves their response rate to platinum re - treatment at a later time point. clinical evidence from ova-301 shows that the toxicity related to the trabectedin + pld combination was acceptable. the most common adverse events with the trabectedin + pld combination were neutropenia and transient increased transaminase levels (box 1 and table 1). the results show that neutropenia followed a predictable pattern of rapid onset and reversibility and, similarly, transaminase increases appear early after administration (during treatment cycles one and two), then generally decrease in incidence and severity with subsequent treatment cycles (fig. 1). hence the clinician can assess the impact of the combination therapy on the patient from the first 2 cycles. the impact of the trabectedin + pld combination was also assessed on patients ' qol via qlq - c30, a standardised qol instrument developed by the eortc to assess the quality of life of cancer patients. results from ova-301 showed no difference in patients ' qol between the 2 study arms (fig. during the course of their illness, patients with ovarian cancer may undergo multiple cycles of treatment, with alternating multiple episodes of remission and relapse. therefore roc patients are treated over a continuum in which therapeutic choices and strategies may impact the efficacy and safety of future therapies. platinum rechallenge in combination or monotherapy regimes are often limited by hypersensitivity reactions and cumulative long - term toxicities, regardless of the regimen. desensitisation protocols may be useful so as to allow treatment to continue, albeit with significant burden on staffing and resources and with varying outcomes. the safety data from ova-301 show that trabectedin + pld can be considered as a treatment option at any relapse of ovarian cancer, including fully platinum - sensitive patients unsuited to receive subsequent platinum, with a manageable safety profile and equivalent efficacy. therefore, trabectedin + pld is a non - platinum alternative that is well tolerated. furthermore, increasing evidence indicates that by extending the platinum - free interval with effective non - platinum agents such as trabectedin + pld, patients are given some extra time to recover from any of the adverse effects of their prior platinum - based therapy with no detriment in qol ; and with even potential induction of their response to any subsequent platinum application, hence preserving their future treatment options. professor fotopoulou received speaker fees from pharmamar for her participation at the bgcs 2014 symposium. | most patients with recurrent ovarian cancer (roc) undergo a series of remissions and recurrences, therefore the additive or cumulative toxicity of chemotherapy must be factored into their treatment plan. there are challenges in defining tailored therapeutic approaches, including optimal timing and drug sequencing management strategies to treat patients with roc. this is particularly relevant as new cytotoxic drugs and biological agents become available. many of these drugs are associated with increased toxicity and with no observable survival advantage. therefore current treatment options for the heavily - pretreated relapsing oc patient population are frequently guided by safety considerations and convenience. rechallenge with platinum - based combination regimes is commonly limited by the risk of cumulative long - term toxicities. not all patients can continue with platinum at second - line or, indeed, further relapses due to loss of activity or toxicity - related issues including hypersensitivity, neurotoxicity, alopecia and ototoxicity. in particular, hypersensitivity reactions are a concern and have been reported in approximately 15 - 20% of women receiving the drug. trabectedin + pld is a non - platinum combination that is well tolerated, with a manageable safety profile, which is independent of age. |
short anagen syndrome (sas) is a recently recognized congenital disease clinically characterized by persistently short fine hair since birth, due to a decreased duration of the anagen phase. the common complaint is that the hair does not grow long and that it has never been cut. only a few cases have been published in the literature, mainly in caucasian blond - haired girls. although some authors claim that short anagen is rare, others believe that it is just under - recognized. we here present a case of sas in a hispanic, dark - haired girl. a healthy hispanic 3-year - old girl presented with a history of short hair since birth. the mother reported that there was excessive shedding and poor hair growth, and that the girl had never needed a haircut. the patient was well- developed and did not present any abnormality except for short, dark, and thin hair [figures 1 and 2 ]. note the short length hair in a 3-year - old child telogen hair with tipped point dermoscopy of pulled hairs showed 4.7 cm long telogen hairs with tipped points, which indicates that the hair had never been cut. short anagen hair syndrome, as the name indicates, is a condition where the anagen phase is shortened and subsequently there is an increase in the number of telogen hairs. the disease is congenital and is thought to be sporadic, even though familial cases have been reported which may suggest an autosomal dominant inheritance. clinically, although the hair shaft is normal without signs of breakage the patients are not able to grow long hair and present with increased shedding. the condition is usually benign, nevertheless associations with tricho - dental syndrome, synchronized pattern of scalp hair growth, and micronychia have been reported. the characteristic clinical image and the finding of short (less than 6 cm long) telogen hairs with a tipped point at the pull test or trichogram makes the diagnosis. the main differential diagnosis has to be made with loose anagen syndrome, which also presents with short hair, but the hair is shed during anagen. treatment is not necessary as this condition tends to improve after puberty, however improvement with minoxidil and cyclosporine has been reported. for the exception of a case reported in an african - american patient, three others in caucasians with dark brown hair, and one in an asian patient most cases have occurred in caucasians with fine blond hair. to our knowledge, based on a search of pubmed, (lilacs) latin american and caribbean health sciences literature, and google scholar from 1999 to the present, using the terms short anagen and sas as key words, this is the first case published in a hispanic dark - haired girl in the english medical literature. | short anagen syndrome (sas) is a condition in which hair does not grow long. it usually perceived by parents in children around 2 - 4 years of age. it is a benign disease of the hair cycle. the condition is relevant from the standpoint of differential diagnosis with loose anagen syndrome. we report a case of sas in a hispanic 3-year - old girl. |
rheumatoid arthritis (ra) is one of the most prevailing inflammatory arthropathies that affects primarily able - to - work persons what attributes a high social significance to the disease. the development of ra is associated with the formation of a wide spectrum of autoantibodies, including rheumatoid factors (rfs) and anti - citrullinated protein antibodies (acpas) (antiperinuclear factor, antikeratin antibodies, antibodies to cyclic citrullinated peptide (anti - ccp), modified citrullinated vimentin, anti - mcv, citrullinated fibrinogen, etc.), the presence of which contributes substantially to the course and prognosis of disease [2, 3 ]. at present, acpa - positive and acpa - negative ra subtypes are identified, which differ in molecular mechanisms of pathogenesis, severity course and approaches to the treatment administered [46 ]. acpa - positive ra is characterized by accelerated radiologic progression, a severe course of the disease with higher total mortality [6, 7 ]. hyperproduction of proinflammatory cytokines (interleukins (il)-1, 6, 17, tumor necrosis factor tnf-) and growth factors in ra promotes the liberation of proteolytic enzymes (matrix metalloproteinases mmps) by synovial cells causing the destruction of connective tissue of the joint and stimulates an increased production of receptor activator of nuclear factor nf-b ligand (rankl) [9, 10 ]. mmps present a group composing over 20 proteolytic enzymes responsible for the cleavage of protein components of extracellular matrix. the family of mmps includes collagenases (mmp-1, 8, and 13), which induce the degradation of collagen i, ii, and iii type, and stromelysins (mmp-3, 10, and 11), ensuring the proteolysis of noncollagen proteins (fibronectin, elastin). at present, mmp-3 is considered as one of key mediators of articular destruction in ra [12, 13 ]. rankl presents a soluble rank ligand that occupies a central place in molecular regulation of bone tissue remodeling. when binding with a rank specific receptor located on osteoclasts precursors, rankl stimulates osteoclastogenesis and bone resorption [14, 15 ]. an important factor in the formation of osteoclasts and acceleration of bone resorption in ra is presented by anti - mcv. the studies in vitro and in vivo have demonstrated the ability of anti - mcv to specifically interact with cv (citrullinated vimentin) expressing upon the membrane of osteoclast precursors [1618 ]. the process induces the production of tnf- and mediated by this cytokine expression of macrophage colony stimulating factor (mcsf) and rankl, thus increasing osteoclast precursor sensitivity for differentiation into mature osteoclasts. one of the markers of cartilage tissue destruction is the cartilage oligomeric matrix protein (comp). comp is a noncollagen glycoprotein of the thrombospondin family that is present in extracellular matrix of articular cartilage, synovium, and tendons. the serum level of comp [20, 21 ] correlates with radiologic changes in the joints, the concentration of c - reactive protein (crp) and mmp-3 in blood and is a prognostic factor of severe destructive lesion to the joints in ra. the objective of the study was to evaluate the relationships between the level of autoantibodies (igm rf, anti - ccp, and anti - mcv) in blood serum, radiologic changes, and laboratory markers of bone and cartilage destruction in ra patients. a total of 114 patients, with definite diagnosis of ra, who were followed up at nasonova research institute of rheumatology, moscow, within the period from 2009 till 2012, were examined. candidate patients fulfilled the following criteria : (1) they were available for first posteroanterior radiographs of hands and anteroposterior radiographs of feet, (2) they had been treated with nonbiological dmards for more than 3 months before entry, and (3) they were not treated with biological dmards throughout the study. the control group included healthy donors matched for sex, age at the time of blood sampling, and area of residence. all participating sites received approval from their governing institutional review board (or equivalent) and all patients provided written informed consent. we determined the das28, the simplified disease activity index (sdai) score, and the clinical disease activity index (cdai) score, using the following equations : das28 = 0.56 (tjc28) + 0.28 (sjc28) + 0.70 ln(esr) + 0.014 gh ; sdai score = sjc28 + tjc28 + pga + ega + crp ; and cdai score = sjc28 + tjc28 + pga + ega, whereby the tender joint count (tjc) and the swollen joint count (sjc) were evaluated using 28 joints, global health (gh) was customarily replaced by the patient 's global assessment (pga) of disease activity (in millimeters on a visual analog scale [vas ]), scores for the pga and the evaluator 's global assessment (ega) were measured in centimeters on a 0100 mm vas, the erythrocyte sedimentation rate (esr) was measured as mm / hour, and the crp level was measured as mg / dl. for all 3 composite measures, cut points for the disease activity states of remission (rem), low disease activity (lda), moderate disease activity (mda), and high disease activity (hda) were determined. these established cut points are as follows : for the das28, rem 3 uln ; > 45.0 iu / ml), low - positive (13 uln ; 15.045.0 iu / ml), and negative (uln ; 15.0 iu / ml) levels of igm rf were identified. the quantitative estimation of anti - ccp was made by an electrochemiluminescent technique (uln 17.0 u / ml) and enzyme - linked immunosorbent assay (elisa) (uln 5.0 u / ml). high - positive (> 3 uln ; > 50.0 u / ml by an electrochemiluminescent technique, and > 15 u / ml by elisa), low - positive (13 uln ; 17.050.0 u / ml and 5.015.0 u / ml, resp.), and negative (uln ; 17.0 u / ml and 5 u / ml, resp.) levels of anti - ccp were identified. the concentration of anti - mcv in blood serum was measured using elisa kit. according to the manufacturer 's instructions, high - positive (> 3 uln ; > 60.0 u / ml), low - positive (13 uln ; 20.060.0 u / ml), and negative (uln ; 20.0 u / ml) levels of anti - mcv were identified. comp concentration in blood serum was measured in 34 ra patients by elisa (uln 5.1) and laboratory (esr > 35 mm / h, crp > 20 mg / l) disease activity, x - ray phase ii (43.9%). the patients received disease - modifying antirheumatic drugs ; the main of which were methotrexate and glucocorticoides (gc) (61.4%). among ra patients, das 28 : 5.9 (5.26.7), sdai : 34.4 (23.545.5), and cdai : 30.5 (20.941) corresponded to a high disease activity. an increased level of crp and esr values was recorded in 100 (87.7%) and 70 (61.4%) patients, respectively. a positive correlation of the level of igm rf with das 28 (r = 0.3 ; p = 0.02), sdai (r = 0.3 ; p = 0.02), and esr (r = 0.3 ; p = 0.003) and the concentration of anti - mcv with sdai (r = 0.25, p = 0.02) and cdai (r = 0.3, p = 0.02) were identified. no significant correlation relationships of the anti - ccp level with indicators of disease activity have been identified. the median of total modified sharp score (mtss), erosion score, and joint space narrowing score (jsn) amounted to 89 (57117), 9 (327), and 76 (5199), respectively. radiographic changes were analyzed in groups of patients depending on the level of igm rf, anti - ccp, and anti - mcv. no differences in radiologic signs of articular destruction in groups of ra patients high - positive and negative/ low positive for igm rf and anti - ccp have been identified (p > 0,05) (data not shown). among patients with high - positive level for anti - mcv, the higher values of mtss and jsn were observed as compared with these indicators in anti - mcv negative / low positive ra patients (p 0.05. alongside with that, a positive correlation between comp concentration and anti - ccp levels (r = 0.4, p = 0.02), anti - mcv (r = 0.5, p = 0.002), x - ray phase (r = 0.4, p = 0.02) have been identified. in the group of patients with radiologic stages i and ii, the level of comp was lower than that in patients with ra stages iii and iv, p 0.05) (data not shown). among high - positive anti - mcv patients (n = 79), the higher values of mtss and jsn and the increased levels of comp, esr, igm rf and anti - ccp were observed as compared with these indicators in anti - mcv negative / low - positive (n = 27) ra patients (p 0.05). among high - positive anti - mcv patients, higher levels of igm rf and anti - ccp were recorded significantly more frequently, what may provide an additional impact on the rate and intensity of destruction of the joints in this group of patients. the association between acpa status and radiographic progression is well established, [2226 ] but the mechanisms behind this association are not known. acpas have been shown to contribute to disease progression by amplifying inflammation and damage in an animal disease model. alternative explanations for a more aggressive disease course in patients with high levels are that high acpa levels could reflect either a more evolved immunological response or, as reported from a genome wide association study, a different genetic background. according to our data, anti - mcv affects bone and cartilage destruction in a higher degree as compared with anti - ccp. vimentin presents a natural citrullinated protein that is synthesized and modified in macrophages of a synovial sheath affected by proinflammatory cytokines. the evidence of pathogenetic relationships between anti - mcv and activity of osteoclasts in ra patients has also been obtained. harre. [16, 17 ] have demonstrated a decreased level of pad4 with a parallel increase in expression of pad2 and cv in the process of differentiation of osteoclast precursors into mature cells. the laser scanning microscopy technique has revealed a direct binding of serum anti - mcv with cv on the surface of osteoclast precursors inducing a dose - related stimulation of bone resorption and osteoclastogenesis. an adaptive transfer of human affine - purified anti - mcv induced in mice rag 1 an increased number of osteoclasts in metaphyses of bones, an increased serum concentration of tnf- and ctx1 with the unchanged level of osteocalcin in blood, and an increased number of cd11bcd14 osteoclast precursors of the spleen with hyperexpression of membrane receptors for mcsf and rankl. the level of comp in our ra patients did not differ from that in healthy donors. recently, serum comp was evaluated in patients with ra and was found to be preferentially elevated not in late - stage ra but in early - stage ra. we have found a positive correlation between comp concentration, anti - ccp levels (r = 0.4, p = 0.02), anti - mcv (r = 0.5, p = 0.002), and radiologic stage of disease (r = 0.4, p = 0.02) and higher levels of comp in patients with iii and iv x - ray phases. n = 98), comp values were statistically high in subjects positive for bone erosions on mri (12.0 u / l) compared with the subjects who were negative for bone erosions (10.16 u / l, p = 0.017). also several authors have found a positive correlation between comp level and delta larsen score [20, 21 ]. besides, a number of authors have demonstrated correlations between the level of anti - mcv and disease activity [33, 34 ]. also hyperproduction of anti - mcv is associated with higher clinic - laboratory activity of disease (das 28, esr, crp) and an increased rate of exacerbations per year. anti - ccp is less dependent on the clinical and laboratory disease activity [5, 36 ]. our study has revealed a positive correlation of the anti - mcv level with indicators of disease activity, sdai and cdai, and an increase in esr values in patients who are high - positive for anti - mcv. this is not a longitudinal study ; data and blood samples were only collected and analyzed at one time point. it may be assumed that the detection of anti - mcv could be a consequence of destructive events, rather than a cause. however, given the data of other authors (that have looked at anti - mcv as a predictive marker of erosions), this alternative hypothesis could likely be discarded. thus, the results of our study have suggested that there is relationship between the level of autoantibodies in blood serum and the development of destructive changes in the joints. anti - mcv affects bone and cartilage destruction in a higher degree as compared with anti - ccp and igm rf. patients who are anti - mcv positive are especially prone to rapid radiographic progression and need early aggressive treatment. | objective. to make individualised decisions regarding treatment is one of the most important challenges in clinical practise, and identification of sensitive and specific markers of prognosis is an important research question. the main objective of this study was to evaluate relationships between the level of autoantibodies, radiographic changes and laboratory markers of bone, and cartilage destruction. methods. a total of 114 ra patients were examined. the serum concentration of igm rf, antibodies to cyclic citrullinated peptide (anti - ccp), modified citrullinated vimentin (anti - mcv), matrix metalloproteinase 3 (mmp-3), and cartilage oligomeric matrix protein (comp, ng / ml) were measured. the van der heijde - modified sharp score was used to quantify the radiologic changes. results. among the patients who were high - positive for anti - mcv, the value of total modified sharp score (mtss) (96.5 ; 66120) was higher as well as the joint space narrowing (82 ; 60.5105.5), and a higher level of mmp-3 was recorded more frequently (56%) in comparison with negative / low - positive patients (57 ; 3188, 50 ; 2982, 31% resp., p < 0.05). the level of comp was also higher among patients high - positive for anti - mcv (9.7 ; 8.113.1 and 6.8 ; 5.410.7, resp., p = 0.02). conclusion. a high positive level of anti - mcv as contrasted with anti - ccp and igm rf is associated with more pronounced destructive changes in the joints. |
pregestational type 1 diabetes mellitus is clearly associated with an increased incidence of adverse maternal, fetal, and neonatal outcome [14 ], and several studies in the last two decades have shown that pregestational type 2 diabetes poses an emerging problem, with pregnancy outcomes at least as poor as in women with type 1 diabetes [59 ]. this gains even more importance in view of the global diabetes epidemic which leads to ever increasing numbers of women in the childbearing age with pregestational type 2 diabetes [5, 10 ]. to add to the problem, pregestational type 2 diabetes is encountered frequently in specific subpopulations in north - western europe, such as recently migrated women from africa, asia, and the middle east. these women are possibly more prone to suboptimal participation in the health care system because of frequently existing language barriers, generally less financial resources, and low education levels in those immigrant groups. the netherlands can be considered as a representative developed european country with ethnic minorities reflecting economic and social history. data from an epidemiological study from the netherlands revealed that perinatal mortality is increased in women with a nonnative dutch origin. therefore, the question rises if ethnicity plays a role in the development of pregnancy complications in women with type 2 diabetes, since the incidence of type 2 diabetes is more common in specific ethnicities. unfortunately, the possible relation between ethnicity and pregnancy complications in women with type 2 diabetes has received limited attention in western europe. data from two british studies showed no differences in pregnancy outcome between native british and one single other ethnic (i.e., afro - caribbean or indo - asian) women with type 2 diabetes [14, 15 ]. however, these studies were not nationwide and compared no mixture of ethnicities. therefore, to elucidate more comprehensively the potential impact of ethnic origin (and their possible accompanying problems) on pregnancy outcome, we performed a retrospective multicentre study to assess maternal, fetal, and neonatal outcome in a large group of native and a mixture of nonnative dutch women with pregestational type 2 diabetes from different hospitals spread throughout the netherlands. we hypothesized that nonnative dutch women with type 2 diabetes had a more unfavourable pregnancy outcome as compared to native dutch women with type 2 diabetes. a multicentre, retrospective study was performed involving seven large hospitals (university medical centre groningen, university medical centre utrecht, academic medical centre amsterdam, erasmus medical centre rotterdam, medical centre haaglanden the hague, atrium medical centre heerlen, and meander medical centre amersfoort) spread around the netherlands. all women with pregestational type 2 diabetes 272 women in whom a singleton pregnancy progressed beyond 20 weeks of gestation and who delivered between january 1997 and august 2009 were included in an anonymised database and subsequently evaluated. the diagnosis pregestational type 2 diabetes was accepted when patients were anti - gad antibody negative and/or had never experienced a keto - acidotic episode and the diabetes was being managed with or oral blood glucose - lowering agents and/or insulin. in the latter case, the diagnosis of type 2 diabetes was accepted when treatment with insulin was initiated > 6 months after the initial diagnosis. patients were divided into native and nonnative dutch groups, which was representative for the socioeconomic history of the netherlands. patients in the latter group were of north - african, hindu, afro - caribbean, asian, or other nonnative origin. we retrospectively reviewed all charts and recorded maternal characteristics (age, body mass index, ethnic origin, alcohol use, smoking habits, and parity), duration of diabetes, presence of chronic complications, and preconceptional treatment of diabetes. to assess the potential impact of ethnicity on pregnancy outcome, the origin of the pregnant women was classified as (1) native dutch and (2) nonnative dutch. the size of the different ethnic groups (i.e., north - african, hindu or afro - caribbean, etc.) was too small for adequate statistical analysis of these specific ethnicities. hba1c (mmol / mol) values were recorded and median hba1c was calculated in the periods one year before pregnancy and during the first, second, and third trimesters. preeclampsia was defined as a diastolic blood pressure 90 mmhg on two occasions at least four hours apart in the second half of pregnancy in previously normotensive women and de novo albuminuria (300 mg/24 h). in women with preexisting hypertension, pre - eclampsia hellp syndrome was defined as platelet count 100 10/l, elevated liver enzymes (serum alanine aminotransferase > 70 u / l and/or serum aspartate aminotransferase > 70 u / l), and haemolysis characterised by serum lactic dehydrogenase level > 600 only major congenital malformations, defined as abnormalities that were fatal, required major surgery or resulted in severe organ malfunction or cosmetic defects were recorded and classified as related to the cardiovascular, central nervous, urogenital system, or other systems. perinatal mortality was defined as fetal loss after 22 weeks of gestation or neonatal loss during the first 28 days after delivery. large for gestational age (lga) was defined as a birth weight above the 90th percentile corrected for gestational age, sex, and parity. continuous parameters were expressed as mean sd when normally distributed and as median (q1q3) in case of a skewed distribution. the appropriate (non)parametric tests (i.e., independent t - test when normally distributed and mann - whitney u - test in case of a skewed distribution) were used to compare differences between groups for continuous data and the chi - square or fisher 's exact test for categorical variables. a p value < 0.05 was considered as statistically significant. all statistical analyses were performed using pasw for windows version 18.0 (spss, inc., chicago, il, usa). a total of 287 singleton pregnancies in women with pregestational type 2 diabetes were referred to our centers. 15 pregnancies ended before 20 weeks of gestation, leaving 272 ongoing pregnancies and deliveries (11 in amersfoort, 28 in groningen, 72 in utrecht, 65 in amsterdam, 39 in rotterdam, 10 in heerlen, and 47 in the hague). 80 pregnancies (29.4%) were from women of native dutch origin and 192 pregnancies were from women of nonnative dutch origin (70.6%). the specific origin of nonnative women with pregestational type 2 diabetes was north african in 73 (26.8%), hindu in 59 (21.7%), afro - caribbean in 43 (15.8%), asian in ten (3.7%) and, other nonnative in seven (2.6%) women. the mean age at conception was 33.0 5.6 (range 1848 years) and comparable between native and nonnative dutch women (32.9 4.8 versus 33.1 5.9 ; p = 0.829). the median gestational age (ga) at first booking was significantly higher in the nonnative group compared to the native group (11.5 (8.019.0) versus 8.0 (6.010.3) weeks ; p < 0.001). there was a significant difference in the use of medication between the two groups : about half of the native women were on insulin, whereas about three - quarters of the nonnative women were on oral glucose - lowering drugs or diet only at presentation (p < 0.001). the glycemic control was less favourable in nonnative women as compared to native women before and during pregnancy (p values 0.04 and < 0.001, resp.), although in itself quite reasonable. pregnancy outcome of the total study population and between native and nonnative dutch women is shown in table 2. in the total study population, median ga at delivery was 38 weeks ; no data of delivery mode was available in one woman because she was referred to another hospital while in labour. delivery started spontaneously in 75 (27.7%) women, labour was induced in 131 (48.3%), women and a primary caesarean section (cs) was performed in 65 (24.0%) women. 49 of the 206 women (23.7%) who were planned to deliver vaginally had a secondary cs. the incidence of cs was significantly higher in native dutch women as compared to nonnative dutch women. this difference was caused by a higher incidence of primary cs in native dutch women with no difference in the incidence of secondary cs between the two groups. the other maternal outcome variables were not different between native and nonnative dutch women. 13 perinatal deaths (4.8%) occurred in the total study group, five fetal and eight neonatal deaths (table 3). four (31%) deaths were associated with congenital malformations ; four (31%) infants died before or at a ga of 25 weeks and the remaining five (38%) deaths were due to infection (n = 1), premature rupture of the membranes (n = 2), tension pneumothorax (n = 1), or remained unexplained (n = 1). major congenital malformations occurred in 17 (6.3%) infants of the total study population ; the incidence in native and nonnative dutch women was similar. major congenital malformations involved the cardiovascular system (n = 7), the central nervous system (n = 4), urogenital system anomalies (n = 2), or other structures (n = 4). three of these women were on oral glucose - lowering drugs during the first trimester. 85 (31.3%) infants were large for gestational age in the total study population, with no differences between native and nonnative dutch women. in contrast to our hypothesis, we showed no differences in pregnancy outcome between native and nonnative dutch women with pregestational type 2 diabetes, with the exception of a higher cs rate in native dutch women. however, a high incidence of adverse pregnancy outcomes in the complete group of women with pregestational type 2 diabetes living in the netherlands was found. pregnancy outcome of women with pregestational type 2 diabetes has been studied before and our results are generally in accordance with those reports [5, 79 ]. according to the results of a study on ethnic differences in perinatal mortality in the netherlands, we expected that outcome in nonnative women with type 2 diabetes would be poorer as compared to native dutch women, for example, linked to cultural or knowledge barriers in some ethnic minorities affecting access to and effectiveness of care. however, we did not find significant differences in pregnancy outcome (except for the incidence of cs). these data are in accordance with studies from new zealand and europe [14, 15, 20, 21 ]. in the previous european studies,. from new zealand, three main ethnicities (i.e., polynesian, asian, and european women) were compared. firstly, we included all ethnicities, although the limited group size forced us to treat them statistically as one group. secondly, none of these studies were truly nationwide, while the hospitals participating in our study were spread throughout the netherlands. from these studies and from our data, it can be concluded that in a setting of easy access to and compliance with the local health care system, outcome in nonnative women with pregestational type 2 diabetes can be similar to that in native women with pregestational type 2 diabetes. this easy access to care might be positively affected by the fact that medical care, for example, in the netherlands is fully reimbursed with insurance coverage for basically all inhabitants, resulting in an absence of any financial barriers to receive medical care. apparently, potential negative effects of slightly poorer glycemic control and later presentation are offset by the care system. the only parameter which differed between native and nonnative dutch women with pregestational type 2 diabetes was the higher incidence of a (primary) cs in the native group. this difference might partly be due to a higher incidence of maternal overweight, which according to us papers is related to the cs incidence [22, 23 ]. it may also be that women of native dutch origin more often opted and negotiated for a primary cs. the proportion native / nonnative dutch (i.e., 29.4% versus 70.6%) was not representative for the normal dutch population. firstly, this can be explained by a higher incidence of type 2 diabetes among specific non - secondly, the hospitals that cooperated to this study are mainly located in the urban areas of the netherlands, where the majority of nonnative dutch people are living. nevertheless, the pregnancy outcome of all studied women still seems worse than the outcome of the general population. according to our study possibly, other factors play a pathophysiological role in this process and offer opportunities to improve this adverse outcome. firstly, general awareness of the necessity of pregnancy planning in women with type 2 diabetes may be less than optimal in the netherlands, since these women are generally seen in long - term community care and only referred to secondary care when already pregnant. unfortunately, we were unable to report about the preconceptional care of the studied women, due to the retrospective nature of our study. therefore, a prospective study about the effects of preconceptional care and pregnancy planning on pregnancy outcome in women with type 2 diabetes is needed. secondly, postprandial spikes of the blood glucose levels contribute to a high - normal or a minimally elevated, but acceptable (4253 mmol / mol), hba1c level. nowadays, there are novel technical options to improve the glycemic control, like continuous glucose monitoring and insulin pumps [1, 27 ]. thirdly, several studies indicate that a low socioeconomic status (ses) is associated with adverse pregnancy outcomes [13, 28, 29 ]. since the incidence of type 2 diabetes is the highest in populations with a low ses, an etiological role of this risk factor may be expected in the development of complications during pregnancy among women with pregestational type 2 diabetes. unfortunately, ses was not sufficiently recorded in our retrospective study and ses should, therefore, be analyzed in future prospective studies. limitations of our study are the retrospective nature, the limited size of the total study population and of the different ethnic groups, and the possibility of selection bias. only 272 pregnancies were studied from seven large hospitals covering a representative area in the netherlands, but possibly not fully representative, since four centres are tertiary care centres and three are larger secondary care centres. this might have led to a selection bias, since patients with a poor obstetrical history are referred to tertiary centres. in conclusion, this study has demonstrated that ethnicity seems not to be a major issue in the cause of pregnancy complications in women with pregestational type 2 diabetes with easy access to medical care. more prospective research is needed to validate these results and to pay more attention to possible causative factors like preconceptional care, avoiding postprandial spikes of the blood glucose and socio - economic status, in the development of adverse pregnancy outcome in women with pregestational type 2 diabetes. | objective. to assess the incidence of adverse pregnancy outcome in native and nonnative dutch women with pregestational type 2 diabetes (t2d) in a multicenter study in the netherlands. methods. maternal characteristics and pregnancy outcome were retrospectively reviewed and the influence of ethnicity on outcome was evaluated using independent t - test, mann - whitney u - test, and chi - square test. results. 272 pregnant women (80 native and 192 non - native dutch) with pregestational t2d were included. overall outcome was unfavourable, with a perinatal mortality of 4.8%, major congenital malformations of 6.3%, preeclampsia of 11%, preterm birth of 19%, birth weight > 90th percentile of 32%, and a caesarean section rate of 42%. in nonnative dutch women, the glycemic control was slightly poorer and the gestational age at booking somewhat later as compared to native dutch women. however, there were no differences in incidence of preeclampsia / hellp, preterm birth, perinatal mortality, macrosomia, and congenital malformations between those two groups. conclusions. a high incidence of adverse pregnancy outcomes was found in women with pregestational t2d, although the outcome was comparable between native and non - native dutch women. this suggests that easy access to and adequate participation in the local health care systems contribute to these comparable outcomes, offsetting potential disadvantages in the non - native group. |
the vascular system is not only essential for the maintenance of tissue homeostasis, but also important for managing a wide variety of physiological processes. blood vessels are actively remodeled and reorganized depending on the tissue oxygen demand, suggesting that the maintenance of blood vessels is an active process achieved by a specific biological program. while the biology leading to new vessel formation has been extensively investigated in the last couple of decades, the stabilization of the newly formed vessel and the maintenance of the existing vasculature have not received significant attention. part of the reason for this under appreciation is the general assumption that blood vessel patency is maintained as a passive process relying on continuous blood flow. this idea has been widely accepted as clinical observations clearly indicate that hemodynamic changes leading to a decrease or cessation of blood flow results in vessel regression. there is little doubt that blood flow is critically important for determining the vessel fate ; however, the recent advance of vascular biology strongly argues for the autonomous fate control achieved by blood vessels. although this is primarily mediated by endothelial cells which appear to have intrinsic mechanisms to sense environmental changes and accordingly remodel blood vessels, recent studies indicate pivotal contribution made by vascular mural cells especially pericytes which can stabilize vessels in part through modulating the endothelial phenotype. the notion of active signaling required to maintain the vasculature is supported by several lines of evidence : (1) as angiogenic growth factors can promote new vessel formation, inhibition of those growth factors in tumor or tissue has been demonstrated to cause vessel regression with variable sensitivity [46 ], (2) disruption of endothelial junctions causes endothelial cell apoptosis leading to disintegration of blood vessels [7, 8 ], and (3) numerous mouse knockout studies describe vascular insufficiency in the later stage of development at which initial vascular development has been completed and blood circulation is established. these embryos show lethality at e9.5 or later due to vascular fragility leading to uncontrolled hemorrhage and edema. the list of the genes that are implicated in the maintenance of vessel integrity has been provided in the recent review, and the number of studies describing the similar phenotype is still growing. in these animals, while blood vessels form initially with normal vasculogenesis and angiogenesis, the maintenance of developing or once - formed vessels is impaired at the later stage of development. this observation implies that vessel formation and maintenance are differently regulated and equally important for producing sustainable tissue perfusion. furthermore, these studies collectively demonstrated that many cellular and noncellular components in the blood vessel coordinately regulate the maintenance of vessel integrity at varying degrees in different vascular beds. these include endothelial cells, pericytes, smooth muscle cells, fibroblasts, glia cells, inflammatory cells, and the extracellular matrix (ecm). the discoveries of angiogenic growth factors considerably advanced our understanding on biology underlying new vessel formation. this subsequently laid the foundation for new possibilities in the treatment of ischemic diseases over the conventional medical management and invasive procedures such as catheter - based angioplasty and coronary bypass surgery. the potential impact of therapeutic angiogenesis in clinical medicine is significant because it will enable us to achieve tissue perfusion by manipulating intrinsic blood vessel growth. however, over the past decades, many attempts to develop biological interventions for therapeutic angiogenesis have not produced significant clinical benefits despite the initial success in preclinical studies. double - blind randomized clinical trials aiming at biological revascularization in patients with ischemic heart diseases have failed to demonstrate therapeutic efficacy of any single growth factor delivery [1012 ]. these studies led to the conclusions that (1) administration of a single growth factor triggering only the initial phase of angiogenesis is not sufficient to drive the whole angiogenic process, (2) vascular maintenance is a crucial process to establish functionally meaningful perfusion, and (3) reevaluation of the detailed biological mechanism of new vessel formation is imperative for the successful clinical application. as currently understood, new vessel formation is triggered by endothelial cell activation and sprouting coordinated with controlled detachment of the surrounding mural cells, and proteolytic remodeling of the basement membrane and the extracellular matrix. this initial step is followed by assembly into a nascent vascular structure, lumen formation, and, finally, maturation of the newly formed endothelial tube and expression of endothelial cell survival factors. throughout the process, proangiogenic factors such as vascular endothelial growth factor (vegf) and fibroblast growth factor (fgf) differentially regulate defined subpopulations of endothelial cells in the angiogenic sprout, promoting endothelial migration at specialized tip cells and proliferation of the stalk cells. mural cells are then recruited by platelet - derived growth factor (pdgf) and transforming growth factor-1 (tgf-1) to stabilize the nascent blood vessels. in contrast with the initial phase of vessel sprouting, endothelial cells stop migration and proliferation in the maturation step and restore the barrier function of vessels. it is, therefore, reasonably assumed that to counteract the action of proangiogenic factors, vessel stabilization requires activation of distinct cellular signaling pathways different from those that initiate vascular cell mobilization. in fact, vegf - a, one of the most potent growth factors that initiates strong vascular growth, is known to increase vascular permeability, which, in turn, can compromise the barrier function when its action persists. by contrast, other angiogenic growth factors such as fibroblast growth factor 2 (fgf2) and angiopoietin 1 (angpt-1) do not cause vascular hyperpermeability. while both fgf2 and vegf - a are potent angiogenic stimulators, capillaries generated by fgf2 are tightly sealed and morphologically different from vegf - induced capillaries which have multiple fenestra and transendothelial channels. this strongly suggests that vegf and fgf induce expression of unique subsets of genes by activating distinct signaling programs in the angiogenic process. in view of this, it is considered that understanding of the specific role of each growth factor at specific stages of vascular development is important to successfully control the angiogenic process. thus, the approach to control vascular stabilization and maintenance needs to be also considered in the practical application of therapeutic angiogenesis in the future. endothelial intercellular junctions, critically important for the maintenance of vascular integrity, are comprised of distinct adhesion structures including adherens, tight, and gap junctions. unlike epithelial junctions which are vertically polarized, endothelial adherens and tight junctions, contributing to the structural maintenance of the endothelium, are intermingled and do not assume stratified localization. interestingly, in endothelial cells, although ve - cadherin can form a complex with polarity proteins such as par6 and par3, this complex lacks atypical - pkc which is required for polarity formation in epithelial cells. it is generally regarded that adherens junctions are primarily important for the control of endothelial permeability whereas tight junctions are dependent on the formation of adherent junctions and implicated in blocking the movement of lipids and integral membrane proteins between the apical and basolateral surfaces of the cell [16, 19 ]. in endothelial cells, ve - cadherin, a calcium - dependent adhesion protein mediating transhomophilic interactions, localizes at cell - cell contacts, regulating the formation of adherens junctions, and linking the site of the junction to the actin cytoskeleton. in the quiescent endothelium, ve - cadherin - based junctions are subjected to continuous reorganization, which renders endothelial junctions highly dynamic and sensitive to extracellular stimuli. in fact, during the process of new vessel formation, endothelial cells undergo dynamic rearrangement upon angiogenic stimuli while continuously reorganizing cell - cell junctions and maintaining barrier function at the same time. this coordination is largely regulated by adhesion molecules at intercellular junctions and is particularly important for tube stabilization and restoration of the full barrier function. thus, junction proteins play a critical role in controlling vascular integrity both in developing and existing vessels. among these, ve - cadherin is crucial for this regulation for its capability to remodel actin cytoskeleton via modifying the function of small gtpases. disruption of ve - cadherin function in the developing vasculature or the adult vasculature results in severe outcomes manifested by significant defects in the vasculature due to vessel collapse, regression, and endothelial apoptosis, leading to extensive hemorrhages [7, 8 ]. mouse embryos lacking cdh5, encoding ve - cadherin, die at e9.5 due to defects in vessel remodeling. pericytes are mesenchymal - derived cells which are positioned around microvessels and cover gaps between endothelial cells [3, 21 ]. although pericytes and endothelial cells are embedded within the same basement membrane, they make special interfaces at peg and socket contacts where the basement membrane is absent and gap and adherens junction constituents are deposited. besides forming physical contacts, the peg - socket contact is thought to facilitate communication between these two cell types, in which pericytes and endothelial cells respond to signals generated by the counterpart. as has been described extensively in numerous mouse genetic models, failure of pericyte - endothelial cell communication results in severe and often lethal vascular defects at the later stage of embryogenesis exemplified by impaired vascular formation, stabilization, and remodeling. while in these mice, vascular development is initiated by vasculogenesis and angiogenesis, subsequent vessel maturation and stabilization are defective with reduced pericyte coverage. pericyte - endothelial cell interaction is mediated by multiple growth factor systems including pdgf, tgf-, sphingosine-1-phosphate, tie-2, and notch, which are required for pericyte differentiation, recruitment, and expansion. on the other hand, alterations in pericyte density or the stable attachment of pericytes to endothelial cells are associated with human pathological conditions such as diabetic microangiopathy, venous malformation, and hereditary stroke and dementia syndrome cadasil. moreover, pericytes of tumor vessels present multiple abnormalities, including an abnormal shape and altered gene expression of more immature and less - contractile markers. these pericytes show loose association with the endothelium and extend cellular processes into the stroma. aberrations in pericyte - endothelial cell communication are attributed to the aggressive nature of tumor angiogenesis and the high incidence of metastasis. signaling systems and pathological conditions related to pericyte functions are discussed in the following sections. as a major structural component of resistant vessels, the ecm plays a substantial role in the maintenance of vessel integrity. the ecm of the blood vessel wall exists mainly in three forms : the interstitial matrix which fills intercellular spaces of the media and the adventitia, elastic laminae of artery, and the basement membrane which is a sheet - like structure that localizes underneath the endothelium and supports both endothelial cells and pericytes [25, 26 ]. reduction of the ecm component in the vascular wall predisposes to compromised vascular structural integrity. for example, patients with osteogenesis imperfecta and ehlers - danlos syndrome caused by genetic disorders of major fibrillar collagens such as type i and type iii collagen, respectively, often present aortic aneurysm and its rupture in early adult life. aortic aneurysm also occurs as a result of atherosclerotic changes in which normal architecture of aorta is progressively destructed by increased activity of matrix degrading enzymes, potentially leading to devastating rupture as the disease advances. besides the degenerated vessel wall, aortic aneurysm features chronic inflammation and a loss of ecm such as medial elastin. increased matrix metalloproteinase (mmp) production / activity in the vascular wall is thought to be responsible for ecm degradation of this disease. specifically mmp-2 and mmp-9 have been implicated in the pathogenesis of aneurysm due to their capability to degrade elastin and increased expression levels in the aneurysm tissue and the plasma of patients. moreover, the imbalance of mmps and tissue inhibitors of mmps (timps) is believed to promote disease progression. in the mouse genetic model, whereas either mmp-2 or mmp-9 deficiency is resistant to aneurysm formation [30, 31 ], disruption of timp-1, an mmp-9 inhibitor, leads to exaggerated aneurysm growth. these results confirmed the contribution of mmps to aneurysm formation and progression through ecm degradation and subsequent impairment of structural integrity of large vessels. as ecm degradation by mmps activates quiescent endothelial cells and thus triggers angiogenesis, constitutive endothelial - matrix interaction is thought to maintain vascular homeostasis and thereby prevent angiogenic vascular growth. therefore, endothelial attachment to the basement membrane is important for the establishment of stable and mature blood vessels. besides the basement membrane, the adventitial matrix mainly containing fibrillar collagens and fibronectin produced by fibroblasts also contributes to the maintenance of vascular integrity not only as structural scaffold, but also as a mediator of vascular remodeling in response to flow changes or vascular injury. recent study revealed the dynamic nature of adventitial fibroblasts which are capable of modulating vascular functions through a paracrine mechanism. fibroblasts secrete cytokines, growth factors, and reactive oxygen species (ros), actively controlling endothelial cell interaction with leukocytes and thus the inflammatory process of the vascular wall. the importance of the ecm in embryonic vascular development has also been shown by an observation of mice lacking histone deacetylase 7 (hdac7). hdac7, exclusively expressed in the vascular endothelium in the early embryonic stage, maintains vascular integrity by repressing mmp-10 expression. null mice show embryonic lethality after e11.0 due to a failure of endothelial cell adhesion, leading to vessel dilatation and rupture. although physiological levels of ros, serving as a signaling mediator in the vascular system, are important for the maintenance of vascular homeostasis and vascular integrity, oxidative stress induced by elevated levels of ros may contribute to the initiation and development of vascular dysfunction associated with hypertension and diabetes. the mechanism of action by which ros cause vascular dysfunction includes protein oxidation by oxidants such as peroxynitrate anion (oono) and h2o2. these ros can increase tyrosine nitration (ny), cysteine (cys), and zinc thiolate (zns4) oxidation, affecting important cardiovascular proteins such as enos, prostacycline synthase, mnsod, and sarcoendoplasmic reticulum calcium atpase (serca). protein oxidation may also lead to junction instability by targeting important components of the junction structure. increased vascular permeability is usually reversible and does not deteriorate vascular integrity because endothelial cells can quickly restore the barrier function by reestablishing ve - cadherin - based junctions ; however, prolonged permeability - inducing stimuli may cause a more profound effect such as accumulation of ros. ros can irreversibly inactivate protein tyrosine phosphatases (ptps) by oxidizing a cys residue in the catalytic center, thereby affecting the function of ptps. given the importance of ve - ptp and other tyrosine phosphatases in the stabilization of the ve - cadherin complex, accumulation of ros in endothelial cells may impair endothelial barrier function by hyperphosphorylation of ve - cadherin, thus deteriorating vascular homeostasis [39, 40 ]. the vegf gene family consisting of vegf - a, b, c, d, and placenta growth factor (plgf) in humans is essential for a wide variety of vascular functions. multiple isoforms of vegf generated by alternative splicing differ in their ability to bind heparan sulfate, which determines their bioavailability, and may play distinct roles in vascular development. vegf - a is a potent angiogenic factor originally identified as a factor capable of increasing vascular permeability through disruption of endothelial junctions. endothelial junction stability is primarily achieved by ve - cadherin homophilic interactions which form intercellular adherens junctions. vegf - a induces phosphorylation of y658 and y731 at the ve - cadherin cytoplasmic domain via src activation, disrupting binding of p120-catenin (p120) and -catenin, respectively. dissociation of catenins especially p120 increases endothelial permeability by dismantling ve - cadherin - based junctions, which is sufficient to maintain endothelial cells in a mesenchymal - like state with a promigratory phenotype [44, 45 ]. while vegf controls ve - cadherin stability by inducing catenin uncoupling and ve - cadherin internalization, signaling from cell - cell junctions can also regulate growth factor signaling leading to actin reorganization in fact, ve - cadherin modulates vegf - induced erk1/2 and akt activation and shear stress response [46, 47 ]. although vegf - a disrupts endothelial junctions, which can potentially compromise vascular integrity when the action is prolonged, vegf signaling is indispensable for physiological endothelial functions and vascular homeostasis. apart from the angiogenic property, vegf is widely recognized as a vascular protective factor with its ability to increase nitric oxide (no) and prostacyclin (pgi2) production in endothelial cells [48, 49 ]. of these, no has been implicated in mediating the effects of vegf on vasodilatation and the maintenance of antiapoptotic signaling through vegfr2-induced pi3k activation leading to akt and enos phosphorylation. while endothelium - derived no exerts antioxidant and anti - inflammatory properties, no produced by both endothelial cells and platelets no has pleiotropic antiatherogenic actions by preventing endothelial dysfunction and smooth muscle cell proliferation which are both required for the initiation and progression of atherosclerosis. endothelial cell activation, critical to triggering an inflammatory response, induces the fusion of weibel - palade bodies containing von willebrand factor (vwf), tissue plasminogen activator (tpa), p - selectin, il-8, and endothelin with the plasma membrane. this process transfers these molecules to the cell surface where they promote the recruitment of leukocytes and platelets. no acts in an anti - inflammatory manner by reducing leukocyte adhesion on the endothelium through inhibiting exocytosis of weibel - palade bodies. this process is regulated by the activity of n - ethylmaleimide - sensitive factor (nsf), which is a major component of the exocytotic trafficking machinery. no appears to inhibit weibel - palade body exocytosis from endothelial cells through inhibiting nsf disassembly activity by nitrosylating critical cysteine residues of nsf. moreover, a recent study demonstrated that autocrine vegf signaling is required for endothelial cell survival in a cell - autonomous manner under nonpathological conditions. endothelial - specific deletion of the vegf - a gene in mice leads to progressive endothelial degeneration and sudden death by 25 weeks of age due to vascular insufficiency, suggesting that endogenous vegf produced by endothelial cells is crucial for vascular homeostasis. precise regulation of differential vegf signaling that can promote both endothelial survival and junction disruption is not well understood at this point ; however, accumulating evidence points that vegf coreceptor systems are capable of modifying vegf signaling outcomes in a context - dependent manner. several studies described an essential role of ve - cadherin in vegf - induced akt activation required for endothelial cell survival [7, 54 ]. ve - cadherin, sensing cellular spatial information depending on cell density, modulates vegf signaling by controlling vegfr2 endocytosis that is required for erk1/2 activation [46, 55 ]. thus, endothelial cells are able to differently respond to vegf stimuli in order to accommodate the environment and tissue demands. the family of roundabout (robo) transmembrane receptors is the canonical receptor for the signaling molecule slit, regulating commissural axon guidance. robo4 is an endothelial cell - specific member of the robo receptor family, which is another signaling system that has the capacity to modulate vegf signaling. robo4 inhibits vegf - induced vascular permeability and angiogenesis by binding and signaling through unc5b. thus, robo4-unc5b signaling in endothelial cells plays a role in the maintenance of vascular integrity by antagonizing the action of vegf. observations of tumor vasculature provided insightful information with regard to mechanisms of vessel fragility and leakiness, which is potentially applicable to anticancer therapy. vegf is recognized as a key factor required for the growth of tumors with its inherent overproduction in many human malignancies. the tumor microenvironment is characterized by hypoxia, low ph, and high interstitial fluid pressure, all of which underlie exaggerated production of proangiogenic factors such as vegf through hif1 activation in the tumor milieu. unregulated vegf expression causes vascular hyperpermeability that leads to leakage of plasma proteins into the stroma, further stimulating persistent angiogenesis and creating a self - reinforcing vicious cycle. as a result, the abnormal tumor vasculature showing chaotic networks composed of heterogeneous immature vessels is formed. structurally, in these tumor vessels, endothelial cells have wide junctions and multiple fenestra with loose association with pericytes and the basement membrane, resulting in hemorrhage and edema which limit efficient perfusion. therefore, normalization of tumor vessels using anti - vegf strategies has emerged as a new approach to cancer therapy [58, 61 ]. vegf inhibition prunes the immature vasculature including excess branches and enhances pericyte coverage, stabilizing vessels and decreasing tumor vessel permeability. these anti - vegf effects lead to improvement of tumor perfusion and oxygenation, resulting in increased sensitivity to chemotherapeutic agents [62, 63 ]. fgfs comprise one of the most versatile and complex signaling families in vertebrates, playing critical roles in a wide variety of biological processes. fgfs are broad - spectrum mitogens that stimulate various cellular functions including migration, proliferation, and differentiation. fgf2 is recognized as a cell survival factor that inhibits apoptosis in many cell types including endothelial cells. the expression pattern of fgfs is very variable, ranging from nearly ubiquitous (fgf1 and 2) to highly restricted to particular cell subsets at specific developmental stages (fgf3, 4, 8, 17, and 19). in pathological conditions such as angiogenesis, inflammation, and malignancies, fgfs are abundantly secreted from various cell types including monocytes, tissue macrophages, endothelial cells, stroma cells, and tumor cells. despite the recognition of fgf as a strong in vivo angiogenic promoter, deciphering its precise functions in the vascular system has suffered from the lack of information obtained from mouse knockout studies [67, 68 ]. disruption of fgfr1 or fgfr2 in mouse embryos results in embryonic lethality at very early stages of development, precluding further evaluation of their contribution to vascular development. on the other hand, knockout studies of angiogenic fgf ligands such as fgf1 and fgf2 failed to show abnormalities in embryonic vascular development, implying that extensive redundancy exists in the ligand system. one of the difficulties investigating the fgf system arises from its promiscuous actions to a variety of cell types and tissues ; however, using tissue - specific promoters, recent studies began to uncover roles played by fgfs in the cardiovascular system. while endothelial fgf signaling is dispensable for mouse coronary vascular development, myocardial fgf signaling appears to be essential for triggering hedgehog activation that is required for vegf expression and coronary vessel formation. furthermore, analyses of fgf signaling in the adult vasculature revealed significant contribution of fgf to vascular development as well as vascular integrity maintenance. in the adult mice, basal endothelial fgf signaling is required for vascular homeostasis. inhibition of fgf signaling leads to disassembly of endothelial junctions, progressing to severe deterioration of vascular integrity. in clear contrast to vegf which induces disruption of ve - cadherin - based junctions by src activation, fgf fortifies junction adhesiveness via enhancing coupling of ve - cadherin with p120 catenin. the critical role of fgf signaling to new vessel formation is also demonstrated in a more recent study in which the mechanism of signaling crosstalk between fgf and vegf is described. vegfr2 expression levels are tightly controlled by endothelial fgf signaling capable of upregulating vegfr2 transcription via an ets - dependent manner ; thereby fgf promotes neovascularization indirectly by regulating endothelial responsiveness to vegf. it has been repeatedly shown that while fgf - induced new vessel formation is often disrupted by vegf inhibition in various in vivo angiogenic models, vegf - induced vascular formation is not as much affected by fgf signaling depletion. together, these studies suggest the hierarchic control of new vessel formation by which the fgf system promotes new vessel growth via controlling vegf signaling. the angiopoietin- (angpt-)tie2 signaling system has crucial roles in vascular functions including angiogenesis and vessel maintenance. angpt-1 is a ligand of the endothelial cell receptor tie2, and activation of tie2 signaling enhances endothelial cell barrier integrity and endothelial - pericyte interaction, thereby promoting vascular stabilization. while expression of tie2 is largely specific to the endothelium, angpt-1 production by mural and perivascular cells facilitates basal tie2 signaling in quiescent endothelial cells that, in turn, is required for endothelial homeostasis. in contrast, angpt-2, produced and stored in weibel - palade bodies in endothelial cells, normally functions as an angpt-1 antagonist. overexpression of angpt-2 in the mouse endothelium attenuates physiological tie2 signaling and thus increases vascular permeability, suggesting that angpt-2 inhibits tie2 signaling and counteract the angpt-1 action. mice lacking angpt-1 or tie2 have similar cardiovascular defects, indicating the importance of the angpt-1-tie2 signaling system in cardiovascular development. these mice die mid gestation (e10.5e12.5) due to the absence of hierarchical vascular development and perturbed vascular integrity, manifested by reduced pericyte coverage and detachment of pericytes from the endothelium. although tie2 signaling is thought to be indispensable for vascular development as previous studies indicated, a recent study using the tissue - specific gene deletion strategy in mice, however, reached the conclusion suggesting that it may not be the case. detailed analysis of angpt-1 embryos showed that the earliest detectable defect seen at e9.5 is loss of myocardial trabeculations while the vascular system appears normal. when angpt-1 is specifically excised from cardiomyocytes using the nkx-2.5 cre - driver line, the vascular defects reported in the global knockout are recapitulated, indicating that vascular phenotypes are dependent on the cardiac defect and resulting from impaired blood circulation. moreover, deletion of angpt-1 in the later stage of vascular development does not affect pericyte number or vascular mural cell recruitment. together, authors concluded that angpt-1 is not required for embryonic vascular development or maintenance of vascular quiescence ; however, it functions as a protective factor, modulating responses to tissue injury and microvascular abnormality in diabetes. angpt-1 inhibits vegf - induced src activation through rhoa activation which leads to src association with mdia (a rhoa downstream target) and sequestration of src from vegfr2. moreover, angpt-1 induces translocation of tie2 to cell - cell contacts and bridges tie2 proteins, resulting in the formation of the transdimer of tie2. although functional contribution of this tie2 homophilic interaction to junctional stability and permeability control is unclear, angpt-1 preferably transmits pi3k - akt signaling in quiescent cells in the presence of tie2 transdimers with their close association with enos. in contrast, in isolated endothelial cells, erk1/2 signaling which promotes cell migration and proliferation is activated by angpt-1 [78, 79 ]. sphingosine-1-phosphate (s1p), a sphingolipid metabolite found in high concentrations in platelets and blood, is a lipid mediator that interacts with gpcrs (s1p1s1p5) to induce a variety of biological responses [80, 81 ]. it is also able to enhance endothelial barrier integrity through s1p1 receptor (edg1) signaling by promoting rac1 activation and adherens junction assembly. administration of the s1p receptor agonist fty720 in vivo potently blocks vegf - induced vascular permeability, suggesting that s1p receptor on endothelial cells is able to regulate vascular permeability. furthermore, the s1p1 receptor is essential for normal vascular function since systemic antagonism of s1p1 receptor under basal physiological conditions causes s1p1 receptor internalization and degradation through receptor phosphorylation, leading to enhanced pulmonary vascular leakage [85, 86 ]. s1p1 knockout mice die between e12.5 and e14.5 due to severe hemorrhage resulting from a defect in the vascular stabilization process. in mice in which s1p1 is specifically deleted from endothelial cells, the phenotype mimics the one obtained from the embryos globally deficient in s1p1 whereas vascular smooth muscle cell - specific knockout has no effect. while these data demonstrate that s1p1 signaling in the endothelium is critical for the regulation of vascular maturation, the lack of vascular maturation in these animals is also attributed to impaired endothelial - pericyte interaction mediated by n - cadherin. although structural and functional basis of endothelial - pericyte interaction has not been well characterized, n - cadherin is reported to localize at the interface of these two cell types in the embryonic brain vasculature. this n - cadherin - based junction mediates pericyte adhesion to endothelial cells, thereby contributing to vessel maturation and stabilization. s1p stimulation of endothelial cells results in activation of rac1, promoting forward trafficking of n - cadherin to the plasma membrane and the formation of the n - cadherin - catenin complex. inhibition of n - cadherin profoundly attenuates the process of vascular stabilization in vitro and in vivo, suggesting the specific contribution of s1p signaling to n - cadherin - induced pericyte attachment. the role of platelet - derived growth factor (pdgf) signaling in the vascular system is established as an important player to mediate pericyte - endothelial interaction. pdgfs are major mitogens for mesoderm - derived cells such as fibroblasts, pericytes, and smooth muscle cells, and for some cell populations of neuroectodermal origin. in the mouse embryo, perivascular mesenchymal cells expressing pdgfr respond to pdgf - bb (a pdgf - b homodimer) produced by the angiogenic endothelium. paracrine pdgf signaling is thus required for mural cell recruitment and expansion as has been demonstrated that pdgf - b expression is particularly abundant in tip cells of angiogenic vessels and in the endothelium of growing arteries. the significant contribution of pdgf signaling promoting mural cell investment to vascular barrier integrity has been shown by mouse genetic studies. both pdgf - b and pdgfr null mutant mice die perinatally, displaying lethal hemorrhage and edema caused by a pericyte loss in microvessels. the results of endothelium - specific knockout of pdgf - b further confirmed that pdgf - bb generated from angiogenic endothelial cells is critically important for recruitment and proliferation of mural cell progenitors in vicinity. the absence of pericytes in capillaries increases their diameters and generates microaneurysms by endothelial hyperplasia, suggesting that the pericyte coverage negatively control endothelial cell proliferation. the detailed analysis of pdgfr-deficient mice revealed that vessel instability observed in these mice is modified by systemic upregulation of vegf. the endothelial junction structure is slightly altered in pdgfr-deficient mice, and this is attributed to the vegf effect, since the onset of endothelial hyperplasia precedes the endothelial junction abnormality. it appears that initial induction of pericyte differentiation from mesenchymal progenitors is independent of pdgf signaling and is most likely mediated by other factors such as tgf-. pericyte populations in different tissues are affected in varying degrees by the loss of pdgf signaling in developing pdgfr embryos. therefore, pdgf signaling is thought to be important in the subsequent maturation process in the angiogenic vessels where pdgf - bb released from endothelial cells drives pericyte migration and expansion. this endothelium - pericyte interplay is particularly crucial for the formation of the blood - brain barrier (bbb), a specific physical barrier of the brain capillaries. using pericyte - deficient mouse mutants which have defective pdgf signaling in the embryonic or adult vasculature, recent studies clearly showed that pericytes are necessary for the formation of the bbb, and that absolute pericyte coverage determines the extent of vascular permeability. interestingly, in the central nervous system vasculature, the formation of tight junctions and endothelial vesicle transport by transcytosis are the critical regulator of vascular permeability which is increased by pericyte deficiency [98, 99 ]. tgf- family includes tgf-s, activins, inhibins, nodal, bone morphogenetic proteins (bmp), and growth differentiation factors (gdf), comprising one of the largest growth factor / cytokine families in vertebrates. although tgf- family members are known to play crucial roles in the vascular development, their precise role especially in endothelial functions is still controversial. this is probably attributable to the remarkable diversity of their actions and complex regulation of the signaling system. in fact, tgf- effects are highly context - dependent with in vitro observations suggesting that tgf- is stimulatory to endothelial cell functions at low concentrations whereas it can be inhibitory at high concentrations. moreover, the presence of pericytes in the endothelial cell culture leads to tgf- activation, which, in turn, inhibits endothelial proliferation and migration through vegfr2 downregulation [101, 102 ]. therefore, one of the important roles of tgf- signaling in mural cells is to attenuate the endothelial cell response to angiogenic stimuli, thereby limiting vessel overgrowth and resolving the active angiogenic process. this appears to form a negative feedback loop by which endothelial cells promotes pericyte differentiation and expansion that lead to vessel stabilization and, consequently, vessel maturation. genetic studies in mice have indicated the essential role of tgf- in pericyte functions and vascular development. disruption of the tgf-1 gene in mice leads to extraembryonic vascular defects exemplified by the failure of endothelial differentiation accompanied with fragile yolk sac vasculature. inactivation of tgfbr2 in the smooth muscle cell lineage results in vascular defects in the yolk sac and embryonic lethality between e12.5 and e16.5, suggesting impairment of mural cell recruitment. the importance of tgf- signaling in mural cell function is further endorsed by the observation that mice with neural crest - specific deletion of tgfbr2 develop a phenotype similar to that of digeorge syndrome due to the failure of neural crest derivative differentiation into smooth muscle cells in the cardiac outflow tract. moreover, mouse phenotypes resulting from targeted disruption of tgf-rii, endoglin or activin receptor - like kinase 1(alk1) are highly reminiscent of tgf-1 null mice, showing vascular abnormalities characterized by systemic vascular dysplasia and recurrent hemorrhage caused by telangiectases and arteriovenous malformations. these studies collectively demonstrate the importance of tgf family genes in the various aspects of vascular development. the notch signaling pathway is an evolutionarily conserved, intercellular signaling system that plays important roles in a wide variety of biological processes. during vascular development, notch signaling plays essential roles in endothelial cell specification including angioblast specification, arteriovenous differentiation, and tip / stalk cell formation [106, 107 ]. the critical contribution of notch signaling to the endothelium and new vessel formation has been well characterized in mouse and zebrafish studies. genetic ablation of notch1 or a notch ligand, delta - like ligand 4 (dll4) causes vascular deformation and embryonic lethality due to defective arterial and venous specification of endothelial cells [108, 109 ]. in this process, notch is a downstream of vegf signaling, inducing arterial differentiation through activation of plc, mapk, and ephrinb2/ephb4 signaling. ephrinb2, which is an arterial endothelial marker, is a direct transcriptional target of notch signaling. as signaling components of the notch pathway are critically involved in arteriovenous specification, deregulation of notch signaling causes aberrant direct communication between an artery and a vein, leading to arteriovenous malformation (avm). in mouse and zebrafish genetic models, both loss and gain of function mutations can result in the formation of arteriovenous malformation and embryonic vascular remodeling defects. when dll4 binds notch1, the intracellular domain of notch1 (nicd) is cleaved by -secretase and translocates to the nucleus where it regulates gene transcription. in the vascular sprout of the retina, while the vegf - a gradient generated by astrocytes in the ischemic area induces endothelial cells at the leading edge to adopt a tip cell phenotype, dll4 prominently expressed in the tip cell signals to following stalk cells through notch1 to downregulate vegfr2 and inhibit filopodia formation [111, 112 ]. thus, during vascular patterning, notch signaling is required for stalk cell specification by actively suppressing the tip cell phenotype, controlling the number of tip cells and vascular sprouts. in fact, ectopic activation of notch signaling in the mouse retina reduces tip cell filopodia and the vascular density. notch signaling inhibits endothelial cell proliferation and motility, thus preventing excessive vessel sprouting and stabilizing newly formed vessels. although increased notch signaling does not necessarily induce vessel instability, recent study revealed that coordination of notch and wnt signaling plays a role in stabilization of nascent vessels. notch - regulated ankyrin repeat protein (nrarp), directly induced by notch signaling, promotes canonical wnt signaling and controls the stability of newly formed vessel anastomosis. notch signaling in vascular smooth muscle cells also contributes to arterial specification / differentiation and thus to vessel stabilization. genetic disruption of notch3, expressed in smooth muscle cells of arteries, leads to arterial defects including enlargement of arteries with thin smooth muscle layers. interestingly, endothelium - specific knockout of jagged1 causes defects in smooth muscle cell differentiation, whereas endothelial notch signaling and arteriovenous differentiation occur normally. this suggests that endothelial - mural cell crosstalk mediated by notch signaling is necessary for proper arterial development. endothelial jagged1 promotes the development of neighboring vascular smooth muscle through notch3 activation, and the loss of jagged1 in the endothelium causes a deficiency of smooth muscle cell recruitment and differentiation, thus leading to vascular instability [116, 117 ]. the semipermeable compartmentalization achieved by blood vessels especially by the endothelium is critically important for normal organ functions and tissue homeostasis. the proximal disorders resulting from the breakdown of the barrier function are bleeding and tissue edema. the impairment of the barrier integrity can result from prolonged stimuli of permeability - inducing agents such as vegf or genetic abnormalities that lead to fragile vessels causing by unstable endothelial junctions, impaired vessel specification / stratification, and pericyte insufficiency. these congenital vascular abnormalities are collectively referred to as vascular malformation which includes several different types according to the type of the blood vessel predominantly affected. among them, venous malformations are the most common form. impaired vessel specification leads to an aberrant direct communication of an artery and a vein, which is termed as arteriovenous malformation (avm). the majority of vascular malformations is sporadic and has no genetic components ; however, recent studies identified a variety of genetic abnormalities which provide clues to pathogenesis of vessel abnormalities and mechanisms of vascular maintenance [119122 ]. venous malformations, the most common type of vascular malformation, comprise either superficial or deep veins that are abnormally formed and dilated in the skin, mucous membrane, or in any organ system characterized by lesions composed of enlarged, tortuous venous channels. although venous malformations are usually present at birth, due to the slow nature of disease progression, they may not be diagnosed at young ages. genetic abnormalities contribute only 2% of reported venous malformations ; however, recent studies revealed mutations in the tek gene, encoding tie2, in the families with dominant inheritance of venous malformations. the mutations identified thus far have been located at the cytoplasmic domain of tie2, resulting in ligand - independent phosphorylation and activation of the receptor. these autosomal - dominant venous malformations, termed venous malformations, multiple cutaneous, and mucosal (vmcm), tend to involve small multifocal, bluish mucocutaneous lesions. in addition to the inherited form of vmcm, somatic mutations causing loss of function of tie2 is suggested to have a role in the etiology of solitary or multiple sporadic venous malformations, which are the more common form of the disease. hereditary hemorrhagic telangiectasia (hht), also known as osler - weber - rendu syndrome, is a genetic disorder that is inherited as an autosomal dominant trait and affects 1 - 2 in 10,000 individuals [120, 125 ]. hht causes abnormal blood vessel formation in the skin, mucous membranes, and often in organs such as the gastrointestinal tract, lungs, liver, and brain. the lesions are characterized by mucocutaneous telangiectases, and in some cases, life - threatening visceral arteriovenous malformations. recent genetic studies identified responsible genes associated with hht, all of which are components of tgf- family signaling pathways. hht type i results from mutations in eng, coding for endoglin, a coreceptor of tgf- receptors. hht type ii causes by mutations in acvrl1 which encodes alk1, a type i receptor for the tgf- superfamily ligands. while mutations of either of the two genes account for most clinical cases, a small subset of patients carry mutations in madh4 encoding smad4, a transcription factor that mediates tgf- signaling. mouse phenotypes resulting from targeted disruption of components of tgf- signaling pathway such as tgf-1, tgf-rii, endoglin, or alk1 are all leading to severe vascular abnormality and embryonic lethality at mid gestation, in some part recapitulating hht phenotypes. detailed analysis of hht gene functions using mouse models of tissue - specific or haploinsufficient gene inactivation has begun to reveal the mechanism of hht development [120, 126 ]. current understanding of hht pathogenesis indicates that hht results from endoglin or alk-1 haploinsufficiency, where the remaining wild - type allele is unable to express sufficient protein for normal function. hht mutations are thought to modify predominantly endothelial cell responses to tgf family ligands mediated by endoglin and alk1 since they are relatively endothelial specific genes. however, because the vast majority of blood vessels in hht patients appear to develop and function normally, perturbation of a pathological process that requires finely - tuned tgf- signaling such as wound healing and angiogenesis might play as an extra trigger. while endothelial alk1 is essential for the establishment of proper arteriovenous (av) connections during vascular development, in the adult endothelium lacking alk1, avm forms following activation of the quiescent endothelium by wound that elicits angiogenesis. in hht, the augmented angiogenic response including excessive endothelial proliferation and exaggerated vessel sprouting is partially attributable to impaired recruitment of mural cells to the newly formed vessel due to reduced endothelial cell secretion of tgf-1 or reduced tgf-1-induced responses. in hht type i patients, circulating levels of tgf-1 are reduced. moreover, paracrine tgf- signaling is defective in mice with endothelial - specific deletion of tgf-rii or alk5 as evidenced by reduced phosphorylation of smad2 in the adjacent mesothelial cell layer of the yolk sac. phosphorylation of smad2 and differentiation of smooth muscle cells can be rescued by exogenous tgf-1 in the yolk sac culture, consistent with the idea that lower levels of tgf- or reduced responsiveness required for receptor activation play a role in hht pathogenesis. in this regard, the recent discovery of thalidomide as a potential therapeutic agent for hht confirmed the importance of mural cell dysfunction responsible for the formation of fragile vessels. in mice heterozygous for a null mutation in the endoglin gene, thalidomide treatment stimulates mural cell recruitment by increasing pdgf - b expression in endothelial cells and thus rescues vessel defects. cerebral cavernous malformations (ccms) are sporadic or inherited vascular malformations in the central nervous system characterized by dilated, thin - walled capillary - like channels without intervening brain parenchyma. they are one of the commonest vascular malformations in the brain, affecting roughly 0.10.5% of the general population. genetic studies have identified autosomal dominant mutations associated with ccms : krit1 (ccm1), ccm2 (mgc4607, malcavernin, osm), and pdcd10 (ccm3). although neither of these ccm proteins are structurally related to each other nor have been implicated as angiogenesis inducers, recent studies indicated possible mechanisms leading to vascular malformation. krit1, originally identified as a rap1a interacting protein, is partially localized at cell - cell contacts and the loss of krit1 accounts for the unstable endothelial junctions. it has been shown that ccm proteins exist as a large protein complex, and a defect of one protein can affect the function of other ccm proteins. krit1 or ccm2 is capable of inhibiting rhoa and rock which can disassemble endothelial junctions and cause hemorrhage. since rap1 is able to enhance junction stability and krit1 is a rap1 effector, there is a possibility that the loss of krit1 directly affects endothelial junctions in a rap1-dependent manner. rap1 regulates the junctional localization of krit1, which is required for rap1-mediated endothelial junction stabilization. disruption of heart of glass (heg1), a transmembrane receptor previously implicated in ccm functions in zebrafish, results in defective integrity of the heart and the vasculature in mice. heg1 coupled with ccm proteins through krit1 is required for vascular development and endothelial junction formation. haploinsufficiency of ccm2 in mice, a genotype equivalent to that in human ccm, results in impaired endothelial barrier function. interestingly, loss of ccm2 leads to activation of rhoa, and the impaired barrier function in heterozygous mice is restored by simvastatin, a drug known to inhibit rho gtpases. moreover mice with global or endothelial cell - specific inactivation of ccm3 show defects in embryonic angiogenesis and die at an early embryonic stage. in response to vegf stimulation, ccm3 is recruited to and stabilizes vegfr2, thereby playing a pivotal role in vegfr2 signaling. capillary malformation - arteriovenous malformation (cm - avm) is a recently discovered hereditary disorder characterized with atypical cutaneous multifocal capillary malformations often in association with high - flow lesions such as arteriovenous malformations of the cerebrospinal and visceral organs or arteriovenous fistulas. genetic studies identified loss of function mutations in rasa1, which encodes p120-rasgap [121, 139 ]. as gtpase activity leads to, by hydrolysis of gtp to gdp, inactivation of small gtpases, loss of p120-rasgap function suggests the possibility to increased ras and downstream mapk activation. furthermore, p120-rasgap has been shown to interact with p190-rhogap, which inhibits rhoa through a p120 catenin - dependent mechanism and is required for adherens junction formation [140, 141 ]. the clinical relevance of delta - notch signaling is manifested by the cerebral small vessel disease : cerebral autosomal - dominant arteriopathy with subcortical infarcts and leukoencephalopathy (cadasil), now recognized as the most common cause of inherited stroke and vascular cognitive impairment in adults [106, 142 ]. cadasil is inherited as an autosomal dominant trait, resulting from mutations in notch3, which causes degeneration of smooth muscle cells of cerebral small vessels and accumulation of the notch extracellular domain (necd) at the surface of residual smooth muscle cells. these changes lead to thickening of the vessel wall and a reduction of cerebral blood flow [142, 143 ]. genetic studies revealed that among 33 exons, all cadasil mutations occur in exon 224 of the notch3 gene within 34 egf - like repeats, leading to an odd number of cysteine residues. genetic disruption of notch3 in mice causes structural defects of small arteries because of impaired differentiation and maturation of arterial smooth - muscle cells ; however, total loss of notch3 does not cause cadasil pathology. furthermore, cadasil - causing mutations of notch3 can activate rbp - j transcription comparable to wild - type levels and do not seem to affect notch signaling per se [144, 145 ]. therefore, although the precise cause of the disease is still unclear, recent studies suggest that gain of novel function of the mutant protein, possibly arising from novel protein - protein interactions rather than defective notch3 function, is a likely mechanism for the cadasil pathogenesis. as discussed earlier, notch pathway is important for arteriovenous differentiation and vessel patterning during embryonic vascular development, and deficiency of notch signaling can cause arteriovenous malformations [146, 147 ]. expression of constitutively active notch4 (int3) in the mouse endothelium develops features of brain arteriovenous malformations characterized by cerebral arteriovenous shunting and vessel enlargement [148, 149 ]. conditional activation of the notch1 gene in endothelial cells of mouse embryos also causes defects in vascular remodeling progressing to arteriovenous malformations. analysis of brain arteriovenous malformations revealed that notch1 signaling is upregulated in smooth muscle and endothelial cells of the lesions surgically resected from human patients. regulation of the vascular barrier function is crucial for tissue homeostasis. recently, our understanding of molecular mechanisms leading to vessel stabilization has significantly expanded owing to the advance of basic research followed by critical appraisal of therapeutic angiogenesis trials. it has also become apparent that the maintenance of existing vessels requires active cellular signaling which share common features with signaling mechanisms involved in the vessel maturation process of new vessel formation. components of signaling pathways involved in vascular stability often cause vascular malformations as disease genes. conversely, genes identified through genetic studies of vascular diseases most likely play a role in physiological regulation of vascular integrity. as has been discussed, vessel maintenance is successfully achieved by orchestrated actions of growth factors and cytokines that are capable of modifying the function of vascular cells especially endothelial cells and mural cells. although indispensability of these cell types and signaling pathways required for the maintenance of vessel integrity have been unequivocally demonstrated by numerous studies, it is important to further elucidate detailed molecular mechanisms of signaling interactions between different cell types in the vasculature. the endothelial - pericyte junctions are anatomically identified ; however, signals exchanged during new vessel formation and vessel maintenance between these cells are not clearly understood. besides genetic components leading of vascular instability, epigenetic factors are also playing an important role in modifying disease manifestations such as the location and the severity of the vascular abnormality in the presence of ubiquitous, germline mutations. further investigations exploring this aspect including the two - hit mechanism for disease development and presentation should provide significant insights into our understanding to vascular maintenance. as regulation of vessel maintenance is a fundamental vascular function associated with a wide variety of vascular disorders and disease conditions, elucidating the precise mechanisms will benefit the development of new approaches for therapeutic angiogenesis and vascular malformations as well as cancer treatment. | as our understanding of molecular mechanisms leading to vascular formation increases, vessel maintenance including stabilization of new vessels and prevention of vessel regression began to be considered as an active process that requires specific cellular signaling. while signaling pathways such as vegf, fgf, and angiopoietin - tie2 are important for endothelial cell survival and junction stabilization, pdgf and tgf- signaling modify mural cell (vascular smooth muscle cells and pericytes) functions, thus they fortify vessel integrity. breakdown of these signaling systems results in pathological hyperpermeability and/or genetic vascular abnormalities such as vascular malformations, ultimately progressing to hemorrhage and edema. hence, blood vessel maintenance is fundamental to controlling vascular homeostasis and tissue functions. this paper discusses signaling pathways essential for vascular maintenance and clinical conditions caused by deterioration of vessel integrity. |
black pakistani cobra (naja naja karachiensis.) is a highly venomous snake that is commonly found in southern parts of south asia. like other naja members, this cobra species found in southern pakistan produces potent venom, which is a major contributor in snake bite - related deaths in south asia. additional complications can arise as snake bite injury can result in wound infections and tissue necrosis leading to sepsis / necrotizing fasciitis [1, 2 ]. thus, there is a need for clinicians to consider appropriate antibiotics in the presence of or likelihood of wound infection ; however, there is no single report of the bacterial flora of cobra found in south asia. in addition to the bacterial flora, several other factors determine the outcome of snake bite wound infection including microbial contamination from the environment, handling of the snake bite, and the health of the patient. previous studies have determined the oral bacterial flora in cobra. a wide range of bacteria were isolated including pathogenic coagulase - negative staphylococcus, enterococcus species, morganella morganii, enterobacter species, pseudomonas, aeromonas hydrophila, citrobacter freundii, proteus species, providencia rettgeri, and clostridium species. goldstein. have suggested that the snake oral flora is most likely faecal in nature, as the live prey may defecate in the snake 's mouth while being ingested. later, lam. addressed this concern by determining oral bacterial flora from snakes that were freshly captured from the wild and fasted before sample collection, but even then contamination from prey - bacteria can not be ruled out. for example, theakston. studied oral swabs from newly captured malayan pit vipers (calloselasma rhodostoma) and found gut - related gram negative rods such as enterobacter and pseudomonas species and some staphylococci and clostridia. thus, the overall aim of the present study was to determine the gut bacterial flora of black pakistani cobra (naja naja karachiensis) found in southern pakistan. during the period of june to october 2012, three black cobra snakes were captured from the interior sindh (tharparkar region), pakistan, and brought to dow university of health sciences for species identification by a qualified team with over 10 years of herpetological experience in the territory. the genus of snake, together with the health condition of the snake was identified by the veterinarian who has extensive expertise of herpetology. as in routine procedures, snakes were anesthetized using chloroform and the surface disinfected with 70% ethanol and subsequently dissected to expose the distal part of the gut. the distal part of the gut from the end of the small intestine and up to cloaca was further dissected open and the greenish semisolid gut contents were collected into 15 ml sterile falcon tubes. in the laboratory, snake gut contents (~3 to 5 g) were resuspended in 5 ml of phosphate buffered saline (pbs) and approximately one ml was streaked onto nutrient agar plates (100 l per plate). one set of plates was incubated at 25c whereas other at 37c for 48 h. bacterial colonies exhibiting growth upon repeated subculturing on nutrient agar plates were selected from all three snakes and streaked onto nutrient agar plates in order to obtain pure cultures. these pure cultures were maintained in nutrient broth containing 25% glycerol and stored at 80c until used. bacterial colony characteristics such as shape, margin, elevation, color, opacity, and consistency were recorded. gram positive bacterial cultures were tested for spore formation, catalase production, and oxygen tolerance, whereas gram negative cultures were inoculated on citrate utilization medium, sulfide - indole - motility (sim) medium, triple sugar iron (tsi) slants, and n, n - dimethyl - p - phenylenediamine (dmpd) discs for oxidase test. based on oxidase activity, bacterial cultures were identified further using analytical profile index (api) strips -e and -ne (biomrieux inc.). oxidase positive cultures were inoculated on api - ne and oxidase negative cultures on api - e strips. the results were then interpreted and bacteria were identified using information provided by the manufacturer (biomrieux inc.). shewanella putrefaciens and ochrobactrum anthropi were tested further for dnase production using dna agar medium (biomrieux). identification of aeromonas hydrophila was confirmed using the 0129 disc diffusion test (oxoid). production of pyocyanin and pyoverdin pigments from pseudomonas aeruginosa was tested using pseudomonas agar - p and agar - f (bd biosciences), respectively. salmonella isolates were characterized further using salmonella group specific antisera (denka seiken co.). the gut contents of the black cobra snake were inoculated onto two sets of nutrient agar plates, which were incubated at 25c and 37c, respectively, for 48 h. nineteen culturable bacterial strains (c1c19) were selected (based on growth at 25c as well as at 37c and repeated culturing), which were conserved among three snakes, while the remaining ones were either unidentifiable, could not be subcultured, duplicate, or doubtful contaminant. gram staining revealed that all of the bacterial cultures were gram negative rods, except for c14 which was found to be gram positive rods with spores. the colony morphology of the bacterial isolates revealed variable morphology as summarized in table 1. bacterial isolates encoded c2, c8, and c10 produced a brownish pink pigment, which diffused into the medium (table 1). based on the morphological and biochemical analysis, nine different bacterial types were identified, that are representative of three snakes (table 2). among the nineteen different bacterial isolates tested, five (c1, c3, c12, c16, and c18) were identified as serratia marcescens. serratia marcescens is known to produce a red pigment known as prodigiosin, but only one of the five isolates tested, that is, c1, produced bright red pigment, while the rest of the four isolates were nonpigmented. these results are consistent with previous findings where isolation of nonpigmented serratia marcescens was reported from clinical samples. three of the bacterial isolates tested (c2, c8, and c10) were identified as pseudomonas aeruginosa. these three strains were positive for the production of the pigments pyocyanin and pyoverdin. among other isolates, c4, c6, and c7 were identified as shewanella putrefaciens, while c9 and c11 were identified as aeromonas hydrophila, and c13 and c15 were identified as salmonella species. the serological typing results show that both of the salmonella strains belong to the subgroup iii (data not shown). the remaining four bacterial isolates tested were identified as follows : c5 was identified as moraxella sp. ; c14 was identified as bacillus sp. ; c17 was identified as ochrobactrum anthropi ; and c19 was identified as providencia rettgeri. table 2 collates the results of the biochemical analysis and identification of the different bacterial isolates. the comparative analysis of the snake gut flora that we have isolated with other metazoan gut flora revealed that members of the genera bacillus and pseudomonas are common inhabitant of insects, fishes, amphibians, reptiles, birds, and the mammalian gut [7, 8 ]. as the cobra mostly feed on rodents, frogs, toads, small birds, and other small snakes, it is possible that the bacteria from within their prey may colonize the gut of the snake ; however, a study in the burmese python showed that most of the gut flora of snake was native rather than derived from their prey. nonetheless, in the present study, snakes were fasted for 3 days to ensure that the gut bacterial flora is not derived from their prey. it is interesting to note that bacterial isolates derived from the cobra gut were mostly gram negative (18 out of 19) ; however, this may not represent the true diversity of the black cobra gut microbiota as the culture methods employed in this study tend to facilitate the isolation of aerobes and facultative anaerobes only. for example, previous studies have identified obligate anaerobes and unculturable bacteria from the gut of burmese pythons using culture - free molecular techniques. consequently, future studies can address this issue together with determination of bacterial flora and their antibiotic susceptibility to propose the empirical antibiotic treatment of choice in the initial snake bite management of snakes in south asia and elsewhere. it is interesting to note that all bacterial isolates identified in this study are known to infect humans. for example, in the case of serratia marcescens, human infections are often associated with the use of contaminated blood products, infusion solutions, and hospital stays. an extensive canadian community - based study showed that a large fraction of s. marcescens infections are community acquired. on the other hand, pseudomonas aeruginosa is an important opportunistic human pathogen that can produce hospital - acquired infections and infections in immunocompromised and cystic fibrosis patients. similarly shewanella putrefaciens, aeromonas hydrophila, ochrobactrum anthropi, and providencia rettgeri are also opportunistic human pathogens, known to cause different human infections in healthy or immunocompromised or hospitalized patients. as the black cobra harbors a number of important human pathogens in their gut, it is tempting to speculate that it may transmit these infectious agents to people who share the same habitat with these reptiles, or during snake bite wound infections, albeit the oral flora of the snake was not determined in the present study. clinicians should seek the advice of clinical microbiologists to consider appropriate antibiotics in the presence of or likelihood of wound infections. future studies should determine the oral flora of black cobra in order to further investigate the possibility of connection between snake bite and related wound infection. tribal culture may prove to be additional risk factors and expose local communities to potentially pathogenic bacteria. for example, cobra meat and organs serve as a source of food and are used in the folk medicine. based on our findings, it is recommended that users remove the gut part while obtaining snake oil or properly cook the meat before consumption ; otherwise, they may risk exposure to different pathogenic bacteria, which may be residing in the gut of snakes. in summary, for the first time we have shown gut bacterial flora (aerobes and facultative anaerobes) in the venomous black cobra from south asia. the results revealed that these snakes inhabit potentially pathogenic bacteria, which may in turn complicate snake bite wound infections and/or expose consumers of snake meat / medicine in the local community. | using morphological analysis and biochemical testing, here for the first time, we determined the culturable gut bacterial flora (aerobes and facultative anaerobes) in the venomous black cobra (naja naja karachiensis) from south asia. the findings revealed that these snakes inhabit potentially pathogenic bacteria including serratia marcescens, pseudomonas aeruginosa, shewanella putrefaciens, aeromonas hydrophila, salmonella sp., moraxella sp., bacillus sp., ochrobactrum anthropi, and providencia rettgeri. these findings are of concern, as injury from snake bite can result in wound infections and tissue necrosis leading to sepsis / necrotizing fasciitis and/or expose consumers of snake meat / medicine in the community to infections. |
cardiovascular diseases (cvds) are the prevalent cause of death in europe and the united states. this is despite general acceptance that a healthy lifestyle and risk factor management can prevent the development of cvd. furthermore, two - thirds of women who die suddenly from cvd have no previously recognized symptoms. thus, it is essential to find effective and broadly applicable indicators of cardiovascular risk that may prompt timely intervention. current noninvasive modalities for imaging atherosclerosis are radiographs, ultrasound, computed tomography (ct), and magnetic resonance imaging (mri). ultrasound is used to visualize the carotid intima - media thickness (imt) because carotid imt has been shown to be associated with atherosclerosis and is thus a marker for cvd. multislice ct is able to quantify the degree of coronary artery calcification (cac) with good reproducibility [6, 7 ], which provides a strong measure of cardiovascular risk independently from, and potentially more powerful than, traditional risk factors such as smoking. however, due to the relatively large exposure to ionizing radiation, use of clinical dose ct is not advisable in large - scale screening, but only to aid interventional treatment of patients at intermediate risk. low - dose ct, on the contrary, could be used to evaluate coronary calcifications for screening purposes, and only its cost is a limiting factor. however, mri measurements are challenged by the size of the smaller arteries, and especially assessment of the coronary arteries is difficult due to cardiac and respiratory motion artefacts. furthermore, also mri still has to prove its cost - effectiveness for screening purposes. an alternative to examining coronary arteries for calcification is to assess the abdominal aorta, since it is contrary to the coronary arteries accessible through radiographs. abdominal aortic calcifications (aacs) are strong predictors of cardiovascular morbidity and mortality, correlate strongly with coronary artery calcifications, and may hence predict the risk of coronary artery problems [13, 14 ]. the state of the art methodology to estimate cvd risk from lumbar aortic radiographs is the abdominal aortic calcification score (ac24) proposed by the framingham study group. a big advantage is that such a aac scoring can, for example, in the case of postmenopausal women, be performed without additional ionising radiation exposure or cost as these images are readily available from osteoporosis screening [16, 17 ]. we investigated if the morphometric aspects of the information that can be made available from ct, mri, or ultrasound as described above could also be obtained from novel markers of aac quantified from plain radiographs. due to the semiquantitative grading of the ac24 score, such markers could potentially be more sensitive in particular with respect to investigating the potential significance of smaller calcifications. for this, we outlined the boundaries of the calcified deposits in the lumbar aortic region and quantified the number of calcified deposits as well as the percentage of the abdominal aorta covered by calcifications in terms of area, simulated - plaque area, thickness, wall coverage, and length. these potential aac markers were evaluated for precision and their ability to predict cvd - related mortality. 308 females were selected from those who took part in the multicentre perf study who were examined radiologically in 1992, and examined again in 2001 in the followup epi study. we chose those whose interval between their first and second clinic visit was 8 - 9 years, with known alive / mortality status, who were postmenopausal, and whose lumbar aorta was visible on a single radiograph at baseline and at followup. information about the mortality status was obtained via the central registry of the danish ministry of health, and the death causes were grouped into three groups : cvd, cancer, and other causes. the studies were approved by the local ethics committee, and the patients signed informed consent forms. at baseline, demographic information and cvd risk parameters such as age, weight, height, body mass index (bmi), waist and hip circumferences, systolic and diastolic blood pressure (bp), treated hypertension, treated diabetes, smoking, regular alcohol and daily coffee consumption, and weekly fitness activity were collected. using a blood analyzer (cobas mira plus, roche diagnostics systems, hoffman - la roche, basel, switzerland), measurements of fasting glucose and lipid profile (total cholesterol, triglycerides, ldl - cholesterol (ldl - c), hdl - cholesterol (hdl - c), and apolipoprotein (apoa and apob)) were obtained. on the basis of these measurements, the composite risk markers, systemic coronary risk evaluation (score) and framingham score, the score is a combination of the age, smoking status, levels of total cholesterol, and systolic blood pressure, while the framingham score is comprised of the same variables plus the hdl - c and the hypertension treatment status. the lateral x - ray images of the lumbar aorta (l1-l4) were acquired on film in 1992 and 2001 [18, 19 ], respectively, and digitized in 2007/2008 using a dosimetryproadvantage scanner (vidar, herndon, usa), providing an image resolution of 9651 4008 pixels on a 12-bit gray scale with a pixel size of 44.6 m 44.6 m. three trained radiologists without prior knowledge of the patients ' conditions annotated the corner and mid points of the vertebrae (l1-l4), the corresponding abdominal aorta walls, and their calcifications in the digitized images manually. they used radiological reading units (sectra, linkping, sweden) and annotation software specifically implemented for that task in matlab (the mathworks, natick, usa), which allowed them to change brightness and contrast, zoom in and out, and to edit outlines, as seen in figure 1. then, the aortic sections adjacent to each vertebra l1-l4 were graded by the degree of lesion occupation : 0 for no aac, 1 for aacs occupying less than 1/3 of the wall they were projected onto, 2 for aacs occupying more than 1/3, but less than 2/3 in the projection, and 3 for a 2/3 or more occupation of the wall. in addition to the ac24 scores provided by the radiologists, the outlines of the calcifications were used in an alternative computer - based computation of the ac24. for all the images with calcifications, annotations were performed by one of the three different radiologists. for a subset of 8 images, annotations by two radiologists reoutlining was performed blinded to earlier outlines and separated by approximately six to eight weeks. the proposed aac markers were automatically computed from the radiologist 's computer - assisted outlines of calcified deposits in the radiographs. area percentage : the percentage of the area of the lumbar aorta adjacent to l1-l4 occupied by aacs. simulated area percentage : we tried to estimate the size of the underlying atherosclerotic inflammation from the area and shape of the observed aacs since x - ray analysis can only visualize the calcified core of the aacs. the extent of the atherosclerotic inflammation was simulated by a morphological dilation with a circular structuring element of radius 200 pixels (approximately 8.9 mm). the size of the structuring element was derived by a parameter study on a subset of the data, and it was confirmed to be biologically sensible by comparing with histology and image analysis observations which estimated the size of the atherosclerotic inflammation surrounding the calcified plaque to be between 3 mm and 510 mm. an illustration of this computer - based simulation of the full plaque area is given in figure 3. the simulated area percentage is the percentage of the lumbar aorta covered by the simulated plaques including both calcified core and simulated inflamed area. thickness percentage : the average thickness of the aacs along the aorta wall relative to the aorta width. wall percentage : the percentage of the anterior and posterior lumbar aorta wall covered by aacs. length percentage : the fraction of the length of the aorta where aacs were present at any position (anterior, posterior, or internal). number of calcified deposits : the number of distinct aacs visible between l1 and l4 in each radiograph. we examined the degree to which these markers could be reliably established on the basis of manual annotations of x - ray images and evaluated their association to mortality, also when adjusted for metabolic or physical markers. kendall 's coefficient of concordance was used to assess the level of agreement between ac24 scorings of calcified images made by radiologists directly on the original x - rays and ac24 scorings by the computer, based on the radiologist 's annotation outlines. to measure the inter- and intraobserver variability of the manual annotations of the radiologists on the 8 images allocated specifically for this purpose we computed the ratio of the area identified as calcified in two outlines, divided by the area identified as calcified in at least one outline : (1)a=|a1a2||a1a2|, where a1 and a2 are a binary annotations. typically, cohen 's would be used to measure the inter - rater agreement for categorical items like pixels. however, the statistics will be dominated by the very large class of non - calcified pixels, and individual pixel scorings can not be considered statistically independent. the inter- and intraobserver variability of the aac markers computed from the radiologist 's outlines was analysed on the 8 images by the mean coefficients of variation (cv). the predictive power of mortality in terms of hazard ratio per standard deviation change (hr) of the individual aac scorings was analysed by cox regression [28, 29 ], where time of death was the outcome variable and survivors were right - censored. this analysis was performed on unadjusted markers as well as markers adjusted with three different sets of biological variables : (a) a model consisting of age, smoking status, and triglyceride levels, (b) the score, and (c) framingham scores. we adjusted by combining the biological variables of each set into one new variable by a linear weighing with their -weights derived by a cox regression. this new variable was then included in another cox regression model for the imaging marker we wanted to adjust. the resulting -weight for the imaging marker determines the biologically adjusted prognostic power. to analyse the complementarity of the aac markers, a backwards stepwise deletion cox regression model with all aac markers least significant markers were successively deleted until only markers with significant -values (p < 0.05) were left. this way, single markers that complemented each other and gave supplementary information were identified. the data consisted of baseline images taken in 1992 from 308 subjects. of these, 121 subjects had no calcifications at baseline or followup. of the remaining 187 subjects, 52 had died before followup due to cancer (n = 27), cvd (n = 20), or other causes (n = 5), and 135 surviving subjects had varying degrees of abdominal aortic calcification at baseline or followup. a schematic overview of the study population is given in figure 4, while an overview of the physical and metabolic measurements is given in table 1. the radiologist and computer - based ac24 scores for the 135 calcified images were in excellent agreement (kendall 's = 0.97, p < 0.0001). on the set of 8 images with four annotations each, the mean jaccard index between the radiologists ' aac outlines was 0.56 0.14 (0.240.79) for the intraobserver variation and 0.51 0.13 (0.290.73) for the interobserver variation, for an example, see figure 5. the two radiologists had an intraobserver variability of 0.53 0.14 (0.240.65) and 0.59 0.14 (0.380.79), respectively. the cv values for the aac marker precision on the same set of 8 images were between 12.5% and 24.9% (table 2). the mean values and respective standard deviations of each of the aac markers can be found in table 3. there was a clear difference between the means in the cvd - death and cancer - death groups compared to the survivors. table 4 shows that the simulated area percentage and number of calcification (ncd) have the largest individual predictive power (hr = 2.96, p < 0.001 and hr = 2.44, p < 0.001) for cvd mortality. their hazard ratio is between 2.0 and 2.96 and 1.76 and 2.44, respectively, for the cvd - death group and between 1.68 and 2.32 and 1.69 and 2.28, respectively, for the combined cvd / cancer - death group. all hazard ratios are significantly different from unity (p < 0.01) both before and after adjusting for three different biological models. ac24 's unadjusted individual predictive power is lower (hr = 1.66, p < 0.001). after adjustment for the three different biological models, the significance of the hazard ratios for ac24 is reduced and in some cases removed, leading to a hazard ratio between 0 and 1.66 for the cvd - death group and between 1.29 and 1.64 for the cvd / cancer - death group. the results of the combined predictive power of the seven imaging markers can be seen for the cvd and the cvd / cancer group in table 5. when combining the markers in a cox regression model, only area percentage and ncd remained significant (parea < 0.001, pncd < 0.001). we evaluated whether a radiologist 's manual scoring of the ac24 correlated with a computer - based scoring of the ac24 derived from a radiologist 's manual outline of the calcifications on a digitized radiograph. further we evaluated inter- and intraobserver variability of manual annotations using the jaccard index and coefficients of variation of the aac markers, including the ac24. although the jaccard index showed that the variation in the outlined calcified deposits was high, the coefficients of variation for the ac24 and the other aac markers based on the outlines were relatively low. these results demonstrated that, even though the outlining of the individual plaques is a challenging task, the resulting markers based on the annotations provided reasonably precise measurements. in the course of the 8 - 9-year - long study, 52 people died, of whom 20 died from cvd - related causes and 27 from cancer. the cox regression models showed similar correlations to cvd and cvd / cancer mortality for the different markers. since cancer and cvd the simulated area percentage and the number of calcified deposits could individually predict cvd and cvd / cancer death and contained additional information for cvd mortality even after adjustments for age, triglycerides and cholesterol, and the score model and framingham score. hence, in this post hoc study, they predicted cvd mortality independently from traditional risk factors, in contrast to ac24. a reason for this could be that the ac24 does not discriminate between severity and spread of individual calcifications. the risk of death due to myocardial infarct (mi) may be related to the number of active plaques. during plaque development, smaller plaques develop into larger complicated lesions that either rupture or become stable plaques [31, 32 ]. smaller lipid - laden plaques with high turnover have been identified as those most likely to rupture and consequent in mi [31, 32 ]. thus, a large number of smaller calcifications may indicate a higher risk of rupture than few large, stable, calcifications in the same area. techniques for measuring different aspects of plaques, such as size, distribution, and number, are in part captured by the simulated area percentage and the number of calcified deposits. this higher emphasis on the number of calcifications, rather than the total calcium burden, may reflect aspects of vulnerability that help improve the cvd - mortality prediction as observed in this work. the cox regression combination model showed that, when combining all the aac markers into one model and deleting the markers that do not significantly contribute to the combined marker, only area percentage and the number of calcified deposits remained. this shows that these two aac markers offer complementary and highly significant information about the risk of death. the complementarity of area percentage and number of calcifications suggests that size and spread of the calcifications both play important roles in atherosclerosis. the sample size is a limitation of the present study. the relatively small population with only 20 cvd deaths, a limited representation of ethnicity and gender, and a mixture of death causes may limit the generalizability of our results.. a limitation of the proposed markers could be the cost of manual annotations, but efforts have been made to automate annotations of calcified deposits [33, 34 ]. compared to markers of cvd obtained with other imaging modalities, such as carotid imt or cac, a clear advantage of using standard radiographs is the availability of large, long duration osteoporosis screening studies [1618 ]. for example, such historical data was used to verify the developed aac markers and can improve understanding of cvd death risk factors. the clinical applicability of aac markers can be increased if the same radiographs are used for osteoporosis screening and cvd risk assessment. while ac24 captures essential information about aac, the results demonstrate that some of these novel morphometric markers of aac may capture complementary information. therefore, the proposed radiographic aac markers may enable improved screening for and monitoring of cvd mortality risk. | abdominal aortic calcifications (aacs) correlate strongly with coronary artery calcifications and can be predictors of cardiovascular mortality. we investigated whether size, shape, and distribution of aacs are related to mortality and how such prognostic markers perform compared to the state - of - the - art ac24 marker introduced by kauppila. methods. for 308 postmenopausal women, we quantified the number of aac and the percentage of the abdominal aorta that the lesions occupied in terms of their area, simulated plaque area, thickness, wall coverage, and length. we analysed inter-/intraobserver reproducibility and predictive ability of mortality after 8 - 9 years via cox regression leading to hazard ratios (hrs). results. the coefficient of variation was below 25% for all markers. the strongest individual predictors were the number of calcifications (hr = 2.4) and the simulated area percentage (hr = 2.96) of a calcified plaque, and, unlike ac24 (hr = 1.66), they allowed mortality prediction also after adjusting for traditional risk factors. in a combined cox regression model, the strongest complementary predictors were the number of calcifications (hr = 2.76) and the area percentage (hr = 3.84). conclusion. morphometric markers of aac quantified from radiographs may be a useful tool for screening and monitoring risk of cvd mortality. |
nineteenth and twentieth century biology was completely based on misleading ideological imposition that living entities are particular states of matter and in that era biologists have only made several attempts to deny the living organism of its veracity as an immortal soul, in favor of genes, molecules, atoms and so on. twenty first century biology realizes that living entities (animate objects) do things, which are intentional and purposeful (internal teleology) and nonliving objects (inanimate objects) have things done to them (external teleology or design). from bacterial antibiotic resistance we can see that even the tiny bacterium displays the sign of great intelligence (natural genetic engineering). unlike inanimate objects, all living cells (and all living organisms) create and maintain order. to create and maintain this order every cell has to work like a tiny chemical factory, performing many millions of reactions every second. thus, the proponents of creationist movement/intelligent design argue that an intelligent being is necessary for the creation / design of factory (example of external teleology) from the basic elements or parts. this is a more reasonable argument as compared to the imprudent materialistic view that the cell / life is a product of mere accumulation of inert chemicals. however, vednta philosophy is not based on creationist movement/intelligent design or reductionistic materialistic views. according to vednta philosophy an organic whole (life comes from life or every cell comes from a cell biogenesis) and an organic whole (prna) can not come from the mechanical and chemical additive sum of the parts (khanda). caetano - anolls completely missed this main point, which the paper life and consciousness it is empirically observable that every day sun rises in the east and hence it is logical to conclude that the first sun rise was in the east. there is no problem, if someone wants to dedicate his whole life to do a rigorous scientific research to prove the opposite first sun rise was in the west. the problem arises when keeping such illogical views in mind someone wants to critique those who have the conviction on the obvious vedntic explanation that independent supreme cognizant being is the source of everything is founded on 2 scientifically verifiable axiomatic facts : (1) life comes from life, and (2) matter comes from life. consciousness arises from consciousness, or life comes from life. where there is life there is consciousness. consciousness does not originate from that which is unconscious or impersonal, and life is not a product of insentient matter. the conception that life comes from life (biogenesis) is the only scientific idea that has ever been verified by experiment and observation. matter comes from life are 2 scientifically observable deductions from vednta. on the other hand, materialism (life originates from matter) is an unverified ideological presupposition that has no scientific or observation - based evidence to support it. for a detailed critique of darwinian objective evolution of bodies and abiogenesis (material origin of life), and a further elaboration on vedntic view for soul hypothesis, consciousness and subjective evolution of consciousness one can refer author 's recent paper why biology is beyond physical sciences?, the published book chapter and the article sorry, darwin : chemistry never made the transition to biology. | abstractthe author would like to thank professor gustavo caetano - anolls from department of crop sciences, university of illinois for his interest in his work. we may sometimes observe that there is a noticeable difference between the anecdote people narrate about the implications of a scientific paper and the real conclusion of the paper. prof. gustavo caetano - anolls 's response1 is an ideal example of the same, where he has tried to make great hay about the implications of the article life and consciousness the vedntic view.2 the vedntic view subscribes neither to the views of creationist movement/intelligent design, nor it supports some splendid anti - science proposal. vedntic view refutes the dominant reductionistic view of life in modern biology by proposing a viable alternative concept of organic whole and thus serves a scientific critique to the nescience (avidy) that is practiced on the name of science. |
numerous short and thick hairs set obliquely in the fibers of orbicularis oculi, corrugator and frontal part of occipito frontalis are inserted in the dermis of eye brows. the head of the eyebrows overlies the frontal sinus and the tail is usually in the region of zygomatiocofrontal suture. it is an appendage of the hair bearing scalp rather than an extension of facial tissue. the head of the eyebrows overlies the frontal sinus and the tail is in the region of the zygomaticofrontal suture. the female eye brow is more arched and rest slightly higher than male eyebrow, which usually rests at the level of superior orbital rim. male eye brows are more irregular. the natural direction of hair of the eyebrow is varied. inferoateral hair direction is found in the upper and lateral parts. in medial and lower eyebrows play an important function in facial identification and may be at least as important as the eyes. human beings have a single eye brow above each eye, but this article presents a case report of a child with double eye brows on the left side. the present case report is about a 6-year - old girl presented to department of pediatric and preventive dentistry, guru nanak institute of dental science and research, kolkata for routine dental check - up. there was no relevant medical history with normal built, gait and intelligence. on facial examination, there was no gross asymmetry of face or any abnormal swelling. dermatological examination revealed that the eyebrows on the both the sides were sparser on the medial sides when compared with the lateral sides. the eye brows were free from any pathology except a double layer of eyebrow was present above the left eye. second layer of eye brow was just present above the first layer [figures 1 and 2 ]. family history revealed no systemic disease or any extra layer of eye brow in either of maternal or paternal individual. no cosmetic / aesthetic or therapeutic treatment has been received by the child for double eyebrow. routine laboratory tests (complete blood count, liver function test, urine analysis, abdominal ultrasound) were all normal. considering the clinical features, a diagnosis of double eyebrow was concluded. the eyebrow is a transverse elevation of hair, which starts medially just inferior to the orbital margin and ends laterally above the orbital margin. they are formed by the transverse elevation of the superciliary ridge of the frontal bone. the superficial muscles of the head develop as mesodermal laminae which begin at the second branchial arch. from infraorbital lamina orbicularis, oculi, corrugator, these laminae join above the eye and form the interdigitating muscular structure of the brow. at 8 - 10 week of fetal development formation of primitive hair starts as a focal crowding of basal cell nuclei in the fetal epidermis. when the basal cell germ enlarges it becomes asymmetric and extends obliquely downward as a solid column. contracting the orbital sections of orbicularis oculi lowers the eyebrows and contracting the corrugators supercilia muscle draws the eyebrows together medially. there are three types of hair found in the eyebrow : (1) fine vellus hair ; (2) slightly larger and lightly pigmented hair and (3) large terminal hair known as the supercilia. the fine hairs form an effective moisture barrier to keep sweat from running downward into the eye. main function of eyebrows is to protect eyes and prevent flowing of salty sweat to eyes. the position and curvature of the eyebrow allows it to shields the eyes from bright light and it is an effective barrier to liquids running from the forehead into the eye. abundant sensory innervations are present in the large hairs of the eyebrow, which are very sensitive to tactile stimulation. the eye brows also function to depict the expression of an individual, like the depression of the medial portion of the eyebrow depicts anger or concern. eye brows abnormalities have a close relation with genomic disorders. in facial esthetics, sexual dimorphism, emotional expression and nonverbal communication eyebrows recent research suggests that eyebrows play an important function in facial identification and may be at least as important as the eyes. main physical function is to prevent flowing of salty sweat to eyes ; henceforth protection of eyes is the main function of eyebrows. eyebrows variation found in various syndromes such as chr1p36.33 microdeletion syndrome, chr2q21 - 23 microdeletion, mowat - wilson syndrome, chr3q26.3-q27 microdeletion, with sparse and broad - based eyebrow, chr7p15.3 duplication : extreme sparseness of the lateral portion of the eyebrows, chr9q34.3 terminal deletion, arched eyebrows and synophrys, chr10q22.3 - 23.2 duplication, with medial flaring eyebrows etc. ip 36.33 microdelation has a close relationship with deep - set eyes and horizontal eyebrows. eye brows abnormalities are useful diagnostic aids of chromosomal phenotype syndrome along with syndromic learning disability and developmental delay. suggested are diagnostic sign of genomic disorder. according to them array based comparative genomic hybridization cause multisystemic developmental diseases in human beings along with learning disability and developmental delay along with learning disability and developmental delay are responsible for most genomic disorder along with craniofacial skeletal and behavioral changes. the study done by berkenstadt. observed partial duplication of the eyebrows with other anomalies in a 7-year - old son. there was excess hair on the forehead and long eyelashes as well as excessive wrinkling of the periorbital skin when the eyes were closed. he had bilateral syndactyly involving the second to the fourth fingers and the second and third toes. gross - kieselstein and har - even also observed the same disorder in brother and sister of north african jewish descent. we could not detect any systemic disorder in our case with physical and laboratory investigations. it is clear that, new reports are still needed to enhance our knowledge about this rare entity. | eye brows are essential for esthetic and functional purposes. various kinds of eye brows are found in human species. protective function is one of the important functions of eye brows. double eye brow is a very rare condition found in human. this case report describes one of the rare cases of double eye brow. |
involvement of the central nervous system (cns) in patients with carcinoma of the uterine cervix is rare and published studies state that only 1% of patients with cervical carcinoma develop cns metastases. most of the reports in the literature on cns metastases from cervical carcinoma are metastases to cerebral or cerebellar parenchyma. meningeal metastases from the uterine cervix with histological variants of squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma and neuroendocrine carcinoma have been reported. metastasis to the dura is less frequent and cervical carcinoma metastatic to dura is extremely rare. in this report, we describe a case of isolated pachymeningeal metastases (dural metastases) from squamous cell carcinoma of the uterine cervix with involvement of dura as well as the optic nerve. a 50-year - old woman presented to our hospital with the chief complaint of bleeding per vaginum for 2 months. she also complained of severe headache, which had worsened in the weeks preceding presentation. pelvic examination revealed a bulky, friable growth in the cervix and a presumptive diagnosis of carcinoma of the cervix was made. no neurological defects were detected and ophthalmic evaluation revealed a normal fundus with no impairment of ocular movement. routine haematological evaluation revealed low haemoglobin levels ; the rest of the parameters were normal. as part of the routine work up, ultrasound scan of the abdomen and pelvis was performed. had increased in severity, contrast enhanced computed tomography (ct) scan of the brain was performed (fig. a lobulated enhancing lesion was also seen in the left orbit involving the dural surface of the optic nerve (fig. no lesions were seen in the cerebral or cerebellar parenchyma nor was there any cerebral oedema, mass effect, or midline shift. cerebrospinal fluid (csf) was obtained for cytological analysis and this revealed the presence of malignant squamous cells, thus confirming the diagnosis of pachymeningeal metastases. figure 1(a) ultrasound image showing the mass in the cervix ; (b) contrast enhanced ct showing enhancement along the falx cerebri ; (c) contrast enhanced ct showing a lobulated mass in the left orbit involving the optic nerve. (a) ultrasound image showing the mass in the cervix ; (b) contrast enhanced ct showing enhancement along the falx cerebri ; (c) contrast enhanced ct showing a lobulated mass in the left orbit involving the optic nerve. brain metastases from squamous cell carcinoma of the uterine cervix are uncommon and isolated dural metastases extremely rare. carcinomatous meningitis occurs when cancer cells gain access to csf pathways, travel to cns sites, settle there and grow. the proposed routes for metastases to get access to the dura include perineural, perivascular, and haematogenous spread or through direct invasion from bony deposits. an isolated case of meningeal metastasis secondary to advanced squamous cell carcinoma has been reported. describe the cytological features of meningeal metastases from cervical squamous cell carcinoma. in both cases, contrast enhanced ct and gadolinium enhanced magnetic resonance (mr) imaging techniques have been used in the diagnosis of leptomeningeal metastases and all neuroimaging features of the disease have been discussed in detail. gadolinium enhanced mr is superior to contrast enhanced ct in detecting meningeal abnormalities when most of the enhancement lies against the skull vault.calvarial metastases from cervical carcinoma inducing a dural tail adjacent to the tumour have also been studied using gadolinium enhanced mr imaging. gadolinium enhanced mri studies of carcinomatous meningitis secondary to large cell cervical neuroendocrine carcinoma have shown diffuse pachymeningeal enhancement. benign conditions may cause leptomeningeal (pia and arachnoid) enhancement with a similar appearance to leptomeningeal metastases and these include infective and inflammatory meningitis, and iatrogenic causes (surgery and chemotherapy). in contrast, carcinomatous meningitis causes pachymeningeal enhancement that is intense and linear, and thickening of the meninges along the inner surface of the calvarium, falx or tentorium without extension into the cortical gyri and basal cisterns. in our case, contrast enhanced ct exhibited thickening of the dura and intense linear enhancement along the falx cerebri suggesting pachymeningeal metastasis. the enhancing mass in the left orbit all these features are suggestive of pachymeningeal metastasis from squamous cell carcinoma of the uterine cervix involving the dural covering of the brain and optic nerve. methotrexate (mtx) is the most commonly used intrathecal chemotherapeutic agent for the treatment of carcinomatous meningitis from cervical carcinoma in addition to radiation therapy. | abstractthis report describes the case of a 50-year - old woman with carcinomatous meningitis from squamous cell carcinoma of the uterine cervix. ultrasound showed an irregular hypoechoic mass in the cervix. contrast enhanced computed tomography imaging revealed intense linear enhancement along the falx cerebri suggestive of pachymeningeal metastasis with involvement of the optic nerve. to our knowledge this is the first reported case of imaging features of isolated metastatic dural involvement from cervical carcinoma. moreover, this is the first case of its kind in which dural involvement was diagnosed at presentation in a locally confined tumour. |
the great saphenous vein (gsv) is the most commonly used conduit for coronary artery bypass grafting (cabg). the referenced standard method of harvesting the gsv is by way of the open surgical technique. depending on the required graft length, the average incision will vary between 20 and 40 centimetres. however, in the population of patients undergoing cabg, risk factors for impaired wound healing are overrepresented. evaluation of 1577 patients that underwent open gsv harvest for cabg in maastricht, revealed that in 1.5% of patients donor - site infections were diagnosed before discharge. remarkably this figure increases to 4.6% at 30 days followup and 7.3% at 90 days followup. therefore 80% of the donor - site infections are diagnosed after discharge, for instance by the patients general physician or cardiologist. furthermore wound complications are often found to be responsible for additional surgery, prolonged hospital stay, increased hospital costs, and in some cases permanent disability [1, 2 ]. the introduction of endoscopic vein harvesting (evh) in 1996 seemed a welcome contribution to achieve goals set in modern cardiac surgery, namely, making cardiac surgery less invasive and promote earlier recovery after surgery. now approximately 15 years after its introduction evh has conquered an important place in standard clinical care (80% of the cabg procedures conducted in the usa use evh). nevertheless, recent publications have raised doubts whether the quality and durability of an endoscopically harvested vein is comparable with a vein harvested in the traditional open fashion [4, 5 ]. is evh indeed a blessing in the attempts to make cardiac surgery less invasive or do the recent publications reveal hidden dangers ? this nonsystematic review article aims to give an overview of 15 years of clinical experience with evh, with appropriate attention for graft quality. based on a pubmed search all original papers, review articles, systematic reviews, and meta - analysis were collected. for the search several combinations of the terms endoscopic, minimal invasive, vessel, vein, conduit, harvest, and harvesting were used. reports investigating radial artery harvest, noncardiac use of vein conduits, the bridging technique, or other nonendoscopic techniques were excluded. during evh disposable or reusable systems are used to harvest approximately 35 cm of the upper leg gsv through a 3 cm incision on the medial side of the knee (figure 1). by repeating the same procedure in the other direction, the entire gsv (70 cm) several systems are available, most frequently used are the disposable systems, namely, vasoview by maquet, virtuosaph by terumo, and clearglide by sorin. even though evh systems have become more user - friendly over the years, a significant personal as well as institutional learning curve persists. analysis of 1348 patients undergoing evh revealed that the average procedural time of the first 50 cases was 68 minutes, while the procedural time dropped significantly to 23 minutes in the last 200 cases. in general one can expect a personal learning curve of 20100 procedures, depending on surgical experience. based on the review of available literature, evh can be considered a save operative procedure. the few case reports describing evh specific complications relate to the use of co2 insufflation, namely, severe hypercapnia, subclinical co2 embolization (10.4%), and massive co2 embolization (0.22%) [6, 7 ]. an important preventive measure is reduction of the co2 insufflation pressure, most importantly not allowing the co2 pressure to surpass the central venous pressure. the most complete overview article investigating noninfectious wound healing disturbances is by athanasiou.. this meta - analysis included 27 studies republished between 1995 and 2002, within total 4953 patients. analysis of 12 randomised controlled trials showed a significant reduction of noninfectious wound healing disturbances (wound drainage, haematoma, oedema, dehiscence, necrosis, need for surgical debridement, and seroma formation) from 13% in open vein harvest to 4% after evh. the number needed to treat in order to prevent one complication was only 2 for oedema and 13 for the other complications. diabetes, hyperlipidemia, obesity, female gender, advanced age, and peripheral vascular disease are known risk factors for wound healing disturbances after open gsv harvest [2, 14 ]. research has however shown that particularly these high - risk patients benefit most from minimally invasive harvest techniques. for example, in patients with diabetes and obesity no additional risk of wound healing disturbances can be found anymore if evh is used [14, 15 ]. it is assumed that evh reduces the number of donor - site infections because it causes less trauma to the surrounding tissue, preserves tissue perfusion, and is less likely to create vital tissue flaps. a meta - analysis from 2003 included all available randomised controlled trials that investigated the incidence of donor - site infections (drainage of pus from the wound, positive wound cultures, and requirement for additional treatment surgical or antibiotic treatment). this meta - analysis revealed that the overall wound infection rate dropped from 13% in the open harvest group to 3% in the evh group. postoperative pain quantified using the visual analogue scale, is significantly reduced after evh compared to the traditional open harvest method [1720 ]. demonstrated that patients undergoing evh rate their experience of pain 2 points lower (on a 010 scale) throughout the whole postoperative period and label themselves pain - free days earlier than their counterparts that underwent open harvest. it is therefore not surprising that a number of studies show that patients undergoing evh are able to mobilize earlier and are also more mobile at hospital discharge and 6 weeks after surgery [17, 19, 21 ]. with the reduction of wound complications and earlier mobilisation of patients, it is likely that recovery time and therefore los is reduced. two independent studies have indeed showed reduction in los in patients undergoing evh, respectively, 31 and 34% reduction compared to traditional conduit harvest [21, 22 ]. another study found a nonsignificant increase of los in the evh group. the remaining 7 available studies investigating this aspect of evh, found trends towards reduced los, without significant differences [17, 18, 2428 ]. a meta - analysis from 2004 with in total 1757 patients confirmed a reduction of los if evh is used. in the netherlands an average disposable - system costs 400 per procedure. so a legitimate question is whether the additional costs of evh are justified by the potential benefits. a controlled trial compared the total hospital costs of 100 patients undergoing either evh or conventional harvest and found no significant differences. the reduction in costs by evh that the authors had expected, was not found because of the longer use of the operating room on the one hand and failure to reduced los on the other. in 2008, a study became available which investigated cost - effectiveness. because data on health - related quality of life was lacking, postoperative pain and mobilisation was used as a measure of effectiveness. procedural costs were calculated based on previous publications on los, operation - time, and prices of the most expensive evh system. so it is important to note that costs of readmissions, outpatient visits related to wound problems and costs of wound treatments were not included in his model. nevertheless, evh was found to be the most cost - effective method of vein harvest. since the scare after evh is considerably smaller, it is not surprising that patients are significantly more satisfied with the cosmetic result after evh then after the traditional harvest [17, 23 ]. however this difference in appreciation, measured using a visual analog scale from 010, is mostly notable during the first postoperative period. whatever the case may be, no improvement in quality of life, measured using a short form 36 questionnaire, has been demonstrated at 2 and 4 weeks after surgery. suture repairs because of holes or torn side branches of the endoscopically harvested vein are 35 times more often necessary then after open vein harvest [12, 32 ]. the presence of these macroscopic lesions make one presume that the endoscopic technique inflicts more trauma to the vessel then the conventional open harvest technique. furthermore, concerns exist with regards to thermal spread due to diathermic coagulation of side branches, detrimental effects of co2 insufflation, and formation of microscopic clots in the collapsed gsv due to the pressurized working tunnel. it has been suggested that this last concern can be dealt with by early systemic heparinization. initial studies addressing graft histology did not find differences in injury to the vascular wall and found comparable endothelial integrity after evh compared to open harvest [17, 19, 27, 34 ]. however, in 2009 boston researchers showed evidence of injury to the saphenous vein endothelium during endoscopic harvest. using three independent techniques (immunohistochemistry, western blot, and multiphoton microscopy), they demonstrated reduced calcium mobilization, nitric oxide production, and esterase activity and reduced levels of von willebrand factor, all signs of impaired structural and functional viability of saphenous vein endothelium. the authors state that the found detrimental effects on saphenous vein endothelium may lead to decreased graft patency and worse patient outcome. only a limited number of studies are available that give insights into angiographic implication of possible histological vein injury. two randomised controlled trials were not able to show significant differences in patency rate between patient undergoing evh or ovh [10, 12 ]. in both studies followup was however short, namely, 3 and 6 months. davis. on the other hand, compared patients who had undergone evh 3.7 years prior, to patients that had undergone open harvest 0.7 years earlier. contrast - enhanced ct showed good patency rate after evh, which did not differ from the control group with the shorter followup. studies that investigate clinical outcome after evh (e.g., recurrent angina, number of reinterventions, recurrent acute myocardial infarction, and survival) are scarcely available. allen. conducted an rct with a fairly small number of patients (112 isolated cabg patients) and found comparable 5 years event - free survival between the evh and open harvest group. if a proper power analysis was conducted remains unclear, since no description is provided. several months after the boston group published about inferior histological properties of endoscopically harvested vein an important clinical paper was published in the new england journal of medicine. conducted a secondary analysis on 3000 patients that had been included in the prevent iv trial. this phase 3 trial had investigated the effect of ex vivo treatment of saphenous vein conduits with edifoligide and had not found any beneficial effect on graft patency. in 1753, study subjects the vein graft had been harvested endoscopically and in the remaining 1247 through open surgery. comparing the two groups revealed that patients who underwent evh had higher rates of vein graft failure at 12 to 18 months. at 3 years, evh was associated with higher death (7.4% versus 5.8%), myocardial infarction, or repeat revascularization. it is important to note that the harvest method was not randomly assigned and that details on harvest technique (e.g., used system, experience of the endoscopist, upper or lower leg harvest, and heparin administration) were missing. a recent observational study compared 5825 patients of whom 34% had undergone evh. at a followup of 2.6 years no correlation between harvest method and recurrent angina, number of reinterventions, acute coronary syndrome, heart failure or survival could be found. this study was also not able to find differences in the rates of freedom from angina, readmission, need for further antianginals or overall survival. for an overview of the available clinical studies see table 1. the aim of minimally invasive conduit harvest techniques is to reduce the morbidity and recovery time associated with the procedure, whilst preserving the quality of the conduit. a series of benefits are well founded adequately : evh results in less wound healing disturbances, less donor - site infections, less postoperative pain, earlier postoperative mobilisation, reduced length of hospital stay, and is likely to be cost - effective. however, recent publications question whether the condition of preserved conduit quality is met. all currently available publication addressing graft quality have important shortcomings, including nonrandomised designs, nonstandardized harvest methods, small sample sizes, retrospective nature, or having only short - term followup. since evh has reached an advanced stage of implementation, and has become accepted and by patients highly appreciated by part of standard care, thorough randomised evaluation has become more difficult. nevertheless a moral obligation exists to ensure without any doubt safety and durability of the endoscopically harvested conduit, since conduit quality is likely to influence clinical outcome of cabg, the golden standard for a considerable portion of patients with multivessel coronary artery disease. therefore randomised aim of such a trial should be to show noninferiority of evh versus open harvest. since we now have reason to suspect clinical implications of possible inferior graft quality based on the findings of lopes., the only relevant endpoint is mace (major adverse cardiac events). the high number of covariates influencing in the primary endpoint will demand hundreds of study subjects in each study arm. the study of lopes. also learned us that followup should be at least one year and that the most interesting secondary endpoint would be graft patency at one year. obviously such a study would be costly, while in this field profit margins are considerably smaller then in the pci industry. willingness of companies to invest large amount of money in such studies will probably be less. nevertheless a randomised controlled trial was initiated by a manufacturer of evh systems, namely, the option study. this study, that is, a single centre, 100 patient trial, investigating graft patency among other endpoint, one year after cabg surgery. results of this and future initiatives will be eagerly awaited, since they will determine the future of endoscopic vein harvesting. | conventional open harvest of the great saphenous vein (gsv) during cabg results in approximately 7% donor - site complications. using endoscopic vein harvesting (evh) the full gsv length can be harvested through a 3 cm incision. this nonsystematic review discusses several key issues concerning evh, based on an extensive pubmed search. found studies show that evh results in reduced number of wound complications, less postoperative pain, earlier postoperative mobilisation, reduced length of hospital stay, and is more cost - effective. initial studies did not find significant differences in graft histology, patency, or clinical outcome. however, in 2009 convincing evidence of inferior histological graft properties became available. furthermore, an observational study showed that evh resulted in significantly more graft stenosis, was associated with higher mortality, more myocard infarction, and more reinterventions. most recent publications could not confirm these findings, however larger randomised controlled trials focusing on graft quality are being awaited. |
gout is one of the oldest recognized afflictions in humans, with a documented history reaching back to the egyptians in 2640 bc. described by hippocrates as the arthritis of the rich due to its association with food and alcohol, gout is an inflammatory arthritis caused by precipitation of monosodium urate (msu) in articular joints and bursal tissues of individuals with hyperuricemia and is characterized by abrupt, self - limiting attacks of joint inflammation. although msu was identified in gout in the 1700s1 and shown to be the causative agent in 1899,2 the mechanisms by which msu crystals trigger acute inflammation have only recently begun to be understood. proinflammatory cytokines undoubtedly have a critical role in orchestrating the inflammatory reaction to msu crystals. recent studies have implicated interleukin-1 (il-1) as a key regulatory proinflammatory cytokine in gout, promoting a neutrophil influx into the synovium and joint fluid that is the pathological hallmark of an acute inflammatory attack.3 compelling evidence for il-1 s role in gout - associated pain and inflammation is provided by studies in both animals and man. in a murine gout model, inflammation following msu injection into the mouse ankle joint was significantly reduced both in mice deficient for the il-1 receptor and in wildtype mice treated with the il-1 inhibitor il1 trap (rilonacept).4,5 in clinical studies, a rapid response was observed in patients with acute gouty arthritis following treatment with a number of anti - il-1 agents (il-1ra [anakinra ], il-1trap, and anti - il-1 monoclonal antibody [canakinumab ]) thereby reinforcing the argument for a pivotal role for il-1 in gout pathogenesis.68 it is currently unclear whether il-1 is also involved in gout pathogenesis since early il-1 inhibitors (anakinra and rilonacept) inhibit both members of the il-1 family ; however the rapid and sustained response of gout patients to canakinumab in a recent phase ii study9 suggests that il-1 may be the more essential of the two il-1 cytokines. il-1 is produced as an inactive pro - molecule by immune cells, including macrophages, monocytes, and dendritic cells, following immune stimulation and is then cleaved to its active, p17 form by caspase-1 (also termed il-1-converting enzyme or ice), itself existing as a proform until the initiation of the innate immune response. the mechanism of caspase-1 and il-1 activation following msu deposition has been the focus of a number of recent high profile studies and is now understood to involve the formation of macromolecular complexes termed inflammasomes. since its discovery less than a decade ago, the nlrp3 inflammasome complex has been implicated as a key regulator of the innate inflammatory phenotype of several diseases, including gout and type 2 diabetes. in addition, mutations in nlrp3 inflammasome components have been shown to be responsible for the dysregulated il-1 production observed in a number of (auto) inflammatory disorders, including muckle wells syndrome (mws), familial cold autoinflammatory syndrome (fcas), and chronic infantile neurologic, cutaneous, and articular syndrome / neonatal onset multisystem inflammatory disease (cinca / nomid). in this review we discuss the mechanisms and pathophysiology of the inflammasome complex and, in particular, its role in mediating the innate immune inflammatory response to msu crystal deposition in gout. the innate immune system is characterized by its ability to recognize and respond to an array of pathogens (including viruses, bacteria, and fungi) and to endogenous molecules released by host cells as a result of necrosis, infections, membrane,10 and lysosomal11,12 damage, injury, or certain pathological conditions (including mammalian dsdna, extracellular atp, and msu crystals),1316 through detection of pathogen - associated and danger - associated molecular patterns (pamps and damps respectively). this response is mediated by germline - encoded receptors termed pattern - recognition receptors (prrs). among the prr families that have been described to date include the toll - like receptors (tlrs), which are associated with extracellular (tlrs 1, 2, 5, 6, and 10) or endosomal (tlrs 3, 7, 8, and 9) membranes and the intracellular rig - like helicases (rlhs) and nod - like receptors (nlrs). tlrs, of which there are 10 known in humans (tlr 110) and 12 in mice (tlr 19 and 1113), are type 1 transmembrane proteins composed of three major domains and characterized by leucine - rich repeats (lrrs) in the ectodomain which mediate pamp / damp recognition. tlrs are expressed on many cell types and are known to respond to a variety of pamps and damps, including bacterial dna,13 lipopolysaccharide (lps),16 peptidoglycan,17 teichoic acids,17 flagellin18 pilin,19,20 viral dsrna,21 and fungal zymosan.22 upon activation they differentially trigger signaling cascades that mediate production of the transcription factors nf-, ap-1, and interferon - regulatory factor (irf)-3, which, in turn, mediate transcription of proinflammatory cytokines, such as interleukins, interferons, and tnf, that drive the inflammatory response. the tlrs have been the subject of extensive investigation and review over recent years and will therefore not be discussed in depth in this review. the two known rlhs, rig-1 and mda5, are viral rna sensors which, upon stimulation, activate nf-b and irf3/7 leading to transcription of type i interferons.23 the intracellular nlrs, like tlrs, are capable of recognizing both pamps and damps, and have become a focal point for investigation of the immune response to foreign agonists and endogenous molecules. known activators of the nlrs include bacterial muramyl dipeptide (mdp),24 microbial toxins (such as alpha hemolysin of staphylococcus aureus),25 rna of bacterial and viral origin,26,27 msu and calcium pyrophosphate dihydrate (cppd) crystals,15 alum,28 asbestos,29,30 silica,29,30 and extracellular atp14 (table 1). to date, at least 23 human and 34 murine nlr genes have been identified, although the physiological functions of many of these are still poorly understood.31 nlrs are characterized by three domains : a c - terminal lrr sensor domain, a central nucleotide - binding domain (nod, also called nacht domain) which regulates self - oligomerization and an n - terminal interaction domain which mediates protein protein interactions with downstream signaling intermediates (figure 1). the n - terminal domain can also be used to categorize the nlrs into 5 subfamilies : nlra (containing an acidic transactivation domain), nlrb (containing a baculovirus inhibitor of apoptosis protein repeat [bir ]), nlrc (containing a caspase - recruitment domain [card ]), nlrp (containing a pyrin domain), and nlrx (containing an unknown domain). of these the best characterized, and the most important in the context of this review, are the nlrps and, in particular, nlrp3. inflammasomes ; multimeric cytoplasmic protein complexes which act as molecular platforms for the activation of inflammatory caspases following stimulation by foreign agonists.32 a typical inflammasome is composed of an nlr, an adaptor protein such as apoptosis - associated speck - like protein containing a card (asc) and an effector caspase that activates proinflammatory cytokines, in particular il-132 (figure 1). to date three nlr proteins have been identified to form inflammasomes : nlrp1, nlrp3, and nlrc4 (also known as ipaf). nlrp3 (nalp3, also known as cryopyrin or pypaf1 [pyrin - containing apaf1-like protein 1 ]), probably the best understood nlrp, has been shown to be involved in the recognition of numerous exogenous and host ligands, including bacterial rna, atp and microbial toxins.33 stimulation of the nlrp3 lrr domain by a foreign agonist is postulated to unfold the nlrp3 molecule enabling recruitment of the asc adaptor proteins and pro - caspase-1. this process of inflammasome assembly results in cleavage of pro - caspase-1 to produce active caspase-1, which in turn cleaves pro - il-1 to produce il-1 and also activates il-18. notably no study has yet demonstrated direct interaction between the lrrs of nlrs and their respective activators, suggesting that indirect mechanisms of sensing and activation may be involved. conceptually, given the diverse array of molecules now known to activate nlrs it follows that additional indirect mechanisms may play a role in mediating inflammasome activation. although the precise molecular details have not yet been fully elucidated, current evidence points to the potential roles of three signaling pathways in mediating agonist recognition and inflammasome activation. firstly, recent studies suggest a central role for p2x7 receptor - mediated k+ efflux in nlrp3 activation, with prevention of k+ efflux abolishing nlrp3 in response to almost all known activators.3436 secondly, activation of the nlrp3 inflammasome by crystals and particulates appears to involve the phagocytic pathway, with particle uptake disrupting the phagolysosome acidic compartment and subsequent release of capthepsin b.12,37,38 finally, reactive oxygen species (ros) generation has been suggested to be critical for inflammasome activation in response to a number of stimuli. inhibition of ros generation, either through pharmacological inhibitors of napdh oxidase or sirna - mediated knockdown, prevents il-1 release in response to msu and a number of other stimuli.29,30 however, the role of ros is still unclear since further studies suggest that increased ros production may actually inhibit caspase-1 activation.39 it is conceivable that the mechanisms involved in inflammasome - activation may differ according to both agonist and cell type. a recent study supports this hypothesis, demonstrating that whilst il-1 release by in vitro monocyte cultures required only a single exogenous stimulus, two distinct exogenous stimuli were required for il-1 production in macrophage cultures.40 the difference between these two cell types is understood to be due to the presence of constitutively active caspase-1 in monocytes and also the ability of monocytes to release endogenous atp, which can act as a second stimulus to trigger il-1 release. in contrast, macrophages require a primary signal to induce transcription and translation and a second distinct signal for caspase activation, which leads to il-1 processing and release. recently an additional inflammasome sensor was described, which triggers caspase-1 and il-1 cleavage in an asc - dependent, nlr - independent manner. the aim2 inflammasome contains a hin200 domain that binds cytosolic dna, including that of bacterial, viral, and mammalian origin, leading to recruitment of the asc adaptor protein to its pyd and subsequent activation of caspase-1 and il-1.4144 negative regulation of inflammasomes is mediated by a family of small proteins composed of a card or pyd only domain. iceberg, pseudo - ice, caspase-12, inca (inhibitory card), and nod2-s which, with the exception of the rip2-interacting nod2-s protein, regulate nlrp3 activity through interaction with the caspase-1 card domain, thereby preventing its interaction with other inflammasome components.4548 two pyd - only proteins, pop1 and pop2, interact with asc or nlrp thereby preventing their recruitment into inflammasome complexes. limited data currently exist as to how these negative regulators are themselves regulated, their different roles in mediating inflammasome response and their involvement in inflammatory disease. notably, caspase-12 deficient mice show increased il-1 secretion49 whilst caspase-12 gene polymorphisms have been linked to the modulation of inflammatory and innate immune responsiveness.50 in addition to these molecular mechanisms of regulation, cd4 + effector and memory t - cells were shown to negatively regulate inflammasome activity in a cd40l - dependent manner.51 inhibition of the nlrp3 inflammasome was found to be dependent on both t - cell activation and antigenic stimulation, suggesting that migration of t - lymphocytes to inflammatory sites may provide a feedback loop to regulate the production of inflammatory mediators during the later phases of inflammatory responses. inflammasomes are now understood to have a fundamental role in the development of autoinflammatory diseases and additional roles in infection control, development of immune pathologies, and recognition of tissue damage. the last decade has seen the emergence of evidence supporting this concept, with the first established link to pathological processes provided by genetic studies of autoinflammatory diseases. cryopyrinopathies (also referred to as hereditary periodic fever syndromes or cyropyrin - associated periodic syndromes) form a clinical continuum of autoinflammatory disorders characterized by recurrent fevers and inflammation. most cases of these three disorders (fcas, mws, and nomid in order of increasing severity) are now understood to be caused by autosomal dominant or de novo gain - of - function mutations in nlrp3. over 40 mutations have been identified in nlrp35256 each producing a constitutively active form of nlrp3, most likely due to spontaneous nlrp3 oligomerization, and hence elevated levels of secreted il-1, which drive the inflammation. in keeping with this, gene - targeted mice harboring mutations equivalent to those found in mws exhibit hyperactive nlrp3 inflammasomes leading to elevated il-1.57 this discovery has revolutionized the clinical management of these diseases, with patients responding well to il-1 antagonism, for example by anakinra,5860 next - generation il-1 antagonists,6165 or caspase-1 inhibitors.66 two additional autoinflammatory diseases, familial mediterranean fever (fmf) and pyogenic arthritis, pyodermagangrenosum, and acne (papa) are also caused by mutations in the inflammasome pathway ; fmf by mutations in the pyrin - encoding mefv gene67 and the syndrome of papa by mutations in the gene encoding proline - serine - threonine - phosphatase- interacting protein (pstpip1), which binds pyrin through its src - homology-3 domain.68,69 genetic variants in nlrp3 and other inflammasome - related genes have also been related to susceptibility and disease severity in a number of other chronic inflammatory disorders, including rheumatoid arthritis,70,71 crohn s disease,72 gout,73 and the recently identified deficiency of the interleukin-1-receptor antagonist (dira).74 in addition to causal mutations in the nlrp3 inflammasome pathway, the pathology of a number of inflammatory diseases has been linked to the activation of this inflammasome by disease - specific inflammasome agonists. for example, the fibrillar peptide amyloid-, which accumulates to form the senile plaques responsible for the pathogenesis of alzheimer s disease,37 is now known to be an inflammasome activator. studies suggest that nlrp3, caspase-1, and il-1 are essential for the recruitment of microglia to exogenous amyloid- in the brain and for microglial synthesis of proinflammatory and neurotoxic factors, indicating a critical role for the inflammasome pathway in the innate immune response to amyloid- and the subsequent tissue damage that is the hallmark of alzheimer s disease. in type 2 diabetes, islet amyloid polypeptide, which forms amyloid deposits in the pancreas, activates nlrp3, resulting in il-1 release and inducing apoptosis of pancreatic beta cells75 whilst cholesterol crystals have been shown to trigger inflammasome activation in atherosclerosis.76,77 late - stage human melanoma cells exhibit autoinflammatory characteristics, spontaneously secreting active il-1 due to constitutive assembly and activation of nlrp3.78 il-1 mediated autoinflammation drives macrophage chemotaxis and angiogenesis, thereby directly contributing to disease development and progression.78 the inflammasome has also been linked to the progression of chronic kidney disease79 and to a number of viral infections.8082 as the latter have been reviewed in depth recently,83 they will not be discussed further here. although the above examples point clearly to a role for nlrp3 inflammasome activation in an array of human diseases, the involvement of this pathway in various disease processes is far from straightforward, as most notably highlighted by a recent paper demonstrating that in colitis - associated cancer the nlrp3 inflammasome plays a protective role, with asc/ and caspase-1/ mice showing increased disease severity and morbidity.84 although the causal agent of gout has been understood for more than a century, the mechanisms underlying msu crystal - induced inflammation have only recently begun to be unraveled. the pathological hallmark of a gout attack is considerable neutrophil influx into the synovium and joint fluid,3 however since neutrophils are absent in the normal joint the primary event following precipitation of msu within the joint is believed to be the interaction of msu crystals with resident joint cells, principally the synovial lining cells, which in turn trigger neutrophil ingress. recent in vitro studies implicate mononuclear phagocytes as playing a central role in the initial response to msu precipitation. the exposure of monocyte cell lines to msu crystals leads to the production of proinflammatory cytokines, in particular il-1,85,86 and it is now understood that phagocytosis of msu crystals is central to this process12 (figure 2). following the recognition that msu crystals may act as a danger signal to cells in a similar way to microbial pathogens,87 martinon demonstrated the nlrp3 inflammasome to be critical in sensing msu deposition and subsequent activation of the downstream innate immune response.15 macrophages from mice deficient in various components of the inflammasome, including caspase-1, asc, and nlrp3, were unable to activate il-1 in response to msu stimulation. notably, these mice also showed impaired neutrophil influx following intraperitoneal msu injection, demonstrating the nlrp3 inflammasome as a critical link between the well - established causal stimulus of gout and the subsequent pathological hallmark of an acute gout attack. similar results were also demonstrated with cppd crystals, the causal agent of pseudogout, suggesting that similar mechanisms are involved in regulating the inflammatory response in both of these diseases. despite much progress in elucidating the biological pathways coordinating a gout attack, the precise mechanisms by which endocytosed msu crystals activate the nlrp3 inflammasome remain unclear and it is possible that sensing may occur through intermediary protein(s). interestingly, the reduced severity of the inflammatory response triggered by cppd deposition in pseudogout and the inflammasome neutrality of the chemically and structurally similar allopurinol, suggests that subtle differences in the physical properties of different crystals, including surface charge and size, may be important in determining the intensity of the inflammatory response. a recent study has shed light on the biological mechanisms underlying the association between excessive consumption of food and onset of gout with the demonstration of a synergistic relationship between free fatty acids (ffa) and msu for the activation of il-1.88 in this study joosten and colleagues demonstrated that msu crystals alone were unable to induce il-1 release from peripheral blood mononuclear cell (pbmc) isolated from healthy subjects ; however in the presence of ffa, large amounts of active il-1 were detected. notably, il-1 release in response to msu and ffa injection was significantly reduced in caspase-1 and asc deficient mice, but not in nlrp3 gene deficient mice.88 how these data fit with earlier studies indicating dependency on nlrp3 for msu - induced il-1 production is unclear and may point to the involvement of alternative inflammasome complexes in the response to msu or relate to differences in cell lineage, cell priming, and/or crystal structure between the studies. in addition to the intracellular nlrp3 receptor, the extracellular tlr2 and tlr4 receptors may also play a role in the innate immune response to msu deposition.89 macrophages isolated from tlr and tlr mice show impaired uptake of msu crystals and reduced proinflammatory cytokine production, suggesting these tlrs to be essential for msu crystal - induced inflammation.89 however conflicting results from a peritoneal model of acute gout suggest tlr2 and tlr4 to be dispensable in the immune response to msu crystals.90 nevertheless, as mentioned briefly above, it is conceivable that the contradictory results of these two studies may be attributable to cells in different tissues responding to msu stimulation through different mechanisms. more recent studies now suggest that rather than having a role in the recognition of msu crystals, the tlrs may play a more indirect part in responding to msu deposition through regulation of the synthesis of pro - il-1 (figure 2).90,91 together these studies allow a picture to be painted of our current understanding of the inflammatory process of an acute gout attack (figure 2). during the initiation phase, msu crystals precipitated within the joint stimulate extracellular tlr receptors expressed by resident monocytes, leading to transcription of pro - il-1. msu crystals are also phagocytosed by resident monocytes, an activity which is thought to be positively regulated by tlr activation,89 resulting in oligomerization of the nlrp3 inflammasome, activation of caspase-1, and subsequent cleavage of pro - il-1 to produce active il-1. as discussed above, k+ efflux, ros, and/or cathepsin b may also be involved in mediating nlrp3 activation. following cleavage, active il-1 is released by the resident monocytes and binds to il-1 receptors expressed by endothelial cells and resident macrophages. during the amplification phase these cells respond to il-1 by producing an array of proinflammatory cytokines and chemokines, which act together to coordinate the recruitment and activation of leukocytes into the joint. one of the characteristic features of gout is that regardless of treatment, gout attacks are self - limiting. however, although great strides have been made in unraveling the molecular mechanisms of gout onset, our understanding of the mechanisms leading to resolution of a gout attack remain far less advanced. a range of possible mechanisms have been suggested, including the binding of crystals to inhibitory proteins,9294 maturation of monocytes to macrophages, which express the anti - inflammatory tgf-1 in response to msu,95,96 and clearance of apoptotic leukocytes by macrophages.97,98 the balance between the multiple regulatory mechanisms that appear to be involved in mediating activation of the inflammasome pathway by msu crystals, including ros and k+ efflux, and inhibitors of the inflammasome pathway, including caspase-12 and cd4 + memory t - cells, may also be important in the down regulation of the inflammatory response that occurs during the resolution of an inflammatory gout attack. the british society for rheumatology recommends nonsteroidal anti - inflammatories (nsaids) as the first drug of choice for an acute gout attack, followed by systemic or intra - articular glucocorticosteroids where nsaids are contraindicated. 99 the third line of treatment is oral colchicine, which is highly effective when given early in an acute gouty attack, but is poorly tolerated because of predictable gastrointestinal side effects. notably, colchicine has been shown to suppress msu crystal - induced nlrp3 inflammasome protein complex assembly, most likely through microtubule inhibition and subsequent impaired delivery of msu crystals to the nlrp3 inflammatory protein complex in the cytosol, although this effect requires relatively high (micromolar) concentrations of colchicine.15 recent animal model studies, using anakinra (an il-1r antagonist) and mil-1 trap (an il-1 inhibitor), clearly demonstrate the potential of targeting il-1 for the management of msu - induced inflammation.4,5 moreover, early clinical studies of the il-1 inhibitors, rilonacept (il-1 trap),6 canakinumab (monoclonal anti - il-1 antibody),9 and anakinra,7,8,100 have demonstrated efficacy in the treatment of patients with acute and chronic gout and have been generally well tolerated, although there are always concerns about increased incidence of infections when proinflammatory cytokines are inhibited and it should be noted that this has been observed in a subset of patients in a number of il-1 inhibitor trials. these studies, coupled to in vitro evidence of the key role of the inflammasome pathway in activating il-1 in gout, point to il-1 and the upstream inflammasome regulatory pathways as key targets for the future treatment of gout. gout is the most common form of inflammatory arthritis in older men, affecting 1%2% of adults in developed countries. despite its prevalence, treatment options for gout have remained static for almost half a century, with the approval of allopurinol in 1965 representing the last drug approved by the us food and drug administration for treating gout. however, the last year has seen the approval of a new gout therapy, the xanthine oxidase inhibitor, febuxostat, and with several new drugs now in the late stages of clinical testing6,9,101 coupled with our enhanced level of understanding of the pathophysiology of the inflammatory process involved, we are entering a new era for the treatment of gout. although the precise molecular details of the nlrp3 inflam masome pathway and its response to msu crystal deposition remain to be determined, the importance of inflammasomes and il-1 activation to the pathology of gout is now firmly established. notably, current strategies for targeting il-1 have proved successful for alleviating the symptoms of gout in small clinical studies suggesting that targeting this and other components of the pathway may have an important place in gout and pseudogout treatment.69 moreover, the impressive results of il-1 inhibitors for the treatment of autoinflammatory syndromes confirm the potential of these agents for diseases such as gout, which involve excessive il-1 production. despite significant advances in our understanding of the inflammatory processes involved in an acute gout attack, many questions remain to be addressed. the precise mechanism by which crystal recognition is achieved remains elusive, whilst the role of additional cytokines, which may be activated by the nlrp3 inflammasome, such as il-18, must also be evaluated. moreover, msu may trigger other pathways aside from the nlrp3 inflammasome and it will be important to identify such pathways and understand their interaction with the nlrp3 inflammasome and il-1 production in the acute and resolution phases of gout. answers to these questions will no doubt provide important insights into the mechanisms underlying msu - induced inflammation and resolution, but may also reveal novel biologically relevant targets for the development of new, more effective therapeutic options for the treatment of gout. | gout is an inflammatory arthritis characterized by abrupt self - limiting attacks of inflammation caused by precipitation of monosodium urate crystals (msu) in the joint. recent studies suggest that orchestration of the msu - induced inflammatory response is dependent on the proinflammatory cytokine il-1, underlined by promising results in early il-1 inhibitor trials in gout patients. this il-1-dependent innate inflammatory phenotype, which is observed in a number of diseases in addition to gout, is now understood to rely on the formation of the macromolecular nlrp3 inflammasome complex in response to the msu danger signal. this review focuses on our current understanding of the nlrp3 inflammasome and its critical role in msu - crystal induced inflammatory gout attacks. it also discusses the management of treatment - resistant acute and chronic tophaceous gout with il-1 inhibitors ; early clinical studies of rilonacept (il-1 trap), canakinumab (monoclonal anti - il-1 antibody), and anakinra have all demonstrated treatment efficacy in such patients. |
the classic symptom of spinal canal stenosis is pseudoclaudication or neurogenic claudication.16 the classification of spinal stenosis proposed by arnoldi. (1976) remains useful.7 patients typically complain of pain, paresthesia, weakness, or heaviness in the buttocks radiating into lower extremities with walking or prolonged standing, relieved with forward bending and sitting. patients with severe spinal canal stenosis either do not improve with conservative measures or have frequent recurrent symptoms. the long term outcomes of decompressive surgery for relief of pain and disability are still unclear. this study evaluates the outcome of surgical management of secondary degenerative lumbar canal stenosis and analyzes the effect on different outcome variables using the joa score. this prospective study was conducted at our hospital between august 2002 to october 2010 after obtaining clearance from the institutional ethical committee. during this period, 46 patients of degenerative lumbar canal stenosis were deemed eligible for operative treatment based on the inclusion and exclusion criteria out of which 14 patients did not consent and thus a total of 32 patients underwent surgical treatment. patients who had posture - related radicular pain with claudication distance less than 100 m and who could not carry out their routine daily activities were assessed with magnetic resonance imaging (mri) [figure 1a and 1b ]. surgery was performed if the central canal diameter on mri was found to be less than or equal to 10 mm. spinal instability was assessed using flexion and extension lateral radiographs using posner 's criteria.8 patients with primary bony canal stenosis, traumatic lumbar canal stenosis, stenosis due to tumors and infection, and patients not medically fit for surgery due to comorbidities were excluded from the study. instrumentated stabilization was done in all cases with preoperative instability and when laminectomy was done at more than one level. all procedures were performed by senior orthopaedic surgeon. according to this protocol, laminectomy with decompression was done in 2 cases, laminectomy and disectomy was done in 23 patients, laminectomy, disectomy with instrumented stabilization was done in 5 cases, and laminectomy, disectomy with posterior lumbar interbody fusion was performed in 2 patients. pre and posttreatment assessment of the patients was done according to joa evaluation system for low back pain. the joa score was determined by direct questioning to assess subjective symptoms, clinical signs, and restriction of activities of daily living. the recovery rate of the patients following treatment was calculated by using the description of hirabayashi. recovery rate was classified using a four - grade scale : excellent, > 90% ; good, 7589% ; fair, 5074% ; and poor, below 49%.9 (a) preoperative ap and (b) lateral x - rays of a 52 years old male patient who presented with secondary degenerative lcs at l3 - 4, l4 - 5 with retrolisthesis of l4 over l5. his preoperative joa score was 5 (a) preoperative t2 sagittal mri section of the same patient showing degenerative lcs l3 - 4, l4 - 5 with degenerated disc at l2 - 3, l3 - 4, l4 - 5. discs at l3 - 4 and l4 - 5 were found to be soft and bulging intraoperatively and hence were removed along with posterior decompression, laminectomy and pedicle screw fixation from l2 to l5, (b) preoperative t2 axial mri section showing large herniated disc at l3 - 4. the patient attained joa score of 28 at 3 months followup which was maintained till last followup of 7 months preoperative and postoperative joa scores at immediate, 3 month, 6 month, and 1 year followup were compared using wilcoxon 's test for nonparametric data. preoperative and postoperative joa scores at immediate, 3 month, 6 month, and 1 year followup were compared using wilcoxon 's test for nonparametric data. complete data of all the 32 patients along with their joa scores are presented [table 1 ]. distribution of patients in all the variables of joa scoring system was assessed before and after treatment [table 2 ]. clinical details and outcome of all patients distribution of variables of joa score in surgically managed lcs patients (preoperative vs. three months postoperative) preoperatively, 71.87% of patients (n=23) presented with continuous severe low backache, 25% (n=8) with occasional severe low backache, and 3.13% (n=1) presented with occasional mild low backache. three months postoperatively, 62.5% patients (n=20) had no back pain and 37.5% (n=12) had occasional mild low back pain. most of the patients (87.5%, n=28) had presented to us with posture related severe leg pain, but postoperatively 96.87% patients (n=31) had no leg pain. all patients had preoperative claudication distance less than 100 m, but 93.75% patients (n=30) had normal gait with walking distance more than 500 m and no claudication symptoms postoperatively. the most common level of involvement was l4-l5(81.82% patients, n=27) followed by l5-s1(54.55% patients, n=18). 93.74% patients (n=30) had abnormal straight leg raising test [46.87% patients (n=15) had straight leg raising positive below 30 and 46.87% patients (n=15) had between 30 and 70 ], but postoperatively all patients had normal straight leg raising test. sensations were diminished in l4 dermatome in 3 patients, l5 dermatome in 14 patients and s1 dermatome in 8 patients. overall, 20 patients (62.5%) had shown sensory disturbance preoperatively, but postoperatively 19 of these 20 patients recovered normal sensory function. motor weakness was present in 15 patients (46.87%) preoperatively, but postoperatively only 6 patients (18.75%) showed motor deficit. overall, 93.75% patients (n=30) in our study showed improvement in all variables of the joa scoring system postoperatively. at 3 month followup, 18.75% (n=6) patients showed excellent outcome, 62.50% (n=20) showed good outcome, and 18.75% (n=6) patients showed fair outcome [table 3 ]. at 6 month followup, 38.46% patients (n=10) showed excellent outcome and 61.54% patients (n=16) showed good outcome. at 1 year followup, 64.00% patients (n=16) showed excellent outcome and 36.00% patients (n=9) showed good outcome. at final followup, 64% patients (n=16) showed excellent outcome, 28% (n=7) showed good outcome, and 8% (n=2) showed fair outcome. outcome of the patients improved as the time after surgery increased till 1 year and was sustained thereafter till the last followup (only two patients showed decrease in their recovery rate which was due to prolapsed disc or canal stenosis at a different level). statistically significant improvement was seen in all variables except running and lifting heavy weight [table 2 ]. outcome of 32 surgically managed patients assessed by pre and posttreatment (joa) score on comparison of preoperative and three months postoperative joa scores using wilcoxon 's test for nonparametric data, p value is < 0.001 which meant that outcomes were extremely significant postoperatively. further, joa scores significantly improved even postoperatively till 1 year (p<0.05). after 1 year, the joa scores did not change significantly with time till the last followup. in our study, 40.63% (n=13) patients were in the age group 5059 years, followed by 4049 year age group, and the average age was 45.1 years, with similar age and sex distribution reported by others.10 64% patients showed excellent and 28% showed good outcome at the end of 1 year followup, while ganz. (1990) reported almost similar result showing 86% good outcome in their series of 33 patients treated by decompressive surgery. in their patients whose preoperative symptoms were relieved by postural changes, the success rate was 96% compared to only 50% in those unchanged by postural changes.11 weinstein. (2010) showed that patients with degenerative spondylolisthesis and spinal stenosis treated surgically showed substantially greater improvement in pain and function during a period of 2 years than those treated nonsurgically.12 in our study, finally, 62.5% patients had no back pain and 37.5% had occasional mild pain, 96.87% had no leg pain, 93.75% had normal gait, 100% had normal straight leg raising, and 95% had sensory improvement (i.e. 19 patients out of 20 who presented with sensory impairment). (1991) with average leg pain improvement of 82% and average back pain improvement of 71%.13 weinstein. (2010) in their prospective multicentre sport study of 654 patients concluded that patients with symptomatic spinal stenosis treated surgically compared to those treated nonoperatively maintain substantially greater improvement in pain and function through 4 years. all patients in their study were surgical candidates with a history of at least 12 weeks of neurogenic claudication or radicular leg symptoms and spinal stenosis without spondylolisthesis (as confirmed on imaging). they were enrolled in either a randomized cohort (289 patients) or an observational cohort (365 patients) at 13 u.s. spine clinics and were treated by either standard decompressive laminectomy (414 patients) or usual nonsurgical care (240 patients).14 no patient in our series had poor result. this could be due to the fact that all patients underwent at least a 12 weeks trial of adequate conservative treatment and were only operated after clinicoradiological correlation of their symptoms with imaging was confirmed. decompressive laminectomy was also adequately supplemented with pedicle screw fixation (in cases of preoperative instability or when more than one level laminectomy was performed) and/or posterior lumbar interbody fusion (in cases of degenerative listhesis) and/or discectomy (cases with a soft bulging disc). the failure of surgery to completely relieve pain in these two patients may be attributed to the widespread degeneration. outcome was also affected due to some variables in scoring system like running and heavy weight lifting in which female patients and patients over 50 years scored less despite being free of pain. getty (1980) personally reviewed 31 patients (age range 18 to 75 years) who had been treated surgically for lumbar spinal stenosis between 1968 and 1978 and followed them for an average of 42 months. in 28 (90%) patients, degenerative changes in the lumbar spine had been the principal etiological factor ; the other 3 had idiopathic developmental lumbar spinal stenosis. in 17 (55%) patients, the result was classified as good, although a total of 26 (84%) patients were satisfied. this compares well with our study in which 93.75% patients (n=30) were satisfied with their surgery. good results of operation for lumbar spinal stenosis in series of getty were characterized by rapid resolution of pain in the leg. the most important reason for failure to relieve symptoms in his series was stated to be inadequate decompression.15 postacchini. (1992) noted bone regrowth in 88% of 40 patients who had laminectomy or laminotomy for spinal stenosis at an average of 8.6 years of followup. bone regrowth was noted in all patients with associated spondylolisthesis.16 we did not observe any case of bone regrowth in our series. a possible explanation could be that interbody fusion was done in all cases of listhesis and pedicle screw fixation to supplement any case with preoperative instability or multilevel decompression in our series. moreover, we performed a wide laminectomy with medial facetectomy in all our patients as compared to narrow laminotomy in some cases of postacchini. postacchini (1999) described that 7080% of patients of lumbar canal stenosis had satisfactory result from surgery, but the outcome tended to deteriorate in the long term.17 our outcomes improved postoperatively till 1 year but therafter neither showed any improvement or deterioration till last followup. the authors are of the opinion that operative treatment in patients of degenerative lumbar canal stenosis yields excellent long term functional results as observed on the basis of joa scoring system provided that patients are properly selected and decompressive surgery is performed simultaneously addressing the associated instability or listhesis. all activities of daily living which were assessed using joa score showed significant improvement except for running and lifting heavy weight. | background : the long term outcomes of decompressive surgery on relief of pain and disability in degenerative lumbar canal stenosis are unclear. the aim of our study was to evaluate the outcome of surgical management of secondary degenerative lumbar canal stenosis and to analyze the effect on outcome variables using japanese orthopaedic association (joa) score.materials and methods : thirty - two patients of degenerative lumbar canal stenosis managed surgically were included in this study. laminectomy (n=2), laminectomy with disectomy (n=23), laminectomy and disectomy with instrumental stabilization (n=5), and laminectomy, disectomy with posterior interbody fusion (n=2) were performed. joa scoring system for low backache was used to assess the patients. the recovery rate was calculated as described by hirabayashi. (1981). surgical outcome was assessed based on the recovery rate and was classified using a four - grade scale : excellent, improvement of > 90% ; good, 7589% improvement ; fair, 5074% improvement ; and poor, below 49% improvement. the patients were evaluated at 3 months, one year and at last followup.results:at 3-month followup, 18.75% patients showed excellent outcome, 62.50% patients showed good outcome, and 18.75% showed fair outcome. at 1-year followup, 64% patients showed excellent outcome and 36% patients showed good outcome. at > 1 year followup (average 34.2 months, range : 2110 months), 64% patients showed excellent outcome, 28% showed good outcome, and 8% showed fair outcome. no patient had poor outcome. outcome of the patients improved as the time after surgery increased till 1 year and was sustained thereafter till the last followup.conclusion:operative treatment in patients of degenerative lumbar canal stenosis yields excellent results as observed on the basis of joa scoring system. no patient got recurrence of symptoms of nerve compression. |
temporomandibular joint ankylosis is defined as the fusion of joint surfaces by bone or fibrous tissue. inability to open the mouth in temporomandibular joint ankylosis results in inability to maintain oral hygiene, inability to chew properly and esthetic problems.[13 ] this leads to dental caries, malocclusion, weight loss and growth retardation. immediate and delayed postoperative pain at the operative site is distressing to the patient and has considerable morbidity. pain at the operative site prevents patients from opening their mouth and it is a major problem that hinders the early active physiotherapy. adequate pain relief at the operative site becomes most important to restore early movements at the temporomandibular joint. this can be achieved by using systemic opioid analgesics or long - acting local anesthetic injection at the operative site. the aim of the present study was to assess the postoperative pain control at the operated site and mouth opening by either placing an epidural catheter in the incision wound and infusing ropivacaine 0.25% or by using a transdermal fentanyl patch. after getting a written informed consent from all the subjects, 80 patients, between 18 and 32 years, of american society of anesthesiologists physical status grade i or ii, average height of 150160 cm and 20% of the ideal body weight, admitted for temporomandibular joint ankylosis surgery, were enrolled in the study as per protocol. inclusion criteria included the subjects with unilateral and/or bilateral bony temporomandibular joint ankylosis, scheduled for temporomandibular joint interpositional gap arthroplasty. obstinate patients, patients with any cardiac and/or respiratory disease and patients allergic to local anesthetic agents were excluded from the study. the visual analog scale (vas) for scoring pain was explained to all the subjects. subjects were also informed that some treatment will be given, which might be helpful in decreasing the postoperative pain. subjects were selected using simple randomized sampling and allocated to either of the 2 groups, using sealed opaque envelopes, by the numbers randomly generated by computer table. all the subjects were fasted for 12 h to eliminate the risk of aspiration during surgery. standard monitoring with non - invasive blood pressure, electrocardiography and pulse oximetry was done. flexible fiber optic laryngoscope and instruments for tracheostomy were kept ready for emergency access to a definitive airway. all subjects received injection glycopyrrolate 0.004 mg / kg body weight iv and injection ondansetron 0.08 mg / kg body weight iv and were preoxygenated for 34 min. this was followed by slow injection fentanyl 2 g / kg iv plus injection midazolam 0.05 mg / kg body weight iv. anesthesia was induced with propofol 2 mg / kg body weight iv and rocuronium 0.9 mg / kg body weight iv. anesthesia was maintained with 1% isoflurane and nitrous oxide / oxygen combination (60/40). at the end of interpositional gap arthroplasty, ropivacaine group (g rop) patients received an epidural catheter in the incision wound at the operated site just above the galea aponeurotica with its tip in the temporomandibular joint and this was followed by the closure of surgical wound in layers [figure 1 ]. after the closure of surgical wound, these patients received injection ropivacaine 0.25% infusion @ 1.6 ml / h through the epidural catheter. visual analogue score during postoperative period transdermal fentanyl group (g tf) patients did not receive epidural catheter instead a fentanyl patch (duragesic, johnson and johnson ltd, mumbai, release at 25 g / h) was applied to a clean, dry area on the upper arm, before conclusion of the surgery. the patch was pressed firmly for 30 s to make sure that the patch stays in place. at the conclusion of surgery, neuromuscular block was antagonized with neostigmine 0.5 mg / kg body weight and glycopyrolate 0.01 mg / kg body weight iv, patients were extubated and transferred to the postanesthesia care unit (pacu). all subjects remained in the pacu for at least 8 h to observe respiratory depression. postoperative pain was assessed by a senior resident (nonanesthetist) blinded to group allocation, using vas scoring (horizontal line from 0 to 100, with 0 representing no pain and 100 representing maximal unbearable pain). the patients were asked to draw a point in the horizontal line denoting the present intensity of their pain. pain was assessed 2, 4 and 8 h after surgery and each morning, until the 7th day after surgery and after that the epidural catheter and fentanyl patch were removed. mouth opening was assessed in the immediate postoperative period, on day 4 and day 10. sample size was calculated using power and sample size calculator by the department of biostatics, vanderbilt university, usa. with 80% power (20% beta error) and 95% confidence level (5% alpha error), the sample size calculated was 34, in each group. patient characteristics, duration of surgery, time to first analgesic requirements, number of diclofenac tablets consumed by each group of patients during the first 4 days after surgery and vas scoring at rest were compared using paired and unpaired student 's t test. patient characteristics and duration of surgery were found to be similar among both the groups [table 1 ]. patient characteristics the time to first analgesic requirement was found to be significantly (p 0.001), but it was significantly better in g rop at 4th and 10th postoperative days (p < 0.001) as compared with g tf [table 3 ]. sensitization of dorsal horn neurons has been demonstrated in acute postoperative pain and this may also play a role in the development of chronic pain after surgery. by reducing the hyperexcitability of dorsal horn neurons or by stabilizing the neuronal membrane, fentanyl and ropivacaine may have roles in the treatment of postoperative pain. therefore, systemic opioid analgesics or long - acting local anesthetic injection at the operative site can be used for postoperative pain relief. hypothesis of infiltrating the wound with bupivacaine in the operated site leads to significant reduction of pain. also with ropivacaine and morphine, similar results were obtained.[912 ] our results demonstrate that the drugs administered as per protocol of the study, significantly reduced the requirement of analgesics throughout the postoperative period. fentanyl, a synthetic opioid agonist interacts primarily with the (mu) receptor. the low molecular weight, high potency and lipid solubility of fentanyl make it a suitable opioid analgesic. the use of fentanyl is a well established and accepted indication in the treatment of pain, as indicated by recent review articles.[1417 ] in our study, transdermal fentanyl patch was applied once before the conclusion of surgery (t 1/2 after transdermal administration : 1322 h) and the patch was replaced at every 24 h for 3 days. our aim was to achieve more consistent serum levels of fentanyl and to reduce analgesic requirement during the early postoperative period. as with other opioid agents, the most frequently observed adverse event during transdermal fentanyl treatment is hypoventilation. it has occurred in 3 (2%) of the 153 patients with cancer pain during the previous premarketing trial. clinically relevant fentanyl - induced respiratory depression, however, was not observed during the previous 3 randomized trials. in our study, for the doses given along with the duration of administration of transdermal fentanyl patch, there was no observable respiratory depression. furthermore, any expected incidence of nausea and vomiting was prevented by the prophylactic use of ondansetron, as per the protocol of our study. ropivacaine is a new amide type long - acting local anesthetic agent mainly used for providing anesthesia and postoperative analgesia. at higher doses it provides surgical anesthesia and at lower doses it provides analgesia. the drug at clinically relevant concentrations reduces the membrane voltage - gated sodium currents in nerve fibers. it produces analgesia by reversible blockade of impulse propagation along the nerve fibers by preventing the inward movement of sodium ions across the nerve cell membrane. unlike bupivacaine side effects of ropivacaine include nausea, paresthesia, dizziness, dyspnea, syncope and cardiac arrhythmias. we did not observe intolerable side effects for the daily dose and the duration of treatment, as determined by the protocol of the study. this analgesic treatment was not associated with adverse effects and may be appropriate for patients who have undergone temporomandibular joint interpositional gap arthroplasty. in our study, ropivacaine infusion provided superior analgesia and better mouth opening throughout the postoperative period as compared with transdermal fentanyl patch. it is concluded that placing epidural catheter at the incision wound and continuously infusing with ropivacaine 0.25% effectively controls the postoperative pain and provides better mouth opening in patients who have undergone temporomandibular joint interpositional gap arthroplasty. | aimthe purpose of this study was to evaluate the postoperative pain control and mouth opening in patients undergoing temporomandibular joint interpositional gap arthroplasty by either placing an epidural catheter in the incision wound and infusing ropivacaine 0.25% or by using a transdermal fentanyl patch.materials and methods : the study was prospective, randomized and double blind. eighty patients belonging to american society of anesthesiologists grade i and ii, 1832 years of age, scheduled for temporomandibular joint interpositional gap arthroplasty were randomized into 2 groups ; ropivacaine group (g rop) : to receive 0.25% ropivacaine infusion and transdermal fentanyl group (g tf) : to receive transdermal fentanyl patch. for postoperative pain (visual analog score [vas ]) and analgesic requirements were assessed 2, 4 and 8 h after surgery and each morning, until and 4 days after surgery.results:time to first analgesic requirement was found to be significantly (p < 0.0001) higher in g rop (49 6.7) as compared with g tf (32 9.1) vas were also significantly lower in g rop throughout the postoperative period. postoperatively, mouth opening was better in g rop as compared with g tf, which was statistically significant.conclusion:it was concluded that by placing an epidural catheter at the incision wound and continuously infusing with ropivacaine 0.25% effectively controls the postoperative pain in patients undergoing temporomandibular joint interpositional gap arthroplasty and provides better postoperative mouth opening. |
metabolic and functional changes that derive from the natural aging process increase the number of diseases, fractures, and falls experienced by individuals, thereby causing patients to experience brief periods of inactivity, which culminate in joint immobilization and may directly influence the development of age - related sarcopenia1. studies show that after 48 hours of joint immobilization, skeletal muscle develops atrophy2, 3, and that six hours after immobilization, a 37% reduction of protein synthesis occurs3, 4. upon joint immobilization, type i fibers (slow) are more vulnerable to hypotrophy due to their oxidative metabolic profile and to the anti - gravitational characteristics of postural or tonic muscles5, 6. prior strength training induces hypertrophy and protects the structural and mechanical properties of skeletal muscle during a disuse period, through the preservation of new cell nuclei that are developed in the muscle tissue during the training period9. therefore, the previous physical training load may influence muscle recovery after a period of inactivity10. aerobic training performed regularly favors the skeletal muscle cellular response through an improvement in capillary density11. high - intensity resistance training is an effective method for the treatment of atrophic muscles12. however, the effect of regular exercise without load on atrophy also needs to be investigated. based on the negative effects of joint immobilization on the aging process and on the cellular alterations derived from regular aerobic training, the hypothesis tested in the present study was that, training performed during a long period prior to immobilization might minimize the effects of muscle disuse and favor post - immobilization muscle recovery. thus, the objective of this study was to evaluate the efficacy of pre - exercise on immobilization and subsequent recovery of the white gastrocnemius (wg) and soleus (sol) muscles of female rats. thirty, 8-month - old, female wistar rats were maintained under controlled temperature conditions, with 12-hour light / dark cycles, and with ad libitum access to food and water. this study was approved by the ethics commission for animal use (comisso de tica sobre o uso de animais ceua) of the methodist university of piracicaba under the protocol 09/13. female wistar rats were randomly and evenly allocated to six groups : sedentary (s) ; immobilized sedentary (is) ; immobilized / rehabilitated sedentary (irs) ; trained (t) ; immobilized trained (it) ; and immobilized / rehabilitated trained (irt). for four months, the t, it and irt group animals performed swimming exercise (water temperature 30 c 2 c) that lasted for 60 minutes per session, at a frequency of three sessions per week on alternate days, for a total of 48 training sessions. during this time, the s, is and irs group animals were housed in cages and could move freely. after four months of training, the left hindlimbs of the animals in the is, irs, it and irt groups were immobilized for five days using an acrylic resin orthosis that kept the ankle at 90 and the left knee and hip free, following the method described by silva. female rats from the irs and irt groups were physically rehabilitated in swimming sessions starting 24 hours after orthosis removal. remobilization was performed five times per week (60-minute sessions without a load increase) for two weeks, a total of 10 sessions, according to the protocol described by nascimento. all of the animals that performed swimming exercise had previously been adapted to this training. animals in the t group continued the pre - immobilization exercise protocol, as follows : three sessions per week, 60 minutes per session, for an additional six sessions. the choice of females was based on the studies of nascimento.10 and milani.14, which conducted swimming after training immobilization of female wistar rats. the animals were euthanized using 1.16 g/10 ml ketamine hydrochloride and 2 g/100 ml xylazine hydrochloride at a dose of 0.09 ml/100 g body weight. the left wg and sol muscles were collected, weighed, and divided into two equal parts : one part was used to perform light microscopy, and the other part was used to perform immunoblotting. samples were fixed on wooden supports with tragacanth gum and were then frozen in 80 c isopentane and 196 c liquid nitrogen. the muscles were cross - sectioned on a cryostat (microm - hs505e) to a thickness of 8 m and mounted on slides. the sections were then stained with hematoxylin and eosin (he). to analyze the muscle cross - sectional area (csa), the samples were viewed under a light microscope (20 magnification) coupled to a camera and analyzed using image pro - plus 6.0 software (media cybernetics). the transforming growth factor (tgf)-1 protein level (tgf-1 ; mouse monoclonal antibody, sigma - aldrich, t7039, st. louis, mo, usa) was quantified in all of the experimental groups. muscle extracts were centrifuged for 20 minutes at 4 c ; the supernatant was treated with laemmli buffer (10% sds, 50% glycerol, 0.1% bromophenol blue, 100 mm tris - hcl, ph 7.4), and 50 mg of protein was loaded onto a 12% sds - polyacrylamide gel. the proteins were transferred from the gel to a nitrocellulose membrane using electrophoretic transfer equipment from bio - rad. the membranes were blocked for one hour at room temperature (rt) in 5% skim milk diluted in basal solution (10 mm trisma base, 150 mm nacl, and 0.02% tween-20). then, the membranes were incubated in the primary antibody overnight at 4 c, washed in basal solution, and incubated for 2 hours at rt in basal solution containing a peroxidase - conjugated secondary antibody (goat anti - mouse igg - hrp, santa cruz : sc-2005, dallas, tx, usa). to detect immunoreactive bands, the membranes were exposed using a chemiluminescence kit (super signal west pico chemiluminescence, pierce, therm scientific, waltham, ma, usa) for 5 minutes, and the signal was detected using g - box equipment (gene sys). the membranes were then washed in basal solution and incubated for one hour at 60 c in 10 ml of stripping buffer (10 mm tris - hcl ph 7.5, 0.1 m -mercaptoethanol, 8 m urea). then, the membranes were incubated in 1 m tris - hcl (ph 7.5) for 30 minutes, to neutralize the stripping buffer, washed in basal solution, and processed as previously described to detect glyceraldehyde-3-phosphate dehydrogenase (gadph mouse monoclonal, santa cruz, sc-59540, dallas, tx, usa), which is an internal control protein, as its expression is not altered by changes in physiologic conditions. optical densitometry quantification was performed using image j software (the national institute of health, usa). the data of three groups, one - way anova followed by tukey s post - test was performed on the data with a parametric distribution, while the kruskal - wallis test was performed on the data with a non - parametric distribution. to perform a comparative analysis of two groups, student s t - test for independent samples was used for parametric data, and the wilcoxon test was used for non - parametric data. in all cases, training favored the gain of sol muscle weight in t group animals compared with s group animals (p<0.01, table 1table 1.white gastrocnemius (wg) muscle and soleus (sol) muscle mean weights in grams (g) and muscle fiber mean cross - section area (m) with the standard deviation for all groupsgroupsmuscle weight (g)csa (m)wbsolwbsols0.417 0.070.135 0.023,884 5103,446 356is0.412 0.010.132 0.032,720 3442,234 91irs0.363 0.030.121 0.022,773 6062,636 315t0.459 0.280.172 0.015,038 700 3,926 310it0.426 0.110.157 0.033,515 5603,064 302irt0.381 0.060.148 0.033,043 505 3,441 300 s : sedentary ; is : immobilized sedentary ; irs : immobilized / rehabilitated sedentary ; t : trained ; it : immobilized trained ; irt : immobilized / rehabilitated trained. different from group t, p<0.01 ; different from groups is and irs, p<0.05 ; different from groups it and irt, p<0.05 ; different from group irs, p<0.05.). immobilization significantly reduced the wg csa in the animals from the is, irs, it, and irs groups compared with the animals from the control groups (s and t, p<0.05). immobilization also significantly reduced the sol csa in the animals from the is and irs groups compared with the animals from the s group (p<0.05) ; however, no significant difference was found among the trained groups, and the irt group showed a significant increase in csa compared with the irs group (p<0.05, table 1). s : sedentary ; is : immobilized sedentary ; irs : immobilized / rehabilitated sedentary ; t : trained ; it : immobilized trained ; irt : immobilized / rehabilitated trained. different from group t, p<0.01 ; different from groups is and irs, p<0.05 ; different from groups it and irt, p<0.05 ; different from group irs, p<0.05. 1.cross sections of white gastrocnemius (a f) and soleus (g l) muscle stained with he. a and g : sedentary (s) ; b and h : immobilized sedentary (is) ; c and i : immobilized / rehabilitated sedentary (irs) ; d and j : trained (t) ; e and k : immobilized trained (it) ; f and l : immobilized / rehabilitated trained (irt). observe reduction in cross - sectional area of the muscle fibers of immobilized groups (b, c, h, i). 1b, c, h, i) and increased csa of the soleus muscle fibers in the irt group (fig. cross sections of white gastrocnemius (a f) and soleus (g l) muscle stained with he. a and g : sedentary (s) ; b and h : immobilized sedentary (is) ; c and i : immobilized / rehabilitated sedentary (irs) ; d and j : trained (t) ; e and k : immobilized trained (it) ; f and l : immobilized / rehabilitated trained (irt). observe reduction in cross - sectional area of the muscle fibers of immobilized groups (b, c, h, i). bar = 100 micron the level of tgf-1 protein in the wg muscle was higher in the s group than in the is, irs, and t groups (p<0.05), but no significant difference was found among animals from the trained groups. immobilization induced an increase in sol tgf-1 in the is group compared with that in the other sedentary groups (p<0.05). sol tgf-1 was similar among the trained groups, with tgf-1 in the irt group being significantly higher than that of the irs group (p<0.02, fig. 2.white gastrocnemius (wg) muscle and soleus (sol) muscle tgf-1 mean protein levels in arbitrary units, with the standard deviation. s : sedentary ; is : immobilized sedentary ; irs : immobilized / rehabilitated sedentary ; t : trained ; it : immobilized trained ; irt : immobilized / rehabilitated trained. different from group t ; different from group s ; # different from group is ; different from group irs ; p0.02). white gastrocnemius (wg) muscle and soleus (sol) muscle tgf-1 mean protein levels in arbitrary units, with the standard deviation. s : sedentary ; is : immobilized sedentary ; irs : immobilized / rehabilitated sedentary ; t : trained ; it : immobilized trained ; irt : immobilized / rehabilitated trained. different from group t ; different from group s ; # different from group is ; different from group irs ; p0.02 pre - immobilization training was able to prevent hypotrophy of the sol muscle (mainly composed of type i fibers), as immobilization did not reduce the csa in the it group, and during the rehabilitation period, the csa was higher in the irt group than in the irs group. the wg muscle (mainly composed of type ii fibers) exhibited csa reduction after immobilization in both the is and it groups compared with their respective controls, the s and t groups, respectively. a short immobilization period, ranging from four to ten days, is sufficient to cause metabolic and structural changes to muscle tissue2, 13, 15, 16. the results of the present study show that five days of inactivity, even with an orthosis model that allowed for unloading of the animal, were sufficient to induce changes in the muscles analyzed, particularly in the sol. changes in the joint, such as adhesions of fibrous hyperplasia tissue in the synovial membrane and fibrosis in the articular cartilage surface, are also observed in the early hours of immobilization17. these data underline the clinical importance of having a rehabilitation program for patients who undergo joint immobilization, even for short periods of time. in addition, it is noteworthy that according to wall, dirks, and van loon18, the repetitive exposure of individuals to short periods of inactivity might culminate in the development of sarcopenia. pitanga.19 emphasize that reduced levels of physical activity also contribute to the development of sarcopenia and that regular physical exercise is a protective factor against sarcopenia in women in the postmenopausal period. the pre - immobilization, long - term training protocol chosen for use in the present study was an attempt to approximate the protocols used in humans and was performed three times per week, without a load, for a total of 180 minutes every week. the remobilization protocol considered that striated skeletal muscles require more time to recover than to undergo atrophy8, 14. although no significant differences were observed between the csa of sedentary and trained female rats, the sol muscle weight was significantly higher in the trained female rats than in the sedentary animals. the results show that swimming remobilization did not contribute to the recovery of wg muscle csa, which can be justified by the physical training performed. milani.14 compared the biomechanical effects of immobilization for seven days and remobilization in the skeletal muscle of trained and sedentary female rats. training was conducted with swimming (60-minute sessions, five times per week) with a progressive load increase (up to 8% of body mass) for six weeks. milani.14 observed that compared with the sedentary group, the group that underwent pre - immobilization training presented a positive effect on the mechanical behavior of the gastrocnemius muscle after a two - week remobilization swimming plan, without a load increase. notably, coupling an external weight equivalent to 6% of the body mass in experimental models during swimming training is considered high - intensity physical activity10, 21. tanaka.22 observed that the soleus (predominantly type i fibers) and plantaris muscles (predominantly type ii fibers) are able to recover after 14 days of tail suspension and three days of reloading, however, the process leading to recovery seems to differ. the production of hepatocyte growth factor (hgf), a mitogen for satellite cells, in the soleus muscle was approximately 3-fold greater than that of controls. the tgf-1 cytokine family is known to regulate several cellular processes, such as angiogenesis, differentiation, and apoptosis, in addition to stimulating fibroblast proliferation and extracellular matrix production23. following a muscle lesion, tgf-1 increases, which may inhibit regeneration and promote fibrosis24. czarkowska - paczek.25 investigated the effects of training on the formation of tgf-1 in the sol muscle and observed increased mrna expression but reduced protein levels after six weeks of treadmill training. because tgf-1 is a strong stimulator of collagen expression, the increase of tgf-1 in muscle might be harmful, and on the basis of the cellular responses to training, czarkowska - paczek.25 suggested that this reduction might be due to a regulatory mechanism of transcription the results of the present study show there was a similar level of tgf-1 among the trained groups, in both muscles, which suggests that a trained muscle has the capacity to adapt to different stimuli, such as immobilization and remobilization. in groups of sedentary animals, however, immobilization increased the level of tgf-1 in the wg and sol muscles only in the is group. future studies are necessary to compare aerobic and anaerobic training with a similar weekly frequency, and to perform additional molecular analyses, such as the quantification of tnf-, metalloproteinases, free radical markers, collagen and satellite cells, as well as functional tests. in conclusion, the tgf-1 protein level highlights the muscle s adaptive capacity, indicating that regular exercise, even without loading and performed three times per week, can have positive effects on the skeletal muscle of female rats. | [purpose ] to evaluate the efficacy of pre - exercise on immobilization and subsequent recovery of white gastrocnemius (wg) and soleus (sol) muscles of female rats. [subjects and methods ] thirty, 8-month - old, female wistar rats were randomly and evenly allocated to six groups : sedentary (s) ; immobilized sedentary (is) ; immobilized / rehabilitated sedentary (irs) ; trained (t) ; immobilized trained (it) ; and immobilized / rehabilitated trained (irt). for four months, t, it and irt group animals performed swimming exercise (three sessions per week, 60 minutes per session), while s, is and irs groups animals remained housed in cages. after this period, the left hindlimb of the animals from the is, irs, it and irt groups was immobilized for five days, with the ankle at 90. after removal of the orthosis, animals from the irs and irt groups followed a rehabilitation program based on swimming (five sessions per week, 60 minutes per session) for two weeks. [results ] immobilization significantly reduced the cross - sectional area of the white gastrocnemius muscle ; no changes were observed in the soleus muscles of the trained animals. transforming growth factor-1 protein levels were similar among the trained groups. [conclusion ] prior swimming prevents hypotrophy of the soleus muscle after immobilization, and protein levels reflected the adaptive capacity of the skeletal muscle. |
pompe disease is characterized by a deficiency of the glycogen - degrading lysosomal enzyme, acid alpha - glucosidase (gaa),. in classic infantile - onset pompe disease (iopd), glycogen deposition in cardiac, skeletal, and respiratory muscles causes severe cardiomyopathy, hypotonia, and respiratory failure, leading to death by 1 year of age if not treated. in those with onset during childhood or adulthood (late - onset pompe disease [lopd ]), glycogen deposition is mainly in skeletal and respiratory muscles, causing progressive limb - girdle myopathy and respiratory insufficiency,. enzyme replacement therapy (ert) with recombinant human gaa (alglucosidase alfa) has been shown to improve walking distance and stabilize pulmonary function in lopd patients,, and to improve survival and motor outcomes in classic iopd patients. studies have examined the effect of individual mutations in the gaa gene and other genetic factors, with one gene of interest encoding angiotensin converting enzyme (ace). ace catalyzes the conversion of angiotensin i to the active form, angiotensin ii (a potent vasoconstrictor), and degrades bradykinin (a potent vasodilator). half of the variation in human ace activity is attributable to an insertion / deletion (i / d) marker allele in intron 16 of the ace gene,,,. an insertion (i allele) of an alu repeat results in lower ace activity, whereas the absence of the insertion (or deletion, d allele) results in higher activity. the ace i / d polymorphism has been linked to physical performance and exercise duration, and it has been reported that the i allele is associated with a predominance of slow - twitch muscle fibers (type i) and the d allele with fast - twitch muscle fibers (type ii),. taken together, it has been hypothesized that differences in muscle - fiber type and vasodynamics may impact the skeletal muscle response to ert. the d / d genotype in 14 lopd patients was found to be associated with higher serum creatine kinase levels at diagnosis, earlier onset of disease, and the presence of muscle pain. in a larger cohort of pompe patients (n = 85), individuals with the d / d ace genotype presented with an earlier onset of disease and muscle pain, confirming the previous observation. the relationship between ace genotype and clinical parameters in a cohort of lopd patients who received biweekly infusions of alglucosidase alfa for > 2 years also was reported. notably, i / i genotype patients had significantly greater muscle mass measured by quantitative magnetic resonance imaging (mri). muscle strength and endurance variations demonstrated a similar trend, with the i / d genotype resulting in intermediate outcomes between i / i and d / d ; however, these differences were not statistically significant. we tested the hypothesis that ace genotype was a contributing factor to disease progression and clinical response to ert in the pompe disease late - onset treatment study (lots). lots, a randomized, double - blind, placebo - controlled trial involving 90 lopd patients, evaluated the safety and efficacy of biweekly infusions (20 mg / kg) of alglucosidase alfa compared to placebo for 78 weeks in juvenile and adult lopd patients. only patients who completed 26 weeks of the study were included in this analysis (n = 58 alglucosidase alfa - treated ; n = 30 placebo - treated). the i / d allele status of a 287 base - pair alu repeat in intron 16 of the ace gene was determined by polymerase chain reaction (pcr) analysis using methods described previously with slight modifications (2 u platinum taq polymerase, 1 platinum taq pcr buffer - mg, 3 mm mgcl2, 0.25 mm dntp mix, and 0.5 mm of the forward and reverse primers). one - way anova analysis was used to evaluate correlations between ace genotype (i / i, i / d, and d / d) and baseline pompe disease characteristics. student 's t - test was conducted to compare alglucosidase alfa treatment and placebo within each ace genotype. at baseline, patients with the d / d genotype did not present with earlier onset of disease (mean age at first symptoms : 27.4 12.7 years vs. 28.5 11.5 [i / d ] and 28.6 14.4 [i / i ]), longer duration of disease at trial entry (mean duration since symptom onset : 18.3 10.2 years vs. 17.6 11.6 [i / d ] and 20.4 9.9 [i / i ]), or more severe disease as measured by 6-minute walk test (6mwt) (mean : 343.0 144.4 m vs. 339.2 125.6 [i / d ] and 293.1 147.4 [i / i ]), or percent predicted forced vital capacity (fvc) (mean : 53.3 15.4% vs. 56.2 15.6 [i / d ] and 52.4 12.5 [i / i ])., lots patients with the d / d genotype appeared less responsive to alglucosidase alfa treatment than patients with the i / i or i / d genotypes (fig. patients with the i / d and i / i genotype who received ert showed significantly greater improvements in fvc (mean change : 1.516 [p = 0.028 ] and 4.8 [p = 0.007 ], respectively) compared to d / d patients (mean change : 1.412) receiving treatment (fig. similarly, the mean increase in the 6mwt was significantly higher in i / d patients on ert (mean change : 40.16 [p = 0.022 ]) compared to the d / d group (mean change : 1.412), with a trend for an improved response in patients with the i / i genotype (mean change : 32) (fig. 1c presents the change from baseline in patients treated with alglucosidase alfa and also illustrates a trend toward lower responsiveness to alglucosidase alfa in d / d genotype patients. importantly, while the mean changes in fvc and 6mwt do not appear to indicate an overall response to alglucosidase alfa within the d / d genotype group, it is important to note that a variable response to ert was observed, and several patients demonstrated gains in the 6mwt and fvc with this genotype (fig. the d / d genotype was not associated with increases in peak igg titers, sustained antibody titers, or inhibitory antibodies (data not shown), indicating that the correlation between ace genotype and response to ert is not associated with an immune response to alglucosidase alfa. our finding that lopd patients with the ace d / d genotype demonstrate a reduced response to alglucosidase alfa relative to those with i / i or i / d genotypes is consistent with earlier findings. we also demonstrated the presence of this association regardless of baseline disease severity and anti - gaa igg antibody levels. however, unlike patients in earlier studies,, lots patients with the d / d genotype did not present with an earlier onset of disease or a longer duration of disease at study entry. our results suggest that the ace i / d polymorphism may influence the response to alglucosidase alfa treatment independent of the patient 's disease severity at treatment initiation. these findings raise pertinent questions about the mechanisms by which the ace genotype influences the muscle response to alglucosidase alfa treatment. does the d / d genotype result in increased vasoconstriction, and therefore less enzyme delivery to skeletal muscle ? do patients with the d / d genotype have an increase in type ii muscles fibers (fast - twitch, anaerobic, glycogen - rich) that decreases their response to treatment ? type i muscles fibers (slow - twitch, aerobic, mitochondria - rich) have an increased capillary density, which has been correlated with increased glycogen clearance after ert in a mouse model of pompe disease. on the other hand, type ii muscle fibers exhibit increased autophagic pathology in pompe mice, which may be responsible for the mis - targeting of ert within the muscle fiber. finally, it is important to note that individual patient responses to alglucosidase alfa were variable across the three ace genotypes, and many patients with the d / d genotype demonstrated improvements in the functional assessments evaluated. this supports the concept that an individualized treatment approach and careful follow - up are warranted for pompe disease patients receiving ert. in conclusion, pompe disease patients with the ace d / d genotype appeared to show a decreased response to alglucosidase alfa, although the mechanism underlying this observation is not yet known. continued genotyping of pompe disease patients for the ace polymorphism and further investigation of therapeutic response are warranted. all authors are employees of sanofi, and the study was funded by sanofi genzyme. | correlations between angiotensin - converting enzyme (ace) genotype (i / i, i / d, d / d), disease severity at baseline and response to enzyme replacement therapy (ert) were assessed in the pompe disease late - onset treatment study (lots). no correlations were observed between ace genotype and disease severity at baseline. however, d / d patients appeared to have a reduced response to alglucosidase alfa treatment than i / i or i / d patients, suggesting that ace polymorphisms may influence the response to alglucosidase alfa treatment and warrants further investigation. |
the amas was administered to 298 undergraduate students (133 males and 165 females) of tabriz university in iran who majored in different disciplines of human sciences ; they were enrolled in entry - level mathematics courses and voluntarily participated in the study. college research examination board approved the research protocol. while adapting the instruments to iranian culture and writing the persian form, we initially translated the scales into persian. the persian version of the scales was translated into english by the second author from the department of english language teaching, who specializes in motivation research, and then it was independently back - translated by two lecturers. the independent translations resulted in general linguistic agreement, with only minor differences in wording of the items. disagreements over wording were solved through discussion by the two psychologists in consultation with the first author. the instruments used in this study were as follows : abbreviated math anxiety scale (amas) (19) ; is a 9-item measure of mathematics anxiety that yields the lma and mea subscales, which accounted for 70% of the variance from an exploratory factor analysis using principal components extraction with varimax rotation. items on the amas were responded to using a 5-point likert - type scale, ranging from 1 (low anxiety) to 5 (high anxiety), with the total score representing a summation of the nine items. as indexed by cronbach 's, internal consistency in this study was excellent within the amas (=.90), as well as the lma (=.85) and mea subscales (=.88). students motivation - to - learn - mathematics (smot) scale that covered the four dimensions of motivation to learn mathematics. the two scales, which had 12 and 28 items, respectively, were embedded in a 108-item students questionnaire of a major study conducted by one of the researchers (24) on factors related to the motivation to learn mathematics among secondary school students in kenya 's nairobi and rift valley provinces. questionnaire items were reviewed and found to be content valid by a panel of educational researchers in kenya. a pilot test in nyandarua and nyeri districts that have similar students, schools and learning conditions with those in the study area indicated cronbach alpha reliability coefficients of 0.88 and 0.89 for mathematics self - concept (msc) and smot, respectively. the researchers visited the 32-selected schools and arranged with head teachers and heads of mathematics departments to group administer the questionnaires to students. the response rate achieved after a series of follow - ups was 98.3%, which the researchers considered satisfactory for the study. statistics anxiety measure (sam) (25) is a 23-item questionnaire that comprises six discrete subscales : anxiety, performance history and self - concept, expectations, attitude, and fearful behavior. in this study, however, four subscales were utilized. in general, the internal consistent reliability of overall scale (=0.93) as well as sub - scales ranged from high to excellent (a=.82.95). first, which existing factor structure (one and two factor structures) provides an acceptable measurement model for the 9-item amas ? to address this question, cfa was used to impose each of the two factor structures on two data sets to evaluate each model 's goodness - of - fit. second, is there measurement invariance with respect to gender ? to address this question, multigroup cfa was used to test hypotheses about the invariance of the 9-item amas across males and females. one - way analysis of variance (anova) was also used to compare gender differences on the subscale of amas. amos was used to perform the cfas of the amas analyzing the fit of models and their respective parameter estimates in two distinct stages. in stage 1, first, the nine items of the amas were expected to load onto a single latent factor (model 1.). second, run for the two - factor model was suggested by hopko (19) who reported that a two - factor model provided a better fit to the data than a single factor. in order to rule out the possibility that this model is superior because any two - factor model would fit the data better than the one - factor model, an alternative two - factor model was tested, with one factor corresponding to the first 4 items and the second factor corresponding to items 5 (model 2). the five items (1,3,6,7,8, and 9) of the math anxiety subscale of the amas were assigned to the first factor, and one factor corresponded to the four items (items 2, 4, 5, and 8) (model 3). in stage 2, multiple group cfa was used to test whether the two - factor structure of the amas operate equivalently across both male and female students. this involved comparing the goodness - of - fit 2 of two nested cfa models : one constraining the magnitudes of the factor loadings to be equal for male and female students, and the other omitting this invariance constraint. several fit indices were examined to evaluate the overall fit of each model : (tests the hypothesis that an unconstrained model fits the covariance or correlation matrix as well as the given model ; ideally values should not be significant) ; comparative fit index (cfi ; comparison of the hypothesized model with a model in which all correlations among variables are zero, and where values around.90 indicate very good fit) ; root - mean - square error of approximation (rmsea ; values of.08 or below indicate reasonable fit for the model ; tucker - lewis index (tli) and the incremental fit index (ifi), with values close to.95 being indicative of good fit (28) ; akaike information criterion (aic ; aic close to zero reflects good fit and between two aic measures, the lower one reflects the model with the better fit)(27) descriptive statistics for amas items and correlations of individual items and subscales / total scale. the amas was administered to 298 undergraduate students (133 males and 165 females) of tabriz university in iran who majored in different disciplines of human sciences ; they were enrolled in entry - level mathematics courses and voluntarily participated in the study. while adapting the instruments to iranian culture and writing the persian form, we initially translated the scales into persian. the persian version of the scales was translated into english by the second author from the department of english language teaching, who specializes in motivation research, and then it was independently back - translated by two lecturers. the independent translations resulted in general linguistic agreement, with only minor differences in wording of the items. disagreements over wording were solved through discussion by the two psychologists in consultation with the first author. the instruments used in this study were as follows : abbreviated math anxiety scale (amas) (19) ; is a 9-item measure of mathematics anxiety that yields the lma and mea subscales, which accounted for 70% of the variance from an exploratory factor analysis using principal components extraction with varimax rotation. items on the amas were responded to using a 5-point likert - type scale, ranging from 1 (low anxiety) to 5 (high anxiety), with the total score representing a summation of the nine items. as indexed by cronbach 's, internal consistency in this study was excellent within the amas (=.90), as well as the lma (=.85) and mea subscales (=.88). students motivation - to - learn - mathematics (smot) scale that covered the four dimensions of motivation to learn mathematics. the two scales, which had 12 and 28 items, respectively, were embedded in a 108-item students questionnaire of a major study conducted by one of the researchers (24) on factors related to the motivation to learn mathematics among secondary school students in kenya 's nairobi and rift valley provinces. questionnaire items were reviewed and found to be content valid by a panel of educational researchers in kenya. a pilot test in nyandarua and nyeri districts that have similar students, schools and learning conditions with those in the study area indicated cronbach alpha reliability coefficients of 0.88 and 0.89 for mathematics self - concept (msc) and smot, respectively. the researchers visited the 32-selected schools and arranged with head teachers and heads of mathematics departments to group administer the questionnaires to students. the response rate achieved after a series of follow - ups was 98.3%, which the researchers considered satisfactory for the study. statistics anxiety measure (sam) (25) is a 23-item questionnaire that comprises six discrete subscales : anxiety, performance history and self - concept, expectations, attitude, and fearful behavior. in this study, however, four subscales were utilized. in general, the internal consistent reliability of overall scale (=0.93) as well as sub - scales ranged from high to excellent (a=.82.95). the instruments used in this study were as follows : abbreviated math anxiety scale (amas) (19) ; is a 9-item measure of mathematics anxiety that yields the lma and mea subscales, which accounted for 70% of the variance from an exploratory factor analysis using principal components extraction with varimax rotation. items on the amas were responded to using a 5-point likert - type scale, ranging from 1 (low anxiety) to 5 (high anxiety), with the total score representing a summation of the nine items. as indexed by cronbach 's, internal consistency in this study was excellent within the amas (=.90), as well as the lma (=.85) and mea subscales (=.88). students motivation - to - learn - mathematics (smot) scale that covered the four dimensions of motivation to learn mathematics. the two scales, which had 12 and 28 items, respectively, were embedded in a 108-item students questionnaire of a major study conducted by one of the researchers (24) on factors related to the motivation to learn mathematics among secondary school students in kenya 's nairobi and rift valley provinces. questionnaire items were reviewed and found to be content valid by a panel of educational researchers in kenya. a pilot test in nyandarua and nyeri districts that have similar students, schools and learning conditions with those in the study area indicated cronbach alpha reliability coefficients of 0.88 and 0.89 for mathematics self - concept (msc) and smot, respectively. the researchers visited the 32-selected schools and arranged with head teachers and heads of mathematics departments to group administer the questionnaires to students. the response rate achieved after a series of follow - ups was 98.3%, which the researchers considered satisfactory for the study. statistics anxiety measure (sam) (25) is a 23-item questionnaire that comprises six discrete subscales : anxiety, performance history and self - concept, expectations, attitude, and fearful behavior. in this study, however, four subscales were utilized. in general, the internal consistent reliability of overall scale (=0.93) as well as sub - scales ranged from high to excellent (a=.82.95). first, which existing factor structure (one and two factor structures) provides an acceptable measurement model for the 9-item amas ? to address this question, cfa was used to impose each of the two factor structures on two data sets to evaluate each model 's goodness - of - fit. second, is there measurement invariance with respect to gender ? to address this question, multigroup cfa was used to test hypotheses about the invariance of the 9-item amas across males and females. one - way analysis of variance (anova) was also used to compare gender differences on the subscale of amas. amos was used to perform the cfas of the amas analyzing the fit of models and their respective parameter estimates in two distinct stages. in stage 1, first, the nine items of the amas were expected to load onto a single latent factor (model 1.). second, run for the two - factor model was suggested by hopko (19) who reported that a two - factor model provided a better fit to the data than a single factor. in order to rule out the possibility that this model is superior because any two - factor model would fit the data better than the one - factor model, an alternative two - factor model was tested, with one factor corresponding to the first 4 items and the second factor corresponding to items 5 (model 2). the five items (1,3,6,7,8, and 9) of the math anxiety subscale of the amas were assigned to the first factor, and one factor corresponded to the four items (items 2, 4, 5, and 8) (model 3). in stage 2, multiple group cfa was used to test whether the two - factor structure of the amas operate equivalently across both male and female students. this involved comparing the goodness - of - fit 2 of two nested cfa models : one constraining the magnitudes of the factor loadings to be equal for male and female students, and the other omitting this invariance constraint. several fit indices were examined to evaluate the overall fit of each model : (tests the hypothesis that an unconstrained model fits the covariance or correlation matrix as well as the given model ; ideally values should not be significant) ; comparative fit index (cfi ; comparison of the hypothesized model with a model in which all correlations among variables are zero, and where values around.90 indicate very good fit) ; root - mean - square error of approximation (rmsea ; values of.08 or below indicate reasonable fit for the model ; tucker - lewis index (tli) and the incremental fit index (ifi), with values close to.95 being indicative of good fit (28) ; akaike information criterion (aic ; aic close to zero reflects good fit and between two aic measures, the lower one reflects the model with the better fit)(27) descriptive statistics for amas items and correlations of individual items and subscales / total scale. n=298 descriptive statistics for amas items and correlations between each item and their own subscales and between each item and the total scale are illustrated in table 1. almost all correlations between individual items and the total scale ranged from.41 to.66. correlations between individual items and their respective subscales ranged from.42 to.61 for learning mathematics anxiety, from.54 to.66 for mathematics evaluation anxiety. scores obtained from the nine - item amas had a cronbach 's alpha of.82. the internal consistency estimates of the two factors were as follows : learning mathematics anxiety (five items ; =.75), and mathematics evaluation anxiety (four items ; =.79). we used smot and sam to provide estimates of convergent validity for the amas scores. we expected that the scores on the nine - item amas, factor 1- learning mathematics anxiety and factor 2-mathematics evaluation anxiety, would be negatively correlated with scores from subscale of smot. we expected the construct of amas to be more closely aligned with subscales of statistics anxiety measure ; hence, we expected larger correlations between amas scores and subscales of sam scores than between amas scores and smot scores. as predicted, scores obtained from fearful behavior factor (r=.58), attitude factor (r=.55) expectations factor (r=.59), and history and self - concept (r=.48) were positively correlated with scores from amas total. likewise, obtained scores from smot total (r=.40), interest (r=.37), relevance (r=.22) satisfaction (r=.25) and perceived probability of success (r=.46) were also negatively correlated with obtained scores from amas as expected (see table 2). in order to examine possible between - group differences in responses to the amas, we ran a one - way analysis of variance (anova) with the subscales of amas (learning mathematics anxiety, mathematics evaluation anxiety) as the dependent variable and participants sex as independent variable. results showed participants sex was significantly related to mathematics evaluation anxiety, f (1, 296)=14.51, p.05. in other words, male students reported more mathematics evaluation anxiety than female students did. to evaluate the goodness - of - fit of three alternative measurement models for the amas, cfa was first run for a one - factor solution in which all 9 items loaded on to a single general strengths factor (model a) and subsequently, it was run for the two - factor model (b) suggested by hopko. in all the analyses the chi - square goodness of fit statistic is large and significant beyond the 0.001 level, rather than being small and associated with a high probability, which would indicate a close fit between model and data. however, this statistic is sensitive to sample size and does not provide a realistic test of the fit of models (27). confirmatory factor analysis showing the two - factor structure of the amas among undergraduate students goodness - of - fit statistics and their comparisons for three alternative measurement models for the 9-item amas note : model 1=one factor ; model b = two factor split half ; model c = final hypothesized two - factor model the results of the initial estimation of the one factor model did not provide a satisfactory result with a chi - square value of 153.237 (df=26), which was significant at the p15 is considered very significant). these results again strongly support the superiority of the two - factor model over the one factor and two - factor split half models ; subsequently, this model was considered optimal. in order to find out whether the model was invariant across gender, first, the two - factor model was tested separately for the male and female students. a prerequisite for assessing the invariant structure is to first stipulate and test a baseline model for each group individually. such a model, which does not include cross- group constrains, should fit the data well in terms of both parsimony and theoretical relevance (27). from the analyses in stage 1, among the two measurement models evaluated, overall fit indexes revealed the multidimensional amas model to be the best fitting model available (model b). hence, on this basis, the multidimensional amas model consisting of two factors was tested on both groups to see if this measurement model was invariant across gender (see table 4). results of multi group confirmatory factor analyses across gender for females, the two - factor model had goodness - of - fit indexes as follows : (df=25, n=165)=41.60, p.05. in other words, male students reported more mathematics evaluation anxiety than female students did. to evaluate the goodness - of - fit of three alternative measurement models for the amas, cfa was first run for a one - factor solution in which all 9 items loaded on to a single general strengths factor (model a) and subsequently, it was run for the two - factor model (b) suggested by hopko. in all the analyses the chi - square goodness of fit statistic is large and significant beyond the 0.001 level, rather than being small and associated with a high probability, which would indicate a close fit between model and data. however, this statistic is sensitive to sample size and does not provide a realistic test of the fit of models (27). confirmatory factor analysis showing the two - factor structure of the amas among undergraduate students goodness - of - fit statistics and their comparisons for three alternative measurement models for the 9-item amas note : model 1=one factor ; model b = two factor split half ; model c = final hypothesized two - factor model the results of the initial estimation of the one factor model did not provide a satisfactory result with a chi - square value of 153.237 (df=26), which was significant at the p15 is considered very significant). these results again strongly support the superiority of the two - factor model over the one factor and two - factor split half models ; subsequently, this model was considered optimal. in order to find out whether the model was invariant across gender, a multi - group analysis was conducted. first, the two - factor model was tested separately for the male and female students. a prerequisite for assessing the invariant structure is to first stipulate and test a baseline model for each group individually. such a model, which does not include cross- group constrains, should fit the data well in terms of both parsimony and theoretical relevance (27). from the analyses in stage 1, among the two measurement models evaluated, overall fit indexes revealed the multidimensional amas model to be the best fitting model available (model b). hence, on this basis, the multidimensional amas model consisting of two factors was tested on both groups to see if this measurement model was invariant across gender (see table 4). results of multi group confirmatory factor analyses across gender for females, the two - factor model had goodness - of - fit indexes as follows : (df=25, n=165)=41.60, p<.02, cfi=.96, rmsea=.06, ifi=.95. for males, the two - factor model was associated with similar goodness - of - fit indexes : (df=25, n=133)=48.88, p<.003, cfi=.94, rmsea=.08, ifi=.94. the unconstrained model (configural model), where factor loadings are allowed to vary between females and males, provided a good fit ([df=48 ] = 87.70, cfi=.95, rmsea=.05). finally, equality constraints were imposed on factor loadings, variances and covariances. although chi - square difference tests indicated that constraining regression weights and factor variances and covariances are equal across sexes and led to statistically significant increases of the chi - square value, the fit of the model remained virtually unchanged in terms of the other fit. nonetheless, the results in table 3 indicate a satisfactory fit for each subgroup and for each of the constraints in the multi - group analysis. the primary purpose of this study was to use confirmatory factor analytic techniques in a sample of young adult college students to explore the fit of the two - factor model of the amas proposed by hopko, mahadevan, bare and hunt. the second aim of the present study was to evaluate the psychometric properties of an iranian version of the amas and to test measurement invariance across sex. results showed that the amas has high internal consistency, with cronbach 's reaching 0.82. these data are further supported by the parameter estimates of the cfas, and is generally consistent with previous work showing that amas has high internal consistency (19). the correlational analysis supported the construct validity of the instrument, showing negative association among the amas and students motivation - to - learn - mathematics. however, there was a high positive correlation between the math anxiety and statistics anxiety. these results are in accordance with those found by previous works (30, 31, 32 and 33) and they indicate that statistics anxiety is related to test anxiety, math anxiety, and an individual 's history of success and failure experiences in situations involving math. the present study showed that there was a difference between girls and boys with respect to mathematics evaluation anxiety and not in learning mathematics anxiety. more specifically, boys reported higher mathematics evaluation anxiety than girls. whether this finding represents an actual gender difference or is more a function of increased willingness of female students to endorse anxiety symptoms, data suggest that female students are more apt to avoid mathematics courses and careers that require math skills (3). this finding is consistent with that of pajares and miller (34), pajares and kranzler (35), shokrani (36) and kabiri (37) who reported a higher math anxiety for boys. nonetheless, it differs from the findings of pajares and graham (38). in this regard, betz suggests that the differences in math attitudes between the sexes are the consequence of socialization. female students may believe to be poor in mathematics and scientific domains (13). female students who perform poorly may justify their performance as a lack of ability rather than lack of hard work in math. in contrast, male students often may excuse their poor performance in mathematics as lack of hard work (14). moreover, like hopko, mahadevan, bare and hunt (19), the results of our cfa indicate that the most parsimonious best fit to the latent structure of the 9-item amas is a two - factor solution assessing learning mathematics anxiety and mathematics evaluation anxiety. although the two dimensional structure of the amas was invariant across sex, one should be cautious about drawing significant conclusions of such a finding ; and further research is required. future work should also seek to examine the construct validity of the amas, by examining the relationship of math anxiety with related constructs. in particular, it may be useful to investigate the amas in relation to other scales that measure test anxiety that had been validated for use among persian - speaking students. in conclusion, the present results support the amas as a valid and reliable measure of math anxiety among college students within the iranian context. | objectivethe aim of this study is to explore the confirmatory factor analysis results of the persian adaptation of abbreviated math anxiety scale (amas), proposed by hopko, mahadevan, bare & hunt.methodthe validity and reliability assessments of the scale were performed on 298 college students chosen randomly from tabriz university in iran. the confirmatory factor analysis (cfa) was carried out to determine the factor structures of the persian version of amas.resultsas expected, the two - factor solution provided a better fit to the data than a single factor. moreover, multi - group analyses showed that this two - factor structure was invariant across sex. hence, amas provides an equally valid measure for use among college students.conclusionsbrief amas demonstrates adequate reliability and validity. the amas scores can be used to compare symptoms of math anxiety between male and female students. the study both expands and adds support to the existing body of math anxiety literature. |
respiratory syncytial virus (rsv) is one of the most prevalent causes of lower respiratory tract infection (lrti) related hospitalizations among infants younger than 2 years of age. almost all children are infected by rsv at least once before the age of 2 years, with a recurrent infection occurring in more than 50% of cases. premature infants are at particular risk for rsv infection, requiring readmission following discharge after neonatal intensive care. other risk factors include bronchopulmonary dysplasia (bpd) and severe congenital heart disease (chd), which contribute to the severity of a rsv infection while also prolonging hospital stay. palivizumab is a monoclonal anti - rsv globulin that has been developed and marketed as a preventive measure in infants at risk for rsv infection. epidemiological studies from turkey have shown that annually, rsv infections occur most frequently during the period between october - march, which translates to a 6-month window for palivizumab prophylaxis. current international guidelines reserve the use of rsv prophylaxis in infants considered to be at high - risk for developing severe rsv infections, namely those with prematurity, bpd or chd. palivizumab has been shown to reduce the overall rate of hospitalization in premature infants with bpd by 55%. in a previous study, we managed to demonstrate a 31% reduction in the duration of hospital stay in bdp patients treated with palivizumab. rsv prophylaxis is mainly used in high - risk patients with prematurity, bpd or chd, however despite the use of palivizumab, subsequent hospitalization rates are mainly affected by socioeconomic status of families. the aim of this study was to determine the risk factors associated with lrti - related hospitalizations in preterm infants receiving palivizumab throughout the high season for rsv infection. this prospective study was undertaken in zekai tahir burak maternity teaching hospital in ankara, which is located in central anatolia with the approval of the local ethics committee. this hospital has 150 incubators and serves as a referral level iii nicu with about 4000 newborn admissions per year. premature infants who were commenced on palivizumab prophylaxis during the rsv season between october 2011 and march 2012 were included in the study following parental consent. prior to initiation of treatment, information on social and demographic characteristics of the patients families, as well as relevant patient data such as birth weight, gestational age (ga), presence of a medical history of respiratory distress syndrome (rds), patent ductus arteriosus (pda), intracranial hemorrhage (grade iii - iv), necrotizing enterocolitis (nec), bpd and retinopathy of prematurity (rop) were recorded on pre - prepared forms. a diagnosis of bpd was made based on criteria put forth by the us national institutes of health. each form also included information on indication for palivizumab prophylaxis as well as planned and administered number of doses. monthly income was provided in united states dollars (usd) using the official exchange rate of the central bank of the republic of turkey. as per manufacturer 's instructions, palivizumab was administered intramuscularly at a standard dose of 15 mg / kg, repeated every 30 days. in turkey, according to the records of the turkish pharmacist 's association, one 50 mg - vial of palivizumab costs 516 usd. additionally, socioeconomic status of each family based on monthly income (1500 usd = very high), along with details regarding family lodgings were also recorded, taking into account the type of community inhabited (urban, suburban, rural), type of heating (stove, radiator, etc.), exposure to smoke (cigarette smoking by at least one of the parents), number of rooms, and the number of individuals in the household including older siblings (cared for at home or in school). monthly follow - up visits were scheduled in the prematurity outpatient clinic for each patient during which patients were subject to a detailed physical examination followed by intramuscular administration of palivizumab. rsv prophylaxis was administered taking into consideration the current recommendations of the turkish neonatology society, which have been adapted from recommendations of the american academy of pediatrics (aap) and the european palivizumab study group. parents were informed in detail regarding the study protocol, and in the event any of the participants developed symptoms suggestive of a lower respiratory infection, parents were instructed to bring their children to designated health facilities depending on their community of residence (urban, suburban or rural). for infants requiring hospitalization, rsv status was evaluated using a rsv strip test (rsv respi - strip kit, coris bioconcept, belgium). this test is based on the interaction between conjugated rsv complex and anti - rsv antibody, which appears as a second line on the test strip. rsv respi - strip test kits have been reported to have a sensitivity of 92%, specificity of 98% and a diagnostic accuracy of 95%. statistical analyses were carried out using the statistical package for the social sciences (spss) for microsoft windows, version 20.0 (spss inc., differences between two groups were examined by the independent samples t - test for normally distributed variables and mann whitney u test for abnormally distributed variables. following univariate analysis, factors that were found to be associated with an increased risk for lrti - related hospitalization and for the hospitalization to be due to an rsv infection were subjected to odds ratio analysis. a total of 275 premature infants who were given palivizumab prophylaxis were initially included in the study, although 23 were excluded from the final analysis due to non - compliance with follow - up visits. overall, 234 (92.8%) participants did not require hospitalization (group 1) whereas 18 (7.2%) patients were hospitalized at least once due to lrti (group 2) during the study period (fig. 1). demographic and clinical characteristics of the study population are presented in table 1. there was no significant difference between groups in terms of gestational age, birth weight, gender, mode of delivery, comorbid conditions (rds, pda, nec and rop), number of cases with intracranial hemorrhage (grade iii - iv), planned and administered number of palivizumab doses, socioeconomic status, type of community inhabited, type of heating (stove, radiator, etc.), exposure to smoke (cigarette smoking by at least one of the parents), number of rooms, and the number of individuals in the household including older siblings (table 1). the rate of moderate - severe bpd was significantly higher in group 2 compared to group 1 (38.9% vs 16.2% ; p=0.02). flow chart of preterm infants who were received palivizumab prophylaxis for rsv rsv : respiratory syncytial virus demographic and clinical characteristics of the study population values provided as mean standard deviation values provided as number and percentage of patients values provided as median (min - max) ; bpd : bronchopulmonary dysplasia of the 18 infants who were hospitalized, 6 (33.3%) tested positive for rsv while the remaining 12 patients (66.7%) were negative for rsv. demographic and clinical characteristics of rsv - positive and rsv - negative patients are summarized in table 2. five of the 6 (83.3%) patients who were rsv - positive presented with pneumonia, whereas 1 (16.7%) patient was hospitalized due to bronchiolitis. half of the patients who were found to be rsv positive required hospitalization for a second time during the rsv season compared to only 1 (8.3%) patient in the rsv - negative group. the difference in re - hospitalization rates between groups was statistically significant (p=0.04). median duration of hospital stay was significantly longer in the rsv - positive group compared to the rsv - negative group [12 (5 - 23) days vs.4 (2 - 20) days ; p=0.02) ]. the distribution of hospitalized patients with rsv - negative and rsv - positive lrti according to month is depicted in fig. hospitalization rates for both rsv - negative and rsv - positive patients were highest in december and january. odds ratio (or) analysis of several risk factors revealed presence of bpd (or : 3.28 ; 95%ci : 1.19 to 9), a ga < 28 weeks (or : 1.6 ; 95%ci : 0.61 to 4.18), history of exposure to smoke (or : 1.51 ; 95%ci : 0.52 to 4.38), being from a family with low socioeconomic status (or : 3.64 ; 95% ci : 1.08 to 12.3), having older siblings (or : 1.35 ; 95%ci : 0.46 to 3.92) and living in a suburban or rural area (or : 1.65 ; 95%ci : 0.61 to 4.45) to be associated with a higher likelihood of lrti - related hospitalization. results of or analysis have been summarized in table 3. the distribution of hospitalized patients with rsv - negative and rsv - positive lrti according to month for the season between november 2011-march 2012 demographic and clinical characteristics of rsv - positive and rsv - negative patients who were hospitalized values provided as mean standard deviation values provided as number and percentage of patients values provided as median (min - max) ; rsv : respiratory syncytial virus ; bpd : bronchopulmonary dysplasia summary of odds ratio analysis for several risk factors associated with an increased likelihood for lrti - related hospitalizations the main outcome of this study was that it was the most widely conducted study for determining with or without rsv related hospitalization risk factors that increase hospitalization of the preterm newborns under the influence of palivizumab prophylaxis that were closely observed during the season in our country. with this study, it wasdetermined that the preterm newborns who were under the influence of palivizumab prophylaxis during the season would have higher hospitalization rate if such cases had bpd at the outset and low socioeconomic status. lrtis are one of the leading causes of death in children worldwide, with rsv being the most common viral pathogen in young children. the aim of this study was to identify risk factors associated with an increased risk of lrti - related hospitalizations, regardless of causative pathogen, in preterm infants under close follow - up while on palivizumab prophylaxis. preterm infants are at high risk for developing bpd, which makes them susceptible to ventilation - induced volutrauma. oxygen toxicity as well as pre- and post - natal infections triggers a pulmonary inflammatory response, which can eventually lead to impaired lung development. in a retrospective study by greenough, on a cohort of 235 premature infants (< 32 weeks of gestation) it was observed that 19% of neonates who require oxygen treatment beyond 28 days were subsequently re - hospitalized with a proven rsv infection. in our study, 38.9% of patient who were hospitalized for an lrti had bpd, compared to 16.2% in patients who were not hospitalized, a statistically significant difference (p=0.02). patients who were hospitalized were 3.28 times more likely to have bpd compared to infants who were not hospitalized. male gender is a well - established risk factor for severe rsv - related lrti, and a review of relevant studies of the past 30 years revealed a boy - to - girl ratio of 1.425:1. it has been postulated that the increased risk could be attributed to boys having shorter and narrower airways making them more likely to develop bronchial obstruction during an rsv infection. in our study, however, the difference between group 1 and group 2 in terms of male gender distribution was insignificant (56% vs 61.1% ; 0.67). nevertheless, male gender was found to be associated with a 1.23-fold increased risk of lrti - related hospitalization. low socioeconomic status is another frequently reported risk factor for rsv infections. in a study from sweden in 2002, univariate analysis revealed the presence of a significant positive correlation between percentage of immigrants, per capita income and an increased risk of developing rsv. in our study, a low socioeconomic status was associated with a 3.64-fold increased risk for lrti - related hospitalizations. crowded living conditions and the presence of other siblings in the home have also been reported as important risk factors for severe rsv infection and lrti - related hospitalization. the obvious reason behind this is the increased likelihood of exposure to the virus in a crowded home setting. in the canadian picnic study the presence of preschool - aged siblings was found to be associated with a significantly increased risk for rsv - related hospitalization, while a weaker association was observed in the presence of school - aged siblings. in our study, the influence of smoking in the household on the risk of bronchiolitis and hospitalization due to rsv - related lrti has been a focus of research, albeit with conflicting results. bivariate analysis in the flip and flip-2 studies from spain established that the presence of more than two smokers at home was associated with a higher risk of rsv - related hospitalization. a similar finding was reported in the canadian picnic- study in that household exposure to cigarette smoke from 2 or more smokers was found to be a predictive factor for subsequent rsv - related hospitalization. in our study, we considered cigarette smoking by at least 1 parent as indicative of smoke exposure, and the difference between group 1 and group 2 in terms of exposure to smoke was statistically insignificant (p=0.4). overall, exposure to smoke was found to be associated with a 1.51-fold increased risk of lrti - related hospitalization. our study has some limitations, the most important of which is that infants hospitalized for lrtis were not enough positive for rsv infection. nevertheless, our study is the first of its kind as well as being the most comprehensive study from turkey to evaluate risk factors for lrti - related hospitalizations in preterm infants receiving palivizumab prophylaxis who were followed up for a whole rsv - season. although the rate of hospitalization decreases by palivizumab prophylaxis, our data demonstrated that rsv is an important lrti agent and the cause of hospitalization especially in preterm infants with additional risks such as bpd, gestational age of < 28 weeks and low socioeconomic status. we suggest that improving care conditions and decreased bpd with prematurity would help preventing lrti hospitalization. | objectiveto determine the risk factors associated with lower respiratory tract infections (lrti) related hospitalizations in preterm infants receiving palivizumab throughout the high season for respiratory syncytial virus (rsv) infection.methodspremature infants who were commenced on palivizumab prophylaxis during the rsv season were included in the study following parental consent. information on demographic, social, prenatal and postnatal clinical characteristics was recorded and risk factors associated with hospitalization were evaluated for each patient.findingswhile 234 participants (group 1, 92.8%) did not require hospitalization during the study period, 18 patients (group 2, 7.2%) were hospitalized at least once for lrti during the rsv season. the rate of moderate - severe bronchopulmonary dysplasia (bpd) was significantly higher in group 2 compared to group 1 (38.9% vs 16.2% ; p=0.016). of the 18 infants who were hospitalized, 6 (33.3%) tested positive for rsv while the remaining 12 patients (66.7%) were negative for rsv. odds ratio (or) analysis of several risk factors revealed the presence of bpd (or : 3.28 ; 95%ci : 1.19 - 9), being from a family with low socioeconomic status (or : 3.64 ; 95%ci 1.08 - 12.3) to be associated with a higher likelihood of lrti - related hospitalization.conclusionour data demonstrated that rsv is an important lrti agent and cause of hospitalization especially in preterm infants with additional risks such as bpd, gestational age of < 28 weeks and low socioeconomic status. we suggest that improving care conditions and decreased bpd with prematurity would help in prevention of lrti hospitalization. |
clinical trials of disease - modifying treatments for ad have been struck by adverse events involving the immune system. in the clinical trial of the active antiamyloid (a) vaccine an1792, a small but notable number of patients developed meningoencephalitis, and treatment with the passive anti - a immunotherapy aab001 led to vasogenic edema in some individuals. the a immunogens that are now being used in the second generation of active immunization trials have been modified to reduce the risk of meningoencephalitis. however, academia, industry, and regulators are on their toes to identify immunological side effects as early as possible to avoid patient injury. therefore, it is of utmost importance to identify and exclude patients who fulfill clinical ad criteria but who already before the start of therapy show biochemical signs of neuroinflammation. typical laboratory findings in patients with neuroinflammatory / neuroinfectious conditions include impaired blood - brain barrier function, as reflected by an elevated ratio of albumin concentrations in cerebrospinal fluid (csf) and serum / plasma, csf monocytosis and igg and igm bands selectively in csf [3, 4 ]. we here present two clinical ad cases who were diagnosed with lyme neuroborreliosis (lnb) during screening for eligibility to enter an anti - a immunotherapy trial. should the two cases have been included and deteriorated, additional investigations might have led to the erroneous conclusion that therapy - induced meningoencephalitis had occurred. his health history is remarkable for a cholecystectomy in 2001 that was followed by confusion. an epileptic seizure was suspected and the patient was treated with carbamazepine for a short period. the patient has a positive family history of ad with both his grandfather and mother having been diagnosed with the disease. in 1995, no started to complain of cognitive decline that progressed over the years with aphasia, apraxia, and agnosia. he developed spatial deficits with orientation difficulties and a strong tendency to misplace belongings. in 2002, he was diagnosed with ad. a magnetic resonance imaging (mri) scan of the brain was normal with no atrophy or white matter changes. eeg showed slow postcentral rhythm (7 hz), and single positron emission computed tomography (spect) of the brain revealed frontotemporal hypoperfusion with left - side predominance. csf analyses showed slightly elevated albumin ratio of 12 (450). he was heterozygous for the alzheimer - associated 4 allele of the apolipoprotein e gene. neurological examination was normal, and the neuropsychological profile and clinical evaluation were consistent with ad. his cognition improved with the minimental state examination (mmse) score rising from 22 to 28 points. in november 2006, the patient was screened for a clinical trial of active immunization against a. his cognition had been rather stable over the four years on treatment with only a minor decrease of the mmse score from 28 to 25. an mri scan of the brain showed slight atrophy with mild white matter changes consistent with ad. a lumbar puncture was performed at baseline before inclusion in the trial, and the biomarkers for ad, t - tau, phospho - tau181 (p - tau181), and a1 - 42 were 520 (450) the albumin ratio was further elevated, and, surprisingly, csf monocyte counts were high and so was the igm - index (table 1). agarose gel electrophoresis of serum and csf followed by immunoblotting against igm supported ongoing intrathecal igm production. the patient was treated with standard doxycycline 200 mg twice a day for 10 days. he was then followed with repeated csf taps for 1.5 year during which his albumin ratio and cell counts were normalized. however, t - tau and p - tau181 remained pathologically elevated and a1 - 42 turned low indicating an ongoing ad process., she started to suffer from progressive cognitive deterioration, and in february 2006 she fulfilled clinical criteria for ad and started treatment with galantamine. the diagnosis was based on the anamnestic record and supported by findings on mri and spect, as well as neuropsychological test results. to further secure her diagnosis, a csf tap was planned but she declined this investigation. the patient was terrified of her diagnosis and wanted to fight it the best she could. she had heard of ongoing immunization trials at the karolinska university hospital and was referred for being evaluated for eligibility. as a part of this evaluation, the results showed completely normal t - tau, p - tau181, and a1 - 42 concentrations in csf speaking against ad. however, the albumin ratio was strongly elevated, csf monocyte counts high, and both the igg- and igm - indices elevated (table 1) with oligoclonal igg and igm bands selectively in csf as a further sign of intrathecal igg and igm production. she was treated with standard doxycycline 200 mg twice a day for 10 days. she was then followed with repeated csf taps for 1.5 year showing normalization of the blood - brain barrier function and cell counts. the case histories are of relevance both to clinicians who evaluate and treat patients with cognitive problems and also to the pharmaceutical industry that designs trials of drug candidates that might impact the immune system. for one of the two cases, bs, the spinal tap was essential with the csf test results radically changing the diagnosis from an incurable, ultimately fatal disease to a treatable spirochetal infection. no suffers from ad but during the disease process he contracted lnb that could have been mistaken for a side effect of the anti - a immunization should he have been included in the trial. lnb is a multisystem disease caused by infection with the spirochete borrelia burgdorferi and the most common infectious cause of chronic neuroinflammation. neurologic complications of lnb include encephalitis, meningitis, and dementia, as well as a broad range of peripheral neuropathies. the many symptoms of lnb may mimic neurodegenerative diseases, such as ad and amyotrophic lateral sclerosis (als). although there is no strong evidence that these diseases are caused by spirochetal infections, diagnosis of each must rule out the possibility of lnb. to the best of our knowledge, there are no published studies on the prevalence of lnb in patients attending a memory clinic. at least four studies have examined the prevalence of anti - borrelia burgdorferi antibodies in serum in ad cases and controls without finding evidence of an association with ad [69 ]. two cases of lnb were diagnosed in 1,089 consecutive patients (aged 2389 years) who underwent lumbar puncture as a part of the routine diagnostic evaluation between february 2001 and march 2007 at the specialized memory clinic in malm, but it should be noted that these patients were selected referral patients. the prevalence of intrathecal immunoglobulin production in ad patients ranges from 5 to 20% in different studies [1113 ]. in conclusion, analysis of csf taken from patients at baseline, before treatment, could be useful in clinical trials for identifying and excluding individuals with chronic infectious or inflammatory cns disorders that can mimic or aggravate the symptoms of ad. the inclusion of such cases in trials could result in the wrong conclusion that an adverse effect, such as encephalitis, was related to the drug being tested. baseline csf samples can also be used in comparisons with csf collected after treatment initiation. the benefit of such comparisons is that even minor inflammatory activation within the cns, as a result of adverse effects of the drug, can be identified. longitudinal csf sampling during the treatment period might indicate whether a certain drug induces harmful immune activation over the long term. considering csf analyses in a broader perspective, we think a spinal tap should be considered in every patient who seeks medical advice for cognitive problems, at least in areas where lnb is endemic and in particular if the clinical presentation is atypical. the safety and acceptability of the procedure support this view [10, 14, 15 ]. | due to side effects in the form of meningoencephalitis in the interrupted phase ii an1792 trial of active antiamyloid (a) immunization against alzheimer 's disease (ad), there has been concern that anti - a immunization may cause destructive neuroinflammation. here, we report on two patients fulfilling clinical ad criteria who were diagnosed with lyme neuroborreliosis during screening before inclusion in anti - a immunotherapy trials. the two cases illustrate the necessity of careful biochemical screening for neuroinflammatory / neuroinfectious conditions before an ad diagnosis is made and before clinical ad patients are included in trials of therapy that could impact the immune system. should the two cases have been included and deteriorated, additional investigations might have led to the erroneous conclusion that therapy - induced meningoencephalitis had occurred. |
the bioflavonoids hesperetin and naringenin are widely distributed in citrus fruits and juices as their glycosides, hesperidin and naringin, respectively. they are present in citrus aurantium as well as a wide variety of other citrus species 1. the sugar moieties for both flavonoids consist of rhamnose plus glucose, which must be removed in the intestinal tract before the flavonoids can be absorbed 2, 3. the absorption of these flavonoids in human subjects has been extensively studied and depends in part upon the forms in which they are ingested (juice or fruit, glycoside or aglycone, capsule, tablet, etc.) 4 - 8. experiments involving human subjects, animals and cell culture systems have shown that the two flavonoids exhibit a wide range of potentially beneficial physiological and biochemical effects. naringen and/or hesperidin or their aglycones have been shown to improve insulin sensitivity and glucose tolerance 9, prevent accumulation of triglycerides 9, inhibit cholesterol biosynthesis 10, 11, and serve as antioxidants and anti - inflammatory agents 12, 13 as well as hepatoprotectants 14 and neuroprotectants 15. however, the effects of naringin and hesperidin on metabolic rate and energy utilization have not been previously assessed. studies have suggested that p - synephrine has thermogenic and lipolytic activities 16 - 18, increasing energy metabolism 19, 20 as well as athletic performance 21. p - synephrine is the primary protoalkaloid in bitter orange extract which is derived from the immature fruits of citrus aurantium 22 - 25, and is widely distributed in other citrus species 22, 23, 26. the safety of p - synephrine and bitter orange extract has recently been reviewed 27, and based on the available studies in animals, humans and cell culture systems, it has been concluded that when taken orally and in recommended amounts both are safe. p - synephrine is widely used in combination with caffeine and other ingredients in products designed to support weight management 19, 28. however, no studies have been reported comparing the thermogenic effects of p - synephrine when combined with the flavonoids hesperidin and naringin. this pilot study was designed to examine these effects using a comparative effectiveness research (cer) study design 29 in a double - blinded, randomized, placebo - controlled protocol to compare the effects of p - synephrine alone and in combination with different amounts of hesperidin and naringin on : (1) resting metabolism, (2) blood pressure, (3) resting heart - rate, and (4) self - reported mood / energy levels in healthy human subjects. a total of 50 participants gave written informed consent in compliance with the helsinki declaration and approved by integrative health technologies ' ethics committee. participants fasted for 8 - 10 hours without consuming caffeinated beverages, nicotine, exercising or participating in vigorous physical activities. upon reporting to the research center after remaining seated in an isolated area for 10 - 15 min., participants completed measurements of their blood pressure, resting heart - rate and resting metabolism using micro life 's medgem indirect calorimeter (microlife medical home solutions, golden, co. 80401). the medgem is a hand - held, self - calibrating calorimeter that measures oxygen consumption (vo2) to determine resting metabolic rate (rmr). in conjunction with the study, test - retest reliabilities of the instrument were measured on 41 subjects from test - retest periods ranging from 1 - 17 days between tests. the average reliability coefficient was 90.2 %, a coefficient that was consistent over the four test - retest periods. p - synephrine was administered in the form of the patented bitter orange extract advantra zwhich contained 60 % of this active material. subsequent to rmr measurements, participants were randomly assigned to one of the five double - blinded treatment conditions in which they consumed one ounce of v-8 juice containing the following ingredient variations : group 1 : placebo (v-8 juice only) group 2 : advantra z (50 mg of p - synephrine) group 3 : advantra z with 0 mg hesperidin and 600 mg naringin group 4 : advantra z with 100 mg hesperidin and 600 mg naringin group 5 : advantra z with 1,000 mg hesperidin and 600 mg naringin after remaining seated and resting for 45 min. after 75 min., a third and final self report rating scale was completed, and measurements of blood pressure, heart - rate and rmr were determined. anovas were calculated between the groups ' baseline, ending and change scores for each treatment group in addition to dunnett 's t - test between each of the treatment groups and the placebo group. anovas were calculated between the groups ' baseline, ending and change scores for each treatment group in addition to dunnett 's t - test between each of the treatment groups and the placebo group. figure 1 shows the changes in resting metabolic rates (rmrs) from baseline expressed as kcals for each of the treatment groups (n=10 per group). as expected, a small decrease occurred in the rmr of the placebo group since these participants continued their 8 - 10 hour fast (group1). as compared to placebo, 50 mg p - synephrine (advantra z) alone (group 2) more than doubled consumption by 65 kcals over placebo. in group 3, adding 600 mg naringin to the 50 mg p - synephrine further increased calorie consumption more than three - fold to 122 kcals over placebo. the addition of 100 mg hesperidin to the p - synephrine plus naringin in group 4 resulted in a further increase in calorie consumption by 5-fold to 183 kcal over placebo. however, in group 5, when the amount of hesperidin consumed in conjunction with the 50 mg p - synephrine plus 600 mg naringin was increased to 1000 mg, the increase in calorie consumption over placebo was only 79 kcal, an increase between calorie consumption in the absence of hesperidin and in the presence of 100 mg hesperidin. in spite of the low levels of statistical power in these groups of only 10 subjects, the increases in rmr in group 4 was significantly greater than placebo (p=0.039) and advantra z alone (group 2). it is also worth noting the a repeated - measures students ' t - test revealed that the changes from baseline in group 3 and group 4 were both statistically significant (p=0.001 and 0.030, respectively). table 2 shows changes in blood pressures and resting heart - rates from baseline to the ending test for each of the five study groups. the average baseline blood pressures and resting heart - rates for the study cohort were well within the normal ranges of 121/74 mm hg and 70 bpm. at the conclusion of the study (75 min.), no increases in either systolic or diastolic blood pressures or heart rate were observed. none of the baseline - ending changes approached statistical significance nor were there any statistically significant differences between any of the five study groups. table 3 depicts the results of the self - report ratings for the 10 symptoms. as compared to the placebo group, none of the changes in these 10 self - reported symptoms were statistically significant either 45 or 75 min. from baseline. no significant effects relative to the placebo group were observed by the participants in the four treatment groups for symptoms including anxiety, hunger, tension, sleepiness, energy, nervousness, headache, upset stomach, concentration or general discomfort. using a double - blinded, randomized, placebo - controlled protocol, this study compared the effects of 50 mg p - synephrine (as advantra z) alone with three different amounts of hesperidin and naringin on : (1) resting metabolism rate, (2) blood pressure, (3) resting heart - rate, and (4) self - reported mood / energy levels in healthy human subjects. test - retest period since subjects continued the 8 - 10 hour fast they began prior to the start of the study (figure 1). the addition of p - synephrine, hesperidin, and naringin led to increases in rmr over placebo. however, while adding 100 mg of hesperidin in group 4 led to an increase in rmr over placebo, an unexpected reduction was observed in the rmr in group 5 which received 1000 mg hesperidin as compared to group 4. these data indicate that increasing the hesperidin from 100 mg to 1,000 mg reversed some of the thermogenic effects of 100 mg hesperidin in combination with 50 mg p - synephrine plus 600 mg naringin. it may be that in this case, more is not better, and increasing the amount of hesperidin constitutes an example of hormesis 30, 31. the increase in the metabolic rate between p - synephrine alone and the placebo control was approximately 6.9 %. this increase in thermogenesis is similar to the increases reported in previous studies involving p - synephrine 17, 18. the increase in rmr between group 4, the combination of p - synephrine with 600 mg naringin and 100 mg hesperidin, and the placebo control is approximately 17.7 %. if one assumes that the product was taken twice a day for one year, the theoretical increase in calorie consumption would amount to over 31 pounds. however, the actual extent of weight loss if the product was consumed under these conditions remains to be determined. the mechanism by which naringin and hesperidin increase the thermogenic effect of p - synephrine may involve enhancing the expression of adiponectin which is known to play a role in lipid and glucose metabolism. 32 have shown that naringenin and hesperetin up - regulate adiponectin expression, and both activate the peroxisome proliferator - activated receptor- (ppar) which has been recognized as the master regulator of adipocyte differentiation. in addition, naringenin also possessed significant ability to activate ppar 32, a major regulator of lipid metabolism in the liver. products with thermogenic properties are frequently associated with elevated blood pressures and heart - rates. p - synephrine is a phenylethylamine derivative with some structural similarities to ephedrine, and is thus assumed to exert cardiovascular effects by many authors [see for example, 20, 21, 33 - 35 ]. however, no effects on heart rate or blood pressure were observed in response to p - synephrine or p - synephrine in combination with naringin and hesperidin (table 2). p - synephrine differs from ephedrine in that it has a hydroxyl group on the para position of the benzene ring and lacks the methyl group on the side chain of the molecule. these structural differences greatly alter the pharmacokinetic and receptor binding properties of p - synephrine, resulting in little or no cardiovascular effects 27, as confirmed by this study. no adverse events have been directly attributable to bitter orange extract and p - synephrine 36, 37. however, caffeine is commonly present in products that contain p - synephrine 19, 28, and is well known to produce cardiovascular effects 38, particularly in caffeine - sensitive individuals 39. the absence of changes in blood pressure, resting heart - rate and self - ratings in the four treatment groups involving p - synephrine, naringin and hesperidin relative to the placebo group is very positive, and in conjunction with the increased thermogenesis may result in the development of beneficial products with respect to weight loss and weight management. the findings were further substantiated by consistency of the results across all four treatment groups. however, longer term studies are required to assess the amount of weight that is actually lost in response to these products, and to provide information concerning safety when used over an extended period of time. a problem faced in studies of thermogenic effects is that noticeable changes in the mood states listed in table 1 often make subjects aware of whether or not they are receiving a placebo, thus undermining blinding of the study. the lack of significant differences between the treatment groups and the control group with respect to the self - report ratings of symptoms increases confidence in the safety of the combination as well as in the blinding procedure used in this study. strengths of the study are the use of randomized placebo - controlled double - blinded study design, the absence of thermogenic changes in the placebo group, the absence of adverse effects on blood pressure, heart - rate and self - reports in the four treatment groups, the consistency of the effects of p - synephrine on metabolism in all treatment groups, and the increases in rmr in the treatment groups. the weakness of this study is the small number (10) of subjects in the placebo and in each of the treatment groups. the study also reveals an interesting effect with respect to hesperidin where an increased amount may lead to a masking of the thermogenic effects found with lesser amounts. this randomized double - blinded placebo control pilot study supports the safety and thermogenic effects of p - synephrine, particularly when combined with 600 mg of naringin and 100 mg of hesperidin. no increase in heart rate or blood pressure was observed over the 75 min. of the study nor were there any differences in a self - reported rating of 10 common symptoms between the treated and placebo groups. the data suggests the combination of the flavonoids naringin and hesperidin with p - synephrine may assist weight management. further studies are required to determine optimal doses as well as safety and efficacy associated with long term use. | bitter orange (citrus aurantium) extract is widely used in dietary supplements for weight management and sports performance. its primary protoalkaloid is p - synephrine. most studies involving bitter orange extract and p - synephrine have used products with multiple ingredients. the current study assessed the thermogenic effects of p - synephrine alone and in conjunction with the flavonoids naringin and hesperidin in a double - blinded, randomized, placebo - controlled protocol with 10 subjects per treatment group. resting metabolic rates (rmr), blood pressure, heart rates and a self - reported rating scale were determined at baseline and 75 min after oral ingestion of the test products in v-8 juice. a decrease of 30 kcal occurred in the placebo control relative to baseline. the group receiving p - synephrine (50 mg) alone exhibited a 65 kcal increase in rmr as compared to the placebo group. the consumption of 600 mg naringin with 50 mg p - synephrine resulted in a 129 kcal increase in rmr relative to the placebo group. in the group receiving 100 mg hesperidin in addition to the 50 mg p - synephrine plus 600 mg naringin, the rmr increased by 183 kcal, an increase that was statistically significant with respect to the placebo control (p<0.02). however, consuming 1000 mg hesperidin with 50 mg p - synephrine plus 600 mg naringin resulted in a rmr that was only 79 kcal greater than the placebo group. none of the treatment groups exhibited changes in heart rate or blood pressure relative to the control group, nor there were no differences in self - reported ratings of 10 symptoms between the treatment groups and the control group. this unusual finding of a thermogenic combination of ingredients that elevated metabolic rates without corresponding elevations in blood pressure and heart - rates warrants longer term studies to assess its value as a weight control agent. |
there is emerging evidence that time to biochemical failure is a robust prognostic factor for prostate cancer mortality and overall mortality in men receiving definitive radiotherapy [13 ]. interval to biochemical failure is also highly prognostic for prostate cancer mortality in men undergoing prostatectomy. one cause of early biochemical failure is occult micrometastatic disease present at time of treatment. regardless of efficacy of local treatment, individuals with occult metastases often have early recurrence with relatively poor prognosis, due to unappreciated stage iv disease at presentation. early biochemical failure can also occur in men with strictly localized disease, if local therapy is inadequate to provide even short term local control. this can be due to radioresistant local disease, inadequate radiation dose delivered, inadequate surgical margin or deficient radiation targeting. as with early failures, late failures can be caused either by more gradual progression of slow growing occult metastatic disease or gradual recurrence of partially treated local disease. in modern radiation therapy literature, there continues to be a relatively large number of late failures (> 4 years), even with dose escalation to 78 gy or over 80 gy [5, 6 ]. the purpose of this paper is to explore whether further dose escalation would lead to a decrease in late failures. if so, particularly for high risk patients, this might suggest that the preponderance of late failures in recent studies may represent incomplete ability to eradicate local disease rather than high incidence of slow growing occult metastases at presentation. in this paper, we examine a cohort of men who received high local radiation doses for clinically localized prostate cancer. consistently, high implant d90s with sizeable margins were utilized for all patients, and the majority of intermediate risk and almost all high risk patients received supplemental external beam radiation. our goal was to explore patterns in time to failure in men receiving high local radiation doses to better understand the role of improved local control and the implications for long term prostate cancer outcomes. we also hope to provide some insight into the question of what portion of patients (particularly high risk patients) are potentially curable, in effect, without viable metastatic disease at presentation. from march 1995 till july 2010, 2234 consecutive patients underwent definitive permanent interstitial brachytherapy by a single brachytherapist (gsm). all biopsy samples were reviewed by a single pathologist (ea) to minimize inconsistencies in pathologic grading. pre- planning technique, intraoperative approach and dosimetric evaluation have been described in detail [7, 8 ]. categorized by national comprehensive cancer network (nccn) risk group, the cohort consisted of 785 men with low risk, 1098 with intermediate risk, and 351 men with high risk disease. of the 2234 patients, 1111 (50.3%) received supplemental external beam radiotherapy and 748 (33.5%) received androgen deprivation therapy. supplemental external beam and androgen deprivation was utilized primarily for intermediate and high risk patients. supplemental external beam was delivered to 69.6% and 96.3% of intermediate and high risk patients, respectively. androgen deprivation therapy (adt) was used in 27.4% and 71.8% of intermediate and high risk patients. when employed, supplemental external beam radiotherapy was generally used in a dose of 45 - 50.4 gy covering prostate, seminal vesicles, and at - risk pelvic nodes. adt, when utilized, was initiated 3 months prior to implantation, and consisted of a luteinizing hormone - releasing hormone (lhrh) agonist and an anti - androgen. indications for adt were prostate cytoreduction prior to implant and for men with higher risk disease. for prostate implants, the target volume (ptv) was the prostate plus a periprostatic margin of 4 - 5 mm, except posteriorly to limit rectal dose. median prostate volume was 32.0 cm, while the median planning volume was 60.6 cm. the primary radionuclide utilized was pd (theragenics model 200), while the other radionuclide i (amersham model 6711 and its stranded form) was not implanted after 2003. the overall population mean d90 (minimum dose received by 90% of the ptv) was 119.1% of prescription dose. these relatively large d90 and v100 values have been associated with improved treatment outcomes [9, 10 ]. biologically effective dose (bed) was calculated using the algorithm recommended by american association of physicist in medicine (aapm) task group 137 to provide consistency in radiobiological comparisons between institutions. this consensus group recommends a uniform set of algorithms and radiobiological indices to facilitate consistency in routine outcome reporting. the task group values used in this work were : = 0.15 gy, = 0.05 gy, / = 3.0 gy, tpot = 42 days, and repair half - life = 16 min. the brachytherapy bed was based on the day 0 d90, and the total bed added any contribution from external beam radiation. patient demographics and treatment details are provided in table 1, which is stratified by national comprehensive cancer network (nccn) risk groups. patients were monitored by physical examination including digital rectal examination, and serum psa determination at 3 and 6 months intervals. patient survival was confirmed for all patients not only by psa reports, but also by frequent quality of life surveys (qol) (by telephone or occasionally mail) at least 25 times during the first five years following brachytherapy, and never less than twice per year thereafter. the primary outcome measures were time to biochemical failure, time to distant metastases, and time to prostate cancer death. we also assessed percent of men with biochemical failure who ultimately progressed to metastases or prostate cancer death, and the time from biochemical failure to those outcomes. patients with metastatic prostate cancer or castrate resistant disease without obvious metastases who died of any cause were classified as dead of prostate cancer. all other deaths were attributed to the immediate cause of death. in our population, 40% of deaths were attributable to cardiovascular disease, 33% to non - prostate cancer deaths, 8% to respiratory disease, 4% to prostate cancer, and 15% to other causes. clinical parameters of the study population, stratified by nccn risk classification psa values only for non - biochemical failures, n = 2131 based on 1431 with testosterone values nccn national comprehensive cancer network, iqr inter quartile range, psa prostate specific antigen, bmi body mass index, bed biologically effective dose based on brachytherapy day zero d90 any external beam, adt androgen deprivation therapy, xrt external beam radiation therapy continuous clinical and treatment variables were compared across groups, using one - way analysis of variance (anova). biochemical survival, metastases free survival, cause specific mortality, and all cause mortality were determined using cumulative incidence and cumulative survival curves. with the population stratified by low, intermediate, and high risk disease, biochemical failure rates were determined by competing risk analysis where non - prostate cancer death was the competitor. significant predictors for prostate specific, biochemical progression free, and overall survival were first determined by univariate regression analysis, and those variables with p 81 gy, the vast majority of failures occurred after four years, whether or not androgen deprivation was used. and overall treatment failure at ten years remained high at approximately 25% for intermediate risk patients and 45% for high risk patients. in high risk patients, the combination of moderate dose external beam radiation therapy, a brachytherapy boost and adt decreases biochemical failure and prostate cancer deaths, when compared to patients treated with external beam and adt. these findings demonstrate that some early failures and many late failures can be eliminated with dose escalation to approximately 80 gy, presumably because many of the failures at lower doses were related to inadequate local disease control. however, the preponderance of late failures even at dose levels of 78 - 86 gy, leaves open the question as to whether men in those studies had occult metastases at presentation or were potentially curable with even further dose escalation. with mean and median brachytherapy day 0 implant d90s of 119%, generous margins with the prostate volume expanded by a factor of 1.9 to the planning volume, and extensive use of supplemental external beam radiotherapy, the patients in our implant population received relatively high dose escalation. using the aapm radiobiological formalism, 86.4 gy of prostate external beam radiation has a bed of 131 gy, while the mean total bed for our population ranged from 133 gy for low risk men to 157 gy, and 164 gy in intermediate and high risk men, respectively (table 1). in patients with positive biopsies at the prostate base treated with brachytherapy monotherapy at m.d. anderson, the 10-year biochemical progression free survival as 93.5%, and all failures occurred prior to 6 years. a much larger population in a recent report by morris. on brachytherapy monotherapy in low and intermediate risk patients with median and maximum follow up of 7.5 years, and 13 years had 16% of failures occurring at 8 years. using only i seeds, their mean d90 was 151 gy which translates to an aapm bed of 116 gy. in our low to intermediate risk cohort, 58.6% (34/58) of failures were within 3 years, while only 2 patients failed after 8 years. our rate of early treatment failures is roughly similar to the rates in the more recent external beam dose escalation studies. however, the rate of late treatment failures is much lower in our series than in those studies. the recent dose escalated external beam series have a high proportion of treatment failures five or more years after treatment ; whereas 91% (84/92) of treatment failures in our series occurred within the first 5 years. this supports the hypothesis that in modern cohorts, even among men with high risk disease, the number of men who experience early failures and are as well, it would appear that most late treatment failures in current dose escalated external beam series are not the result of untreated occult metastases ; but more likely due to inadequate dose escalation. we view this optimistically, that with current combined modality dose escalation techniques, outcomes for high risk and intermediate risk patients can be significantly improved through a reduction of late failures. this builds on a line of reasoning dating back to fuks. in 1991, who found that even in men undergoing retropubic brachytherapy implants, durable local control led to a substantial reduction in development of metastases the strengths of this work are that low, intermediate and high risk patients were treated with a consistent implant philosophy and pattern of dose escalation, that the dose escalation parameters were well documented, and that no patients were lost to follow up. in general, lower risk patients received high quality brachytherapy - alone with day 0 d90s of approximately 120%. higher risk patients received supplemental external beam radiotherapy, followed by equally high quality implants with day 0 d90s of approximately 120%. one limitation is that this study was not a randomized trial comparing ttf between dose escalated brachytherapy with and without external beam radiotherapy versus dose escalated external beam radiotherapy alone. in addition, although the premise that early biochemical failures are due to subclinical distant metastases at diagnosis, this remains unproven due to the inability of current radiographic technologies to identify such early metastatic disease. nevertheless, based on the results above, we feel comfortable with the conclusion that there is a low burden of micro - metastatic disease at presentation in modern day patient cohorts similar to ours ; and that effective dose escalation with brachytherapy can lead to very successful treatment outcomes. the strengths of this work are that low, intermediate and high risk patients were treated with a consistent implant philosophy and pattern of dose escalation, that the dose escalation parameters were well documented, and that no patients were lost to follow up. in general, lower risk patients received high quality brachytherapy - alone with day 0 d90s of approximately 120%. higher risk patients received supplemental external beam radiotherapy, followed by equally high quality implants with day 0 d90s of approximately 120%. one limitation is that this study was not a randomized trial comparing ttf between dose escalated brachytherapy with and without external beam radiotherapy versus dose escalated external beam radiotherapy alone. in addition, although the premise that early biochemical failures are due to subclinical distant metastases at diagnosis, this remains unproven due to the inability of current radiographic technologies to identify such early metastatic disease. nevertheless, based on the results above, we feel comfortable with the conclusion that there is a low burden of micro - metastatic disease at presentation in modern day patient cohorts similar to ours ; and that effective dose escalation with brachytherapy can lead to very successful treatment outcomes. aggressive radiation dose escalation provided by brachytherapy for men with low, intermediate, and high risk disease leads to low levels of late treatment failures. this supports the hypothesis that the great majority of prostate cancer patients, even those with high risk disease are without micro - metastatic disease on presentation, and therefore are potentially curable with adequate dose escalation. | purposeto explore patterns of time to failure in men receiving high doses of permanent seed brachytherapy with or without external beam radiation therapy as a function of risk status.material and methodstwo thousand two hundred and thirty four patients were treated with prostate brachytherapy with median follow up of 8.0 years. the population was 35% low risk, 49% intermediate risk, and 16% high risk (nccn). median day 0 implant d90 was 119% and v100 was 98%. treatment failure was defined as psa > 0.40 ng / ml after nadir. rates of biochemical failure, distant metastases, and prostate cancer death were determined with non - prostate death as a competing risk.resultsfor all patients, the 10-year biochemical failure, distant metastases, and cause - specific mortality were 4.4%, 1.4%, and 1.3%, respectively. the biochemical failure rates were 1.3%, 4.8%, and 10.0% for men with low, intermediate, and high risk disease, respectively. median time to failure was 2.8 years. in men who died from prostate cancer, the median time from treatment failure to death was 4.2 years. overall, 83% of biochemical failures and 97% of metastases occurred within the first 4 years after treatment.conclusionswith the dose escalation achieved by high quality brachytherapy dosimetry, even high - risk prostate cancer patients have excellent long term biochemical outcomes. treatment failures occur early, and one third become metastatic and progress rapidly to prostate cancer death. the low frequency and pattern of failures suggest the presence of micrometastatic disease prior to treatment is rare, even in high risk patients. |
traditional systems of medicines have been in use for promotive, preventive, and curative health services since centuries in many parts of the world. being the oldest traditional system of medicine in india, ayurveda caters to about 80% of the population in developing countries as per the estimate of world health organization (who). despite their wide usage, research in this field ashes or bhasmas used in traditional system of medicine contain heavy metal particles in varying proportions. it is not easy to write off the usage of these preparations just by presuming that heavy metals are toxic. proper scientific documentation is required to validate the risks and benefits associated with use of such metallic ayurvedic preparations. there are some specific methods for their detoxification and bhasma preparation, making them suitable for clinical use in therapeutic doses, as claimed by rasa shastra experts. there is a need to ascertain whether the conventional shodhan (purification) process of ayurveda is being properly followed or not. silver is one among the heavy metals which is considered to be a non - essential accumulative trace element with wide distribution in the body, including the central nervous system, but with no known biological and physiological function. in ayurveda, preparations like raupya bhasma and kusta nukras have been used to treat many clinical conditions such as pain, inflammation, insomnia, neuralgias, anxiety disorders, convulsions, memory loss, heat stroke, infections, pro - myelocytic leukemias, sexual disorders, etc. metal bhasmas of gold (e.g., gold disodium thiomalate and auranofin) and silver (raupya bhasma, kusta nukras, etc.) have been used for the treatment of rheumatoid arthritis, acute pro - myelo cytic leukemias, immunostimulation, and as analgesics in painful inflammatory conditions, and are prescribed with accompaniments such as ginger or cumin water, tulsi extract, lemon extract, etc., that have been shown to protect against unwanted toxicity due to various reasons which include high proportions of trace elements and have synergistic or protective effects due to buffering between various constituents or free radical scavenging property. since the safety of ash of silver has already been established, reverse pharmacological studies are required to confirm the established facts regarding usage, safety, and efficacy in various clinical conditions mentioned above. since raw silver ore is considered hazardous for health as mentioned in ayurvedic literature, it needs to be converted into non - harmful form (ash of silver) by the process of trituration, pulverization, and repeated calcinations (at 300c) for 14 times. reduced form of silver thus obtained acquires spherical nanostructure with a size of 16 nm without any change in the morphology of silver, and is now called as ash of silver. nanosize of the silver particle is probably responsible for improving the penetration of silver in brain ; hence, ash of silver had been used in the past for treatment of various neurological conditions, viz. being a heavy metal preparation, ash of silver bears cumulative potential after prolonged use and in overdoses, as seen in preliminary animal studies. after certain controversial reports of toxicity due to use of metallic / elemental drugs, it has now been made mandatory (who guidelines) that ayurvedic drugs in any form should be tested for their heavy metal content prior to export, so that heavy metals remain within permissible limits. this study was conducted with an aim (a) to explore the sedative - hypnotic effect of ash of silver, if any, as claimed in ayurvedic literature. further, if ash of silver showed sedative - hypnotic effect, the study aimed (b) to observe whether the test drug ash of silver was potentiating the sedative - hypnotic effect of pentobarbitone at the doses used in mice when given 30 min prior to the standard drug. swiss albino mice of either sex weighing between 20 and 30 g were screened for the study, after obtaining approval from the institutional animal ethics committee. mice were fed on a standard pellet diet and water ad libitum, and were housed in polypropylene cages under similar environmental conditions in an animal room that was maintained at 24 1c and 55 5% humidity with a 12 h light the test drug ash of silver was procured from m / s baidyanath ayurved bhawan ltd (jhansi, india). ash of silver [50 mg / kg per os (p.o.) ] was suspended in 1% solution of gum acacia. gum acacia (1% p.o.), procured from arora pharmacy (new delhi, india), was labeled as cont rol and was administered in a volume of 1 ml/100 g. ash of silver and gum acacia were administered orally using infant feeding pipe with a 1 ml syringe attached at the other end. ] was procured from nembutal dainippon pharmaceutical co. (osaka, japan) and was administered as i.p. animals (mice) were divided into four groups consisting of six animals in each. study protocol was as follows : group 1 : received vehicle gum acacia (1% p.o.) as control, given in a volume of 1 ml/100 g p.o. group 2 : received pentobarbitone (50 mg / kg i.p.) as the standard drug group 3 : received the test drug ash of silver (50 mg / kg p.o.) suspended in 1% solution of gum acacia group 4 : received the test drug ash of silver (50 mg / kg p.o.) suspended in 1% solution of gum acacia, following which the standard drug pentobarbitone (50 mg / kg i.p.) was given after 30 min the responses of all drugs [in terms of time of onset, time of recovery, and total duration of loss of righting reflex (lorr) in mice ] were assessed by continuous observation of animals throughout the experiments from the time of administration of drug in the plexiglass chamber, using a stop watch. the duration of lorr was measured according to the procedures described by marley. when the mice became ataxic, they were placed on their backs on a pre - warmed (37c) pad and the onset, recovery, and total duration of lorr [starting at the time of administration of the test drug (ash of silver), the standard drug pentobarbitone (50 mg / kg), and the test drug (ash of silver) followed 30 min later by the standard drug pentobarbitone (50 mg / kg) ] were noted until they regained their righting reflexes. mice were presumed to have regained the righting response when they could right themselves three times within 30 sec. the test drug ash of silver was procured from m / s baidyanath ayurved bhawan ltd (jhansi, india). ash of silver [50 mg / kg per os (p.o.) ] was suspended in 1% solution of gum acacia. gum acacia (1% p.o.), procured from arora pharmacy (new delhi, india), was labeled as cont rol and was administered in a volume of 1 ml/100 g. ash of silver and gum acacia were administered orally using infant feeding pipe with a 1 ml syringe attached at the other end. ] was procured from nembutal dainippon pharmaceutical co. (osaka, japan) and was administered as i.p. animals (mice) were divided into four groups consisting of six animals in each. study protocol was as follows : group 1 : received vehicle gum acacia (1% p.o.) as control, given in a volume of 1 ml/100 g p.o. group 2 : received pentobarbitone (50 mg / kg i.p.) as the standard drug group 3 : received the test drug ash of silver (50 mg / kg p.o.) suspended in 1% solution of gum acacia group 4 : received the test drug ash of silver (50 mg / kg p.o.) suspended in 1% solution of gum acacia, following which the standard drug pentobarbitone (50 mg / kg i.p.) was given after 30 min the responses of all drugs [in terms of time of onset, time of recovery, and total duration of loss of righting reflex (lorr) in mice ] were assessed by continuous observation of animals throughout the experiments from the time of administration of drug in the plexiglass chamber, using a stop watch. the duration of lorr was measured according to the procedures described by marley. when the mice became ataxic, they were placed on their backs on a pre - warmed (37c) pad and the onset, recovery, and total duration of lorr [starting at the time of administration of the test drug (ash of silver), the standard drug pentobarbitone (50 mg / kg), and the test drug (ash of silver) followed 30 min later by the standard drug pentobarbitone (50 mg / kg) ] were noted until they regained their righting reflexes. mice were presumed to have regained the righting response when they could right themselves three times within 30 sec. effects of various drug treatments on sleep in mice all values were expressed as mean sem and analyzed using analysis of variance (anova) followed by dunnett 's t test. in the present study, we tried to explore the pharmacological effect of ash of silver as a sedative - hypnotic and its secondary effect to potentiate the sedative - hypnotic effect of pentobarbitone. such studies help to fast track drug discovery and development when carried out in selected animal models through screening. thus, ayurvedic knowledge and experimental database are able to provide new functional leads, thereby reducing the toxicity of drugs and saving time and money. in the present study, ash of silver was observed to possess sedative effect at a dose of 50 mg / kg (p.o.) in mice. the sedative effect was significant (p < 0.01) when compared with the vehicle gum acacia (1% p.o.) in mice. the sedative effect of the test drug ash of silver was significantly less (p < 0.01) compared to pentobarbitone (50 mg / kg i.p.), the standard drug (p < 0.001). significant potentiation (p < 0.001) of the sedative - hypnotic effect of pentobarbitone (50 mg / kg i.p.) was observed with 30 min prior administration of the test drug ash of silver (50 mg / kg p.o.). the above findings reveal that ash of silver had sedative effect at the doses used ; therefore, it can be proposed that ash of silver might be acting as a sedative - hypnotic owing to its pharmacological effects probably mediated by inhibition of neuropeptide s (nps) or n - methyl - d - aspartate (nmda)/histamine/5-ht3/dopamine or potentiation of effects mediated through gamma - aminobutyric acid (gaba)/glycine or benzodiazepines (bzds)/opioid receptors. nps was recently identified as the endogenous ligand of an orphan receptor, now referred to as the nps receptor. in vivo, nps produces a unique behavioral profile by increasing wakefulness and exerting anxiolytic - like effects. to explore the mechanism of action of ash of silver as a sedative, antagonists / blockers need to be administered against the above - mentioned mediators. in our previous study, we tried to explore the analgesic activity of ash of silver, which is probably mediated through opioid receptors as it was observed after administering naloxone, the opioid antagonist, although the role of other mediators can not be ruled out. one of the studies has proposed that ashes of heavy metals used in traditional systems of medicine function as a catalyzer by their presence in intestine, plasma, and blood, thereby acting as free radical scavengers. ash particles of heavy metals (gold, silver) in calcined form, being insoluble, exist as nanoparticles (16 nm), which are very tiny particles and biocompatible, and therefore can cross the blood brain barrier to exert various central actions as claimed in ayurvedic literature, viz. analgesic, anti - inflammatory, sedative, anti - anxiety, cognitive, neuroleptic, and antiepileptic. kim. have proved the distribution of nanosized silver particles in the central nervous system as well as in other tissues, e.g., liver, kidney and spleen, and intestine, when administered by the oral route. as the findings of our study have shown significant potentiation of sedative - hypnotic effect of pentobarbitone with 30 min prior administration of ash of silver at the dose used, it can be hypothesized that there is a possibility of ash of silver acting as hepatic cytochrome p450 microsomal enzyme inhibitor. further, it can be hypothesized that ash of silver increases the plasma levels of pentobarbitone by inhibiting its hepatic metabolism, so as to potentiate the sedative / anesthetic effect of pentobarbitone. further studies are required to confirm and establish this fact also by intracerebroventricular injection of ash of silver in the brain of mouse / rats. till date, hardly any studies have been conducted to explore the pharmacological effects of ash of silver on sedation / sleep in human body, in spite of its wide use in humans in ayurvedic practice for many centuries. studies conducted by nadeem. on silver preparations showed their interesting anti - anxiety, anti - cataleptic, and anti - aggressive effects. no scientific reports are available to confirm these claims except for some preliminary experimental studies demonstrating slight diminution of discharge frequency in frog nerve - muscle preparation bathed in 3% suspension of ash of silver. the anti - anxiety and anti - aggressive effects observed by nadeem. support the nerve - soothing properties (nervine tonic) of silver preparations. in one study, there is a mention of phytochelatins (pcs), produced from reduced glutathione present in green plants and legumes, which tend to chelate the heavy metals from soil. therefore, it can be postulated that in the presence of vegetarian diet, a fraction of ash of silver is also liable to get chelated if taken with vegetarian food. also, the same theory can be applied to overcome the toxic effects produced by excess dose of ash of silver, as mentioned in the study of inder. pharmacokinetic and pharmacodynamic studies need to be carried out to find if there is any interaction of ash of silver if taken with vegetarian diet, so as to formulate and revise the dose for human use. the role of free radical scavengers needs to be established, which can help to reduce the adverse effects of ash of silver. one of the studies has shown the interaction of ash of silver with some biomolecules, proteins, vitamins, etc., thus affecting various physiological reactions. from the observations and results of the study with the test drug ash of silver, it can be postulated that at the doses used in mice (50 mg / kg p.o.), it acts as a mild to moderate sedative and owing to this property, it might have potentiated the duration of lorr effect of pentobarbitone (50 mg / kg i.p.), i.e., synergistic effect. but as both these drugs are metabolized by the liver to a greater extent, inhibition of cytochrome p450 enzymes system in the liver by ash of silver can not be ruled out, which is probably responsible for inhibiting the hepatic metabolism of pentobarbitone resulting in high plasma levels of pentobarbitone, which might have potentiated the sedative - hypnotic effect of pentobarbitone at the doses used in mice. ash of silver possesses significant sedative hypnotic potential at a dose used in present animal study. therefore it can serve as a better alternative as sleep inducing drug with a better safety profile compared to conventional hypnotics. | ash of silver is used in traditional systems of medicine for various neurological conditions like insomnias, neuralgias, anxiety disorders, and convulsions. the present study was conducted to evaluate the sedative - hypnotic activity of ash of silver in comparison to pentobarbitone (standard drug) in albino mice. the mice were divided into four groups as follows : group 1 (control) : gum acacia [ga ; 1% per os (p.o.) ], group 2 (standard) : pentobarbitone [50 mg / kg intraperitoneal (i.p.) ], group 3 (test) : ash of silver (50 mg / kg p.o.), and group 4 : ash of silver (50 mg / kg p.o.) given 30 min prior to administration of pentobarbitone (50 mg / kg i.p.). time of onset, recovery, and total duration of loss of righting reflex were studied. ash of silver (test) produced significant sedation (p < 0.01) compared to control (ga 1%), but the effect was significantly less compared to that of standard pentobarbitone at the doses used. also, significant potentiation (p < 0.001) of the sedative - hypnotic effect of pentobarbitone was observed with the test drug. |
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